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Sample records for nadph dehydrogenase mediates

  1. A dehydrogenase-mediated recycling system of NADPH in plant peroxisomes.

    PubMed Central

    Corpas, F J; Barroso, J B; Sandalio, L M; Distefano, S; Palma, J M; Lupiáñez, J A; Del Río, L A

    1998-01-01

    The presence of the two NADP-dependent dehydrogenases of the pentose phosphate pathway has been investigated in plant peroxisomes from pea (Pisum sativum L.) leaves. Both enzymes, glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44), were present in the matrix of leaf peroxisomes, and their kinetic properties were studied. G6PDH and 6PGDH showed a typical Michaelis-Menten kinetic saturation curve, and had specific activities of 12.4 and 29.6 mU/mg protein, respectively. The Km values of G6PDH and 6PGDH for glucose 6-phosphate and for 6-phosphogluconate were 107.3 and 10.2 microM, respectively. Dithiothreitol did not inhibit G6PDH activity. By isoelectric focusing of peroxisomal matrices, the G6PDH activity was resolved into three isoforms with isoelectric points of 5.55, 5.30 and 4.85. The isoelectric point of peroxisomal 6PGDH was 5.10. Immunoblot analyses of peroxisomal matrix with an antibody against yeast G6PDH revealed a single cross-reactive band of 56 kDa. Post-embedment, EM immunogold labelling of G6PDH confirmed that this enzyme was localized in the peroxisomal matrices, the thylakoid membrane and matrix of chloroplasts, and the cytosol. The presence of the two oxidative enzymes of the pentose phosphate pathway in plant peroxisomes implies that these organelles have the capacity to reduce NADP+ to NADPH for its re-utilization in the peroxisomal metabolism. NADPH is particularly required for the ascorbate-glutathione cycle, which has been recently demonstrated in plant peroxisomes [Jiménez, Hernández, del Río and Sevilla (1997) Plant Physiol. 114, 275-284] and represents an important antioxidant protection system against H2O2 generated in peroxisomes. PMID:9480890

  2. Enzymic analysis of NADPH metabolism in beta-lactam-producing Penicillium chrysogenum: presence of a mitochondrial NADPH dehydrogenase.

    PubMed

    Harris, Diana M; Diderich, Jasper A; van der Krogt, Zita A; Luttik, Marijke A H; Raamsdonk, Léonie M; Bovenberg, Roel A L; van Gulik, Walter M; van Dijken, Johannes P; Pronk, Jack T

    2006-03-01

    Based on assumed reaction network structures, NADPH availability has been proposed to be a key constraint in beta-lactam production by Penicillium chrysogenum. In this study, NADPH metabolism was investigated in glucose-limited chemostat cultures of an industrial P. chrysogenum strain. Enzyme assays confirmed the NADP(+)-specificity of the dehydrogenases of the pentose-phosphate pathway and the presence of NADP(+)-dependent isocitrate dehydrogenase. Pyruvate decarboxylase/NADP(+)-linked acetaldehyde dehydrogenase and NADP(+)-linked glyceraldehyde-3-phosphate dehydrogenase were not detected. Although the NADPH requirement of penicillin-G-producing chemostat cultures was calculated to be 1.4-1.6-fold higher than that of non-producing cultures, in vitro measured activities of the major NADPH-providing enzymes were the same. Isolated mitochondria showed high rates of antimycin A-sensitive respiration of NADPH, thus indicating the presence of a mitochondrial NADPH dehydrogenase that oxidises cytosolic NADPH. The presence of this enzyme in P. chrysogenum might have important implications for stoichiometric modelling of central carbon metabolism and beta-lactam production and may provide an interesting target for metabolic engineering.

  3. Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation dr...

  4. Role of apoptosis-inducing factor, proline dehydrogenase, and NADPH oxidase in apoptosis and oxidative stress

    PubMed Central

    Natarajan, Sathish Kumar; Becker, Donald F

    2012-01-01

    Flavoproteins catalyze a variety of reactions utilizing flavin mononucleotide or flavin adenine dinucleotide as cofactors. The oxidoreductase properties of flavoenzymes implicate them in redox homeostasis, oxidative stress, and various cellular processes, including programmed cell death. Here we explore three critical flavoproteins involved in apoptosis and redox signaling, ie, apoptosis-inducing factor (AIF), proline dehydrogenase, and NADPH oxidase. These proteins have diverse biochemical functions and influence apoptotic signaling by unique mechanisms. The role of AIF in apoptotic signaling is two-fold, with AIF changing intracellular location from the inner mitochondrial membrane space to the nucleus upon exposure of cells to apoptotic stimuli. In the mitochondria, AIF enhances mitochondrial bioenergetics and complex I activity/assembly to help maintain proper cellular redox homeostasis. After translocating to the nucleus, AIF forms a chromatin degrading complex with other proteins, such as cyclophilin A. AIF translocation from the mitochondria to the nucleus is triggered by oxidative stress, implicating AIF as a mitochondrial redox sensor. Proline dehydrogenase is a membrane-associated flavoenzyme in the mitochondrion that catalyzes the rate-limiting step of proline oxidation. Upregulation of proline dehydrogenase by the tumor suppressor, p53, leads to enhanced mitochondrial reactive oxygen species that induce the intrinsic apoptotic pathway. NADPH oxidases are a group of enzymes that generate reactive oxygen species for oxidative stress and signaling purposes. Upon activation, NADPH oxidase 2 generates a burst of superoxide in neutrophils that leads to killing of microbes during phagocytosis. NADPH oxidases also participate in redox signaling that involves hydrogen peroxide-mediated activation of different pathways regulating cell proliferation and cell death. Potential therapeutic strategies for each enzyme are also highlighted. PMID:22593641

  5. Characteristics of external and internal NAD(P)H dehydrogenases in Hoya carnosa mitochondria.

    PubMed

    Hong, Hoang Thi Kim; Nose, Akihiro

    2012-12-01

    This study aims at characterizing NAD(P)H dehydrogenases on the inside and outside of the inner membrane of mitochondria of one phosphoenolpyruvate carboxykinase-crassulacean acid metabolism plant, Hoya carnosa. In crassulacean acid metabolism plants, NADH is produced by malate decarboxylation inside and outside mitochondria. The relative importance of mitochondrial alternative NADH dehydrogenases and their association was determined in intact-and alamethicin-permeabilized mitochondria of H. carnosa to discriminate between internal and external activities. The major findings in H. carnosa mitochondria are: (i) external NADPH oxidation is totally inhibited by DPI and totally dependent on Ca(2+), (ii) external NADH oxidation is partially inhibited by DPI and mainly dependent on Ca(2+), (iii) total NADH oxidation measured in permeabilized mitochondria is partially inhibited by rotenone and also by DPI, (iv) total NADPH oxidation measured in permeabilized mitochondria is partially dependent on Ca(2+) and totally inhibited by DPI. The results suggest that complex I, external NAD(P)H dehydrogenases, and internal NAD(P)H dehydrogenases are all linked to the electron transport chain. Also, the total measurable NAD(P)H dehydrogenases activity was less than the total measurable complex I activity, and both of these enzymes could donate their electrons not only to the cytochrome pathway but also to the alternative pathway. The finding indicated that the H. carnosa mitochondrial electron transport chain is operating in a classical way, partitioning to both Complex I and alternative Alt. NAD(P)H dehydrogenases.

  6. Mitochondrial type II NAD(P)H dehydrogenases in fungal cell death

    PubMed Central

    Gonçalves, A. Pedro; Videira, Arnaldo

    2015-01-01

    During aerobic respiration, cells produce energy through oxidative phosphorylation, which includes a specialized group of multi-subunit complexes in the inner mitochondrial membrane known as the electron transport chain. However, this canonical pathway is branched into single polypeptide alternative routes in some fungi, plants, protists and bacteria. They confer metabolic plasticity, allowing cells to adapt to different environmental conditions and stresses. Type II NAD(P)H dehydrogenases (also called alternative NAD(P)H dehydrogenases) are non-proton pumping enzymes that bypass complex I. Recent evidence points to the involvement of fungal alternative NAD(P)H dehydrogenases in the process of programmed cell death, in addition to their action as overflow systems upon oxidative stress. Consistent with this, alternative NAD(P)H dehydrogenases are phylogenetically related to cell death - promoting proteins of the apoptosis-inducing factor (AIF)-family. PMID:28357279

  7. Identification and Characterization of an Inducible NAD(P)H Dehydrogenase from Red Beetroot Mitochondria.

    PubMed Central

    Menz, R. I.; Day, D. A.

    1996-01-01

    Exogenous NADH oxidation of mitochondria isolated from red beetroots (Beta vulgaris L.) increased dramatically upon slicing and aging the tissue. Anion-exchange chromatography of soluble fractions derived by sonication from fresh and aged beetroot mitochondria yielded three NADH dehydrogenase activity peaks. The third peak from aged beetroot mitochondria was separated into two activities by blue-affinity chromatography. One of these (the unbound peak) readily oxidized dihydrolipoamide, whereas the other (the bound peak) did not. The latter was an NAD(P)H dehydrogenase with high quinone and ferricyanide reductase activity and was absent from fresh beet mitochondria. Further affinity chromatography of the NAD(P)H dehydrogenase indicated enrichment of a 58-kD polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that this 58-kD protein is the inducible, external NADH dehydrogenase. PMID:12226415

  8. Identification and Characterization of an Inducible NAD(P)H Dehydrogenase from Red Beetroot Mitochondria.

    PubMed

    Menz, R. I.; Day, D. A.

    1996-10-01

    Exogenous NADH oxidation of mitochondria isolated from red beetroots (Beta vulgaris L.) increased dramatically upon slicing and aging the tissue. Anion-exchange chromatography of soluble fractions derived by sonication from fresh and aged beetroot mitochondria yielded three NADH dehydrogenase activity peaks. The third peak from aged beetroot mitochondria was separated into two activities by blue-affinity chromatography. One of these (the unbound peak) readily oxidized dihydrolipoamide, whereas the other (the bound peak) did not. The latter was an NAD(P)H dehydrogenase with high quinone and ferricyanide reductase activity and was absent from fresh beet mitochondria. Further affinity chromatography of the NAD(P)H dehydrogenase indicated enrichment of a 58-kD polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that this 58-kD protein is the inducible, external NADH dehydrogenase.

  9. Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

    PubMed Central

    Henningsen, Brooks M.; Hon, Shuen; Covalla, Sean F.; Sonu, Carolina; Argyros, D. Aaron; Barrett, Trisha F.; Wiswall, Erin; Froehlich, Allan C.

    2015-01-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter−1 acetate during fermentation of 114 g liter−1 glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter−1, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter−1 and raised the ethanol yield to 7% above the wild-type level. PMID:26386051

  10. Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

    PubMed

    Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

    2015-12-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level.

  11. Purification and properties of an unusual UDP-glucose dehydrogenase, NADPH-dependent, from Xanthomonas albilineans.

    PubMed

    Blanch, María; Legaz, María-Estrella; Vicente, C

    2008-01-01

    Xanthomonas albilineans produces a UDP-glucose dehydrogenase growing on sucrose. The enzyme oxidizes UDP-glucose to UDP-glucuronic acid by using molecular oxygen and NADPH. Kinetics of enzymatic oxydation of NADPH is linearly dependent on the amount of oxygen supplied. The enzyme has been purified at homogeneity. The value of pI of the purified enzyme is 8.98 and its molecular mass has been estimated as about 14 kDa. The enzyme shows a michaelian kinetics for UDP-glucose concentrations. The value of K(m) for UDP-glucose is 0.87 mM and 0.26 mM for NADPH, although the enzyme has three different sites to interact with NADPH. The enzyme is inhibited by UDP-glucose concentrations higher than 1.3 mM. N-Terminal sequence has been determined as IQPYNH.

  12. Nitric oxide degradation by potato tuber mitochondria: evidence for the involvement of external NAD(P)H dehydrogenases.

    PubMed

    de Oliveira, Halley Caixeta; Wulff, Alfredo; Saviani, Elzira Elisabeth; Salgado, Ione

    2008-05-01

    The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O2(-)). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O2(-) generation, besides complex III, stimulated NO degradation. Larger amounts of O2(-) were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O2(-) production were stimulated by free Ca2+ concentration. Together, these results indicate that Ca2+-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O2(-) production that favors NO degradation in potato tuber mitochondria.

  13. Purification, Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase.

    PubMed Central

    Luethy, M. H.; Thelen, J. J.; Knudten, A. F.; Elthon, T. E.

    1995-01-01

    An NADH dehydrogenase activity from red beet (Beta vulgaris L.) root mitochondria was purified to a 58-kD protein doublet. An immunologically related dehydrogenase was partially purified from maize (Zea mays L. B73) mitochondria to a 58-kD protein doublet, a 45-kD protein, and a few other less prevalent proteins. Polyclonal antibodies prepared against the 58-kD protein of red beet roots were found to immunoprecipitate the NAD(P)H dehydrogenase activity. The antibodies cross-reacted to similar proteins in mitochondria from a number of plant species but not to rat liver mitochondrial proteins. The polyclonal antibodies were used in conjunction with maize mitochondrial fractionation to show that the 58-kD protein was likely part of a protein complex loosely associated with the membrane fraction. A membrane-impermeable protein cross-linking agent was used to further show that the majority of the 58-kD protein was located on the outer surface of the inner mitochondrial membrane or in the intermembrane space. Analysis of the cross-linked 58-kD NAD(P)H dehydrogenase indicated that specific proteins of 64, 48, and 45 kD were cross-linked to the 58-kD protein doublet. The NAD(P)H dehydrogenase activity was not affected by ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime] -tetraacetic acid or CaCl2, was stimulated somewhat (21%) by flavin mononucleotide, was inhibited by p-chloromercuribenzoic acid (49%) and mersalyl (40%), and was inhibited by a bud scale extract of Platanus occidentalis L. containing platanetin (61%). PMID:12228370

  14. Response of Chloroplast NAD(P)H Dehydrogenase-Mediated Cyclic Electron Flow to a Shortage or Lack in Ferredoxin-Quinone Oxidoreductase-Dependent Pathway in Rice Following Short-Term Heat Stress

    PubMed Central

    Essemine, Jemaa; Qu, Mingnan; Mi, Hualing; Zhu, Xin-Guang

    2016-01-01

    Cyclic electron flow (CEF) around photosystem I (PSI) can protect photosynthetic electron carriers under conditions of stromal over-reduction. The goal of the research reported in this paper was to investigate the responses of both PSI and photosystem II (PSII) to a short-term heat stress in two rice lines with different capacities of cyclic electron transfer, i.e., Q4149 with a high capacity (hcef) and C4023 with a low capacity (lcef). The absorbance change at 820 nm (ΔA820) was used here to assess the charge separation in the PSI reaction center (P700). The results obtained show that short-term heat stress abolishes the ferredoxin-quinone oxidoreductase (FQR)-dependent CEF in rice and accelerates the initial rate of P700+ re-reduction. The P700+ amplitude was slightly increased at a moderate heat-stress (35°C) because of a partial restriction of FQR but it was decreased following high heat-stress (42°C). Assessment of PSI and PSII activities shows that PSI is more susceptible to heat stress than PSII. Under high temperature, FQR-dependent CEF was completely removed and NDH-dependent CEF was up-regulated and strengthened to a higher extent in C4023 than in Q4149. Specifically, under normal growth temperature, hcef (Q4149) was characterized by higher FQR- and chloroplast NAD(P)H dehydrogenase (NDH)-dependent CEF rates than lcef (C4023). Following thermal stress, the activation of NDH-pathway was 130 and 10% for C4023 and Q4149, respectively. Thus, the NDH-dependent CEF may constitute the second layer of plant protection and defense against heat stress after the main route, i.e., FQR-dependent CEF, reaches its capacity. We discuss the possibility that under high heat stress, the NDH pathway serves as a safety valve to dissipate excess energy by cyclic photophosphorylation and overcome the stroma over-reduction following inhibition of CO2 assimilation and any shortage or lack in the FQR pathway. The potential role of the NDH-dependent pathway during the evolution

  15. Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria 1

    PubMed Central

    Luethy, Michael H.; Hayes, Marianne K.; Elthon, Thomas E.

    1991-01-01

    Mitochondria isolated from the taproot of beet (Beta vulgaris) were used in an effort to identify and partially purify the proteins constituting the exogenous NADH dehydrogenase. Three NAD(P)H dehydrogenases are released from these mitochondria by sonication, and these enzymes were partially purified using fast protein liquid chromatography. One of the enzymes, designated peak I, is capable of oxidizing NADPH and the β form of NADH. The other two activities, peaks II and III, oxidize only β-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component to peak I is a polypeptide with an apparent molecular mass of approximately 42 kilodaltons. Peak I activity was insensitive to platanetin, a specific inhibitor of the exogenous dehydrogenase, and insensitive to added Ca2+ or Mg2+. Peak I displayed a broad pH activity profile with an optimum between 7.5 and 8.0 for both NADPH and NADH. Purified peak II gave a single polypeptide of about 32 kilodaltons, had a pH optimum between 7.0 and 7.5, and was slightly stimulated by Ca2+ and Mg2+. As with peak I, platanetin had no effect on peak II activity. Peak III was not purified completely, but contained two major polypeptides with apparent molecular masses of 55 and 40 kilodaltons. This enzyme was not affected by Ca2+ and Mg2+, but was inhibited by platanetin. The peak III enzyme had a rather sharp pH optimum of approximately 6.5 to 6.6. The above data indicate that peak III activity is likely the exogenous NADH dehydrogenase. ImagesFigure 2Figure 3 PMID:16668549

  16. Glucose regulates enzymatic sources of mitochondrial NADPH in skeletal muscle cells; a novel role for glucose-6-phosphate dehydrogenase.

    PubMed

    Mailloux, Ryan J; Harper, Mary-Ellen

    2010-07-01

    Reduced nicotinamide adenine dinucleotide (NADPH) is a functionally important metabolite required to support numerous cellular processes. However, despite the identification of numerous NADPH-producing enzymes, the mechanisms underlying how the organellar pools of NADPH are maintained remain elusive. Here, we have identified glucose-6-phosphate dehydrogenase (G6PDH) as an important source of NADPH in mitochondria. Activity analysis, submitochondrial fractionation, fluorescence microscopy, and protease sensitivity assays revealed that G6PDH is localized to the mitochondrial matrix. 6-ANAM, a specific G6PDH inhibitor, depleted mitochondrial NADPH pools and increased oxidative stress revealing the importance of G6PDH in NADPH maintenance. We also show that glucose availability and differences in metabolic state modulate the enzymatic sources of NADPH in mitochondria. Indeed, cells cultured in high glucose (HG) not only adopted a glycolytic phenotype but also relied heavily on matrix-associated G6PDH as a source of NADPH. In contrast, cells exposed to low-glucose (LG) concentrations, which displayed increased oxygen consumption, mitochondrial metabolic efficiency, and decreased glycolysis, relied predominantly on isocitrate dehydrogenase (ICDH) as the principal NADPH-producing enzyme in the mitochondria. Culturing glycolytic cells in LG for 48 h decreased G6PDH and increased ICDH protein levels in the mitochondria, further pointing to the regulatory role of glucose. 2-Deoxyglucose treatment also prevented the increase of mitochondrial G6PDH in response to HG. The role of glucose in regulating enzymatic sources of mitochondrial NADPH pool maintenance was confirmed using human myotubes from obese adults with a history of type 2 diabetes mellitus (post-T2DM). Myotubes from post-T2DM participants failed to increase mitochondrial G6PDH in response to HG in contrast to mitochondria in myotubes from control participants (non-T2DM). Hence, we not only identified a matrix

  17. Aqueous soluble tetrazolium/formazan MTS as an indicator of NADH- and NADPH-dependent dehydrogenase activity.

    PubMed

    Dunigan, D D; Waters, S B; Owen, T C

    1995-10-01

    Recently a new tetrazolium was described for the use of monitoring cell viability in culture. This tetrazolium, commonly referred to as MTS [3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], has the unusual property that it can be reduced to a water-soluble formazan. beta-Nicotinamide adenine dinucleotide/reduced (NADH) and beta-nicotinamide adenine dinucleotide phosphate/reduced (NADPH) are examples of physiologically important reducing agents. In cell-free studies, MTS was reduce to the soluble formazan in the presence of NADH and NADPH, and reaction were compared to those with dithiothreitol (DTT) or 2-mercaptoethanol (2-ME). The efficiency of these reactions was enhanced 1000-fold by the presence of phenazine methosulfate. Selectivity in the electron transfer from NADPH was slightly greater than NADH, and NADPH or NADH was much greater than the thiols DTT or 2-ME. Generation of either NADH or NADPH in solution by malate dehydrogenase or isocitrate dehydrogenase, respectively, was monitored by the MTS reduction reaction. The rate of formazan formation was comparable to the formation of NADH or NADPH. This system represents a useful tool for evaluating reaction kinetics in solutions of NAD- or NADP-dependent dehydrogenase enzymes, and these reactions can be performed in typical biological buffers containing reducing agents without significant interference to the MTS/formazan system.

  18. The Ca2+-Regulation of the Mitochondrial External NADPH Dehydrogenase in Plants Is Controlled by Cytosolic pH.

    PubMed

    Hao, Meng-Shu; Jensen, Anna M; Boquist, Ann-Sofie; Liu, Yun-Jun; Rasmusson, Allan G

    2015-01-01

    NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca2+ and pH in vitro, but concentrations of Ca2+ needed are not known. We have determined the K0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O2 and decylubiquinone as electron acceptors. The K0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K0.5(Ca2+) for NADPH oxidation was high (≈100 μM), yet 20-fold lower K0.5(Ca2+) values were determined at pH 6.8. Lower K0.5(Ca2+) values were observed with decylubiquinone than with O2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene.

  19. The Ca2+-Regulation of the Mitochondrial External NADPH Dehydrogenase in Plants Is Controlled by Cytosolic pH

    PubMed Central

    Hao, Meng-Shu; Jensen, Anna M.; Boquist, Ann-Sofie; Liu, Yun-Jun; Rasmusson, Allan G.

    2015-01-01

    NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca2+ and pH in vitro, but concentrations of Ca2+ needed are not known. We have determined the K0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O2 and decylubiquinone as electron acceptors. The K0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K0.5(Ca2+) for NADPH oxidation was high (≈100 μM), yet 20-fold lower K0.5(Ca2+) values were determined at pH 6.8. Lower K0.5(Ca2+) values were observed with decylubiquinone than with O2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene. PMID:26413894

  20. Antimalarial NADPH-Consuming Redox-Cyclers As Superior Glucose-6-Phosphate Dehydrogenase Deficiency Copycats

    PubMed Central

    Bielitza, Max; Belorgey, Didier; Ehrhardt, Katharina; Johann, Laure; Lanfranchi, Don Antoine; Gallo, Valentina; Schwarzer, Evelin; Mohring, Franziska; Jortzik, Esther; Williams, David L.; Becker, Katja; Arese, Paolo; Elhabiri, Mourad

    2015-01-01

    Abstract Aims: Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum were shown to protect G6PD-deficient populations from severe malaria. Here, we investigated the mechanism of a novel antimalarial series, namely 3-[substituted-benzyl]-menadiones, to understand whether these NADPH-consuming redox-cyclers, which induce oxidative stress, mimic the natural protection of G6PD deficiency. Results: We demonstrated that the key benzoylmenadione metabolite of the lead compound acts as an efficient redox-cycler in NADPH-dependent methaemoglobin reduction, leading to the continuous formation of reactive oxygen species, ferrylhaemoglobin, and subsequent haemichrome precipitation. Structure–activity relationships evidenced that both drug metabolites and haemoglobin catabolites contribute to potentiate drug effects and inhibit parasite development. Disruption of redox homeostasis by the lead benzylmenadione was specifically induced in Plasmodium falciparum parasitized erythrocytes and not in non-infected cells, and was visualized via changes in the glutathione redox potential of living parasite cytosols. Furthermore, the redox-cycler shows additive and synergistic effects in combination with compounds affecting the NADPH flux in vivo. Innovation: The lead benzylmenadione 1c is the first example of a novel redox-active agent that mimics the behavior of a falciparum parasite developing inside a G6PD-deficient red blood cell (RBC) giving rise to malaria protection, and it exerts specific additive effects that are inhibitory to parasite development, without harm for non-infected G6PD-sufficient or -deficient RBCs. Conclusion: This strategy offers an innovative perspective for the development of future antimalarial drugs for G6PD-sufficient and -deficient populations. Antioxid. Redox Signal. 22, 1337–1351. PMID:25714942

  1. NADPH-dependent glutamate dehydrogenase in Penicillium chrysogenum is involved in regulation of beta-lactam production.

    PubMed

    Thykaer, Jette; Rueksomtawin, Kanchana; Noorman, Henk; Nielsen, Jens

    2008-04-01

    The interactions between the ammonium assimilatory pathways and beta-lactam production were investigated by disruption of the NADPH-dependent glutamate dehydrogenase gene (gdhA) in two industrial beta-lactam-producing strains of Penicillium chrysogenum. The strains used were an adipoyl-7-ADCA- and a penicillin-producing strain. The gdhA gene disruption caused a decrease in maximum specific growth rate of 26 % and 35 % for the adipoyl-7-ADCA-producing strain and the penicillin-producing strain, respectively, compared to the corresponding reference strains. Interestingly, no beta-lactam production was detected in either of the DeltagdhA strains. Supplementation with glutamate restored growth but no beta-lactam production was detected for the constructed strains. Cultures with high ammonium concentrations (repressing conditions) and with proline as nitrogen source (de-repressed conditions) showed continued beta-lactam production for the reference strains whereas the DeltagdhA strains remained non-productive under all conditions. By overexpressing the NAD-dependent glutamate dehydrogenase, the specific growth rate could be restored, but still no beta-lactam production was detected. The results indicate that the NADPH-dependent glutamate dehydrogenase may be directly or indirectly involved in the regulation of beta-lactam production in industrial strains of P. chrysogenum.

  2. Molecular and structural characterization of NADPH-dependent d-glycerate dehydrogenase from the enteric parasitic protist Entamoeba histolytica.

    PubMed Central

    Ali, Vahab; Shigeta, Yasuo; Nozaki, Tomoyoshi

    2003-01-01

    Putative NADPH-dependent GDH (L-glycerate dehydrogenase) of the protozoan parasite Entamoeba histolytica (EhGDH) has been characterized. The EhGDH gene encodes a protein of 318 amino acids with a calculated isoelectric point of 6.29 and a molecular mass of 35.8 kDa. EhGDH showed highest identities with GDH from epsilon-proteobacteria. This close kinship was also supported by phylogenetic analyses, suggesting possible lateral transfer of the gene from epsilon-proteobacteria to E. histolytica. In contrast with the implications from protein alignment and phylogenetic analysis, kinetic studies revealed that the amoebic GDH showed biochemical properties similar to those of mammalian GDH, i.e. a preference for NADPH as cofactor and higher affinities towards NADPH and beta-hydroxypyruvate than towards NADP+ and L-glycerate. Whereas the amino acids involved in nucleotide binding and catalysis are totally conserved in EhGDH, substitution of a negatively charged amino acid with a non-charged hydroxy-group-containing amino acid is probably responsible for the observed high affinity of EhGDH for NADP+/NADPH. In addition, the amoebic GDH, dissimilar to the bacterial and mammalian GDHs, lacks glyoxylate reductase activity. Native and recombinant EhGDH showed comparable subunit structure, kinetic parameters and elution profiles on anion-exchange chromatography. We propose that the GDH enzyme is likely to be involved in regulation of the intracellular concentration of serine, and, thus, also in controlling cysteine biosynthesis located downstream of serine metabolic pathways in this protist. PMID:12877657

  3. Improved NADPH Regeneration for Fungal Cytochrome P450 Monooxygenase by Co-Expressing Bacterial Glucose Dehydrogenase in Resting-Cell Biotransformation of Recombinant Yeast.

    PubMed

    Jeon, Hyunwoo; Durairaj, Pradeepraj; Lee, Dowoo; Ahsan, Md Murshidul; Yun, Hyungdon

    2016-12-28

    Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19+FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at 30°C. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

  4. Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase

    PubMed Central

    Köpke, Michael; Gerth, Monica L.; Maddock, Danielle J.; Mueller, Alexander P.; Liew, FungMin

    2014-01-01

    Acetogenic bacteria use CO and/or CO2 plus H2 as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogen Clostridium autoethanogenum is known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize the C. autoethanogenum enzymes for lactate and 2,3-butanediol biosynthesis. The putative C. autoethanogenum lactate dehydrogenase was active when expressed in Escherichia coli. The 2,3-butanediol pathway was reconstituted in E. coli by cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resulting E. coli strain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 μM h−1 optical density unit−1), which is comparable to the level produced by C. autoethanogenum during growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, that C. autoethanogenum can act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway from C. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential of C. autoethanogenum as a platform for sustainable chemical production. PMID:24657865

  5. Disruption of the NADPH-dependent glutamate dehydrogenase affects the morphology of two industrial strains of Penicillium chrysogenum.

    PubMed

    Thykaer, Jette; Rueksomtawin, Kanchana; Noorman, Henk; Nielsen, Jens

    2009-02-23

    New morphological aspects of Penicillium chrysogenum were found during physiological characterisation of two NADPH-dependent glutamate dehydrogenase mutant strains. A morphological characterisation of the previously constructed strains, together with the two beta-lactam producing industrial recipient strains, was conducted. The reference strains showed a compact structure with highly branched hyphal elements whereas the morphology of the DeltagdhA strains consisting of long elongated hyphal elements with few branches. On solid medium, the hyphal growth unit (length) increased from an average of 47 microm tip(-1) in the reference strains to 117 microm tip(-1) in the DeltagdhA strains and in submerged cultures a decrease of 18% in branching frequency was measured due to the gdhA deletion. P. chrysogenum Wis 54-1255, the ancestor of most production strains was also characterised and this strain showed morphology similar to the industrial strains. Interestingly, the constructed strains showed morphology similar to wild type Aspergillus nidulans another species carrying the penicillin biosynthetic cluster. Thus, the results showed that elimination of glutamate dehydrogenase activity in high producing strains of P. chrysogenum has a radical impact on morphology.

  6. Glucose-6-Phosphate Dehydrogenase Enhances Antiviral Response through Downregulation of NADPH Sensor HSCARG and Upregulation of NF-κB Signaling

    PubMed Central

    Wu, Yi-Hsuan; Chiu, Daniel Tsun-Yee; Lin, Hsin-Ru; Tang, Hsiang-Yu; Cheng, Mei-Ling; Ho, Hung-Yao

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD)-deficient cells are highly susceptible to viral infection. This study examined the mechanism underlying this phenomenon by measuring the expression of antiviral genes—tumor necrosis factor alpha (TNF-α) and GTPase myxovirus resistance 1 (MX1)—in G6PD-knockdown cells upon human coronavirus 229E (HCoV-229E) and enterovirus 71 (EV71) infection. Molecular analysis revealed that the promoter activities of TNF-α and MX1 were downregulated in G6PD-knockdown cells, and that the IκB degradation and DNA binding activity of NF-κB were decreased. The HSCARG protein, a nicotinamide adenine dinucleotide phosphate (NADPH) sensor and negative regulator of NF-κB, was upregulated in G6PD-knockdown cells with decreased NADPH/NADP+ ratio. Treatment of G6PD-knockdown cells with siRNA against HSCARG enhanced the DNA binding activity of NF-κB and the expression of TNF-α and MX1, but suppressed the expression of viral genes; however, the overexpression of HSCARG inhibited the antiviral response. Exogenous G6PD or IDH1 expression inhibited the expression of HSCARG, resulting in increased expression of TNF-α and MX1 and reduced viral gene expression upon virus infection. Our findings suggest that the increased susceptibility of the G6PD-knockdown cells to viral infection was due to impaired NF-κB signaling and antiviral response mediated by HSCARG. PMID:26694452

  7. Redox-mediated signal transduction by cardiovascular Nox NADPH oxidases.

    PubMed

    Brandes, Ralf P; Weissmann, Norbert; Schröder, Katrin

    2014-08-01

    The only known function of the Nox family of NADPH oxidases is the production of reactive oxygen species (ROS). Some Nox enzymes show high tissue-specific expression and the ROS locally produced are required for synthesis of hormones or tissue components. In the cardiovascular system, Nox enzymes are low abundant and function as redox-modulators. By reacting with thiols, nitric oxide (NO) or trace metals, Nox-derived ROS elicit a plethora of cellular responses required for physiological growth factor signaling and the induction and adaptation to pathological processes. The interactions of Nox-derived ROS with signaling elements in the cardiovascular system are highly diverse and will be detailed in this article, which is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System".

  8. Enhanced production of epsilon-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant Escherichia coli harboring cyclohexanone monooxygenase gene.

    PubMed

    Lee, Won-Heong; Park, Jin-Byung; Park, Kyungmoon; Kim, Myoung-Dong; Seo, Jin-Ho

    2007-08-01

    Whole-cell conversion of cyclohexanone to epsilon-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and epsilon-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to epsilon-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of epsilon-caprolactone from cyclohexanone. A glucose-limited fed-batch Baeyer-Villiger oxidation where a cyclohexanone level was maintained less than 6 g/l resulted in a maximum epsilon-caprolactone concentration of 11.0 g/l. The maximum epsilon-caprolactone concentration was improved further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in epsilon-caprolactone concentration compared with the control experiment performed under the same conditions.

  9. Oxidation of guaiacol by myeloperoxidase: a two-electron-oxidized guaiacol transient species as a mediator of NADPH oxidation.

    PubMed Central

    Capeillère-Blandin, C

    1998-01-01

    The present study was first aimed at a complete steady-state kinetic analysis of the reaction between guaiacol (2-methoxyphenol) and the myeloperoxidase (MPO)/H2O2 system, including a description of the isolation and purification of MPO from human polymorphonuclear neutrophil cells. Secondly, the overall reaction of the oxidation of NADPH, mediated by the reactive intermediates formed from the oxidation of guaiacol in the MPO/H2O2 system, was analysed kinetically. The presence of guaiacol stimulates the oxidation of NADPH by the MPO/H2O2 system in a concentration-dependent manner. Concomitantly, the accumulation of biphenoquinone (BQ), the final steady-state product of guaiacol oxidation, is lowered, and even inhibited completely, at high concentrations of NADPH. Under these conditions, the stoichiometry of NADPH:H2O2 is 1, and the oxidation rate of NADPH approximates to that of the rate of guaiacol oxidation by MPO. The effects of the presence of superoxide dismutase, catalase and of anaerobic conditions on the overall oxidation of NADPH have also been examined, and the data indicated that superoxide formation did not occur. The final product of NADPH oxidation was shown to be enzymically active NADP+, while guaiacol was generated continuously from the reaction between NADPH and oxidized guaiacol product. In contrast, similar experiments performed on the indirect, tyrosine-mediated oxidation of NADPH by MPO showed that a propagation of the free radical chain was occurring, with generation of both O2(-.) and H2O2. BQ, in itself, was able to spontaneously oxidize NADPH, but neither the rate nor the stoichiometry of the reaction could account for the NADPH-oxidation process involved in the steady-state peroxidation cycle. These results provide evidence that the oxidation of NADPH does not involve a free nucleotide radical intermediate, but that this is probably due to a direct electron-transfer reaction between NADPH and a two-electron-oxidized guaiacol intermediate

  10. Leflunomide induces NAD(P)H quinone dehydrogenase 1 enzyme via the aryl hydrocarbon receptor in neonatal mice.

    PubMed

    Shrestha, Amrit Kumar; Patel, Ananddeep; Menon, Renuka T; Jiang, Weiwu; Wang, Lihua; Moorthy, Bhagavatula; Shivanna, Binoy

    2017-03-25

    Aryl hydrocarbon receptor (AhR) has been increasingly recognized to play a crucial role in normal physiological homeostasis. Additionally, disrupted AhR signaling leads to several pathological states in the lung and liver. AhR activation transcriptionally induces detoxifying enzymes such as cytochrome P450 (CYP) 1A and NAD(P)H quinone dehydrogenase 1 (NQO1). The toxicity profiles of the classical AhR ligands such as 3-methylcholanthrene and dioxins limit their use as a therapeutic agent in humans. Hence, there is a need to identify nontoxic AhR ligands to develop AhR as a clinically relevant druggable target. Recently, we demonstrated that leflunomide, a FDA approved drug, used to treat rheumatoid arthritis in humans, induces CYP1A enzymes in adult mice via the AhR. However, the mechanisms by which this drug induces NQO1 in vivo are unknown. Therefore, we tested the hypothesis that leflunomide will induce pulmonary and hepatic NQO1 enzyme in neonatal mice via AhR-dependent mechanism(s). Leflunomide elicited significant induction of pulmonary CYP1A1 and NQO1 expression in neonatal mice. Interestingly, the dose at which leflunomide increased NQO1 was significantly higher than that required to induce CYP1A1 enzyme. Likewise, it also enhanced hepatic CYP1A1, 1A2 and NQO1 expression in WT mice. In contrast, leflunomide failed to induce these enzymes in AhR-null mice. Our results indicate that leflunomide induces pulmonary and hepatic CYP1A and NQO1 enzymes via the AhR in neonatal mice. These findings have important implications to prevent and/or treat disorders such as bronchopulmonary dysplasia in human infants where AhR may play a crucial role in the disease pathogenesis.

  11. NADPH oxidases regulate septin-mediated cytoskeletal remodeling during plant infection by the rice blast fungus

    PubMed Central

    Ryder, Lauren S.; Dagdas, Yasin F.; Mentlak, Thomas A.; Kershaw, Michael J.; Thornton, Christopher R.; Schuster, Martin; Chen, Jisheng; Wang, Zonghua; Talbot, Nicholas J.

    2013-01-01

    The rice blast fungus Magnaporthe oryzae infects plants with a specialized cell called an appressorium, which uses turgor to drive a rigid penetration peg through the rice leaf cuticle. Here, we show that NADPH oxidases (Nox) are necessary for septin-mediated reorientation of the F-actin cytoskeleton to facilitate cuticle rupture and plant cell invasion. We report that the Nox2–NoxR complex spatially organizes a heteroligomeric septin ring at the appressorium pore, required for assembly of a toroidal F-actin network at the point of penetration peg emergence. Maintenance of the cortical F-actin network during plant infection independently requires Nox1, a second NADPH oxidase, which is necessary for penetration hypha elongation. Organization of F-actin in appressoria is disrupted by application of antioxidants, whereas latrunculin-mediated depolymerization of appressorial F-actin is competitively inhibited by reactive oxygen species, providing evidence that regulated synthesis of reactive oxygen species by fungal NADPH oxidases directly controls septin and F-actin dynamics. PMID:23382235

  12. Benzylidenemalononitrile derivatives as substrates and inhibitors of a new NAD(P)H dehydrogenase of erythrocytes. Purification and crystallisation of two forms of the enzyme.

    PubMed

    Ueberschär, K H; Kille, S; Laule, G; Maurer, P; Wallenfels, K

    1979-10-01

    Using the powerful lachrymator (2-chlorobenzylidene)malononitrile as electron acceptor, two types of NAD(P)H dehydrogenases have been isolated from human blood. Crystallisation of the homogenous enzymes was performed in 50% polyethylene glycol solution. The enzymes (average molecular weight 18 000) are composed of only one polypeptide chain and have a very similar amino acid composition. B-side stereospecificity was determined with respect to the cofactor by gas chromatography-mass spectrometry for the reductase. Besides (2-chlorobenzylidene)malononitrile, 2,6-dichloroindophenol, methylene blue, 4-benzoquinone, FMN and FAD are also reduced using NADH or NADPH as hydrogen donor with the rates decreasing in the given order. Reduction of methemoglobin is observed only upon addition of methylene blue, FMN or FAD as carriers. (2-Chlorobenzylidene)malononitrile reduction is inhibited by most of the compounds known to be decouplers of oxidative phosphorylation.

  13. Boost in bioethanol production using recombinant Saccharomyces cerevisiae with mutated strictly NADPH-dependent xylose reductase and NADP(+)-dependent xylitol dehydrogenase.

    PubMed

    Khattab, Sadat Mohammad Rezq; Saimura, Masayuki; Kodaki, Tsutomu

    2013-06-10

    The xylose-fermenting recombinant Saccharomyces cerevisiae and its improvement have been studied extensively. The redox balance between xylose reductase (XR) and xylitol dehydrogenase (XDH) is thought to be an important factor in effective xylose fermentation. Using protein engineering, we previously successfully reduced xylitol accumulation and improved ethanol production by reversing the dependency of XDH from NAD(+) to NADP(+). We also constructed a set of novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis. In the present study, we constructed a set of recombinant S. cerevisiae carrying a novel set of mutated strictly NADPH-dependent XR and NADP(+)-dependent XDH genes with overexpression of endogenous xylulokinase (XK) to study the effects of complete NADPH/NADP(+) recycling on ethanol fermentation and xylitol accumulation. All mutated strains demonstrated reduced xylitol accumulation, ranging 34.4-54.7% compared with the control strain. Moreover, compared with the control strain, the two strains showed 20% and 10% improvement in ethanol production.

  14. Suppression of NDA-type alternative mitochondrial NAD(P)H dehydrogenases in arabidopsis thaliana modifies growth and metabolism, but not high light stimulation of mitochondrial electron transport.

    PubMed

    Wallström, Sabá V; Florez-Sarasa, Igor; Araújo, Wagner L; Escobar, Matthew A; Geisler, Daniela A; Aidemark, Mari; Lager, Ida; Fernie, Alisdair R; Ribas-Carbó, Miquel; Rasmusson, Allan G

    2014-05-01

    The plant respiratory chain contains several pathways which bypass the energy-conserving electron transport complexes I, III and IV. These energy bypasses, including type II NAD(P)H dehydrogenases and the alternative oxidase (AOX), may have a role in redox stabilization and regulation, but current evidence is inconclusive. Using RNA interference, we generated Arabidopsis thaliana plants simultaneously suppressing the type II NAD(P)H dehydrogenase genes NDA1 and NDA2. Leaf mitochondria contained substantially reduced levels of both proteins. In sterile culture in the light, the transgenic lines displayed a slow growth phenotype, which was more severe when the complex I inhibitor rotenone was present. Slower growth was also observed in soil. In rosette leaves, a higher NAD(P)H/NAD(P)⁺ ratio and elevated levels of lactate relative to sugars and citric acid cycle metabolites were observed. However, photosynthetic performance was unaffected and microarray analyses indicated few transcriptional changes. A high light treatment increased AOX1a mRNA levels, in vivo AOX and cytochrome oxidase activities, and levels of citric acid cycle intermediates and hexoses in all genotypes. However, NDA-suppressing plants deviated from the wild type merely by having higher levels of several amino acids. These results suggest that NDA suppression restricts citric acid cycle reactions, inducing a shift towards increased levels of fermentation products, but do not support a direct association between photosynthesis and NDA proteins.

  15. Organochlorine Pesticide-Mediated Induction of NADPH Oxidase and Nitric-Oxide Synthase in Endothelial Cell

    PubMed Central

    Ghosh, Rishila; Siddharth, Manushi; Singh, Neeru; Kare, Pawan Kumar; Banerjee, Basu Dev; Wadhwa, Neelam

    2017-01-01

    Introduction Organochlorine Pesticides (OCPs) are detected ubiquitously in human and have been shown to be associated with Cardiovascular Disease (CVD) and atherosclerosis. Aim To find out the effect of organochlorine pesticides in endothelial cell with regard to oxidative stress and associated expression of enzymes producing superoxide and Nitric Oxide (NO). Materials and Methods Human Umbilical Vein Endothelial Cells (HUVEC) were cultured and treated with four OCPs which were found in human blood at a concentration of 0.1μM. The cells were tested for Reactive Oxygen Species (ROS) generation, NO production and mRNA expression of NAPDH oxidase (p47phox) and endothelial Nitric Oxide Synthase (eNOS). ROS generation was measured by using 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA) method. NO was analysed by Bioxytech nitric oxide assay kit method and mRNA of NADPH oxidase and eNOS was quantified by real time PCR. Data were expressed as the mean±SEM. Comparison between the groups were made by student’s t-test (2-tailed) or one-way ANOVA with Tukey’s post-hoc analysis depending on number of groups. For all statistical tests, p< 0.05 was considered to be significant. Results All the four pesticides generated ROS accompanied by enhanced expression of NADPH oxidase. Maximum effect was observed with β-endosulfan. Level of NO was found to be decreased significantly in endothelial cells treated with these pesticides accompanied by enhanced expression of eNOS. The antioxidant N-acetylcysteine (NAC) reduced ROS generation and enhanced NO formation. Pesticide-mediated ROS generation possibly reacts with NO forming peroxinitrite and thereby reducing the bioavailability of NO although eNOS expression is increased. Conclusion OCPs induce endothelial dysfunction through increased ROS generation via NADPH oxidase expression and reduced bioavailability of nitric oxide. PMID:28273962

  16. RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes.

    PubMed

    Filina, Julia V; Gabdoulkhakova, Aida G; Safronova, Valentina G

    2014-10-01

    Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1μM fMLF and 1μM WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells.

  17. The anorexigenic effect of serotonin is mediated by the generation of NADPH oxidase-dependent ROS.

    PubMed

    Fang, Xin-Ling; Shu, Gang; Yu, Jian-Jian; Wang, Li-Na; Yang, Jing; Zeng, Qing-Jie; Cheng, Xiao; Zhang, Zhi-Qi; Wang, Song-Bo; Gao, Ping; Zhu, Xiao-Tong; Xi, Qian-Yun; Zhang, Yong-Liang; Jiang, Qing-Yan

    2013-01-01

    Serotonin (5-HT) is a central inhibitor of food intake in mammals. Thus far, the intracellular mechanisms for the effect of serotonin on appetite regulation remain unclear. It has been recently demonstrated that reactive oxygen species (ROS) in the hypothalamus are a crucial integrative target for the regulation of food intake. To investigate the role of ROS in the serotonin-induced anorexigenic effects, conscious mice were treated with 5-HT alone or combination with Trolox (a ROS scavenger) or Apocynin (an NADPH oxidase inhibitor) by acute intracerebroventricular injection. Both Trolox and Apocynin reversed the anorexigenic action of 5-HT and the 5-HT-induced hypothalamic ROS elevation. The mRNA and protein expression levels of pro-opiomelanocortin (POMC) were dramatically increased after ICV injection with 5-HT. The anorexigenic action of 5-HT was accompanied by markedly elevated hypothalamic MDA levels and GSH-Px activity, while the SOD activity was decreased. Moreover, 5-HT significantly increased the mRNA expression of UCP-2 but reduced the levels of UCP-3. Both Trolox and Apocynin could block the 5-HT-induced changes in UCP-2 and UCP-3 gene expression. Our study demonstrates for the first time that the anorexigenic effect of 5-HT is mediated by the generation of ROS in the hypothalamus through an NADPH oxidase-dependent pathway.

  18. NADPH oxidase-mediated redox signaling promotes oxidative stress resistance and longevity through memo-1 in C. elegans

    PubMed Central

    Ewald, Collin Yvès; Hourihan, John M; Bland, Monet S; Obieglo, Carolin; Katic, Iskra; Moronetti Mazzeo, Lorenza E; Alcedo, Joy; Blackwell, T Keith; Hynes, Nancy E

    2017-01-01

    Transient increases in mitochondrially-derived reactive oxygen species (ROS) activate an adaptive stress response to promote longevity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases produce ROS locally in response to various stimuli, and thereby regulate many cellular processes, but their role in aging remains unexplored. Here, we identified the C. elegans orthologue of mammalian mediator of ErbB2-driven cell motility, MEMO-1, as a protein that inhibits BLI-3/NADPH oxidase. MEMO-1 is complexed with RHO-1/RhoA/GTPase and loss of memo-1 results in an enhanced interaction of RHO-1 with BLI-3/NADPH oxidase, thereby stimulating ROS production that signal via p38 MAP kinase to the transcription factor SKN-1/NRF1,2,3 to promote stress resistance and longevity. Either loss of memo-1 or increasing BLI-3/NADPH oxidase activity by overexpression is sufficient to increase lifespan. Together, these findings demonstrate that NADPH oxidase-induced redox signaling initiates a transcriptional response that protects the cell and organism, and can promote both stress resistance and longevity. DOI: http://dx.doi.org/10.7554/eLife.19493.001 PMID:28085666

  19. NADPH oxidase-mediated redox signaling promotes oxidative stress resistance and longevity through memo-1 in C. elegans.

    PubMed

    Ewald, Collin Yvès; Hourihan, John M; Bland, Monet S; Obieglo, Carolin; Katic, Iskra; Moronetti Mazzeo, Lorenza E; Alcedo, Joy; Blackwell, T Keith; Hynes, Nancy E

    2017-01-13

    Transient increases in mitochondrially-derived reactive oxygen species (ROS) activate an adaptive stress response to promote longevity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases produce ROS locally in response to various stimuli, and thereby regulate many cellular processes, but their role in aging remains unexplored. Here, we identified the C. elegans orthologue of mammalian mediator of ErbB2-driven cell motility, MEMO-1, as a protein that inhibits BLI-3/NADPH oxidase. MEMO-1 is complexed with RHO-1/RhoA/GTPase and loss of memo-1 results in an enhanced interaction of RHO-1 with BLI-3/NADPH oxidase, thereby stimulating ROS production that signal via p38 MAP kinase to the transcription factor SKN-1/NRF1,2,3 to promote stress resistance and longevity. Either loss of memo-1 or increasing BLI-3/NADPH oxidase activity by overexpression is sufficient to increase lifespan. Together, these findings demonstrate that NADPH oxidase-induced redox signaling initiates a transcriptional response that protects the cell and organism, and can promote both stress resistance and longevity.

  20. Curcumin ameliorated diabetic neuropathy partially by inhibition of NADPH oxidase mediating oxidative stress in the spinal cord.

    PubMed

    Zhao, Wei-Cheng; Zhang, Bin; Liao, Mei-Juan; Zhang, Wen-Xuan; He, Wan-You; Wang, Han-Bing; Yang, Cheng-Xiang

    2014-02-07

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are the main enzymes that produce oxidative stress, which plays an important role in painful diabetic neuropathy. Curcumin has been reported to exert an antinociceptive effect in a rat model of diabetic neuropathy by suppressing oxidative stress in the spinal cord. However, it remains unknown whether the mechanism by which curcumin ameliorates diabetic neuropathy can be attributed to spinal NADPH oxidases. This study was designed to determine the effect of curcumin on diabetic neuropathy and to investigate its precise mechanism in relation to NADPH oxidase-mediating oxidative stress in the spinal cord. Diabetic neuropathy was induced in Sprague-Dawley rats by intraperitoneal injection with 1% streptozotocin (STZ; 60 mg/kg). After the onset of diabetic neuropathy, a subset of the diabetic rats received daily intragastric administrations of curcumin (200mg/kg) or intraperitoneal injections of apocynin (2.5mg/kg) for 14 consecutive days, whereas other diabetic rats received equivalent volumes of normal saline (NS). STZ resulted in diabetic neuropathy with hyperglycemia and a lower paw withdrawal threshold (PWT), accompanied by elevations in the expression of the NADPH oxidase subunits p47(phox) and gp91(phox) and in the levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA) and a reduction in superoxide dismutase (SOD) activity (P<0.05) in the spinal cord. Both curcumin and apocynin ameliorated diabetic neuropathy. In conclusion, curcumin attenuated neuropathic pain in diabetic rats, at least partly by inhibiting NADPH oxidase-mediating oxidative stress in the spinal cord.

  1. Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction.

    PubMed Central

    Larroy, Carol; Fernández, M Rosario; González, Eva; Parés, Xavier; Biosca, Josep A

    2002-01-01

    YMR318C represents an open reading frame from Saccharomyces cerevisiae with unknown function. It possesses a conserved sequence motif, the zinc-containing alcohol dehydrogenase (ADH) signature, specific to the medium-chain zinc-containing ADHs. In the present study, the YMR318C gene product has been purified to homogeneity from overexpressing yeast cells, and found to be a homodimeric ADH, composed of 40 kDa subunits and with a pI of 5.0-5.4. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (linear and branched-chain) and aromatic primary alcohols and aldehydes. Aldehydes were processed with a 50-fold higher catalytic efficiency than that for the corresponding alcohols. The highest k(cat)/K(m) values were found with pentanal>veratraldehyde > hexanal > 3-methylbutanal >cinnamaldehyde. Taking into consideration the substrate specificity and sequence characteristics of the YMR318C gene product, we have proposed this gene to be called ADH6. The disruption of ADH6 was not lethal for the yeast under laboratory conditions. Although S. cerevisiae is considered a non lignin-degrading organism, the catalytic activity of ADHVI can direct veratraldehyde and anisaldehyde, arising from the oxidation of lignocellulose by fungal lignin peroxidases, to the lignin biodegradation pathway. ADHVI is the only S. cerevisiae enzyme able to significantly reduce veratraldehyde in vivo, and its overexpression allowed yeast to grow under toxic concentrations of this aldehyde. The enzyme may also be involved in the synthesis of fusel alcohols. To our knowledge this is the first NADPH-dependent medium-chain ADH to be characterized in S. cerevisiae. PMID:11742541

  2. Thioredoxin-1/peroxiredoxin-1 as sensors of oxidative stress mediated by NADPH oxidase activity in atherosclerosis.

    PubMed

    Madrigal-Matute, Julio; Fernandez-Garcia, Carlos-Ernesto; Blanco-Colio, Luis Miguel; Burillo, Elena; Fortuño, Ana; Martinez-Pinna, Roxana; Llamas-Granda, Patricia; Beloqui, Oscar; Egido, Jesus; Zalba, Guillermo; Martin-Ventura, José Luis

    2015-09-01

    To assess the potential association between TRX-1/PRX-1 and NADPH oxidase (Nox) activity in vivo and in vitro, TRX-1/PRX-1 levels were assessed by ELISA in 84 asymptomatic subjects with known phagocytic NADPH oxidase activity and carotid intima-media thickness (IMT). We found a positive correlation between TRX-1/PRX-1 and NADPH oxidase-dependent superoxide production (r=0.48 and 0.47; p<0.001 for both) and IMT (r=0.31 and 0.36; p<0.01 for both) adjusted by age and sex. Moreover, asymptomatic subjects with plaques have higher PRX-1 and TRX plasma levels (p<0.01 for both). These data were confirmed in a second study in which patients with carotid atherosclerosis showed higher PRX-1 and TRX plasma levels than healthy subjects (p<0.001 for both). In human atherosclerotic plaques, the NADPH oxidase subunit p22phox colocalized with TRX-1/PRX-1 in macrophages (immunohistochemistry). In monocytes and macrophages, phorbol 12-myristate 13-acetate (PMA) induced NADPH activation and TRX-1/PRX-1 release to the extracellular medium, with a concomitant decrease in their intracellular levels, which was reversed by the NADPH inhibitor apocynin (Western blot). In loss-of-function experiments, genetic silencing of the NADPH oxidase subunit Nox2 blocked PMA-induced intracellular TRX-1/PRX-1 downregulation in macrophages. Furthermore, the PMA-induced release of TRX-1/PRX-1 involves the modulation of their redox status and exosome-like vesicles. TRX-1/PRX-1 levels are associated with NADPH oxidase-activity in vivo and in vitro. These data could suggest a coordinated antioxidant response to oxidative stress in atherothrombosis.

  3. NADPH Oxidase-Derived ROS Induced by Chronic Intermittent Hypoxia Mediates Hypersensitivity of Lung Vagal C Fibers in Rats

    PubMed Central

    Yang, Chang-Huan; Zhuang, Wei-Ling; Shen, Yan-Jhih; Lai, Ching Jung; Kou, Yu Ru

    2016-01-01

    stimulants and that this sensitization is mediated via ROS generated by NADPH oxidase. PMID:27242540

  4. Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation.

    PubMed

    Yang, Chul-Su; Lee, Jong-Soo; Rodgers, Mary; Min, Chan-Ki; Lee, June-Yong; Kim, Hee Jin; Lee, Kwang-Hoon; Kim, Chul-Joong; Oh, Byungha; Zandi, Ebrahim; Yue, Zhenyu; Kramnik, Igor; Liang, Chengyu; Jung, Jae U

    2012-03-15

    Phagocytosis and autophagy are two important and related arms of the host's first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicon's actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection.

  5. Reversible phosphorylation regulation of NADPH-linked polyol dehydrogenase in the freeze-avoiding gall moth, Epiblema scudderiana: role in glycerol metabolism.

    PubMed

    Holden, Helen A; Storey, Kenneth B

    2011-05-01

    Larvae of the goldenrod gall moth, Epiblema scudderiana, use a freeze avoidance strategy of cold hardiness to survive the winter. A key metabolic adaption that supports subzero survival is the accumulation of large amounts of glycerol as a colligative antifreeze. Production of glycerol relies on polyol dehydrogenase (PDH) which catalyzes the NADPH-dependent conversion of glyceraldehyde into glycerol. Kinetic analysis of PDH from E. scudderiana revealed significant changes in properties as a result of subzero temperature acclimation; the K(m) for glyceraldehyde in 5°C-acclimated larvae was 7.0 mM and doubled in - 15°C-exposed larvae. This change suggested that PDH is regulated by a state-dependent covalent modification. Indeed, high and low K(m) forms could be interconverted by incubating larval extracts in vitro under conditions that stimulated either endogenous protein kinases or protein phosphatases. Protein kinase incubations doubled the K(m) glyceraldehyde of the 5°C enzyme, whereas protein phosphatase incubations decreased the K(m) of the - 15°C enzyme by about 50%. PDH was purified by ion exchange and affinity chromatography steps and then subjected to electrophoresis. Staining with ProQ Diamond phosphoprotein stain showed a much higher phosphate content of PDH from - 15°C-acclimated larvae, a result that was further confirmed by immunoblotting that showed a much greater phosphoserine content on the - 15°C enzyme. These experiments established that PDH is regulated by state-dependent reversible phosphorylation in E. scudderiana and suggest that this regulatory mechanism makes a significant contribution to controlling the synthesis, maintenance, and degradation of glycerol pools over the winter months.

  6. The Type II NADPH Dehydrogenase Facilitates Cyclic Electron Flow, Energy-Dependent Quenching, and Chlororespiratory Metabolism during Acclimation of Chlamydomonas reinhardtii to Nitrogen Deprivation1[OPEN

    PubMed Central

    Grossman, Arthur R.

    2016-01-01

    When photosynthetic organisms are deprived of nitrogen (N), the capacity to grow and assimilate carbon becomes limited, causing a decrease in the productive use of absorbed light energy and likely a rise in the cellular reduction state. Although there is a scarcity of N in many terrestrial and aquatic environments, a mechanistic understanding of how photosynthesis adjusts to low-N conditions and the enzymes/activities integral to these adjustments have not been described. In this work, we use biochemical and biophysical analyses of photoautotrophically grown wild-type and mutant strains of Chlamydomonas reinhardtii to determine the integration of electron transport pathways critical for maintaining active photosynthetic complexes even after exposure of cells to N deprivation for 3 d. Key to acclimation is the type II NADPH dehydrogenase, NDA2, which drives cyclic electron flow (CEF), chlororespiration, and the generation of an H+ gradient across the thylakoid membranes. N deprivation elicited a doubling of the rate of NDA2-dependent CEF, with little contribution from PGR5/PGRL1-dependent CEF. The H+ gradient generated by CEF is essential to sustain nonphotochemical quenching, while an increase in the level of reduced plastoquinone would promote a state transition; both are necessary to down-regulate photosystem II activity. Moreover, stimulation of NDA2-dependent chlororespiration affords additional relief from the elevated reduction state associated with N deprivation through plastid terminal oxidase-dependent water synthesis. Overall, rerouting electrons through the NDA2 catalytic hub in response to photoautotrophic N deprivation sustains cell viability while promoting the dissipation of excess excitation energy through quenching and chlororespiratory processes. PMID:26858365

  7. Plastidial Expression of Type II NAD(P)H Dehydrogenase Increases the Reducing State of Plastoquinones and Hydrogen Photoproduction Rate by the Indirect Pathway in Chlamydomonas reinhardtii1.

    PubMed

    Baltz, Anthony; Dang, Kieu-Van; Beyly, Audrey; Auroy, Pascaline; Richaud, Pierre; Cournac, Laurent; Peltier, Gilles

    2014-07-01

    Biological conversion of solar energy into hydrogen is naturally realized by some microalgae species due to a coupling between the photosynthetic electron transport chain and a plastidial hydrogenase. While promising for the production of clean and sustainable hydrogen, this process requires improvement to be economically viable. Two pathways, called direct and indirect photoproduction, lead to sustained hydrogen production in sulfur-deprived Chlamydomonas reinhardtii cultures. The indirect pathway allows an efficient time-based separation of O2 and H2 production, thus overcoming the O2 sensitivity of the hydrogenase, but its activity is low. With the aim of identifying the limiting step of hydrogen production, we succeeded in overexpressing the plastidial type II NAD(P)H dehydrogenase (NDA2). We report that transplastomic strains overexpressing NDA2 show an increased activity of nonphotochemical reduction of plastoquinones (PQs). While hydrogen production by the direct pathway, involving the linear electron flow from photosystem II to photosystem I, was not affected by NDA2 overexpression, the rate of hydrogen production by the indirect pathway was increased in conditions, such as nutrient limitation, where soluble electron donors are not limiting. An increased intracellular starch was observed in response to nutrient deprivation in strains overexpressing NDA2. It is concluded that activity of the indirect pathway is limited by the nonphotochemical reduction of PQs, either by the pool size of soluble electron donors or by the PQ-reducing activity of NDA2 in nutrient-limited conditions. We discuss these data in relation to limitations and biotechnological improvement of hydrogen photoproduction in microalgae.

  8. NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

    SciTech Connect

    Liu Qing; He Xiaoqing; Liu Yongsheng; Du Bingbing; Wang Xiaoyan; Zhang Weisheng; Jia Pengfei; Dong Jingmei; Ma Jianxiu; Wang Xiaohu; Li Sha; Zhang Hong

    2008-12-19

    Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91{sup phox} was dose-dependent. Meanwhile, the cytoplasmic subunit p47{sup phox} was translocated to the cell membrane and localized with p22{sup phox} and gp91{sup phox} to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.

  9. Glucose-6-Phosphate Dehydrogenase Protects Escherichia coli from Tellurite-Mediated Oxidative Stress

    PubMed Central

    Sandoval, Juan M.; Arenas, Felipe A.; Vásquez, Claudio C.

    2011-01-01

    The tellurium oxyanion tellurite induces oxidative stress in most microorganisms. In Escherichia coli, tellurite exposure results in high levels of oxidized proteins and membrane lipid peroxides, inactivation of oxidation-sensitive enzymes and reduced glutathione content. In this work, we show that tellurite-exposed E. coli exhibits transcriptional activation of the zwf gene, encoding glucose 6-phosphate dehydrogenase (G6PDH), which in turn results in augmented synthesis of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Increased zwf transcription under tellurite stress results mainly from reactive oxygen species (ROS) generation and not from a depletion of cellular glutathione. In addition, the observed increase of G6PDH activity was paralleled by accumulation of glucose-6-phosphate (G6P), suggesting a metabolic flux shift toward the pentose phosphate shunt. Upon zwf overexpression, bacterial cells also show increased levels of antioxidant molecules (NADPH, GSH), better-protected oxidation-sensitive enzymes and decreased amounts of oxidized proteins and membrane lipids. These results suggest that by increasing NADPH content, G6PDH plays an important role in E. coli survival under tellurite stress. PMID:21984934

  10. Globular adiponectin elicits neuroprotection by inhibiting NADPH oxidase-mediated oxidative damage in ischemic stroke.

    PubMed

    Song, W; Huo, T; Guo, F; Wang, H; Wei, H; Yang, Q; Dong, H; Wang, Q; Xiong, L

    2013-09-17

    Recent studies indicate that adiponectin can attenuate cerebral ischemic lesions via its functional area located in the C-terminal globular domain, which is called globular adiponectin (gAD). However, the mechanisms underlying this action remain unclear. In this study, we investigated the antioxidant properties of gAD during cerebral ischemia. Adult male C57BL/6 mice received an intracerebral injection of gAD with or without tetrabromocinnamic acid (TBCA, a NADPH oxidase activator). Mice were subjected to middle cerebral artery occlusion (MCAO) after gAD injection. Infarct volume, neurological function, the activity of antioxidant enzymes (superoxide dismutase [SOD], catalase), the content of malondialdehyde (MDA), and the expression of Bax, Bcl-2, cleaved caspase-3 and NADPH oxidase 2 (NOX2) were examined at 24h after MCAO. Infarct volume was attenuated in gAD-transduced mice when compared with mice in the MCAO group, with significant improvement in neurological function. In addition, neuronal apoptosis was attenuated, along with the expression of Bax/Bcl-2 and cleaved caspase 3. Furthermore, the activities of SOD and catalase increased, and the content of MDA reduced. However, TBCA blocked the effect of gAD on cerebral protection and its antioxidant abilities. Taken together, these results demonstrate that the neuroprotective action of gAD may result from the promotion of antioxidant capacity by inhibiting the NOX2 signaling system.

  11. Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells

    PubMed Central

    Lee, Su Jeong; Park, Jeen-Woo

    2014-01-01

    Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocytes. This effect manifested as DNA fragmentation, changes in cellular redox status, mitochondrial dysfunction, and modulation of apoptotic marker expression. Based on our findings, we suggest that attenuation of IDPc expression may protect skin from UVB-mediated damage, by inducing the apoptosis of UV-damaged cells. [BMB Reports 2014; 47(4): 209-214] PMID:24286310

  12. Micro-RNA 21 inhibition of SMAD7 enhances fibrogenesis via leptin-mediated NADPH oxidase in experimental and human nonalcoholic steatohepatitis.

    PubMed

    Dattaroy, Diptadip; Pourhoseini, Sahar; Das, Suvarthi; Alhasson, Firas; Seth, Ratanesh Kumar; Nagarkatti, Mitzi; Michelotti, Gregory A; Diehl, Anna Mae; Chatterjee, Saurabh

    2015-02-15

    Hepatic fibrosis in nonalcoholic steatohepatitis (NASH) is the common pathophysiological process resulting from chronic liver inflammation and oxidative stress. Although significant research has been carried out on the role of leptin-induced NADPH oxidase in fibrogenesis, the molecular mechanisms that connect the leptin-NADPH oxidase axis in upregulation of transforming growth factor (TGF)-β signaling have been unclear. We aimed to investigate the role of leptin-mediated upregulation of NADPH oxidase and its subsequent induction of micro-RNA 21 (miR21) in fibrogenesis. Human NASH livers and a high-fat (60% kcal) diet-fed chronic mouse model, where hepatotoxin bromodichloromethane was used to induce NASH, were used for this study. To prove the role of the leptin-NADPH oxidase-miR21 axis, mice deficient in genes for leptin, p47phox, and miR21 were used. Results showed that wild-type mice and human livers with NASH had increased oxidative stress, increased p47phox expression, augmented NF-κB activation, and increased miR21 levels. These mice and human livers showed increased TGF-β, SMAD2/3-SMAD4 colocalizations in the nucleus, increased immunoreactivity against Col1α, and α-SMA with a concomitant decrease in protein levels of SMAD7. Mice that were deficient in leptin or p47phox had decreased activated NF-κB and miR21 levels, suggesting the role of leptin and NADPH oxidase in inducing NF-κB-mediated miR21 expression. Further miR21 knockout mice had decreased colocalization events of SMAD2/3-SMAD4 in the nucleus, increased SMAD7 levels, and decreased fibrogenesis. Taken together, the studies show the novel role of leptin-NADPH oxidase induction of miR21 as a key regulator of TGF-β signaling and fibrogenesis in experimental and human NASH.

  13. NADPH oxidase (NOX) 1 mediates cigarette smoke-induced superoxide generation in rat vascular smooth muscle cells.

    PubMed

    Chang, Kyung-Hwa; Park, Jung-Min; Lee, Chang Hoon; Kim, Bumseok; Choi, Kyung-Chul; Choi, Seong-Jin; Lee, Kyuhong; Lee, Moo-Yeol

    2017-02-01

    Smoking is a well-established risk factor for cardiovascular diseases. Oxidative stress is one of the common etiological factors, and NADPH oxidase (NOX) has been suggested as a potential mediator of oxidative stress. In this study, cigarette smoke (CS)-induced superoxide production was characterized in vascular smooth muscle cells (VSMC). CS was prepared in forms of cigarette smoke extract (CSE) and total particulate matter (TPM). Several molecular probes for reactive oxygen species were trialed, and dihydroethidium (DHE) and WST-1 were chosen for superoxide detection considering the autofluorescence, light absorbance, and peroxidase inhibitory activity of CS. Both CSE and TPM generated superoxide in a VSMC culture system by stimulating cells to produce superoxide and by directly producing superoxide in the aqueous solution. NOX, specifically NOX1 was found to be an important cellular source of superoxide through experiments with the NOX inhibitors diphenyleneiodonium (DPI) and VAS2870 as well as isoform-specific NOX knockdown. NOX inhibitors and the superoxide dismutase mimetic TEMPOL reduced the cytotoxicity of CSE, thus suggesting the contribution of NOX1-derived superoxide to cytotoxicity. Since NOX1 is known to mediate diverse pathological processes in the vascular system, NOX1 may be a critical effector of cardiovascular toxicity caused by smoking.

  14. NAD kinase levels control the NADPH concentration in human cells.

    PubMed

    Pollak, Nadine; Niere, Marc; Ziegler, Mathias

    2007-11-16

    NAD kinases (NADKs) are vital, as they generate the cellular NADP pool. As opposed to three compartment-specific isoforms in plants and yeast, only a single NADK has been identified in mammals whose cytoplasmic localization we established by immunocytochemistry. To understand the physiological roles of the human enzyme, we generated and analyzed cell lines stably deficient in or overexpressing NADK. Short hairpin RNA-mediated down-regulation led to similar (about 70%) decrease of both NADK expression, activity, and the NADPH concentration and was accompanied by increased sensitivity toward H(2)O(2). Overexpression of NADK resulted in a 4-5-fold increase in the NADPH, but not NADP(+), concentration, although the recombinant enzyme phosphorylated preferentially NAD(+). Surprisingly, NADK overexpression and the ensuing increase of the NADPH level only moderately enhanced protection against oxidant treatment. Apparently, to maintain the NADPH level for the regeneration of oxidative defense systems human cells depend primarily on NADP-dependent dehydrogenases (which re-reduce NADP(+)), rather than on a net increase of NADP. The stable shifts of the NADPH level in the generated cell lines were also accompanied by alterations in the expression of peroxiredoxin 5 and Nrf2. Because the basal oxygen radical level in the cell lines was only slightly changed, the redox state of NADP may be a major transmitter of oxidative stress.

  15. Resveratrol inhibits foam cell formation via NADPH oxidase 1-mediated reactive oxygen species and monocyte chemotactic protein-1

    PubMed Central

    Park, Dae-Weon; Baek, Kheewoong; Kim, Jae-Ryong; Lee, Jae-Jin; Ryu, Sang-Ho; Chin, Byung-Rho

    2009-01-01

    Resveratrol is a polyphenolic compound in red wine that has anti-oxidant and cardioprotective effects in animal models. Reactive oxygen species (ROS) and monocyte chemotactic protein-1 (MCP-1) play key roles in foam cell formation and atherosclerosis. We studied LPS-mediated foam cell formation and the effect of resveratrol. Resveratrol pretreatment strongly suppressed LPS-induced foam cell formation. To determine if resveratrol affected the expression of genes that control ROS generation in macrophages, NADPH oxidase 1 (Nox1) was measured. Resveratrol treatment of macrophages inhibited LPS-induced Nox1 expression as well as ROS generation, and also suppressed LPS-induced MCP-1 mRNA and protein expression. We investigated the upstream targets of Nox1 and MCP-1 expression and found that Akt-forkhead transcription factors of the O class (FoxO3a) is an important signaling pathway that regulates both genes. These inhibitory effects of resveratrol on Nox1 expression and MCP-1 production may target to the Akt and FoxO3a signaling pathways. PMID:19293636

  16. Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes

    PubMed Central

    Shmelzer, Zeev; Haddad, Nurit; Admon, Ester; Pessach, Itai; Leto, Thomas L.; Eitan-Hazan, Zahit; Hershfinkel, Michal; Levy, Rachel

    2003-01-01

    Cytosolic phospholipase A2 (cPLA2)–generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets. PMID:12913107

  17. NADPH Oxidase-Dependent Reactive Oxygen Species Mediate Amplified TLR4 Signaling and Sepsis-Induced Mortality in Nrf2-deficient Mice

    PubMed Central

    Kong, Xiaoni; Thimmulappa, Rajesh; Kombairaju, Ponvijay; Biswal, Shyam

    2010-01-01

    Sepsis syndrome is characterized by a dysregulated inflammatory response to infection. NADPH oxidase-dependent reactive oxygen species (ROS) play significant roles in the pathophysiology of sepsis. We previously showed that disruption of Nrf2, a master regulator of antioxidant defenses, caused a dysregulation of innate immune response that resulted in greater mortality in a polymicrobial sepsis and lipopolysaccharide (LPS) shock model; however, the underlying mechanisms are unclear. In the present study, compared to wild-type (Nrf2+/+) macrophages, we observed greater PKC-induced NADPH oxidase-dependent ROS generation in Nrf2-disrupted (Nrf2−/−) macrophages that was modulated by glutathione (GSH) levels. To address the NADPH oxidase-mediated hyper-inflammatory response and sepsis-induced lung injury and mortality in Nrf2−/− mice, we used double knockout mice lacking Nrf2 and NADPH oxidase subunit, gp91phox (Nrf2−/−//Gp91phox−/−). Compared to Nrf2+/+ macrophages, LPS induced greater activation of TLR4 as evident by TLR4 surface trafficking and downstream recruitment of MYD88 and TRIF in Nrf2−/− macrophages that was diminished by ablation of gp91phox. Similarly, phosphorylation of IκB and IRF3 as well as cytokine expression was markedly higher in Nrf2−/− macrophages, while it was similar in Nrf2+/+ and Nrf2−/−//Gp91phox−/−. In vivo studies showed greater LPS-induced pulmonary inflammation in Nrf2−/− mice that was significantly reduced by ablation of gp91phox. Furthermore, LPS shock and polymicrobial sepsis induced early and greater mortality in Nrf2−/− mice, while Nrf2−/−//Gp91phox−/− showed prolong survival. Together, these results demonstrate that Nrf2 is essential for the regulation of NADPH oxidase-dependent ROS-mediated TLR4 activation and lethal innate immune response in sepsis. PMID:20511556

  18. Influence of long-term hyper-gravity on the reactivity of succinic acid dehydrogenase and NADPH-diaphorase in the central nervous system of fish: a histochemical study

    NASA Astrophysics Data System (ADS)

    Anken, R. H.; Rahmann, H.

    In the course of a densitometric evaluation, the histochemically demonstrated reactivity of succinic acid dehydrogenase (SDH) and of NADPH-diaphorase (NADPHD) was determined in different brain nuclei of two teleost fish (cichlid fish Oreochromis mossambicus, swordtail fish Xiphophorus helleri), which had been kept under 3g hyper-gravity for 8 days. SDH was chosen since it is a rate limiting enzyme of the Krebs cycle and therefore it is regarded as a marker for metabolic and neuronal activity. NADPHD reactivity reflects the activity of nitric oxide synthase. Nitric oxide (NO) is a gaseous intercellular messenger that has been suggested to play a major role in several different in vivo models of neuronal plasticity including learning. Within particular vestibulum-connected brain centers, significant effects of hyper-gravity were obtained, e.g., in the magnocellular nucleus, a primary vestibular relay ganglion of the brain stem octavolateralis area, in the superior rectus subdivision of the oculomotoric nucleus and within cerebellar eurydendroid cells, which in teleosts possibly resemble the deep cerebellar nucleus of higher vertebrates. Non-vestibulum related nuclei did not respond to hypergravity in a significant way. The effect of hyper-gravity found was much less distinct in adult animals as compared to the circumstances seen in larval fish (Anken et al., Adv. Space Res. 17, 1996), possibly due to a development correlated loss of neuronal plasticity.

  19. Proinflammatory cytokines provoke oxidative damage to actin in neuronal cells mediated by Rac1 and NADPH oxidase.

    PubMed

    Barth, Brian M; Stewart-Smeets, Shelli; Kuhn, Thomas B

    2009-06-01

    The proinflammatory cytokines TNFalpha and Il-1beta orchestrate the progression of CNS inflammation, which substantially contributes to neurodegeneration in many CNS pathologies. TNFalpha and Il-1beta stimulate actin filament reorganization in non-neuronal cells often accompanied by the formation of reactive oxygen species (ROS). Actin filament dynamics is vital for cellular plasticity, mitochondrial function, and gene expression despite being highly susceptible to oxidative damage. We demonstrated that, in neuronal cells, TNFalpha and Il-1beta stimulate a transient, redox-dependent reorganization of the actin cytoskeleton into lamellipodia under the regulation of Rac1 and a neuronal NADPH oxidase as the source of ROS. The persistent presence of intracellular ROS provoked oxidative damage (carbonylation) to actin coinciding with the loss of lamellipodia and arrest of cellular plasticity. Inhibition of NADPH oxidase activity or Rac1 abolished the adverse effects of cytokines. These findings suggest that oxidative damage to the neuronal actin cytoskeleton could represent a key step in CNS neurodegeneration.

  20. FFA-induced hepatic insulin resistance in vivo is mediated by PKCδ, NADPH oxidase, and oxidative stress.

    PubMed

    Pereira, Sandra; Park, Edward; Mori, Yusaku; Haber, C Andrew; Han, Ping; Uchida, Toyoyoshi; Stavar, Laura; Oprescu, Andrei I; Koulajian, Khajag; Ivovic, Alexander; Yu, Zhiwen; Li, Deling; Bowman, Thomas A; Dewald, Jay; El-Benna, Jamel; Brindley, David N; Gutierrez-Juarez, Roger; Lam, Tony K T; Najjar, Sonia M; McKay, Robert A; Bhanot, Sanjay; Fantus, I George; Giacca, Adria

    2014-07-01

    Fat-induced hepatic insulin resistance plays a key role in the pathogenesis of type 2 diabetes in obese individuals. Although PKC and inflammatory pathways have been implicated in fat-induced hepatic insulin resistance, the sequence of events leading to impaired insulin signaling is unknown. We used Wistar rats to investigate whether PKCδ and oxidative stress play causal roles in this process and whether this occurs via IKKβ- and JNK-dependent pathways. Rats received a 7-h infusion of Intralipid plus heparin (IH) to elevate circulating free fatty acids (FFA). During the last 2 h of the infusion, a hyperinsulinemic-euglycemic clamp with tracer was performed to assess hepatic and peripheral insulin sensitivity. An antioxidant, N-acetyl-L-cysteine (NAC), prevented IH-induced hepatic insulin resistance in parallel with prevention of decreased IκBα content, increased JNK phosphorylation (markers of IKKβ and JNK activation, respectively), increased serine phosphorylation of IRS-1 and IRS-2, and impaired insulin signaling in the liver without affecting IH-induced hepatic PKCδ activation. Furthermore, an antisense oligonucleotide against PKCδ prevented IH-induced phosphorylation of p47(phox) (marker of NADPH oxidase activation) and hepatic insulin resistance. Apocynin, an NADPH oxidase inhibitor, prevented IH-induced hepatic and peripheral insulin resistance similarly to NAC. These results demonstrate that PKCδ, NADPH oxidase, and oxidative stress play a causal role in FFA-induced hepatic insulin resistance in vivo and suggest that the pathway of FFA-induced hepatic insulin resistance is FFA → PKCδ → NADPH oxidase and oxidative stress → IKKβ/JNK → impaired hepatic insulin signaling.

  1. Inhibition of stress mediated cell death by human lactate dehydrogenase B in yeast.

    PubMed

    Sheibani, Sara; Jones, Natalie K; Eid, Rawan; Gharib, Nada; Arab, Nagla T T; Titorenko, Vladimir; Vali, Hojatollah; Young, Paul A; Greenwood, Michael T

    2015-08-01

    We report the identification of human L- lactate dehydrogenase B (LDHB) as a novel Bax suppressor. Yeast heterologously expressing LDHB is also resistant to the lethal effects of copper indicating that it is a general suppressor of stress mediated cell death. To identify potential LDHB targets, LDHB was expressed in yeast mutants defective in apoptosis, necrosis and autophagy. The absence of functional PCD regulators including MCA1, YBH3, cyclophilin (CPR3) and VMA3, as well as the absence of the pro-survival autophagic pathway (ATG1,7) did not interfere with the LDHB mediated protection against copper indicating that LDHB functions independently of known PCD regulators or by simply blocking or stimulating a common PCD promoting or inhibitory pathway. Measurements of lactate levels revealed that short-term copper stress (1.6 mM, 4 h), does not increase intracellular levels of lactate, instead a three-fold increase in extracellular lactate was observed. Thus, yeast cells resemble mammalian cells where different stresses are known to lead to increased lactate production leading to lactic acidosis. In agreement with this, we found that the addition of exogenous lactic acid to growth media was sufficient to induce cell death that could be inhibited by the expression of LDHB. Taken together our results suggest that lactate dehydrogenase is a general suppressor of PCD in yeast.

  2. Genetic and genomic analysis of Rhizoctonia solani interactions with Arabidopsis; evidence of resistance mediated through NADPH oxidases.

    PubMed

    Foley, Rhonda C; Gleason, Cynthia A; Anderson, Jonathan P; Hamann, Thorsten; Singh, Karam B

    2013-01-01

    Rhizoctonia solani is an important soil-borne necrotrophic fungal pathogen, with a broad host range and little effective resistance in crop plants. Arabidopsis is resistant to R. solani AG8 but susceptible to R. solani AG2-1. A screen of 36 Arabidopsis ecotypes and mutants affected in the auxin, camalexin, salicylic acid, abscisic acid and ethylene/jasmonic acid pathways did not reveal any variation in response to R. solani and demonstrated that resistance to AG8 was independent of these defense pathways. The Arabidopsis Affymetrix ATH1 Genome array was used to assess global gene expression changes in plants infected with AG8 and AG2-1 at seven days post-infection. While there was considerable overlap in the response, some gene families were differentially affected by AG8 or AG2-1 and included those involved in oxidative stress, cell wall associated proteins, transcription factors and heat shock protein genes. Since a substantial proportion of the gene expression changes were associated with oxidative stress responses, we analysed the role of NADPH oxidases in resistance. While single NADPH oxidase mutants had no effect, a NADPH oxidase double mutant atrbohf atrbohd resulted in an almost complete loss of resistance to AG8, suggesting that reactive oxidative species play an important role in Arabidopsis's resistance to R. solani.

  3. The three-dimensional structure of Clostridium absonum 7α-hydroxysteroid dehydrogenase: new insights into the conserved arginines for NADP(H) recognition

    PubMed Central

    Lou, Deshuai; Wang, Bochu; Tan, Jun; Zhu, Liancai; Cen, Xiaoxi; Ji, Qingzhi; Wang, Yue

    2016-01-01

    7α-hydroxysteroid dehydrogenase (7α-HSDH) can catalyse the oxidation of C7 α-OH of the steroid nucleus in the bile acid metabolism. In the paper we determined the crystal structure of 7α-HSDH from Clostridium absonum (CA 7α-HSDH) complexed with taurochenodeoxycholic acid (TCDCA) and NADP+ by X-ray diffraction, which, as a tetramer, possesses the typical α/β folding pattern. The four subunits of an asymmetric unit lie in the fact that there are the stable hydrophobic interactions between Q-axis-related subunits. Significantly, we captured an active state of the NADP+, confirming that nicotinamide moiety of NADP+ act as electron carrier in the dehydrogenation. On the basis of crystal structure analysis, site-directed mutagenesis and MD simulation, furthermore, we find that the guanidinium of Arg38 can form the stable cation-π interaction with the adenine ring of NADP+, and the cation-π interaction and hydrogen bonds between Arg38 and NADP+ have a significant anchor effect on the cofactor binding to CA 7α-HSDH. PMID:26961171

  4. ATL9, a RING Zinc Finger Protein with E3 Ubiquitin Ligase Activity Implicated in Chitin- and NADPH Oxidase-Mediated Defense Responses

    PubMed Central

    Berrocal-Lobo, Marta; Stone, Sophia; Yang, Xin; Antico, Jay; Callis, Judy; Ramonell, Katrina M.; Somerville, Shauna

    2010-01-01

    Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 ( = ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst. PMID:21203445

  5. Plastidial Expression of Type II NAD(P)H Dehydrogenase Increases the Reducing State of Plastoquinones and Hydrogen Photoproduction Rate by the Indirect Pathway in Chlamydomonas reinhardtii1[W][OPEN

    PubMed Central

    Baltz, Anthony; Dang, Kieu-Van; Beyly, Audrey; Auroy, Pascaline; Richaud, Pierre; Cournac, Laurent; Peltier, Gilles

    2014-01-01

    Biological conversion of solar energy into hydrogen is naturally realized by some microalgae species due to a coupling between the photosynthetic electron transport chain and a plastidial hydrogenase. While promising for the production of clean and sustainable hydrogen, this process requires improvement to be economically viable. Two pathways, called direct and indirect photoproduction, lead to sustained hydrogen production in sulfur-deprived Chlamydomonas reinhardtii cultures. The indirect pathway allows an efficient time-based separation of O2 and H2 production, thus overcoming the O2 sensitivity of the hydrogenase, but its activity is low. With the aim of identifying the limiting step of hydrogen production, we succeeded in overexpressing the plastidial type II NAD(P)H dehydrogenase (NDA2). We report that transplastomic strains overexpressing NDA2 show an increased activity of nonphotochemical reduction of plastoquinones (PQs). While hydrogen production by the direct pathway, involving the linear electron flow from photosystem II to photosystem I, was not affected by NDA2 overexpression, the rate of hydrogen production by the indirect pathway was increased in conditions, such as nutrient limitation, where soluble electron donors are not limiting. An increased intracellular starch was observed in response to nutrient deprivation in strains overexpressing NDA2. It is concluded that activity of the indirect pathway is limited by the nonphotochemical reduction of PQs, either by the pool size of soluble electron donors or by the PQ-reducing activity of NDA2 in nutrient-limited conditions. We discuss these data in relation to limitations and biotechnological improvement of hydrogen photoproduction in microalgae. PMID:24820024

  6. Inositol 1,4,5-triphosphate receptors and NAD(P)H mediate Ca2+ signaling required for hypoxic preconditioning of hippocampal neurons

    PubMed Central

    Bickler, Philip E.; Fahlman, Christian S.; Gray, Jonathan; McKleroy, William; Warren, Daniel E.

    2009-01-01

    Exposure of neurons to a non-lethal hypoxic stress greatly reduces cell death during subsequent severe ischemia (hypoxic preconditioning, HPC). In organotypic cultures of rat hippocampus, we demonstrate that HPC requires inositol triphosphate (IP3) receptor-dependent Ca2+ release from the endoplasmic reticulum (ER) triggered by increased cytosolic NAD(P)H. Ca2+ chelation with intracellular BAPTA, ER Ca2+ store depletion with thapsigargin, IP3 receptor block with xestospongin, and RNA interference against subtype 1 of the IP3 receptor all blunted the moderate increases in [Ca2+]i (50–100 nM) required for tolerance induction. Increases in [Ca2+]i during HPC and neuroprotection following HPC was not prevented with NMDA receptor block or by removing Ca2+ from the bathing medium. Increased NAD(P)H fluorescence in CA1 neurons during hypoxia and demonstration that NADH manipulation increases [Ca2+]i in an IP3R-dependent manner revealed a primary role of cellular redox state in liberation of Ca2+ from the ER. Blockade of IP3Rs and intracellular Ca2+ chelation prevented phosphorylation of known HPC signaling targets, including MAPK p42/44 (ERK), protein kinase B (Akt) and CREB. We conclude that the endoplasmic reticulum, acting via redox/NADH-dependent intracellular Ca2+ store release, is an important mediator of the neuroprotective response to hypoxic stress. PMID:19217932

  7. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    NASA Astrophysics Data System (ADS)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  8. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    PubMed Central

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-01-01

    Inosine-5′-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches. PMID:26558346

  9. 15-Oxoeicosatetraenoic acid is a 15-hydroxyprostaglandin dehydrogenase-derived electrophilic mediator of inflammatory signaling pathways.

    PubMed

    Snyder, Nathaniel W; Golin-Bisello, Franca; Gao, Yang; Blair, Ian A; Freeman, Bruce A; Wendell, Stacy Gelhaus

    2015-06-05

    Bioactive lipids govern cellular homeostasis and pathogenic inflammatory processes. Current dogma holds that bioactive lipids, such as prostaglandins and lipoxins, are inactivated by 15-hydroxyprostaglandin dehydrogenase (15PGDH). In contrast, the present results reveal that catabolic "inactivation" of hydroxylated polyunsaturated fatty acids (PUFAs) yields electrophilic α,β-unsaturated ketone derivatives. These endogenously produced species are chemically reactive signaling mediators that induce tissue protective events. Electrophilic fatty acids diversify the proteome through post-translational alkylation of nucleophilic cysteines in key transcriptional regulatory proteins and enzymes that govern cellular metabolic and inflammatory homeostasis. 15PGDH regulates these processes as it is responsible for the formation of numerous electrophilic fatty acids including the arachidonic acid metabolite, 15-oxoeicosatetraenoic acid (15-oxoETE). Herein, the role of 15-oxoETE in regulating signaling responses is reported. In cell cultures, 15-oxoETE activates Nrf2-regulated antioxidant responses (AR) and inhibits NF-κB-mediated pro-inflammatory responses via IKKβ inhibition. Inhibition of glutathione S-transferases using ethacrynic acid incrementally increased the signaling capacity of 15-oxoETE by decreasing 15-oxoETE-GSH adduct formation. This work demonstrates that 15PGDH plays a role in the regulation of cell and tissue homeostasis via the production of electrophilic fatty acid signaling mediators.

  10. NADPH OXIDASE 4 MEDIATES TGF-β-INDUCED SMOOTH MUSCLE α-ACTIN VIA p38MAPK AND SRF

    PubMed Central

    Martin-Garrido, Abel; Brown, David I.; Lyle, Alicia N.; Dikalova, Anna; Seidel-Rogol, Bonnie; Lassègue, Bernard; Martín, Alejandra San; Griendling, Kathy K.

    2010-01-01

    In contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle gene expression; however, the underlying mechanisms are incompletely characterized. Using smooth muscle α-actin (SMA) as a prototypical smooth muscle gene and transforming growth factor-β (TGF-β) as a differentiating agent, we examined Nox4-dependent signaling. TGF-β increases Nox4 expression and activity in human aortic smooth muscle cells (HASMC). Transfection of HASMC with siRNA against Nox4 (siNox4) abolishes TGF-β-induced SMA expression and stress fiber formation. siNox4 also significantly inhibits TGF-β-stimulated p38MAPK phosphorylation, as well as that of its substrate, mitogen-activated protein kinase-activated protein kinase-2 (MK-2). Moreover, the p38MAPK inhibitor SB-203580 nearly completely blocks the SMA increase induced by TGF-β. Inhibition of either p38MAPK or NADPH oxidase-derived reactive oxygen species impairs the TGF-β-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Binding of SRF to myocardin-related transcription factor (MRTF) is also necessary, because downregulation of MRTF by siRNA abolishes TGF-β-induced SMA expression. Taken together, these data suggest that Nox4 regulates SMA expression via activation of a p38MAPK/SRF/MRTF pathway in response to TGF-β. PMID:21074607

  11. Resveratrol decreases fructose-induced oxidative stress, mediated by NADPH oxidase via an AMPK-dependent mechanism

    PubMed Central

    Cheng, Pei-Wen; Ho, Wen-Yu; Su, Yu-Ting; Lu, Pei-Jung; Chen, Bo-Zone; Cheng, Wen-Han; Lu, Wen-Hsien; Sun, Gwo-Ching; Yeh, Tung-Chen; Hsiao, Michael; Tseng, Ching-Jiunn

    2014-01-01

    Background and Purpose Oxidative stress is an important pathogenic factor in the development of hypertension. Resveratrol, the main antioxidant in red wine, improves NO bioavailability and prevents cardiovascular disease. The aim of this study was to examine whether resveratrol decreases the generation of reactive oxygen species (ROS), thereby reducing BP in rats with fructose-induced hypertension. Experimental Approach Rats were fed 10% fructose with or without resveratrol (10 mg·kg−1·day−1) for 1 week or for 4 weeks with resveratrol treatment beginning at week 2; systolic BP (SBP) was measured by tail-cuff method. Endogenous in vivo O2− production in the nucleus tractus solitarii (NTS) was determined with dihydroethidium. Real-time PCR and immunoblotting analyses were used to quantify RNA and protein expression levels. Key Results In fructose-fed rats, ROS levels in the NTS were higher, whereas the NO level was significantly decreased. Also, RNA and protein levels of NADPH oxidase subunits (p67, p22-phox) were elevated, superoxide dismutase 2 (SOD2) reduced and AMP-activated PK (AMPK) T172 phosphorylation levels in the NTS were lower in fructose-fed rats. Treatment with the AMPK activator resveratrol decreased levels of NADPH oxidase subunits and ROS, and increased NO and SOD2 levels in the NTS of fructose-fed rats. Administration of resveratrol, in combination with fructose at week 0 and later at week 2, significantly reduced the SBP of fructose-fed rats. Conclusions and Implications Collectively, resveratrol decreased BP through the phosphorylation of AMPK, Akt and neuronal NOS in fructose-fed rats. These novel findings suggest that resveratrol may be a potential pharmacological candidate for the treatment of hypertension. PMID:24547812

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

    PubMed

    Sánchez-Riego, Ana M; Mata-Cabana, Alejandro; Galmozzi, Carla V; 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 the 2-Cys peroxiredoxin (2-Cys Prx) as well as through other functions related to redox enzyme regulation. In cyanobacteria, the Anabaena NTRC has been characterized in vitro, however, nothing was known about its in vivo function. In order to study that, we have generated the first knockout mutant strain (ΔntrC), apart from the previously described in Arabidopsis. Detailed characterization of this strain reveals a differential sensitivity to oxidative stress treatments with respect to the wild-type Anabaena strain, including a higher level of ROS (reactive oxygen species) in normal growth conditions. In the mutant strain, different oxidative stress treatments such as hydrogen peroxide, methyl-viologen or high light irradiance provoke an increase in the expression of genes related to ROS detoxification, including AnNTRC and peroxiredoxin genes, with a concomitant increase in the amount of AnNTRC and 2-Cys Prx. Moreover, the role of AnNTRC in the antioxidant response is confirmed by the observation of a pronounced overoxidation of the 2-Cys Prx and a time-delay recovery of the reduced form of this protein upon oxidative stress treatments. Our results suggest the participation of this enzyme in the peroxide detoxification in Anabaena. In addition, we describe the role of Anabaena NTRC in thermotolerance, by the appearance of high molecular mass AnNTRC complexes, showing that the mutant strain is more sensitive to high temperature treatments.

  13. NAD(P)H oxidase-derived peroxide mediates elevated basal and impaired flow-induced NO production in SHR mesenteric arteries in vivo.

    PubMed

    Zhou, Xiaosun; Bohlen, H Glenn; Miller, Steven J; Unthank, Joseph L

    2008-09-01

    Nitric oxide (NO) and reactive oxygen species (ROS) have fundamentally important roles in the regulation of vascular tone and remodeling. Although arterial disease and endothelial dysfunction alter NO and ROS levels to impact vasodilation and vascular structure, direct measurements of these reactive species under in vivo conditions with flow alterations are unavailable. In this study, in vivo measurements of NO and H2O2 were made on mesenteric arteries to determine whether antioxidant therapies could restore normal NO production in spontaneously hypertensive rats (SHR). Flow was altered from approximately 50-200% of control in anesthetized Wistar-Kyoto rats (WKY) and SHR by selective placement of microvascular clamps on adjacent arteries while NO and H2O2 were directly measured with microelectrodes. Relative to WKY, SHR had significantly increased baseline NO and H2O2 concentrations (2,572 +/- 241 vs. 1,059 +/- 160 nM, P < 0.01; and 26 +/- 7 vs. 7 +/- 1 microM, P < 0.05, respectively). With flow elevation, H2O2 but not NO increased in SHR; NO but not H2O2 was elevated in WKY. Apocynin and polyethylene-glycolated catalase decreased baseline SHR NO and H2O2 to WKY levels and restored flow-mediated NO production. Suppression of NAD(P)H oxidase with gp91ds-tat decreased SHR H2O2 to WKY levels. Addition of topical H2O2 to increase peroxide to the basal concentration measured in SHR elevated WKY NO to levels observed in SHR. The results support the hypothesis that increased vascular peroxide in SHR is primarily derived from NAD(P)H oxidase and increases NO concentration to levels that cannot be further elevated with increased flow. Short-term and even acute administration of antioxidants are able to restore normal flow-mediated NO signaling in young SHR.

  14. PCR-mediated recombination of the amplification products of the Hibiscus tiliaceus cytosolic glyceraldehyde-3-phosphate dehydrogenase gene.

    PubMed

    Wu, Linghui; Tang, Tian; Zhou, Renchao; Shi, Suhua

    2007-03-31

    PCR-mediated recombination describes the process of in vitro chimera formation from related template sequences present in a single PCR amplification. The high levels of genetic redundancy in eukaryotic genomes should make recombination artifacts occur readily. However, few evolutionary biologists adequately consider this phenomenon when studying gene lineages. The cytosolic glyceraldehyde-3-phosphate dehydrogenase gene (GapC), which encodes a NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase in the cytosol, is a classical low-copy nuclear gene marker and is commonly used in molecular evolutionary studies. Here, we report on the occurrence of PCR-mediated recombination in the GapC gene family of Hibiscus tiliaceus. The study suggests that recombinant areas appear to be correlated with DNA template secondary structures. Our observations highlight that recombination artifacts should be considered when studying specific and allelic phylogenies. The authors suggest that nested PCR be used to suppress PCR-mediated recombination.

  15. The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67phox

    NASA Astrophysics Data System (ADS)

    Bechor, Edna; Dahan, Iris; Fradin, Tanya; Berdichevsky, Yevgeny; Zahavi, Anat; Rafalowski, Meirav; Federman-Gross, Aya; Pick, Edgar

    2015-02-01

    The superoxide (O2.-)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b558 (a heterodimer of Nox2 and p22phox), and four cytosolic components, p47phox, p67phox, p40phox, and Rac. The catalytic component, responsible for O2.- generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67phox. Using a peptide-protein binding assay, we found that Nox2 peptides containing a 369CysGlyCys371 triad (CGC) bound p67phox with high affinity, dependent upon the establishment of a disulfide bond between the two cysteines. Serially truncated recombinant Nox2 DHR proteins bound p67phox only when they comprised the CGC triad. CGC resembles the catalytic motif (CGHC) of protein disulfide isomerases (PDIs). This led to the hypothesis that Nox2 establishes disulfide bonds with p67phox via a thiol-dilsulfide exchange reaction and, thus, functions as a PDI. Evidence for this was provided by the following: 1. Recombinant Nox2 protein, which contained the CGC triad, exhibited PDI-like disulfide reductase activity; 2. Truncation of Nox2 C-terminal to the CGC triad or mutating C369 and C371 to R, resulted in loss of PDI activity; 3. Comparison of the sequence of the DHR of Nox2 with PDI family members revealed three small regions of homology with PDIA3; 4. Two monoclonal anti-Nox2 antibodies, with epitopes corresponding to regions of Nox2/PDIA3 homology, reacted with PDIA3 but not with PDIA1; 5. A polyclonal anti-PDIA3 (but not an anti-PDIA1) antibody reacted with Nox2; 6. p67phox, in which all cysteines were mutated to serines, lost its ability to bind to a Nox2 peptide containing the CGC triad and had an impaired capacity to support oxidase activity in vitro. We propose a model of oxidase assembly in which binding of p67phox to Nox2 via disulfide bonds, by virtue of the intrinsic PDI activity of Nox2, stabilizes the primary interaction between the two components.

  16. Effects of various compounds on lipid peroxidation mediated by detergent-solubilized rat liver NADPH-cytochrome C reductase.

    PubMed

    Kamataki, T; Sugita, O; Naminohira, S; Kitagawa, H

    1978-12-01

    A reconstituted lipid peroxidation system containing NADPH-cytochrome c reductase isolated from detergent-solubilized rat liver microsomes was used to determine the effects of several compounds, including drugs, on the lipid peroxidation activity. EDTA and ferrous ion were essential requirements for reconstitution of the activity. The addition of 1,10-phenanthroline to the system containing both EDTA and ferrous ion further enhanced the activity. Pyrocatecol, thymol, p-aminophenol, imipramine, p-chloromercuribenzoate (PCMB) and alpha-tocopherol exhibited strong inhibition, aniline, N-monomethylaniline, aminopyrine, benzphetamine, SKF 525-A and NADP exhibited moderate inhibition, and phenol, benzoic acid, acetanilide and nicotinamide exhibited less or no inhibition at the concentrations lower than 1000 micron M. Metal ions such as Hg+, Hg2+, Co2+, Cu2+, Mn2+ and U6+ inhibited lipid peroxidation strongly. In addition, Cd2+, St2+ and Ca2+ exhibited less potent to moderate inhibition, and Ba2+ and Mg2+ were without effects on the activity. Among sulfhydryl compounds tested, dithiothreitol inhibited lipid peroxidation to a greater extent than did the other three compounds, glutathione, cysteine and mercaptoethanol.

  17. Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of Brassica napus leaf protoplasts.

    PubMed

    Tewari, Rajesh Kumar; Watanabe, Daisuke; Watanabe, Masami

    2012-01-01

    Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation in Brassica napus leaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation in B. napus leaf protoplasts. In vivo localization of H(2)O(2) and enzymes involved in H(2)O(2) generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H(2)O(2) and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H(2)O(2) was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H(2)O(2) in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H(2)O(2) generation probably induced apoptotic-like cell death of B. napus leaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential.

  18. NOX3 NADPH Oxidase Couples Transient Receptor Potential Vanilloid 1 to Signal Transducer and Activator of Transcription 1-Mediated Inflammation and Hearing Loss

    PubMed Central

    Mukherjea, Debashree; Jajoo, Sarvesh; Sheehan, Kelly; Kaur, Tejbeer; Sheth, Sandeep; Bunch, Jennifer; Perro, Christopher; Rybak, Leonard P.

    2011-01-01

    Abstract Transient receptor potential vanilloid 1 (TRPV1) is implicated in cisplatin ototoxicity. Activation of this channel by cisplatin increases reactive oxygen species generation, which contribute to loss of outer hair cells in the cochlea. Knockdown of TRPV1 by short interfering RNA protected against cisplatin ototoxicity. In this study, we examined the mechanism underlying TRPV1-mediated ototoxicity using cultured organ of Corti transformed cells (UB/OC-1) and rats. Trans-tympanic injections of capsaicin produced transient hearing loss within 24 h, which recovered by 72 h. In UB/OC-1 cells, capsaicin increased NOX3 NADPH oxidase activity and activation of signal transducer and activator of transcription 1 (STAT1). Intratympanic administration of capsaicin transiently increased STAT1 activity and expression of downstream proinflammatory molecules. Capsaicin produced a transient increase in CD14-positive inflammatory cells into the cochlea, which mimicked the temporal course of STAT1 activation but did not alter the expression of apoptotic genes or damage to outer hair cells. In addition, trans-tympanic administration of STAT1 short interfering RNA protected against capsaicin-induced hearing loss. These data suggest that activation of TRPV1 mediates temporary hearing loss by initiating an inflammatory process in the cochlea via activation of NOX3 and STAT1. Thus, these proteins represent reasonable targets for ameliorating hearing loss. Antioxid. Redox Signal. 14, 999–1010. PMID:20712533

  19. NOX3 NADPH oxidase couples transient receptor potential vanilloid 1 to signal transducer and activator of transcription 1-mediated inflammation and hearing loss.

    PubMed

    Mukherjea, Debashree; Jajoo, Sarvesh; Sheehan, Kelly; Kaur, Tejbeer; Sheth, Sandeep; Bunch, Jennifer; Perro, Christopher; Rybak, Leonard P; Ramkumar, Vickram

    2011-03-15

    Transient receptor potential vanilloid 1 (TRPV1) is implicated in cisplatin ototoxicity. Activation of this channel by cisplatin increases reactive oxygen species generation, which contribute to loss of outer hair cells in the cochlea. Knockdown of TRPV1 by short interfering RNA protected against cisplatin ototoxicity. In this study, we examined the mechanism underlying TRPV1-mediated ototoxicity using cultured organ of Corti transformed cells (UB/OC-1) and rats. Trans-tympanic injections of capsaicin produced transient hearing loss within 24 h, which recovered by 72 h. In UB/OC-1 cells, capsaicin increased NOX3 NADPH oxidase activity and activation of signal transducer and activator of transcription 1 (STAT1). Intratympanic administration of capsaicin transiently increased STAT1 activity and expression of downstream proinflammatory molecules. Capsaicin produced a transient increase in CD14-positive inflammatory cells into the cochlea, which mimicked the temporal course of STAT1 activation but did not alter the expression of apoptotic genes or damage to outer hair cells. In addition, trans-tympanic administration of STAT1 short interfering RNA protected against capsaicin-induced hearing loss. These data suggest that activation of TRPV1 mediates temporary hearing loss by initiating an inflammatory process in the cochlea via activation of NOX3 and STAT1. Thus, these proteins represent reasonable targets for ameliorating hearing loss.

  20. Intermittent Hypoxia-Induced Parvalbumin-Immunoreactive Interneurons Loss and Neurobehavioral Impairment is Mediated by NADPH-Oxidase-2.

    PubMed

    Yuan, Liang; Wu, Jing; Liu, Jiang; Li, Guowei; Liang, Dong

    2015-06-01

    Obstructive sleep apnea usually contribute to psychiatric diseases and cognitive impairments in adults. Loss of parvalbumin (PV)-immunoreactive interneurons (PV-IN) in the brain cortex is an important feature of psychiatric diseases, such as schizophrenia. Here we investigate the causal contribution of oxidative stress in the brain cortex to neuropathological alterations in a mouse model of sleep apnea. Wild-type (WT) and the NADPH-oxidase-2 (gp91-phox/NOX2) knock-out adult male C57BL/6J mice were exposed to intermittent hypoxia (IH) or standard room air in the same chamber. In vivo we determined the impact (1) of IH exposures on NOX2 expression, (2) of genetic gp91-phox/NOX2 knock-out and (3) of pharmacological NOX2 inhibition on IH-induced neuropathological alterations in adult mice. Endpoints were oxidative stress, PV-IN and neurobehavioral alterations. The results showed IH exposures increased NOX2 expression in the prefrontal cortex of WT mice, which was accompanied with elevations of indirect markers of oxidative stress (HNE, HIF-1α, 8-OHDG). WT mice showed loss of PV-IN in the prefrontal cortex and increased locomotion activity and anxiety levels after exposed to IH, while no change emerged in NOX2 knock-out mice. Treatment of WT mice with the antioxidant/NOX inhibitor apocynin prevented the neuropathological and neurobehavioral alterations induced by IH exposures. Our data suggest that NOX2-derived oxidative stress is involved in the loss of PV-IN in the prefrontal cortex and development of neurobehavioral alterations for adult mice exposed to IH. These results provide a molecular mechanism for the coupling between sleep apnea and brain oxidative stress as well as potential new therapeutic avenues.

  1. A novel role of microglial NADPH oxidase in mediating extra-synaptic function of norepinephrine in regulating brain immune homeostasis.

    PubMed

    Jiang, Lulu; Chen, Shih-Heng; Chu, Chun-Hsien; Wang, Shi-Jun; Oyarzabal, Esteban; Wilson, Belinda; Sanders, Virginia; Xie, Keqin; Wang, Qingshan; Hong, Jau-Shyong

    2015-06-01

    Although the peripheral anti-inflammatory effect of norepinephrine (NE) is well documented, the mechanism by which this neurotransmitter functions as an anti-inflammatory/neuroprotective agent in the central nervous system (CNS) is unclear. This article aimed to determine the anti-inflammatory/neuroprotective effects and underlying mechanisms of NE in inflammation-based dopaminergic neurotoxicity models. In mice, NE-depleting toxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) was injected at 6 months of lipopolysaccharide (LPS)-induced neuroinflammation. It was found that NE depletion enhanced LPS-induced dopaminergic neuron loss in the substantia nigra. This piece of in vivo data prompted us to conduct a series of studies in an effort to elucidate the mechanism as to how NE affects dopamine neuron survival by using primary midbrain neuron/glia cultures. Results showed that submicromolar concentrations of NE dose-dependently protected dopaminergic neurons from LPS-induced neurotoxicity by inhibiting microglia activation and subsequent release of pro-inflammatory factors. However, NE-elicited neuroprotection was not totally abolished in cultures from β2-adrenergic receptor (β2-AR)-deficient mice, suggesting that novel pathways other than β2-AR are involved. To this end, It was found that submicromolar NE dose-dependently inhibited NADPH oxidase (NOX2)-generated superoxide, which contributes to the anti-inflammatory and neuroprotective effects of NE. This novel mechanism was indeed adrenergic receptors independent since both (+) and (-) optic isomers of NE displayed the same potency. We further demonstrated that NE inhibited LPS-induced NOX2 activation by blocking the translocation of its cytosolic subunit to plasma membranes. In summary, we revealed a potential physiological role of NE in maintaining brain immune homeostasis and protecting neurons via a novel mechanism.

  2. Rhinovirus-Induced Barrier Dysfunction in Polarized Airway Epithelial Cells Is Mediated by NADPH Oxidase 1▿

    PubMed Central

    Comstock, Adam T.; Ganesan, Shyamala; Chattoraj, Asamanja; Faris, Andrea N.; Margolis, Benjamin L.; Hershenson, Marc B.; Sajjan, Umadevi S.

    2011-01-01

    Previously, we showed that rhinovirus (RV), which is responsible for the majority of common colds, disrupts airway epithelial barrier function, as evidenced by reduced transepithelial resistance (RT), dissociation of zona occludins 1 (ZO-1) from the tight junction complex, and bacterial transmigration across polarized cells. We also showed that RV replication is required for barrier function disruption. However, the underlying biochemical mechanisms are not known. In the present study, we found that a double-stranded RNA (dsRNA) mimetic, poly(I:C), induced tight junction breakdown and facilitated bacterial transmigration across polarized airway epithelial cells, similar to the case with RV. We also found that RV and poly(I:C) each stimulated Rac1 activation, reactive oxygen species (ROS) generation, and Rac1-dependent NADPH oxidase 1 (NOX1) activity. Inhibitors of Rac1 (NSC23766), NOX (diphenylene iodonium), and NOX1 (small interfering RNA [siRNA]) each blocked the disruptive effects of RV and poly(I:C) on RT, as well as the dissociation of ZO-1 and occludin from the tight junction complex. Finally, we found that Toll-like receptor 3 (TLR3) is not required for either poly(I:C)- or RV-induced reductions in RT. Based on these results, we concluded that Rac1-dependent NOX1 activity is required for RV- or poly(I:C)-induced ROS generation, which in turn disrupts the barrier function of polarized airway epithelia. Furthermore, these data suggest that dsRNA generated during RV replication is sufficient to disrupt barrier function. PMID:21507984

  3. Amyloid β-induced astrogliosis is mediated by β1-integrin via NADPH oxidase 2 in Alzheimer's disease.

    PubMed

    Wyssenbach, Ane; Quintela, Tania; Llavero, Francisco; Zugaza, Jose L; Matute, Carlos; Alberdi, Elena

    2016-10-05

    Astrogliosis is a hallmark of Alzheimer's disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid-β modulates β1-integrin activity and triggers NADPH oxidase (NOX)-dependent astrogliosis in vitro and in vivo. Amyloid-β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1-integrin in cultured astrocytes. This mechanism promotes β1-integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple-transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1-integrin in reactive astrocytes which correlates with the amyloid β-oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1-integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1-integrin were significantly associated with increased amyloid-β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1-integrin which in turn leads to enhancing β1-integrin and NOX2 activity via NOX-dependent mechanisms. These observations may be relevant to AD pathophysiology.

  4. A novel role of microglial NADPH oxidase in mediating extra-synaptic function of norepinephrine in regulating brain immune homeostasis

    PubMed Central

    Jiang, Lulu; Chen, Shih-Heng; Chu, Chun-Hsien; Wang, Shi-Jun; Oyarzabal, Esteban; Wilson, Belinda; Sanders, Virginia; Xie, Keqin; Wang, Qingshan; Hong, Jau-Shyong

    2015-01-01

    Although the peripheral anti-inflammatory effect of norepinephrine (NE) is well-documented, the mechanism by which this neurotransmitter functions as an anti-inflammatory/neuroprotective agent in the central nervous system is unclear. This study aimed to determine the anti-inflammatory/neuroprotective effects and underlying mechanisms of NE in inflammation-based dopaminergic neurotoxicity models. In mice, NE-depleting toxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) was injected at 6 months of lipopolysaccharide (LPS)-induced neuroinflammation. We found that NE depletion enhanced LPS-induced dopaminergic neuron loss in the substantia nigra. This piece of in vivo data prompted us to conduct a series of studies in an effort to elucidate the mechanism as to how NE affects dopamine neuron survival by using primary midbrain neuron-glia cultures. Results showed that sub-micromolar concentrations of NE dose-dependently protected dopaminergic neurons from LPS-induced neurotoxicity by inhibiting microglia activation and subsequent release of pro-inflammatory factors. However, NE-elicited neuroprotection was not totally abolished in cultures from β2-adrenergic receptor (β2-AR) deficient mice, suggesting that novel pathways other than β2-AR are involved. To this end, we found that sub-micromolar NE dose-dependently inhibited NADPH oxidase (NOX2)-generated superoxide, which contributes to the anti-inflammatory and neuroprotective effects of NE. This novel mechanism was indeed adrenergic receptors independent since both (+) and (−) optic isomers of NE displayed the same potency. We further demonstrated that NE inhibited LPS-induced NOX2 activation by blocking the translocation of its cytosolic subunit to plasma membranes. In summary, we revealed a potential physiological role of NE in maintaining brain immune homeostasis and protecting neurons via a novel mechanism. PMID:25740080

  5. E4F1-mediated control of pyruvate dehydrogenase activity is essential for skin homeostasis.

    PubMed

    Goguet-Rubio, Perrine; Seyran, Berfin; Gayte, Laurie; Bernex, Florence; Sutter, Anne; Delpech, Hélène; Linares, Laetitia Karine; Riscal, Romain; Repond, Cendrine; Rodier, Geneviève; Kirsh, Olivier; Touhami, Jawida; Noel, Jean; Vincent, Charles; Pirot, Nelly; Pavlovic, Guillaume; Herault, Yann; Sitbon, Marc; Pellerin, Luc; Sardet, Claude; Lacroix, Matthieu; Le Cam, Laurent

    2016-09-27

    The multifunctional protein E4 transcription factor 1 (E4F1) is an essential regulator of epidermal stem cell (ESC) maintenance. Here, we found that E4F1 transcriptionally regulates a metabolic program involved in pyruvate metabolism that is required to maintain skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetyltransferase (Dlat), a gene encoding the E2 subunit of the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) keratinocytes exhibited impaired PDH activity and a redirection of the glycolytic flux toward lactate production. The metabolic reprogramming of E4f1 KO keratinocytes associated with remodeling of their microenvironment and alterations of the basement membrane, led to ESC mislocalization and exhaustion of the ESC pool. ShRNA-mediated depletion of Dlat in primary keratinocytes recapitulated defects observed upon E4f1 inactivation, including increased lactate secretion, enhanced activity of extracellular matrix remodeling enzymes, and impaired clonogenic potential. Altogether, our data reveal a central role for Dlat in the metabolic program regulated by E4F1 in basal keratinocytes and illustrate the importance of PDH activity in skin homeostasis.

  6. MxaY regulates the lanthanide-mediated methanol dehydrogenase switch in Methylomicrobium buryatense

    PubMed Central

    Chu, Frances; Beck, David A.C.

    2016-01-01

    Many methylotrophs, microorganisms that consume carbon compounds lacking carbon–carbon bonds, use two different systems to oxidize methanol for energy production and biomass accumulation. The MxaFI methanol dehydrogenase (MDH) contains calcium in its active site, while the XoxF enzyme contains a lanthanide in its active site. The genes encoding the MDH enzymes are differentially regulated by the presence of lanthanides. In this study, we found that the histidine kinase MxaY controls the lanthanide-mediated switch in Methylomicrobium buryatense 5GB1C. MxaY controls the transcription of genes encoding MxaFI and XoxF at least partially by controlling the transcript levels of the orphan response regulator MxaB. We identify a constitutively active version of MxaY, and identify the mutated residue that may be involved in lanthanide sensing. Lastly, we find evidence to suggest that tight control of active MDH production is required for wild-type growth rates. PMID:27651996

  7. E4F1-mediated control of pyruvate dehydrogenase activity is essential for skin homeostasis

    PubMed Central

    Goguet-Rubio, Perrine; Seyran, Berfin; Gayte, Laurie; Sutter, Anne; Delpech, Hélène; Linares, Laetitia Karine; Riscal, Romain; Repond, Cendrine; Rodier, Geneviève; Touhami, Jawida; Noel, Jean; Vincent, Charles; Pirot, Nelly; Herault, Yann; Pellerin, Luc; Sardet, Claude; Lacroix, Matthieu; Le Cam, Laurent

    2016-01-01

    The multifunctional protein E4 transcription factor 1 (E4F1) is an essential regulator of epidermal stem cell (ESC) maintenance. Here, we found that E4F1 transcriptionally regulates a metabolic program involved in pyruvate metabolism that is required to maintain skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetyltransferase (Dlat), a gene encoding the E2 subunit of the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) keratinocytes exhibited impaired PDH activity and a redirection of the glycolytic flux toward lactate production. The metabolic reprogramming of E4f1 KO keratinocytes associated with remodeling of their microenvironment and alterations of the basement membrane, led to ESC mislocalization and exhaustion of the ESC pool. ShRNA-mediated depletion of Dlat in primary keratinocytes recapitulated defects observed upon E4f1 inactivation, including increased lactate secretion, enhanced activity of extracellular matrix remodeling enzymes, and impaired clonogenic potential. Altogether, our data reveal a central role for Dlat in the metabolic program regulated by E4F1 in basal keratinocytes and illustrate the importance of PDH activity in skin homeostasis. PMID:27621431

  8. Role of gp91phox-Containing NADPH Oxidase in Mediating the Effect of K Restriction on ROMK Channels and Renal K Excretion

    PubMed Central

    Babilonia, Elisa; Lin, Daohong; Zhang, Yan; Wei, Yuan; Yue, Peng; Wang, Wen-Hui

    2009-01-01

    Previous study has demonstrated that superoxide and the related products are involved in mediating the effect of low K intake on renal K secretion and ROMK channel activity in the cortical collecting duct (CCD). This study investigated the role of gp91phox-containing NADPH oxidase (NOXII) in mediating the effect of low K intake on renal K excretion and ROMK channel activity in gp91(-/-) mice. K depletion increased superoxide levels, phosphorylation of c-Jun, expression of c-Src, and tyrosine phosphorylation of ROMK in renal cortex and outer medulla in wild-type (WT) mice. In contrast, tempol treatment in WT mice abolished whereas deletion of gp91 significantly attenuated the effect of low K intake on superoxide production, c-Jun phosphorylation, c-Src expression, and tyrosine phosphorylation of ROMK. Patch-clamp experiments demonstrated that low K intake decreased mean product of channel number (N) and open probability (P) (NPo) of ROMK channels from 1.1 to 0.4 in the CCD. However, the effect of low K intake on ROMK channel activity was significantly attenuated in the CCD from gp91(-/-) mice and completely abolished by tempol treatment. Immunocytochemical staining also was used to examine the ROMK distribution in WT, gp91(-/-), and WT mice with tempol treatment in response to K restriction. K restriction decreased apical staining of ROMK in WT mice. In contrast, a sharp apical ROMK staining was observed in the tempol-treated WT or gp91(-/-) mice. Metabolic cage study further showed that urinary K loss is significantly higher in gp91(-/-) mice than in WT mice. It is concluded that superoxide anions play a key role in suppressing K secretion during K restriction and that NOXII is involved in mediating the effect of low K intake on renal K secretion and ROMK channel activity. PMID:17538186

  9. Role of gp91phox -containing NADPH oxidase in mediating the effect of K restriction on ROMK channels and renal K excretion.

    PubMed

    Babilonia, Elisa; Lin, Daohong; Zhang, Yan; Wei, Yuan; Yue, Peng; Wang, Wen-Hui

    2007-07-01

    Previous study has demonstrated that superoxide and the related products are involved in mediating the effect of low K intake on renal K secretion and ROMK channel activity in the cortical collecting duct (CCD). This study investigated the role of gp91(phox)-containing NADPH oxidase (NOXII) in mediating the effect of low K intake on renal K excretion and ROMK channel activity in gp91(-/-) mice. K depletion increased superoxide levels, phosphorylation of c-Jun, expression of c-Src, and tyrosine phosphorylation of ROMK in renal cortex and outer medulla in wild-type (WT) mice. In contrast, tempol treatment in WT mice abolished whereas deletion of gp91 significantly attenuated the effect of low K intake on superoxide production, c-Jun phosphorylation, c-Src expression, and tyrosine phosphorylation of ROMK. Patch-clamp experiments demonstrated that low K intake decreased mean product of channel number (N) and open probability (P) (NP(o)) of ROMK channels from 1.1 to 0.4 in the CCD. However, the effect of low K intake on ROMK channel activity was significantly attenuated in the CCD from gp91(-/-) mice and completely abolished by tempol treatment. Immunocytochemical staining also was used to examine the ROMK distribution in WT, gp91(-/-), and WT mice with tempol treatment in response to K restriction. K restriction decreased apical staining of ROMK in WT mice. In contrast, a sharp apical ROMK staining was observed in the tempol-treated WT or gp91(-/-) mice. Metabolic cage study further showed that urinary K loss is significantly higher in gp91(-/-) mice than in WT mice. It is concluded that superoxide anions play a key role in suppressing K secretion during K restriction and that NOXII is involved in mediating the effect of low K intake on renal K secretion and ROMK channel activity.

  10. Enhancing biomass and ethanol production by increasing NADPH production in Synechocystis sp. PCC 6803.

    PubMed

    Choi, Yun-Nam; Park, Jong Moon

    2016-08-01

    This study demonstrates that increased NADPH production can improve biomass and ethanol production in cyanobacteria. We over-expressed the endogenous zwf gene, which encodes glucose-6-phosphate dehydrogenase of pentose phosphate pathway, in the model cyanobacterium Synechocystis sp. PCC 6803. zwf over-expression resulted in increased NADPH production, and promoted biomass production compared to the wild type in both autotrophic and mixotrophic conditions. Ethanol production pathway including NADPH-dependent alcohol dehydrogenase was also integrated with and without zwf over-expression. Excessive NADPH production by zwf over-expression could improve both biomass and ethanol production in the autotrophic conditions.

  11. Critical role of X-box binding protein 1 in NADPH oxidase 4-triggered cardiac hypertrophy is mediated by receptor interacting protein kinase 1.

    PubMed

    Chen, Li; Zhao, Mingyue; Li, Junli; Wang, Yu; Bao, Qinxue; Wu, Siyuan; Deng, Xueqin; Tang, Xiaoju; Wu, Wenchao; Liu, Xiaojing

    2017-02-16

    NADPH oxidase 4 (NOX4) and the NOX4-related redox signaling are implicated in cardiac hypertrophy. NOX4 is interrelated with endoplasmic reticulum stress (ERS). Spliced X-box binding protein 1 (Xbp1s) is a key mediator of ERS while its role in cardiac hypertrophy is still poorly understood. Recently, receptor interacting protein kinase 1(RIPK1) has been increasingly reported to be associated with ERS. Therefore, we aimed to test the hypothesis that Xbp1s mediates NOX4-triggered cardiac hypertrophy via RIPK1 signaling. In the heart tissue of transverse aortic constriction (TAC) rats and in primary cultured neonatal cardiomyocytes(NCMs) treated with angiotensinII(AngII) or isoproterenol (ISO), NOX4 expression and reactive oxygen species (ROS) generation, and expression of Xbp1s as well as RIPK1-related phosphorylation of P65 subunit of NF-κB were elevated. Gene silencing of NOX4 by specific small interfering RNA (siRNA) significantly blocked the upregulation of NOX4, generation of ROS, splicing of Xbp1 and activation of the RIPK1-related NF-κB signaling, meanwhile attenuated cardiomyocyte hypertrophy. In addition, ROS scavenger (N-acetyl-L-cysteine, NAC) and NOX4 inhibitor GKT137831 reduced ROS generation and alleviated activation of Xbp1 and RIPK1-related NF-κB signaling. Furthermore, splicing of Xbp1 was responsible for the increase in RIPK1 expression in AngII or ISO-treated NCMs. Upregulated RIPK1 in turn activates NF-κB signaling in a kinase activity-independent manner. These findings suggest that Xbp1s plays an important role in NOX4-triggered cardiomyocyte hypertrophy via activating its downstream effector RIPK1, which may prove significant for the development of future therapeutic strategies.

  12. Phosphorylation of the pyruvate dehydrogenase complex precedes HIF-1-mediated effects and pyruvate dehydrogenase kinase 1 upregulation during the first hours of hypoxic treatment in hepatocellular carcinoma cells

    PubMed Central

    Zimmer, Andreas David; Walbrecq, Geoffroy; Kozar, Ines; Behrmann, Iris; Haan, Claude

    2016-01-01

    The pyruvate dehydrogenase complex (PDC) is an important gatekeeper enzyme connecting glycolysis to the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Thereby, it has a strong impact on the glycolytic flux as well as the metabolic phenotype of a cell. PDC activity is regulated via reversible phosphorylation of three serine residues on the pyruvate dehydrogenase (PDH) E1α subunit. Phosphorylation of any of these residues by the PDH kinases (PDKs) leads to a strong decrease in PDC activity. Under hypoxia, the inactivation of the PDC has been described to be dependent on the hypoxia-inducible factor 1 (HIF-1)-induced PDK1 protein upregulation. In this study, we show in two hepatocellular carcinoma cell lines (HepG2 and JHH-4) that, during the adaptation to hypoxia, PDH is already phosphorylated at time points preceding HIF-1-mediated transcriptional events and PDK1 protein upregulation. Using siRNAs and small molecule inhibitor approaches, we show that this inactivation of PDC is independent of HIF-1α expression but that the PDKs need to be expressed and active. Furthermore, we show that reactive oxygen species might be important for the induction of this PDH phosphorylation since it correlates with the appearance of an altered redox state in the mitochondria and is also inducible by H2O2 treatment under normoxic conditions. Overall, these results show that neither HIF-1 expression nor PDK1 upregulation is necessary for the phosphorylation of PDH during the first hours of the adaptation to hypoxia. PMID:27800515

  13. NADPH-generating systems in bacteria and archaea

    PubMed Central

    Spaans, Sebastiaan K.; Weusthuis, Ruud A.; van der Oost, John; Kengen, Servé W. M.

    2015-01-01

    Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided. PMID:26284036

  14. Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

    SciTech Connect

    Castro, Miguel E.; Molina, Roberto C.; Diaz, Waldo A.; Pradenas, Gonzalo A.; Vasquez, Claudio C.

    2009-02-27

    Potassium tellurite (K{sub 2}TeO{sub 3}) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.

  15. H2O2 generated by NADPH oxidase 4 contributes to transient receptor potential vanilloid 1 channel-mediated mechanosensation in the rat kidney.

    PubMed

    Lin, Chian-Shiung; Lee, Shang-Hsing; Huang, Ho-Shiang; Chen, Yih-Sharng; Ma, Ming-Chieh

    2015-08-15

    The presence of NADPH oxidase (Nox) in the kidney, especially Nox4, results in H2O2 production, which regulates Na(+) excretion and urine formation. Redox-sensitive transient receptor potential vanilloid 1 channels (TRPV1s) are distributed in mechanosensory fibers of the renal pelvis and monitor changes in intrapelvic pressure (IPP) during urine formation. The present study tested whether H2O2 derived from Nox4 affects TRPV1 function in renal sensory responses. Perfusion of H2O2 into the renal pelvis dose dependently increased afferent renal nerve activity and substance P (SP) release. These responses were attenuated by cotreatment with catalase or TRPV1 blockers. In single unit recordings, H2O2 activated afferent renal nerve activity in response to rising IPP but not high salt. Western blots revealed that Nox2 (gp91(phox)) and Nox4 are both present in the rat kidney, but Nox4 is abundant in the renal pelvis and originates from dorsal root ganglia. This distribution was associated with expression of the Nox4 regulators p22(phox) and polymerase δ-interacting protein 2. Coimmunoprecipitation experiments showed that IPP increases polymerase δ-interacting protein 2 association with Nox4 or p22(phox) in the renal pelvis. Interestingly, immunofluorescence labeling demonstrated that Nox4 colocalizes with TRPV1 in sensory fibers of the renal pelvis, indicating that H2O2 generated from Nox4 may affect TRPV1 activity. Stepwise increases in IPP and saline loading resulted in H2O2 and SP release, sensory activation, diuresis, and natriuresis. These effects, however, were remarkably attenuated by Nox inhibition. Overall, these results suggest that Nox4-positive fibers liberate H2O2 after mechanostimulation, thereby contributing to a renal sensory nerve-mediated diuretic/natriuretic response.

  16. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

    PubMed

    Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

    2015-08-21

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

  17. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

    PubMed Central

    Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

    2015-01-01

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

  18. NAD(P)H oxidase and renal epithelial ion transport

    PubMed Central

    Schreck, Carlos

    2011-01-01

    A fundamental requirement for cellular vitality is the maintenance of plasma ion concentration within strict ranges. It is the function of the kidney to match urinary excretion of ions with daily ion intake and nonrenal losses to maintain a stable ionic milieu. NADPH oxidase is a source of reactive oxygen species (ROS) within many cell types, including the transporting renal epithelia. The focus of this review is to describe the role of NADPH oxidase-derived ROS toward local renal tubular ion transport in each nephron segment and to discuss how NADPH oxidase-derived ROS signaling within the nephron may mediate ion homeostasis. In each case, we will attempt to identify the various subunits of NADPH oxidase and reactive oxygen species involved and the ion transporters, which these affect. We will first review the role of NADPH oxidase on renal Na+ and K+ transport. Finally, we will review the relationship between tubular H+ efflux and NADPH oxidase activity. PMID:21270341

  19. Ethanol-induced erectile dysfunction and increased expression of pro-inflammatory proteins in the rat cavernosal smooth muscle are mediated by NADPH oxidase-derived reactive oxygen species.

    PubMed

    Leite, Letícia N; do Vale, Gabriel T; Simplicio, Janaina A; De Martinis, Bruno S; Carneiro, Fernando S; Tirapelli, Carlos R

    2017-03-15

    Ethanol consumption is associated with an increased risk of erectile dysfunction (ED), but the molecular mechanisms through which ethanol causes ED remain elusive. Reactive oxygen species are described as mediators of ethanol-induced cell toxicity/damage in distinctive tissues. The enzyme NADPH oxidase is the main source of reactive oxygen species in the endothelium and vascular smooth muscle cells and ethanol is described to increase NADPH oxidase activation and reactive oxygen species generation. This study evaluated the contribution of NADPH oxidase-derived reactive oxygen species to ethanol-induced ED, endothelial dysfunction and production of pro-inflammatory and redox-sensitive proteins in the rat cavernosal smooth muscle (CSM). Male Wistar rats were treated with ethanol (20% v/v) or ethanol plus apocynin (30mg/kg/day; p.o. gavage) for six weeks. Apocynin prevented both the decreased in acetylcholine-induced relaxation and intracavernosal pressure induced by ethanol. Ethanol increased superoxide anion (O2(-)) generation and catalase activity in CSM, and treatment with apocynin prevented these responses. Similarly, apocynin prevented the ethanol-induced decreased of nitrate/nitrite (NOx), hydrogen peroxide (H2O2) and SOD activity. Treatment with ethanol increased p47phox translocation to the membrane as well as the expression of Nox2, COX-1, catalase, iNOS, ICAM-1 and p65. Apocynin prevented the effects of ethanol on protein expression and p47phox translocation. Finally, treatment with ethanol increased both TNF-α production and neutrophil migration in CSM. The major new finding of this study is that NADPH oxidase-derived reactive oxygen species play a role on chronic ethanol consumption-induced ED and endothelial dysfunction in the rat CSM.

  20. Role of Quinones in Electron Transfer of PQQ–Glucose Dehydrogenase Anodes—Mediation or Orientation Effect

    SciTech Connect

    Babanova, Sofia; Matanovic, Ivana; Chavez, Madelaine Seow; Atanassov, Plamen

    2015-06-24

    In this study, the influence of two quinones (1,2- and 1,4-benzoquinone) on the operation and mechanism of electron transfer in PQQ-dependent glucose dehydrogenase (PQQ–sGDH) anodes has been determined. Benzoquinones were experimentally explored as mediators present in the electrolyte. The electrochemical performance of the PQQ–sGDH anodes with and without the mediators was examined and for the first time molecular docking simulations were used to gain a fundamental understanding to explain the role of the mediator molecules in the design and operation of the enzymatic electrodes. It was proposed that the higher performance of the PQQ–sGDH anodes in the presence of 1,2- and 1,4-benzoquinones introduced in the solution is due to the shorter distance between these molecules and PQQ in the enzymatic molecule. It was also hypothesized that when 1,4-benzoquinone is adsorbed on a carbon support, it would play the dual role of a mediator and an orienting agent. At the same time, when 1,2-benzoquinone and ubiquinone are adsorbed on the electrode surface, the enzyme would transfer the electrons directly to the support, and these molecules would primarily play the role of an orienting agent.

  1. Quantitative flux analysis reveals folate-dependent NADPH production

    NASA Astrophysics Data System (ADS)

    Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-06-01

    ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

  2. Single-Protein Tracking Reveals That NADPH Mediates the Insertion of Cytochrome P450 Reductase into a Biomimetic of the Endoplasmic Reticulum.

    PubMed

    Barnaba, Carlo; Martinez, Michael J; Taylor, Evan; Barden, Adam O; Brozik, James A

    2017-04-06

    Cytochrome P450 reductase (CPR) is the redox partner for most human cytochrome P450 enzymes. It is also believed that CPR is an integral membrane protein exclusively. Herein, we report that, contrary to this belief, CPR can exist as a peripheral membrane protein in the absence of NADPH and will transition to an integral membrane protein in the presence of stoichiometric amounts of NADPH or greater. All experiments were performed in a solid-supported cushioned lipid bilayer that closely matched the chemical composition of the human endoplasmic reticulum and served as an ER biomimetic. The phase characteristics and fluidity of the ER biomimetic was characterized with fluorescence micrographs and temperature-dependent fluorescence recovery after photobleaching. The interactions of CPR with the ER biomimetic were directly observed by tracking single CPR molecules using time-lapse single-molecule fluorescence imaging and subsequent analysis of tracks. These studies revealed dramatic changes in diffusion coefficient and the degree of partitioning of CPR as a function of NADPH concentration.

  3. Integration of Inhibition Kinetics and Molecular Dynamics Simulations: A Urea-Mediated Folding Study on Acetaldehyde Dehydrogenase 1.

    PubMed

    Xu, Yingying; Lee, Jinhyuk; Lü, Zhi-Rong; Mu, Hang; Zhang, Qian; Park, Yong-Doo

    2016-07-01

    Understanding the mechanism of acetaldehyde dehydrogenase 1 (ALDH1) folding is important because this enzyme is directly involved in several types of cancers and other diseases. We investigated the urea-mediated unfolding of ALDH1 by integrating kinetic inhibition studies with computational molecular dynamics (MD) simulations. Conformational changes in the enzyme structure were also analyzed using intrinsic and 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence measurements. Kinetic studies revealed that the direct binding of urea to ALDH1 induces inactivation of ALDH1 in a manner of mixed-type inhibition. Tertiary structural changes associated with regional hydrophobic exposure of the active site were observed. The urea binding regions on ALDH1 were predicted by docking simulations and were partly shared with active site residues of ALDH1 and with interface residues of the oligomerization domain for tetramer formation. The docking results suggest that urea prevents formation of the ALDH1 normal shape for the tetramer state as well as entrance of the substrate into the active site. Our study provides insight into the structural changes that accompany urea-mediated unfolding of ALDH1 and the catalytic role associated with conformational changes.

  4. Cardiac Ryanodine Receptor (Ryr2)-mediated Calcium Signals Specifically Promote Glucose Oxidation via Pyruvate Dehydrogenase.

    PubMed

    Bround, Michael J; Wambolt, Rich; Cen, Haoning; Asghari, Parisa; Albu, Razvan F; Han, Jun; McAfee, Donald; Pourrier, Marc; Scott, Nichollas E; Bohunek, Lubos; Kulpa, Jerzy E; Chen, S R Wayne; Fedida, David; Brownsey, Roger W; Borchers, Christoph H; Foster, Leonard J; Mayor, Thibault; Moore, Edwin D W; Allard, Michael F; Johnson, James D

    2016-11-04

    Cardiac ryanodine receptor (Ryr2) Ca(2+) release channels and cellular metabolism are both disrupted in heart disease. Recently, we demonstrated that total loss of Ryr2 leads to cardiomyocyte contractile dysfunction, arrhythmia, and reduced heart rate. Acute total Ryr2 ablation also impaired metabolism, but it was not clear whether this was a cause or consequence of heart failure. Previous in vitro studies revealed that Ca(2+) flux into the mitochondria helps pace oxidative metabolism, but there is limited in vivo evidence supporting this concept. Here, we studied heart-specific, inducible Ryr2 haploinsufficient (cRyr2Δ50) mice with a stable 50% reduction in Ryr2 protein. This manipulation decreased the amplitude and frequency of cytosolic and mitochondrial Ca(2+) signals in isolated cardiomyocytes, without changes in cardiomyocyte contraction. Remarkably, in the context of well preserved contractile function in perfused hearts, we observed decreased glucose oxidation, but not fat oxidation, with increased glycolysis. cRyr2Δ50 hearts exhibited hyperphosphorylation and inhibition of pyruvate dehydrogenase, the key Ca(2+)-sensitive gatekeeper to glucose oxidation. Metabolomic, proteomic, and transcriptomic analyses revealed additional functional networks associated with altered metabolism in this model. These results demonstrate that Ryr2 controls mitochondrial Ca(2+) dynamics and plays a specific, critical role in promoting glucose oxidation in cardiomyocytes. Our findings indicate that partial RYR2 loss is sufficient to cause metabolic abnormalities seen in heart disease.

  5. 15-Hydroxyprostaglandin Dehydrogenase Generation of Electrophilic Lipid Signaling Mediators from Hydroxy Ω-3 Fatty Acids*

    PubMed Central

    Wendell, Stacy Gelhaus; Golin-Bisello, Franca; Wenzel, Sally; Sobol, Robert W.; Holguin, Fernando; Freeman, Bruce A.

    2015-01-01

    15-Hydroxyprostaglandin dehydrogenase (15PGDH) is the primary enzyme catalyzing the conversion of hydroxylated arachidonic acid species to their corresponding oxidized metabolites. The oxidation of hydroxylated fatty acids, such as the conversion of prostaglandin (PG) E2 to 15-ketoPGE2, by 15PGDH is viewed to inactivate signaling responses. In contrast, the typically electrophilic products can also induce anti-inflammatory and anti-proliferative responses. This study determined that hydroxylated docosahexaenoic acid metabolites (HDoHEs) are substrates for 15PGDH. Examination of 15PGDH substrate specificity was conducted in cell culture (A549 and primary human airway epithelia and alveolar macrophages) using chemical inhibition and shRNA knockdown of 15PGDH. Substrate specificity is broad and relies on the carbon position of the acyl chain hydroxyl group. 14-HDoHE was determined to be the optimal DHA substrate for 15PGDH, resulting in the formation of its electrophilic metabolite, 14-oxoDHA. Consistent with this, 14-HDoHE was detected in bronchoalveolar lavage cells of mild to moderate asthmatics, and the exogenous addition of 14-oxoDHA to primary alveolar macrophages inhibited LPS-induced proinflammatory cytokine mRNA expression. These data reveal that 15PGDH-derived DHA metabolites are biologically active and can contribute to the salutary signaling actions of Ω-3 fatty acids. PMID:25586183

  6. Urotensin II-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in HepG2 cells

    PubMed Central

    Li, Ying-Ying; Shi, Zheng-Ming; Yu, Xiao-Yong; Feng, Ping; Wang, Xue-Jiang

    2016-01-01

    AIM: To investigated the effects of urotensin II (UII) on hepatic insulin resistance in HepG2 cells and the potential mechanisms involved. METHODS: Human hepatoma HepG2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucose-oxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species (ROS) levels were detected with a multimode reader using a 2′,7′-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase (JNK), insulin signal essential molecules such as insulin receptor substrate -1 (IRS-1), protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β), and glucose transporter-2 (Glut 2), and NADPH oxidase subunits such as gp91phox, p67phox, p47phox, p40phox, and p22phox were evaluated by Western blot. RESULTS: Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption (P < 0.05) and glycogen content (P < 0.01) in HepG2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression (P < 0.01) and phosphorylation of IRS-1 (P < 0.05), associated with down-regulation of Akt (P < 0.05) and GSK-3β (P < 0.05) phosphorylation levels, and the expression of Glut 2 (P < 0.001), indicating an insulin-resistance state in HepG2 cells. Furthermore, UII enhanced the phosphorylation of JNK (P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1 (P < 0.001), phosphorylation of IRS-1 (P < 0.001) and GSK-3β (P < 0.05), and glycogen synthesis (P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation (P < 0.05) and NADPH oxidase subunit expression (P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production (P < 0.05), JNK phosphorylation (P < 0

  7. Dynamin 2 and c-Abl are novel regulators of hyperoxia-mediated NADPH oxidase activation and reactive oxygen species production in caveolin-enriched microdomains of the endothelium.

    PubMed

    Singleton, Patrick A; Pendyala, Srikanth; Gorshkova, Irina A; Mambetsariev, Nurbek; Moitra, Jaideep; Garcia, Joe G N; Natarajan, Viswanathan

    2009-12-11

    Reactive oxygen species (ROS) generation, particularly by the endothelial NADPH oxidase family of proteins, plays a major role in the pathophysiology associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. We examined potential regulators of ROS production and discovered that hyperoxia treatment of human pulmonary artery endothelial cells induced recruitment of the vesicular regulator, dynamin 2, the non-receptor tyrosine kinase, c-Abl, and the NADPH oxidase subunit, p47(phox), to caveolin-enriched microdomains (CEMs). Silencing caveolin-1 (which blocks CEM formation) and/or c-Abl expression with small interference RNA inhibited hyperoxia-mediated tyrosine phosphorylation and association of dynamin 2 with p47(phox) and ROS production. In addition, treatment of human pulmonary artery endothelial cells with dynamin 2 small interfering RNA or the dynamin GTPase inhibitor, Dynasore, attenuated hyperoxia-mediated ROS production and p47(phox) recruitment to CEMs. Using purified recombinant proteins, we observed that c-Abl tyrosine-phosphorylated dynamin 2, and this phosphorylation increased p47(phox)/dynamin 2 association (change in the dissociation constant (K(d)) from 85.8 to 6.9 nm). Furthermore, exposure of mice to hyperoxia increased ROS production, c-Abl activation, dynamin 2 association with p47(phox), and pulmonary leak, events that were attenuated in the caveolin-1 knock-out mouse confirming a role for CEMs in ROS generation. These results suggest that hyperoxia induces c-Abl-mediated dynamin 2 phosphorylation required for recruitment of p47(phox) to CEMs and subsequent ROS production in lung endothelium.

  8. Comparative study of the tissue distribution of NADH and NADPH-dependent chloral hydrate reducing enzymes in the rat

    SciTech Connect

    Ogino, Keiki; Hobara, Tatsuya; Kobayashi, Haruo; Iwamoto, Susumu )

    1990-03-01

    Chloral hydrate (CH), an intermediate metabolite of trichloroethylene, is reduced to trichloroethanol (TCE) by alcohol dehydrogenase and aldehyde reductase. Alcohol dehydrogenase requires reduced nicotinamide adenine dinucleotide (NADH), and aldehyde reductase requires reduced nicotinamide adenine dinucleotide phosphate (NADPH). No reports have appeared concerning comparative studies of the tissue distribution of CH-reducing enzymes. In this report, NADH and NADPH-dependent CH-reducing activities were investigated in various organs of the rat.

  9. NADPH Oxidase 4 is required for interleukin-1β-mediated activation of protein kinase Cδ and downstream activation of c-Jun N-terminal kinase signaling in smooth muscle

    PubMed Central

    Ginnan, Roman; Jourd’heuil, Frances L.; Guikema, Benjamin; Simons, Malorie; Singer, Harold A.; Jourd’heuil, David

    2012-01-01

    Reactive oxygen species (ROS) are generated in the vascular wall upon stimulation by pro-inflammatory cytokines and are important mediators of diverse cellular responses that occur as a result of vascular injury. Member of the NADPH oxidase (NOX) family of proteins have been identified in vascular smooth muscle cells (VSM) as important sources of ROS. In this study, we tested the hypothesis that NOX4 is a proximal mediator of IL-1β-dependent activation of PKCδ and increases IL-1β stimulated c-Jun kinase (JNK) signaling in primary rat aortic VSM cells. We found that stimulation of VSM cells with IL-1β increased PKCδ activity and intracellular ROS generation. SiRNA silencing of NOX4 but not NOX1 ablated the IL-1β-dependent increase in ROS production. Pharmacological inhibition of PKCδ activity as well as siRNA depletion of PKCδ or NOX4 blocked the IL-1β-dependent activation of JNK. Further studies showed that the IL-1β-dependent upregulation of iNOS expression was inhibited through JNK inhibition and NOX4 silencing. Taken together, these results indicate that IL-1β-dependent activation of PKCδ is modulated by NOX4-derived ROS. Our study positions PKCδ as an important redox sensitive mediator of IL-1β-dependent signaling and downstream activation of inflammatory mediators in VSM cells. PMID:23022406

  10. The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly.

    PubMed

    Askenasy, Isabel; Pennington, Joseph M; Tao, Yeqing; Marshall, Alan G; Young, Nicolas L; Shang, Weifeng; Stroupe, M Elizabeth

    2015-07-31

    Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the β subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the α8β4 SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We also report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. From these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme.

  11. The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly*

    PubMed Central

    Askenasy, Isabel; Pennington, Joseph M.; Tao, Yeqing; Marshall, Alan G.; Young, Nicolas L.; Shang, Weifeng; Stroupe, M. Elizabeth

    2015-01-01

    Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the β subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the α8β4 SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We also report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. From these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme. PMID:26088143

  12. Effects of high fat and high carbohydrate diets on liver pyruvate dehydrogenase and its activation by a chemical mediator released from insulin-treated liver particulate fraction: effect of neuraminidase treatment on the chemical mediator activity.

    PubMed

    Begum, N; Tepperman, H M; Tepperman, J

    1983-01-01

    Rats were fed a high fat diet or a high glucose diet for 5-7 days. Basal pyruvate dehydrogenase activity (both the active form and the total enzyme activity) was decreased in liver homogenates from fat diet-adapted rats as compared to those fed the glucose diet. Supernatants from insulin-exposed liver particulate fractions from fat-fed rats showed decreased stimulation of pyruvate dehydrogenase activity as compared to those from glucose-fed rats. There was no difference in the response of the mitochondria from the two groups when they were stimulated by supernatants from insulin-treated liver particulate fractions from stock diet-fed rats. Liver particulate fractions from fat-fed rats showed decreased generation of the chemical activator in response to Concanavalin A and trypsin stimulation. This suggests that fat feeding results in a decrease in membrane protease substrate availability. Treatment of the insulin mediator with neuraminidase and beta-D-galactosidase resulted in inactivation of the mediator. Presence of exogenous enzyme substrates during enzyme digestion protected the mediator from inactivation, suggesting that carbohydrate residues are important in the action of the insulin mediator. This fat diet-induced decrease in the generation of a chemical mediator of insulin action may result from 1) a decrease in insulin binding, shown earlier; 2) a decrease in the amount of protease substrate; and 3) an alteration in its carbohydrate composition, which is important in its ability to activate pyruvate dehydrogenase.

  13. Malate Synthesis and Secretion Mediated by a Manganese-Enhanced Malate Dehydrogenase Confers Superior Manganese Tolerance in Stylosanthes guianensis1

    PubMed Central

    Chen, Zhijian; Sun, Lili; Liu, Pandao; Liu, Guodao; Tian, Jiang; Liao, Hong

    2015-01-01

    Manganese (Mn) toxicity is a major constraint limiting plant growth on acidic soils. Superior Mn tolerance in Stylosanthes spp. has been well documented, but its molecular mechanisms remain largely unknown. In this study, superior Mn tolerance in Stylosanthes guianensis was confirmed, as reflected by a high Mn toxicity threshold. Furthermore, genetic variation of Mn tolerance was evaluated using two S. guianensis genotypes, which revealed that the Fine-stem genotype had higher Mn tolerance than the TPRC2001-1 genotype, as exhibited through less reduction in dry weight under excess Mn, and accompanied by lower internal Mn concentrations. Interestingly, Mn-stimulated increases in malate concentrations and exudation rates were observed only in the Fine-stem genotype. Proteomic analysis of Fine-stem roots revealed that S. guianensis Malate Dehydrogenase1 (SgMDH1) accumulated in response to Mn toxicity. Western-blot and quantitative PCR analyses showed that Mn toxicity resulted in increased SgMDH1 accumulation only in Fine-stem roots, but not in TPRC2001-1. The function of SgMDH1-mediated malate synthesis was verified through in vitro biochemical analysis of SgMDH1 activities against oxaloacetate, as well as in vivo increased malate concentrations in yeast (Saccharomyces cerevisiae), soybean (Glycine max) hairy roots, and Arabidopsis (Arabidopsis thaliana) with SgMDH1 overexpression. Furthermore, SgMDH1 overexpression conferred Mn tolerance in Arabidopsis, which was accompanied by increased malate exudation and reduced plant Mn concentrations, suggesting that secreted malate could alleviate Mn toxicity in plants. Taken together, we conclude that the superior Mn tolerance of S. guianensis is achieved by coordination of internal and external Mn detoxification through malate synthesis and exudation, which is regulated by SgMDH1 at both transcription and protein levels. PMID:25378694

  14. Pyruvate Dehydrogenase Kinase-mediated Glycolytic Metabolic Shift in the Dorsal Root Ganglion Drives Painful Diabetic Neuropathy.

    PubMed

    Rahman, Md Habibur; Jha, Mithilesh Kumar; Kim, Jong-Heon; Nam, Youngpyo; Lee, Maan Gee; Go, Younghoon; Harris, Robert A; Park, Dong Ho; Kook, Hyun; Lee, In-Kyu; Suk, Kyoungho

    2016-03-11

    The dorsal root ganglion (DRG) is a highly vulnerable site in diabetic neuropathy. Under diabetic conditions, the DRG is subjected to tissue ischemia or lower ambient oxygen tension that leads to aberrant metabolic functions. Metabolic dysfunctions have been documented to play a crucial role in the pathogenesis of diverse pain hypersensitivities. However, the contribution of diabetes-induced metabolic dysfunctions in the DRG to the pathogenesis of painful diabetic neuropathy remains ill-explored. In this study, we report that pyruvate dehydrogenase kinases (PDK2 and PDK4), key regulatory enzymes in glucose metabolism, mediate glycolytic metabolic shift in the DRG leading to painful diabetic neuropathy. Streptozotocin-induced diabetes substantially enhanced the expression and activity of the PDKs in the DRG, and the genetic ablation of Pdk2 and Pdk4 attenuated the hyperglycemia-induced pain hypersensitivity. Mechanistically, Pdk2/4 deficiency inhibited the diabetes-induced lactate surge, expression of pain-related ion channels, activation of satellite glial cells, and infiltration of macrophages in the DRG, in addition to reducing central sensitization and neuroinflammation hallmarks in the spinal cord, which probably accounts for the attenuated pain hypersensitivity. Pdk2/4-deficient mice were partly resistant to the diabetes-induced loss of peripheral nerve structure and function. Furthermore, in the experiments using DRG neuron cultures, lactic acid treatment enhanced the expression of the ion channels and compromised cell viability. Finally, the pharmacological inhibition of DRG PDKs or lactic acid production substantially attenuated diabetes-induced pain hypersensitivity. Taken together, PDK2/4 induction and the subsequent lactate surge induce the metabolic shift in the diabetic DRG, thereby contributing to the pathogenesis of painful diabetic neuropathy.

  15. Pyruvate Dehydrogenase Kinase-mediated Glycolytic Metabolic Shift in the Dorsal Root Ganglion Drives Painful Diabetic Neuropathy*

    PubMed Central

    Rahman, Md Habibur; Jha, Mithilesh Kumar; Kim, Jong-Heon; Nam, Youngpyo; Lee, Maan Gee; Go, Younghoon; Harris, Robert A.; Park, Dong Ho; Kook, Hyun; Lee, In-Kyu; Suk, Kyoungho

    2016-01-01

    The dorsal root ganglion (DRG) is a highly vulnerable site in diabetic neuropathy. Under diabetic conditions, the DRG is subjected to tissue ischemia or lower ambient oxygen tension that leads to aberrant metabolic functions. Metabolic dysfunctions have been documented to play a crucial role in the pathogenesis of diverse pain hypersensitivities. However, the contribution of diabetes-induced metabolic dysfunctions in the DRG to the pathogenesis of painful diabetic neuropathy remains ill-explored. In this study, we report that pyruvate dehydrogenase kinases (PDK2 and PDK4), key regulatory enzymes in glucose metabolism, mediate glycolytic metabolic shift in the DRG leading to painful diabetic neuropathy. Streptozotocin-induced diabetes substantially enhanced the expression and activity of the PDKs in the DRG, and the genetic ablation of Pdk2 and Pdk4 attenuated the hyperglycemia-induced pain hypersensitivity. Mechanistically, Pdk2/4 deficiency inhibited the diabetes-induced lactate surge, expression of pain-related ion channels, activation of satellite glial cells, and infiltration of macrophages in the DRG, in addition to reducing central sensitization and neuroinflammation hallmarks in the spinal cord, which probably accounts for the attenuated pain hypersensitivity. Pdk2/4-deficient mice were partly resistant to the diabetes-induced loss of peripheral nerve structure and function. Furthermore, in the experiments using DRG neuron cultures, lactic acid treatment enhanced the expression of the ion channels and compromised cell viability. Finally, the pharmacological inhibition of DRG PDKs or lactic acid production substantially attenuated diabetes-induced pain hypersensitivity. Taken together, PDK2/4 induction and the subsequent lactate surge induce the metabolic shift in the diabetic DRG, thereby contributing to the pathogenesis of painful diabetic neuropathy. PMID:26769971

  16. Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.

    PubMed

    Jungmann, Richard A; Kiryukhina, Olga

    2005-07-01

    Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA

  17. Engineering of Corynebacterium glutamicum with an NADPH-Generating Glycolytic Pathway for l-Lysine Production ▿

    PubMed Central

    Takeno, Seiki; Murata, Ryosuke; Kobayashi, Ryosuke; Mitsuhashi, Satoshi; Ikeda, Masato

    2010-01-01

    A sufficient supply of NADPH is a critical factor in l-lysine production by Corynebacterium glutamicum. Endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of C. glutamicum was replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) of Streptococcus mutans, which catalyzes the reaction of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP+ to NADPH, resulting in the reconstruction of the functional glycolytic pathway. Although the growth of the engineered strain on glucose was significantly retarded, a suppressor mutant with an increased ability to utilize sugars was spontaneously isolated from the engineered strain. The suppressor mutant was characterized by the properties of GapN as well as the nucleotide sequence of the gene, confirming that no change occurred in either the activity or the basic properties of GapN. The suppressor mutant was engineered into an l-lysine-producing strain by plasmid-mediated expression of the desensitized lysC gene, and the performance of the mutant as an l-lysine producer was evaluated. The amounts of l-lysine produced by the suppressor mutant were larger than those produced by the reference strain (which was created by replacement of the preexisting gapN gene in the suppressor mutant with the original gapA gene) by ∼70% on glucose, ∼120% on fructose, and ∼100% on sucrose, indicating that the increased l-lysine production was attributed to GapN. These results demonstrate effective l-lysine production by C. glutamicum with an additional source of NADPH during glycolysis. PMID:20851994

  18. Phytochrome-mediated regulation of plant respiration and photorespiration.

    PubMed

    Igamberdiev, Abir U; Eprintsev, Alexander T; Fedorin, Dmitry N; Popov, Vasily N

    2014-02-01

    The expression of genes encoding various enzymes participating in photosynthetic and respiratory metabolism is regulated by light via the phytochrome system. While many photosynthetic, photorespiratory and some respiratory enzymes, such as the rotenone-insensitive NADH and NADPH dehydrogenases and the alternative oxidase, are stimulated by light, succinate dehydrogenase, subunits of the pyruvate dehydrogenase complex, cytochrome oxidase and fumarase are inhibited via the phytochrome mechanism. The effect of light, therefore, imposes limitations on the tricarboxylic acid cycle and on the mitochondrial electron transport coupled to ATP synthesis, while the non-coupled pathways become activated. Phytochrome-mediated regulation of gene expression also creates characteristic distribution patterns of photosynthetic, photorespiratory and respiratory enzymes across the leaf generating different populations of mitochondria, either enriched by glycine decarboxylase (in the upper part) or by succinate dehydrogenase (in the bottom part of the leaf).

  19. NADPH oxidase-derived H2O2 mediates the regulatory effects of microglia on astrogliosis in experimental models of Parkinson's disease.

    PubMed

    Hou, Liyan; Zhou, Xueying; Zhang, Cong; Wang, Ke; Liu, Xiaofang; Che, Yuning; Sun, Fuqiang; Li, Huihua; Wang, Qingshan; Zhang, Dan; Hong, Jau-Shyong

    2017-02-22

    Astrogliosis has long been recognized in Parkinson's disease (PD), the most common neurodegenerative movement disorder. However, the mechanisms of how astroglia become activated remain unclear. Reciprocal interactions between microglia and astroglia play a pivotal role in regulating the activities of astroglia. The purpose of this study is to investigate the mechanism by which microglia regulate astrogliosis by using lipopolysaccharide (LPS) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse PD models. We found that the activation of microglia preceded astroglia in the substantia nigra of mice treated with either LPS or MPTP. Furthermore, suppression of microglial activation by pharmacological inhibition or genetic deletion of NADPH oxidase (NOX2) in mice attenuated astrogliosis. The important role of NOX2 in microglial regulation of astrogliosis was further mirrored in a mixed-glia culture system. Mechanistically, H2O2, a product of microglial NOX2 activation, serves as a direct signal to regulate astrogliosis. Astrogliosis was induced by H2O2 through a process in which extracellularly generated H2O2 diffused into the cytoplasm and subsequently stimulated activation of transcription factors, STAT1 and STAT3. STAT1/3 activation regulated the immunological functions of H2O2-induced astrogliosis since AG490, an inhibitor of STAT1/3, attenuated the gene expressions of both proinflammatory and neurotrophic factors in H2O2-treated astrocyte. Our findings indicate that microglial NOX2-generated H2O2 is able to regulate the immunological functions of astroglia via a STAT1/3-dependent manner, providing additional evidence for the immune pathogenesis and therapeutic studies of PD.

  20. Dihydroorotate dehydrogenase (DHODH) inhibitors affect ATP depletion, endogenous ROS and mediate S-phase arrest in breast cancer cells.

    PubMed

    Mohamad Fairus, A K; Choudhary, B; Hosahalli, S; Kavitha, N; Shatrah, O

    2017-04-01

    Dihydroorotate dehydrogenase (DHODH) is the key enzyme in de novo biosynthesis of pyrimidine in both prokaryotes and eukaryotes. The de novo pathway of pyrimidine biosynthesis is essential in cancer cells proliferation. Leflunomide is an approved DHODH inhibitor that has been widely used for the treatment of arthritis. Similarly, brequinar sodium is another DHODH inhibitor that showed anti-tumour effect in MC38 colon carcinoma cells when used in combination with fluorouracil. Despite the potential role of DHODH inhibitors in cancer therapy, their mechanisms of action remain obscure and await further elucidation. Here, we evaluated the effect of DHODH inhibitors on the production of ATP and ROS in sensitive and non-sensitive breast cancer cells. Subsequently, the effects of DHODH inhibitors on cell cycle as well as on signalling molecules such as p53, p65 and STAT6 were evaluated in sensitive T-47D and non-sensitive MDAMB-436 cells. The correlations between DHODH protein expression, proliferation speed and sensitivity to DHODH inhibitors were also investigated in a panel of cancer cell lines. DHODH inhibitors-sensitive T-47D and MDAMB-231 cells appeared to preserve ROS production closely to endogenous ROS level whereas the opposite was observed in non-sensitive MDAMB-436 and W3.006 cells. In addition, we observed approximately 90% of intracellular ATP depletion in highly sensitive T-47D and MDAMB-231 cells compared to non-sensitive MDAMB-436 cells. There was significant over-expression of p53, p65 and STAT6 signalling molecules in sensitive cells which may be involved in mediating the S-phase arrest in cell cycle progression. The current study suggests that DHODH inhibitors are most effective in cells that express high levels of DHODH enzyme. The inhibition of cell proliferation by these inhibitors appears to be accompanied by ROS production as well as ATP depletion. The increase in expression of signalling molecules observed may be due to pyrimidine depletion

  1. Cytosolic NADPH Homeostasis in Glucose-starved Procyclic Trypanosoma brucei Relies on Malic Enzyme and the Pentose Phosphate Pathway Fed by Gluconeogenic Flux*

    PubMed Central

    Allmann, Stefan; Morand, Pauline; Ebikeme, Charles; Gales, Lara; Biran, Marc; Hubert, Jane; Brennand, Ana; Mazet, Muriel; Franconi, Jean-Michel; Michels, Paul A. M.; Portais, Jean-Charles; Boshart, Michael; Bringaud, Frédéric

    2013-01-01

    All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H2O2 stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H2O2 stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an ∼10-fold increase of susceptibility to H2O2 stress observed for the Δmec/Δmec/RNAiPGI double mutant when compared with the single mutants, and (iii) the 13C enrichment of glycolytic and PPP intermediates from cells incubated with [U-13C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host. PMID:23665470

  2. A mediated glucose/oxygen enzymatic fuel cell based on printed carbon inks containing aldose dehydrogenase and laccase as anode and cathode.

    PubMed

    Jenkins, Peter; Tuurala, Saara; Vaari, Anu; Valkiainen, Matti; Smolander, Maria; Leech, Dónal

    2012-03-10

    Enzyme electrodes show great potential for many applications, as biosensors and more recently as anodes and cathodes in biocatalytic fuel cells for power generation. Enzymes have advantages over metal catalysts, as they provide high specificity and reaction rates, while operating under mild conditions. Here we report on studies related to development of mass-producible, completely enzymatic printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks containing mediators and laccase, for reduction of oxygen, or aldose dehydrogenase, for oxidation of glucose. Mediator performance in these printed formats is compared to relative rate constants for the enzyme-mediator reaction in solution, for a range of anode and cathode mediators. The power output and stability of fuels cells using an acidophilic laccase isolated from Trametes hirsuta is greater, at pH 5, than that for cells based on Melanocarpus albomyces laccase, that shows optimal activity closer to neutral pH, at pH 6. Highest power output, although of limited stability, was observed for ThL/ABTS cathodes, providing a maximum power density of 3.5 μWcm(-2) at 0.34 V, when coupled to an ALDH glucose anode mediated by an osmium complex. The stability of cell voltage above a threshold of 200 mV under a moderate 75 kΩ load is used to benchmark printed fuel cell performance. Highest stability was obtained for a printed fuel cell using osmium complexes as mediators of glucose oxidation by aldose dehydrogenase, and oxygen reduction by T. hirsuta laccase, maintaining cell voltage above 200 mV for 137 h at pH 5. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells.

  3. Nox4 NADPH Oxidase Mediates Peroxynitrite-dependent Uncoupling of Endothelial Nitric-oxide Synthase and Fibronectin Expression in Response to Angiotensin II

    PubMed Central

    Lee, Doug-Yoon; Wauquier, Fabien; Eid, Assaad A.; Roman, Linda J.; Ghosh-Choudhury, Goutam; Khazim, Khaled; Block, Karen; Gorin, Yves

    2013-01-01

    Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces endothelial nitric-oxide synthase (eNOS) uncoupling with enhanced generation of reactive oxygen species (ROS) and decreased production of NO. Ang II promotes a rapid increase in 3-nitrotyrosine formation, and uric acid attenuates Ang II-induced decrease in NO bioavailability, demonstrating that peroxynitrite mediates the effects of Ang II on eNOS dysfunction. Ang II rapidly up-regulates Nox4 protein. Inhibition of Nox4 abolishes the increase in ROS and peroxynitrite generation as well as eNOS uncoupling triggered by Ang II, indicating that Nox4 is upstream of eNOS. This pathway contributes to Ang II-mediated fibronectin accumulation in MCs. Ang II also elicits an increase in mitochondrial abundance of Nox4 protein, and the oxidase contributes to ROS production in mitochondria. Overexpression of mitochondrial manganese superoxide dismutase prevents the stimulatory effects of Ang II on mitochondrial ROS production, loss of NO availability, and MC fibronectin accumulation, whereas manganese superoxide dismutase depletion increases mitochondrial ROS, NO deficiency, and fibronectin synthesis basally and in cells exposed to Ang II. This work provides the first evidence that uncoupled eNOS is responsible for Ang II-induced MC fibronectin accumulation and identifies Nox4 and mitochondrial ROS as mediators of eNOS dysfunction. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal fibrosis. PMID:23940049

  4. Regulation of G6PD acetylation by SIRT2 and KAT9 modulates NADPH homeostasis and cell survival during oxidative stress#

    PubMed Central

    Wang, Yi-Ping; Zhou, Li-Sha; Zhao, Yu-Zheng; Wang, Shi-Wen; Chen, Lei-Lei; Liu, Li-Xia; Ling, Zhi-Qiang; Hu, Fu-Jun; Sun, Yi-Ping; Zhang, Jing-Ye; Yang, Chen; Yang, Yi; Xiong, Yue; Guan, Kun-Liang; Ye, Dan

    2014-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway (PPP) and plays an essential role in the oxidative stress response by producing NADPH, the main intracellular reductant. G6PD deficiency is the most common human enzyme defect, affecting more than 400 million people worldwide. Here, we show that G6PD is negatively regulated by acetylation on lysine 403 (K403), an evolutionarily conserved residue. The K403 acetylated G6PD is incapable of forming active dimers and displays a complete loss of activity. Knockdown of G6PD sensitizes cells to oxidative stress, and re-expression of wild-type G6PD, but not the K403 acetylation mimetic mutant, rescues cells from oxidative injury. Moreover, we show that cells sense extracellular oxidative stimuli to decrease G6PD acetylation in a SIRT2-dependent manner. The SIRT2-mediated deacetylation and activation of G6PD stimulates PPP to supply cytosolic NADPH to counteract oxidative damage and protect mouse erythrocytes. We also identified KAT9/ELP3 as a potential acetyltransferase of G6PD. Our study uncovers a previously unknown mechanism by which acetylation negatively regulates G6PD activity to maintain cellular NADPH homeostasis during oxidative stress. PMID:24769394

  5. Oxidation of External NAD(P)H by Mitochondria from Taproots and Tissue Cultures of Sugar Beet (Beta vulgaris).

    PubMed Central

    Zottini, M.; Mandolino, G.; Zannoni, D.

    1993-01-01

    The present study compares the exogenous NAD(P)H oxidation and the membrane potential ([delta][psi]) generated in mitochondria isolated from different tissues of an important agricultural crop, sugar beet (Beta vulgaris}. We observed that mitochondria from taproots, cold-stored taproots, and in vitro-grown tissue cultures contain a functional NADH dehydrogenase, whereas only those isolated from tissue cultures displayed a functional NAD(P)H dehydrogenase. It is interesting that the NADH-dependent [delta][psi] of mitochondria from cold-stored taproots and from tissue cultures was not affected by free Ca2+ ions, whereas free Ca2+ was required for the mitochondrial NADPH oxidation by in vitro-grown cells and cytosolic NADH oxidation by mitochondria from fresh taproots. A tentative model accounting for the different response to Ca2+ ions of the NADH dehydrogenase in mitochondria from cold-stored taproots and tissue cultures of B. vulgaris is discussed. PMID:12231847

  6. A comparison of glucose oxidase and aldose dehydrogenase as mediated anodes in printed glucose/oxygen enzymatic fuel cells using ABTS/laccase cathodes.

    PubMed

    Jenkins, Peter; Tuurala, Saara; Vaari, Anu; Valkiainen, Matti; Smolander, Maria; Leech, Dónal

    2012-10-01

    Current generation by mediated enzyme electron transfer at electrode surfaces can be harnessed to provide biosensors and redox reactions in enzymatic fuel cells. A glucose/oxygen enzymatic fuel cell can provide power for portable and implantable electronic devices. High volume production of enzymatic fuel cell prototypes will likely require printing of electrode and catalytic materials. Here we report on preparation and performance of, completely enzymatic, printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks, enzyme and mediator. A comparison of cell performance using a range of mediators for either glucose oxidase (GOx) or aldose dehydrogenase (ALDH) oxidation of glucose at the anode and ABTS and a fungal laccase, for reduction of oxygen at the cathode, is reported. Highest power output, although of limited stability, is observed for ALDH anodes mediated by an osmium complex, providing a maximum power density of 3.5 μW cm(-2) at 0.34 V, when coupled to a laccase/ABTS cathode. The stability of cell voltage in a biobattery format, above a threshold of 200 mV under a moderate 75 kΩ load, is used to benchmark printed fuel cell performance. Highest stability is obtained for printed fuel cells using ALDH, providing cell voltages over the threshold for up to 74 h, compared to only 2 h for cells with anodes using GOx. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells.

  7. Nox4 NAD(P)H Oxidase Mediates Src-dependent Tyrosine Phosphorylation of PDK-1 in Response to Angiotensin II

    PubMed Central

    Block, Karen; Eid, Assaad; Griendling, Kathy K.; Lee, Duck-Yoon; Wittrant, Yohann; Gorin, Yves

    2008-01-01

    Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to hypertrophy and extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces an increase in PDK-1 (3-phosphoinositide-dependent protein kinase-1) kinase activity that required its phosphorylation on tyrosine 9 and 373/376. Introduction into the cells of PDK-1, mutated on these tyrosine residues or kinase-inactive, attenuates Ang II-induced hypertrophy and fibronectin accumulation. Ang II-mediated PDK-1 activation and tyrosine phosphorylation (total and on residues 9 and 373/376) are inhibited in cells transfected with small interfering RNA for Src, indicating that Src is upstream of PDK-1. In cells expressing oxidation-resistant Src mutant C487A, Ang II-induced hypertrophy and fibronectin expression are prevented, suggesting that the pathway is redox-sensitive. Ang II also up-regulates Nox4 protein, and siNox4 abrogates the Ang II-induced increase in intracellular reactive oxygen species (ROS) generation. Small interfering RNA for Nox4 also inhibits Ang II-induced activation of Src and PDK-1 tyrosine phosphorylation (total and on residues 9 and 373/376), demonstrating that Nox4 functions upstream of Src and PDK-1. Importantly, inhibition of Nox4, Src, or PDK-1 prevents the stimulatory effect of Ang II on fibronectin accumulation and cell hypertrophy. This work provides the first evidence that Nox4-derived ROS are responsible for Ang II-induced PDK-1 tyrosine phosphorylation and activation through stimulation of Src. Importantly, this pathway contributes to Ang II-induced MC hypertrophy and fibronectin accumulation. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal hypertrophy and fibrosis. PMID:18559349

  8. Indicaxanthin inhibits NADPH oxidase (NOX)-1 activation and NF-κB-dependent release of inflammatory mediators and prevents the increase of epithelial permeability in IL-1β-exposed Caco-2 cells.

    PubMed

    Tesoriere, L; Attanzio, A; Allegra, M; Gentile, C; Livrea, M A

    2014-02-01

    Dietary redox-active/antioxidant phytochemicals may help control or mitigate the inflammatory response in chronic inflammatory bowel disease (IBD). In the present study, the anti-inflammatory activity of indicaxanthin (Ind), a pigment from the edible fruit of cactus pear (Opuntia ficus-indica, L.), was shown in an IBD model consisting of a human intestinal epithelial cell line (Caco-2 cells) stimulated by IL-1β, a cytokine known to play a major role in the initiation and amplification of inflammatory activity in IBD. The exposure of Caco-2 cells to IL-1β brought about the activation of NADPH oxidase (NOX-1) and the generation of reactive oxygen species (ROS) to activate intracellular signalling leading to the activation of NF-κB, with the over-expression of inflammatory enzymes and release of pro-inflammatory mediators. The co-incubation of the cells with Ind, at a nutritionally relevant concentration (5-25 μM), and IL-1β prevented the release of the pro-inflammatory cytokines IL-6 and IL-8, PGE2 and NO, the formation of ROS and the loss of thiols in a dose-dependent manner. The co-incubation of the cells with Ind and IL-1β also prevented the IL-1β-induced increase of epithelial permeability. It was also shown that the activation of NOX-1 and NF-κB was prevented by Ind and the expression of COX-2 and inducible NO synthase was reduced. The uptake of Ind in Caco-2 cell monolayers appeared to be unaffected by the inflamed state of the cells. In conclusion, our findings suggest that the dietary pigment Ind may have the potential to modulate inflammatory processes at the intestinal level.

  9. NecroX-7 prevents oxidative stress-induced cardiomyopathy by inhibition of NADPH oxidase activity in rats

    SciTech Connect

    Park, Joonghoon; Park, Eok; Ahn, Bong-Hyun; Kim, Hyoung Jin; Park, Ji-hoon; Koo, Sun Young; Kwak, Hyo-Shin; Park, Heui Sul; Kim, Dong Wook; Song, Myoungsub; Yim, Hyeon Joo; Seo, Dong Ook; Kim, Soon Ha

    2012-08-15

    Oxidative stress is one of the causes of cardiomyopathy. In the present study, NecroXs, novel class of mitochondrial ROS/RNS scavengers, were evaluated for cardioprotection in in vitro and in vivo model, and the putative mechanism of the cardioprotection of NecroX-7 was investigated by global gene expression profiling and subsequent biochemical analysis. NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced death of H9C2 rat cardiomyocytes at EC{sub 50} = 0.057 μM. In doxorubicin (DOX)-induced cardiomyopathy in rats, NecroX-7 significantly reduced the plasma levels of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) which were increased by DOX treatment (p < 0.05). Microarray analysis revealed that 21 genes differentially expressed in tBHP-treated H9C2 cells were involved in ‘Production of reactive oxygen species’ (p = 0.022), and they were resolved by concurrent NecroX-7 treatment. Gene-to-gene networking also identified that NecroX-7 relieved cell death through Ncf1/p47phox and Rac2 modulation. In subsequent biochemical analysis, NecroX-7 inhibited NADPH oxidase (NOX) activity by 53.3% (p < 0.001). These findings demonstrate that NecroX-7, in part, provides substantial protection of cardiomyopathy induced by tBHP or DOX via NOX-mediated cell death. -- Highlights: ► NecroX-7 prevented tert-butyl hydroperoxide-induced in vitro cardiac cell death. ► NecroX-7 ameliorated doxorubicin-induced in vivo cardiomyopathy. ► NecroX-7 prevented oxidative stress and necrosis-enriched transcriptional changes. ► NecroX-7 effectively inhibited NADPH oxidase activation. ► Cardioprotection of Necro-7 was brought on by modulation of NADPH oxidase activity.

  10. Pharmacological activation of the pyruvate dehydrogenase complex reduces statin-mediated upregulation of FOXO gene targets and protects against statin myopathy in rodents.

    PubMed

    Mallinson, Joanne E; Constantin-Teodosiu, Dumitru; Glaves, Philip D; Martin, Elizabeth A; Davies, Wendy J; Westwood, F Russell; Sidaway, James E; Greenhaff, Paul L

    2012-12-15

    We previously reported that statin myopathy is associated with impaired carbohydrate (CHO) oxidation in fast-twitch rodent skeletal muscle, which we hypothesised occurred as a result of forkhead box protein O1 (FOXO1) mediated upregulation of pyruvate dehydrogenase kinase-4 (PDK4) gene transcription. Upregulation of FOXO gene targets known to regulate proteasomal and lysosomal muscle protein breakdown was also evident. We hypothesised that increasing CHO oxidation in vivo, using the pyruvate dehydrogenase complex (PDC) activator, dichloroacetate (DCA), would blunt activation of FOXO gene targets and reduce statin myopathy. Female Wistar Hanover rats were dosed daily for 12 days (oral gavage) with either vehicle (control, 0.5% w/v hydroxypropyl-methylcellulose 0.1% w/v polysorbate-80; n = 9), 88 mg( )kg(-1) day(-1) simvastatin (n = 8), 88 mg( )kg(-1) day(-1) simvastatin + 30 mg kg(-1) day(-1) DCA (n = 9) or 88 mg kg(-1) day(-1) simvastatin + 40 mg kg(-1) day(-1) DCA (n = 9). Compared with control, simvastatin reduced body mass gain and food intake, increased muscle fibre necrosis, plasma creatine kinase levels, muscle PDK4, muscle atrophy F-box (MAFbx) and cathepsin-L mRNA expression, increased PDK4 protein expression, and proteasome and cathepsin-L activity, and reduced muscle PDC activity. Simvastatin with DCA maintained body mass gain and food intake, abrogated the myopathy, decreased muscle PDK4 mRNA and protein, MAFbx and cathepsin-L mRNA, increased activity of PDC and reduced proteasome activity compared with simvastatin. PDC activation abolished statin myopathy in rodent skeletal muscle, which occurred at least in part via inhibition of FOXO-mediated transcription of genes regulating muscle CHO utilisation and protein breakdown.

  11. NADPH Oxidases in Vascular Pathology

    PubMed Central

    Konior, Anna; Schramm, Agata; Czesnikiewicz-Guzik, Marta

    2014-01-01

    Abstract Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of “kindling radicals,” which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794–2814. PMID:24180474

  12. NADP+-Preferring d-Lactate Dehydrogenase from Sporolactobacillus inulinus

    PubMed Central

    Zhu, Lingfeng; Xu, Xiaoling; Wang, Limin; Ma, Yanhe

    2015-01-01

    Hydroxy acid dehydrogenases, including l- and d-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD+ as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d-lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn174 was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases. PMID:26150461

  13. Mediated electron transfer of cellobiose dehydrogenase and glucose oxidase at osmium polymer-modified nanoporous gold electrodes.

    PubMed

    Salaj-Kosla, Urszula; Scanlon, Micheál D; Baumeister, Tobias; Zahma, Kawah; Ludwig, Roland; Ó Conghaile, Peter; MacAodha, Domhnall; Leech, Dónal; Magner, Edmond

    2013-04-01

    Nanoporous and planar gold electrodes were utilised as supports for the redox enzymes Aspergillus niger glucose oxidase (GOx) and Corynascus thermophilus cellobiose dehydrogenase (CtCDH). Electrodes modified with hydrogels containing enzyme, Os-redox polymers and the cross-linking agent poly(ethylene glycol)diglycidyl ether were used as biosensors for the determination of glucose and lactose. Limits of detection of 6.0 (±0.4), 16.0 (±0.1) and 2.0 (±0.1) μM were obtained for CtCDH-modified lactose and glucose biosensors and GOx-modified glucose biosensors, respectively, at nanoporous gold electrodes. Biofuel cells composed of GOx- and CtCDH-modified gold electrodes were utilised as anodes, together with Myrothecium verrucaria bilirubin oxidase (MvBOD) or Melanocarpus albomyces laccase as cathodes, in biofuel cells. A maximum power density of 41 μW/cm(2) was obtained for a CtCDH/MvBOD biofuel cell in 5 mM lactose and O2-saturated buffer (pH 7.4, 0.1 M phosphate, 150 mM NaCl).

  14. Role of quinones in electron transfer of PQQ–glucose dehydrogenase anodes—mediation or orientation effect

    DOE PAGES

    Babanova, Sofia; Matanovic, Ivana; Chavez, Madelaine Seow; ...

    2015-06-16

    In this study, the influence of two quinones (1,2- and 1,4-benzoquinone) on the operation and mechanism of electron transfer in PQQ-sGDH anodes has been determined. Benzoquinones were experimentally explored as mediators present in the electrolyte. The electrochemical performance of the PQQ–sGDH anodes with and without the mediators was examined and for the first time molecular docking simulations were used to gain a fundamental understanding to explain the role of the mediator molecules in the design and operation of the enzymatic electrodes. It was proposed that the higher performance of the PQQ–sGDH anodes in the presence of 1,2- and 1,4-benzoquinones introducedmore » in the solution is due to the shorter distance between these molecules and PQQ in the enzymatic molecule. It was also hypothesized that when 1,4-benzoquinone is adsorbed on a carbon support, it would play the dual role of a mediator and an orienting agent. At the same time, when 1,2-benzoquinone and ubiquinone are adsorbed on the electrode surface, the enzyme would transfer the electrons directly to the support, and these molecules would primarily play the role of an orienting agent.« less

  15. Role of quinones in electron transfer of PQQ–glucose dehydrogenase anodes—mediation or orientation effect

    SciTech Connect

    Babanova, Sofia; Matanovic, Ivana; Chavez, Madelaine Seow; Atanassov, Plamen

    2015-06-16

    In this study, the influence of two quinones (1,2- and 1,4-benzoquinone) on the operation and mechanism of electron transfer in PQQ-sGDH anodes has been determined. Benzoquinones were experimentally explored as mediators present in the electrolyte. The electrochemical performance of the PQQ–sGDH anodes with and without the mediators was examined and for the first time molecular docking simulations were used to gain a fundamental understanding to explain the role of the mediator molecules in the design and operation of the enzymatic electrodes. It was proposed that the higher performance of the PQQ–sGDH anodes in the presence of 1,2- and 1,4-benzoquinones introduced in the solution is due to the shorter distance between these molecules and PQQ in the enzymatic molecule. It was also hypothesized that when 1,4-benzoquinone is adsorbed on a carbon support, it would play the dual role of a mediator and an orienting agent. At the same time, when 1,2-benzoquinone and ubiquinone are adsorbed on the electrode surface, the enzyme would transfer the electrons directly to the support, and these molecules would primarily play the role of an orienting agent.

  16. Influence of human serum albumin on the bile acid-mediated inhibition of liver microsomal type 1 11β-hydroxysteroid dehydrogenase.

    PubMed

    Maeda, Yorio; Funagayama, Mayumi; Shinohara, Akio; Koshimoto, Chihiro; Furusawa, Hidemi; Nakahara, Hiroshi; Yamaguchi, Yukiko; Saitoh, Tomokazu; Yamamoto, Takashi; Komaki, Kansei

    2014-09-01

    The influence of human serum albumin (HSA) on the bile acid-mediated inhibition of liver microsomal type 1 11β-hydroxysteroid dehydrogenase (11β-HSD1) was studied in vitro. A rat liver microsomal fraction was prepared, and the 11β-HSD1 enzyme activity in the presence of various concentrations of bile acids and HSA was determined using hydrocortisone as the substrate. The products of the reaction were extracted and analyzed using high-performance liquid chromatography. The magnitude of the inhibition decreased with the addition of HSA in a dose-dependent manner. Four percent human albumin decreased the inhibitory effects of 100 μM chenodeoxycholic acid and lithocholic acid from 89.9 ± 5.6 to 54.5 ± 6.1% and from 83.8 ± 4.8 to 20.8 ± 4.2%, respectively. In contrast, ursodeoxycholic acid and deoxycholic acid showed no inhibitory effect on the enzyme activity in the presence of 4% human serum albumin, and the addition of 1% γ-globulin to the assay mixture in the presence of bile acids did not affect the enzyme activity. Our in vitro study showed that the addition of HSA ameliorated the inhibition of 11β-HSD1 and that the magnitude of the change is dependent on the species of bile acid, presumably based on the numbers of hydroxyl groups. These results suggest that HSA seems to protect the bile acid-mediated inhibition of 11β-HSD1 in the healthy subject. On the other hand, in the patients with obstructive biliary diseases, not only elevated serum bile acid but also the accompanying hypoalbuminemia is important to evaluate the pathophysiology of the bile acid-mediated inhibition of 11β-HSD1 of the disease.

  17. Targeting NADPH oxidase and phospholipases A2 in Alzheimer's disease.

    PubMed

    Simonyi, Agnes; He, Yan; Sheng, Wenwen; Sun, Albert Y; Wood, W Gibson; Weisman, Gary A; Sun, Grace Y

    2010-06-01

    Alzheimer's disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Abeta) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Abeta. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Abeta in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A(2) (PLA(2)) and secretory PLA(2). In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Abeta NADPH oxidase and PLA(2) can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.

  18. Nox NADPH Oxidases and the Endoplasmic Reticulum

    PubMed Central

    Araujo, Thaís L.S.; Abrahão, Thalita B.

    2014-01-01

    Abstract Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between

  19. The interaction of the Arabidopsis response regulator ARR18 with bZIP63 mediates the regulation of PROLINE DEHYDROGENASE expression.

    PubMed

    Veerabagu, Manikandan; Kirchler, Tobias; Elgass, Kirstin; Stadelhofer, Bettina; Stahl, Mark; Harter, Klaus; Mira-Rodado, Virtudes; Chaban, Christina

    2014-10-01

    As the first and rate-limiting enzyme of proline degradation, PROLINE DEHYDROGENASE1 (PDH1) is tightly regulated during plant stress responses, including induction under hypoosmolarity and repression under water deficit. The plant receptor histidine kinases AHKs, elements of the two-component system (TCS) in Arabidopsis thaliana, are proposed to function in water stress responses by regulating different stress-responsive genes. However, little information is available concerning AHK phosphorelay-mediated downstream signaling. Here we show that the Arabidopsis type-B response regulator 18 (ARR18) functions as a positive osmotic stress response regulator in Arabidopsis seeds and affects the activity of the PDH1 promoter, known to be controlled by C-group bZIP transcription factors. Moreover, direct physical interaction of ARR18 with bZIP63 was identified and shown to be dependent on phosphorylation of the conserved aspartate residue in the ARR18 receiver domain. We further show that bZIP63 itself functions as a negative regulator of seed germination upon osmotic stress. Using reporter gene assays in protoplasts, we demonstrated that ARR18 interaction negatively interferes with the transcriptional activity of bZIP63 on the PDH1 promoter. Our findings provide new insight into the function of ARR18 and bZIP63 as antagonistic regulators of gene expression in Arabidopsis.

  20. Group II intron-mediated deletion of lactate dehydrogenase gene in an isolated 1,3-propanediol producer Hafnia alvei AD27.

    PubMed

    Celińska, Ewelina; Drożdżyńska, Agnieszka; Wita, Agnieszka; Juzwa, Wojciech; Białas, Wojciech; Czaczyk, Katarzyna; Grajek, Włodzimierz

    2016-03-03

    Our previous studies showed that glycerol fermentation by Hafnia alvei AD27 strain was accompanied by formation of high quantities of lactate. The ultimate aim of this work was the elimination of excessive lactate production in the 1,3-propanediol producer cultures. Group II intron-mediated deletion of ldh (lactate dehydrogenase) gene in an environmental isolate of H. alvei AD27 strain was conducted. The effect of the Δldh genotype in H. alvei AD27 strain varied depending on the culture medium applied. Under lower initial glycerol concentration (20 gL(-1)), lactate and 1,3-propanediol production was fully abolished, and the main carbon flux was directed to ethanol synthesis. On the other hand, at higher initial glycerol concentrations (40 gL(-1)), 1,3-propanediol and lactate production was recovered in the recombinant strain. The final titers of 1,3-propanediol and ethanol were similar for the recombinant and the WT strains, while the Δldh genotype displayed significantly decreased lactate titer. The by-products profile was altered upon ldh gene deletion, while glycerol utilization and biomass accumulation remained unaltered. As indicated by flow-cytometry analyses, the internal pH was not different for the WT and the recombinant Δldh strains over the culture duration, however, the WT strain was characterized by higher redox potential.

  1. Cytosolic NADPH balancing in Penicillium chrysogenum cultivated on mixtures of glucose and ethanol.

    PubMed

    Zhao, Zheng; Kuijvenhoven, Karel; van Gulik, Walter M; Heijnen, Joseph J; van Winden, Wouter A; Verheijen, Peter J T

    2011-01-01

    The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary (13)C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602-618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD(+) dependent. Metabolic modeling shows that changing the NAD(+)-aldehyde dehydrogenase to NADP(+)-aldehyde dehydrogenase can increase the penicillin yield on substrate.

  2. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis

    PubMed Central

    Bataller, Ramón; Schwabe, Robert F.; Choi, Youkyung H.; Yang, Liu; Paik, Yong Han; Lindquist, Jeffrey; Qian, Ting; Schoonhoven, Robert; Hagedorn, Curt H.; Lemasters, John J.; Brenner, David A.

    2003-01-01

    Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II–induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47phox, a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen α1(I) mRNA expression, and secretion of TGF-β1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redox-sensitive manner, as assessed by microarray analysis. HSCs isolated from p47phox–/– mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47phox–/– mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle α-actin and expression of TGF-β1 were reduced in p47phox–/– mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis. PMID:14597764

  3. The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

    NASA Astrophysics Data System (ADS)

    DeCoursey, Thomas E.; Morgan, Deri; Cherny, Vladimir V.

    2003-04-01

    The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (Ie) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that Ie is voltage-independent from -100mV to >0mV, but is steeply inhibited by further depolarization, and is abolished at about +190mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates Ie, because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.

  4. The role of nicotinamide–adenine dinucleotide phosphate-dependent malate dehydrogenase and isocitrate dehydrogenase in the supply of reduced nicotinamide–adenine dinucleotide phosphate for steroidogenesis in the superovulated rat ovary

    PubMed Central

    Flint, A. P. F.; Denton, R. M.

    1970-01-01

    1. Superovulated rat ovary was found to contain high activities of NADP–malate dehydrogenase and NADP–isocitrate dehydrogenase. The activity of each enzyme was approximately four times that of glucose 6-phosphate dehydrogenase and equalled or exceeded the activities reported to be present in other mammalian tissues. Fractionation of a whole tissue homogenate of superovulated rat ovary indicated that both enzymes were exclusively cytoplasmic. The tissue was also found to contain pyruvate carboxylase (exclusively mitochondrial), NAD–malate dehydrogenase and aspartate aminotransferase (both mitochondrial and cytoplasmic) and ATP–citrate lyase (exclusively cytoplasmic). 2. The kinetic properties of glucose 6-phosphate dehydrogenase, NADP–malate dehydrogenase and NADP–isocitrate dehydrogenase were determined and compared with the whole-tissue concentrations of their substrates and NADPH; NADPH is a competitive inhibitor of all three enzymes. The concentrations of glucose 6-phosphate, malate and isocitrate in incubated tissue slices were raised at least tenfold by the addition of glucose to the incubation medium, from the values below to values above the respective Km values of the dehydrogenases. Glucose doubled the tissue concentration of NADPH. 3. Steroidogenesis from acetate is stimulated by glucose in slices of superovulated rat ovary incubated in vitro. It was found that this stimulatory effect of glucose can be mimicked by malate, isocitrate, lactate and pyruvate. 4. It is concluded that NADP–malate dehydrogenase or NADP–isocitrate dehydrogenase or both may play an important role in the formation of NADPH in the superovulated rat ovary. It is suggested that the stimulatory effect of glucose on steroidogenesis from acetate results from an increased rate of NADPH formation through one or both dehydrogenases, brought about by the increases in the concentrations of malate, isocitrate or both. Possible pathways involving the two enzymes are discussed

  5. Glucose-6-Phosphate Dehydrogenase Deficiency.

    PubMed

    Luzzatto, Lucio; Nannelli, Caterina; Notaro, Rosario

    2016-04-01

    G6PD is a housekeeping gene expressed in all cells. Glucose-6-phosphate dehydrogenase (G6PD) is part of the pentose phosphate pathway, and its main physiologic role is to provide NADPH. G6PD deficiency, one of the commonest inherited enzyme abnormalities in humans, arises through one of many possible mutations, most of which reduce the stability of the enzyme and its level as red cells age. G6PD-deficient persons are mostly asymptomatic, but they can develop severe jaundice during the neonatal period and acute hemolytic anemia when they ingest fava beans or when they are exposed to certain infections or drugs. G6PD deficiency is a global health issue.

  6. Molecular basis for thermoprotection in Bemisia: structural differences between whitefly ketose reductase and other medium-chain dehydrogenases/reductases.

    PubMed

    Wolfe, G R; Smith, C A; Hendrix, D L; Salvucci, M E

    1999-02-01

    The silverleaf whitefly (Bemisia argentifolii, Bellows and Perring) accumulates sorbitol as a thermoprotectant in response to elevated temperature. Sorbitol synthesis in this insect is catalyzed by an unconventional ketose reductase (KR) that uses NADPH to reduce fructose. A cDNA encoding the NADPH-KR from adult B. argentifolii was cloned and sequenced to determine the primary structure of this enzyme. The cDNA encoded a protein of 352 amino acids with a calculated molecular mass of 38.2 kDa. The deduced amino acid sequence of the cDNA shared 60% identity with sheep NAD(+)-dependent sorbitol dehydrogenase (SDH). Residues in SDH involved in substrate binding were conserved in the whitefly NADPH-KR. An important structural difference between the whitefly NADPH-KR and NAD(+)-SDHs occurred in the nucleotide-binding site. The Asp residue that coordinates the adenosyl ribose hydroxyls in NAD(+)-dependent dehydrogenases (including NAD(+)-SDH), was replaced by an Ala in the whitefly NADPH-KR. The whitefly NADPH-KR also contained two neutral to Arg substitutions within four residues of the Asp to Ala substitution. Molecular modeling indicated that addition of the Arg residues and loss of the Asp decreased the electric potential of the adenosine ribose-binding pocket, creating an environment favorable for NADPH-binding. Because of the ability to use NADPH, the whitefly NADPH-KR synthesizes sorbitol under physiological conditions, unlike NAD(+)-SDHs, which function in sorbitol catabolism.

  7. A sensitive radioisotopic method for the measurement of NAD(P)H: Its application to the assay of metabolites and enzymatic activities

    SciTech Connect

    Sener, A.; Malaisse, W.J. )

    1990-05-01

    A radioisotopic method for the assay of NADH or NADPH is presented, which is based on the conversion of 2-(U-{sup 14}C)ketoglutarate to {sup 14}C-labeled glutamate in the reaction catalyzed by glutamate dehydrogenase. The efficiency of the method is close to 75%, its precision (coefficient of variation) close to 5%, and its sensitivity close to 0.1 pmol/sample. This simple and rapid method can be applied to the measurement of several metabolites and enzymatic activities. In the present study, its application to the assay of sorbitol, 3-hydroxybutyrate, glutamate dehydrogenase, 3-hydroxybutyrate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase is documented.

  8. Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas

    PubMed Central

    Chung, Taemoon; Na, Juri; Kim, Young-il; Chang, Da-Young; Kim, Young Il; Kim, Hyeonjin; Moon, Ho Eun; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key; Kim, Sung-Soo; Suh-Kim, Haeyoung; Paek, Sun Ha; Youn, Hyewon

    2016-01-01

    We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy. PMID:27446484

  9. Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas.

    PubMed

    Chung, Taemoon; Na, Juri; Kim, Young-Il; Chang, Da-Young; Kim, Young Il; Kim, Hyeonjin; Moon, Ho Eun; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key; Kim, Sung-Soo; Suh-Kim, Haeyoung; Paek, Sun Ha; Youn, Hyewon

    2016-01-01

    We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy.

  10. Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy consuming redox circuit

    PubMed Central

    Fisher-Wellman, Kelsey H.; Lin, Chien-Te; Ryan, Terence E.; Reese, Lauren R.; Gilliam, Laura A. A.; Cathey, Brook L.; Lark, Daniel S.; Smith, Cody D.; Muoio, Deborah M.; Neufer, P. Darrell

    2015-01-01

    SUMMARY Cellular proteins rely on reversible redox reactions to establish and maintain biological structure and function. How redox catabolic (NAD+:NADH) and anabolic (NADP+:NADPH) processes integrate during metabolism to maintain cellular redox homeostasis however is unknown. The present work identifies a continuously cycling, mitochondrial membrane potential-dependent redox circuit between the pyruvate dehydrogenase complex (PDHC) and nicotinamide nucleotide transhydrogenase (NNT). PDHC is shown to produce H2O2 in relation to reducing pressure within the complex. The H2O2 produced however is effectively masked by a continuously cycling redox circuit that links, via glutathione/thioredoxin, to NNT, which catalyzes the regeneration of NADPH from NADH at the expense of the mitochondrial membrane potential. The net effect is an automatic fine tuning of NNT-mediated energy expenditure to metabolic balance at the level of PDHC. In mitochondria, genetic or pharmacological disruptions in the PDHC-NNT redox circuit negate counterbalance changes in energy expenditure. At the whole animal level, mice lacking functional NNT (C57BL/6J) are characterized by lower energy expenditure rates, consistent with their well known susceptibility to diet-induced obesity. These findings suggest the integration of redox sensing of metabolic balance with compensatory changes in energy expenditure provides a potential mechanism by which cellular redox homeostasis is maintained and body weight is defended during periods of positive and negative energy balance. PMID:25643703

  11. The NADPH oxidase inhibitor apocynin (acetovanillone) induces oxidative stress

    SciTech Connect

    Riganti, Chiara . E-mail: dario.ghigo@unito.it

    2006-05-01

    Apocynin (acetovanillone) is often used as a specific inhibitor of NADPH oxidase. In N11 glial cells, apocynin induced, in a dose-dependent way, a significant increase of both malonyldialdehyde level (index of lipid peroxidation) and lactate dehydrogenase release (index of a cytotoxic effect). Apocynin evoked also, in a significant way, an increase of H{sub 2}O{sub 2} concentration and a decrease of the intracellular glutathione/glutathione disulfide ratio, accompanied by augmented efflux of glutathione and glutathione disulfide. Apocynin induced the activation of both pentose phosphate pathway and tricarboxylic acid cycle, which was blocked when the cells were incubated with glutathione together with apocynin. The cell incubation with glutathione prevented also the apocynin-induced increase of malonyldialdehyde generation and lactate dehydrogenase leakage. Apocynin exerted an oxidant effect also in a cell-free system: indeed, in aqueous solution, it evoked a faster oxidation of the thiols glutathione and dithiothreitol, and elicited the generation of reactive oxygen species, mainly superoxide anions. Our results suggest that apocynin per se can induce an oxidative stress and exert a cytotoxic effect in N11 cells and other cell types, and that some effects of apocynin in in vitro and in vivo experimental models should be interpreted with caution.

  12. Protein engineering reveals ancient adaptive replacements in isocitrate dehydrogenase

    PubMed Central

    Dean, Antony M.; Golding, G. Brian

    1997-01-01

    Evolutionary analysis indicates that eubacterial NADP-dependent isocitrate dehydrogenases (EC 1.1.1.42) first evolved from an NAD-dependent precursor about 3.5 billion years ago. Selection in favor of utilizing NADP was probably a result of niche expansion during growth on acetate, where isocitrate dehydrogenase provides 90% of the NADPH necessary for biosynthesis. Amino acids responsible for differing coenzyme specificities were identified from x-ray crystallographic structures of Escherichia coli isocitrate dehydrogenase and the distantly related Thermus thermophilus NAD-dependent isopropylmalate dehydrogenase. Site-directed mutagenesis at sites lining the coenzyme binding pockets has been used to invert the coenzyme specificities of both enzymes. Reconstructed ancestral sequences indicate that these replacements are ancestral. Hence the adaptive history of molecular evolution is amenable to experimental investigation. PMID:9096353

  13. A specific radiochemical assay for pyrroline-5-carboxylate dehydrogenase.

    PubMed

    Small, C; Jones, M E

    1987-03-01

    Previous studies of pyrroline-5-carboxylate dehydrogenase have been conducted using a spectrophotometric method to monitor substrate-dependent NAD(P)H production. For the assay of the mammalian enzyme, the spectrophotometric assay was found to be unacceptable for kinetic studies as the production of NAD(P)H was nonlinear with time and protein concentration. An assay which measures radiolabeled glutamate production by this enzyme in the presence of NAD+ from radiolabeled pyrroline-5-carboxylate has been developed. Separation of substrate from product is achieved by column chromatography using Dowex 50 cation-exchange resin. The product isolated by this procedure was identified as glutamate. This new assay is linear with time and protein concentration and gives reproducible results. The assay is not influenced by competing enzyme activities, such as glutamate dehydrogenase, in a liver homogenate so that quantitative conversion of pyrroline-5-carboxylate to glutamate is observed.

  14. Activity of the glutathione antioxidant system and NADPH-generating enzymes in blood serum of rats with type 2 diabetes mellitus after administration of melatonin-correcting drugs.

    PubMed

    Agarkov, A A; Popova, T N; Verevkin, A N; Matasova, L V

    2014-06-01

    We studied the effects of epifamin and melaxen on serum content of reduced glutathione and activities of glutathione peroxidase, glutathione reductase, and NADPH-generating enzymes (glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase) in rats with type 2 diabetes mellitus. The concentration of reduced glutathione was decreased in rats with this disease (by 1.8 times), but increased after treatment with epifamin and melaxen (by 1.6 and 1.7 times, respectively). Activities of glutathione peroxidase, glutathione reductase, and NADPH-generating enzymes returned to the control level. Correction of melatonin concentration after treatment with the test drugs was probably followed by inhibition of free radical processes. The observed changes were accompanied by normalization of activity of the glutathione antioxidant system and NADPH-generating enzymes required for normal function of this system.

  15. Yeast surface display of dehydrogenases in microbial fuel-cells.

    PubMed

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems.

  16. Lactate dehydrogenase test

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003471.htm Lactate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Lactate dehydrogenase (LDH) is a protein that helps produce energy ...

  17. NADPH oxidase plays a crucial role in the activation of pancreatic stellate cells.

    PubMed

    Masamune, Atsushi; Watanabe, Takashi; Kikuta, Kazuhiro; Satoh, Kennichi; Shimosegawa, Tooru

    2008-01-01

    Activated pancreatic stellate cells (PSCs) play an important role in pancreatic fibrosis and inflammation, where oxidative stress is implicated in the pathogenesis. NADPH oxidase might be a source of reactive oxygen species (ROS) in the injured pancreas. This study aimed to clarify the expression and regulation of cell functions by NADPH oxidase in PSCs. PSCs were isolated from rat and human pancreas tissues. Expression of NADPH oxidase was assessed by reverse transcription-PCR and immunostaining. Intracellular ROS production was assessed using 2',7'-dichlorofluorescin diacetate. The effects of diphenylene iodonium (DPI) and apocynin, inhibitors of NADPH oxidase, on key parameters of PSC activation were evaluated in vitro. In vivo, DPI (at 1 mg.kg body wt(-1).day(-1)) was administered in drinking water to 10-wk-old male Wistar Bonn/Kobori rats for 10 wk and to rats with chronic pancreatitis induced by dibutyltin dichloride (DBTC). PSCs expressed key components of NADPH oxidase (p22(phox), p47(phox), NOX1, gp91(phox)/NOX2, NOX4, and NOX activator 1). PDGF-BB, IL-1beta, and angiotensin II induced ROS production, which was abolished by DPI and apocynin. DPI inhibited PDGF-induced proliferation, IL-1beta-induced chemokine production, and expression of alpha-smooth muscle actin and collagen. DPI inhibited transformation of freshly isolated cells to a myofibroblast-like phenotype. In addition, DPI inhibited the development of pancreatic fibrosis in Wistar Bonn/Kobori rats and in rats with DBTC-induced chronic pancreatitis. In conclusion, PSCs express NADPH oxidase to generate ROS, which mediates key cell functions and activation of PSCs. NADPH oxidase might be a potential target for the treatment of pancreatic fibrosis.

  18. A novel approach to improve poly-γ-glutamic acid production by NADPH Regeneration in Bacillus licheniformis WX-02.

    PubMed

    Cai, Dongbo; He, Penghui; Lu, Xingcheng; Zhu, Chengjun; Zhu, Jiang; Zhan, Yangyang; Wang, Qin; Wen, Zhiyou; Chen, Shouwen

    2017-02-23

    Poly-γ-glutamic acid (γ-PGA) is an important biochemical product with a variety of applications. This work reports a novel approach to improve γ-PGA through over expression of key enzymes in cofactor NADPH generating process for NADPH pool. Six genes encoding the key enzymes in NADPH generation were over-expressed in the γ-PGA producing strain B. licheniformis WX-02. Among various recombinants, the strain over-expressing zwf gene (coding for glucose-6-phosphate dehydrogenase), WX-zwf, produced the highest γ-PGA concentration (9.13 g/L), 35% improvement compared to the control strain WX-pHY300. However, the growth rates and glucose uptake rates of the mutant WX-zwf were decreased. The transcriptional levels of the genes pgsB and pgsC responsible for γ-PGA biosynthesis were increased by 8.21- and 5.26-fold, respectively. The Zwf activity of the zwf over expression strain increased by 9.28-fold, which led to the improvement of the NADPH generation, and decrease of accumulation of by-products acetoin and 2,3-butanediol. Collectively, these results demonstrated that NADPH generation via over-expression of Zwf is as an effective strategy to improve the γ-PGA production in B. licheniformis.

  19. A novel approach to improve poly-γ-glutamic acid production by NADPH Regeneration in Bacillus licheniformis WX-02

    PubMed Central

    Cai, Dongbo; He, Penghui; Lu, Xingcheng; Zhu, Chengjun; Zhu, Jiang; Zhan, Yangyang; Wang, Qin; Wen, Zhiyou; Chen, Shouwen

    2017-01-01

    Poly-γ-glutamic acid (γ-PGA) is an important biochemical product with a variety of applications. This work reports a novel approach to improve γ-PGA through over expression of key enzymes in cofactor NADPH generating process for NADPH pool. Six genes encoding the key enzymes in NADPH generation were over-expressed in the γ-PGA producing strain B. licheniformis WX-02. Among various recombinants, the strain over-expressing zwf gene (coding for glucose-6-phosphate dehydrogenase), WX-zwf, produced the highest γ-PGA concentration (9.13 g/L), 35% improvement compared to the control strain WX-pHY300. However, the growth rates and glucose uptake rates of the mutant WX-zwf were decreased. The transcriptional levels of the genes pgsB and pgsC responsible for γ-PGA biosynthesis were increased by 8.21- and 5.26-fold, respectively. The Zwf activity of the zwf over expression strain increased by 9.28-fold, which led to the improvement of the NADPH generation, and decrease of accumulation of by-products acetoin and 2,3-butanediol. Collectively, these results demonstrated that NADPH generation via over-expression of Zwf is as an effective strategy to improve the γ-PGA production in B. licheniformis. PMID:28230096

  20. Activated barrier crossing dynamics in the non-radiative decay of NADH and NADPH

    NASA Astrophysics Data System (ADS)

    Blacker, Thomas S.; Marsh, Richard J.; Duchen, Michael R.; Bain, Angus J.

    2013-08-01

    In live tissue, alterations in metabolism induce changes in the fluorescence decay of the biological coenzyme NAD(P)H, the mechanism of which is not well understood. In this work, the fluorescence and anisotropy decay dynamics of NADH and NADPH were investigated as a function of viscosity in a range of water-glycerol solutions. The viscosity dependence of the non-radiative decay is well described by Kramers and Kramers-Hubbard models of activated barrier crossing over a wide viscosity range. Our combined lifetime and anisotropy analysis indicates common mechanisms of non-radiative relaxation in the two emitting states (conformations) of both molecules. The low frequencies associated with barrier crossing suggest that non-radiative decay is mediated by small scale motion (e.g. puckering) of the nicotinamide ring. Variations in the fluorescence lifetimes of NADH and NADPH when bound to different enzymes may therefore be attributed to differing levels of conformational restriction upon binding.

  1. Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior.

    PubMed

    Campbell, Elliot; Wheeldon, Ian R; Banta, Scott

    2010-12-01

    Cofactor specificity in the aldo-keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site-directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild-type. Results of previous pre-steady-state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2'-phosphate of the cofactor. Pre-steady-state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2'-phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex.

  2. Mechanism of NADPH oxidation catalyzed by horse-radish peroxidase and 2,4-diacetyl-[2H]heme-substituted horse-radish peroxidase.

    PubMed

    De Sandro, V; Dupuy, C; Kaniewski, J; Ohayon, R; Dème, D; Virion, A; Pommier, J

    1991-10-15

    The mechanism of NADPH oxidation catalyzed by horse-radish peroxidase (HRP) and 2,4-diacetyl-[2H]heme-substituted horse-radish peroxidase (DHRP) was studied. The roles of the different H2O2/peroxidase compounds were examined by spectral studies. The oxidized NADPH species were identified using the superoxide dismutase effect and by measuring the stoichiometry between NADPH oxidized and H2O2 used. In the presence of a mediating molecule, like scopoletin, both enzymes acted via a similar mechanism, producing only NADP degrees, which in turn reacted with O2 producing O2-. Consequently H2O2 was completely regenerated in the presence of superoxide dismutase and partially regenerated in its absence. In the absence of a mediating molecule, the H2O2 complex of both enzymes (compound I) catalysed NADPH oxidation by single-electron transfer, producing NADP degrees; compound II of these enzymes catalyzed NADPH oxidation more slowly by a direct two-electron transfer, producing NADPH+. There were difference between HRP and DHRP. HRP compound II was produced by the oxidation of 1 mol NADPH/mole compound I, while DHRP compound II was formed by the spontaneous conversion of compound I to compound II. The NADPH oxidation catalyzed by DHRP compound I did not lead to the formation of compound II. When H2O2 was produced slowly by the glucose/glucose-oxidase system, compound II was never formed and a pure O2- adduct of DHRP (compound III) accumulated.

  3. Biphasic Regulation of the NADPH Oxidase by HGF/c-Met Signaling Pathway in Primary Mouse Hepatocytes

    PubMed Central

    Clavijo-Cornejo, Denise; Enriquez-Cortina, Cristina; López-Reyes, Alberto; Domínguez-Pérez, Mayra; Nuño, Natalia; Domínguez-Meraz, Marcela; Bucio, Leticia; Souza, Verónica; Factor, Valentina M.; Thorgeirsson, Snorri S.; Gutiérrez-Ruiz, María Concepción; Gómez-Quiroz, Luis E.

    2013-01-01

    Redox signaling is emerging as an essential mechanism in the regulation of biological activities of the cell. The HGF/c-Met signaling pathway has been implicated as a key regulator of the cellular redox homeostasis and oxidative stress. We previously demonstrated that genetic deletion of c-met in hepatocytes disrupts redox homeostasis by a mechanism involving NADPH oxidase. Here, we were focused to address the mechanism of NADPH oxidase regulation by HGF/c-Met signaling in primary mouse hepatocytes and its relevance. HGF induced a biphasic mechanism of NADPH oxidase regulation. The first phase employed the rapid increase in production of ROS as signaling effectors to activate the Nrf2-mediated protective response resulting in up-regulation of the antioxidant proteins, such as NAD(P)H quinone oxidoreductase and γ-glutamylcysteine synthetase. The second phase operated under a prolonged HGF exposure, caused a suppression of the NADPH oxidase components, including NOX2, NOX4, p22 and p67, and was able to abrogate the TGFβ-induced ROS production and improve cell viability. In conclusion, HGF/c-Met induces a Nrf2-mediated protective response by a double mechanism driven by NADPH oxidase. PMID:23333744

  4. Bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by human NAD(P)H quinone oxidoreductase 2: a novel co-substrate-mediated antitumor prodrug therapy.

    PubMed

    Knox, R J; Jenkins, T C; Hobbs, S M; Chen, S; Melton, R G; Burke, P J

    2000-08-01

    A novel prodrug activation system, endogenous in human tumor cells, is described. A latent enzyme-prodrug system is switched on by a simple synthetic, small molecule co-substrate. This ternary system is inactive if any one of the components is absent. CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] is an antitumor prodrug that is activated in certain rat tumors via its 4-hydroxylamine derivative to a potent bifunctional alkylating agent. However, human tumor cells are resistant to CB 1954 because they are unable to catalyze this bioactivation efficiently. A human enzyme has been discovered that can activate CB 1954, and it has been shown to be commonly present in human tumor cells. The enzyme is NQO2 [NAD(P)H quinone oxidoreductase 2], but its activity is normally latent, and a nonbiogenic co-substrate such as NRH [nicotinamide riboside (reduced)] is required for enzymatic activity. There is a very large (100-3000-fold) increase in CB 1954 cytotoxicity toward either NQO2-transfected rodent or nontransfected human tumor cell lines in the presence of NRH. Other reduced pyridinium compounds can also act as co-substrates for NQO2. Thus, the simplest quaternary salt of nicotinamide, 1-methyl-3-carboxamidopyridinium iodide, was a co-substrate for NQO2 when reduced to the corresponding 1,4-dihydropyridine derivative. Increased chain length and/or alkyl load at the 1-position of the dihydropyridine ring improved specific activity, and compounds more active than NRH were found. However, little activity was seen with either the 1-benzyl or 1-(2-phenylethyl) derivatives. A negatively charged substituent at the 3-position of the reduced pyridine ring also negated the ability of these compounds to act as cosubstrates for NQO2. In particular, 1-carbamoylmethyl-3-carbamoyl-1,4dihydropyridine was shown to be a co-substrate for NQO2 with greater stability than NRH, with the ability to enter cells and potentiate the cytotoxicity of CB 1954. Furthermore, this agent is synthetically

  5. Stereo-specificity for pro-(R) hydrogen of NAD(P)H during enzyme-catalyzed hydride transfer to CL-20

    SciTech Connect

    Bhushan, Bharat; Halasz, Annamaria; Hawari, Jalal . E-mail: jalal.hawari@nrc.ca

    2005-12-02

    A dehydrogenase from Clostridium sp. EDB2 and a diaphorase from Clostridium kluyveri were reacted with CL-20 to gain insights into the enzyme-catalyzed hydride transfer to CL-20, and the enzyme's stereo-specificity for either pro-R or pro-S hydrogens of NAD(P)H. Both enzymes biotransformed CL-20 at rates of 18.5 and 24 nmol/h/mg protein, using NADH and NADPH as hydride-source, respectively, to produce a N-denitrohydrogenated product with a molecular weight of 393 Da. In enzyme kinetics studies using reduced deuterated pyridine nucleotides, we found a kinetic deuterium isotopic effect of 2-fold on CL-20 biotransformation rate using dehydrogenase enzyme against (R)NADD as a hydride-source compared to either (S)NADD or NADH. Whereas, in case of diaphorase, the kinetic deuterium isotopic effect of about 1.5-fold was observed on CL-20 biotransformation rate using (R)NADPD as hydride-source. In a comparative study with LC-MS, using deuterated and non-deuterated NAD(P)H, we found a positive mass-shift of 1 Da in the N-denitrohydrogenated product suggesting the involvement of a deuteride (D{sup -}) transfer from NAD(P)D. The present study thus revealed that both dehydrogenase and diaphorase enzymes from the two Clostridium species catalyzed a hydride transfer to CL-20 and showed stereo-specificity for pro-R hydrogen of NAD(P)H.

  6. Exploiting algal NADPH oxidase for biophotovoltaic energy.

    PubMed

    Anderson, Alexander; Laohavisit, Anuphon; Blaby, Ian K; Bombelli, Paolo; Howe, Christopher J; Merchant, Sabeeha S; Davies, Julia M; Smith, Alison G

    2016-01-01

    Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.

  7. The role of the NAD-dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant.

    PubMed

    Boles, E; Lehnert, W; Zimmermann, F K

    1993-10-01

    Phosphoglucose isomerase pgi1-deletion mutants of Saccharomyces cerevisiae cannot grow on glucose as the sole carbon source and are even inhibited by glucose. These growth defects could be suppressed by an over-expression on a multi-copy plasmid of the structural gene GDH2 coding for the NAD-dependent glutamate dehydrogenase. GDH2 codes for a protein with 1092 amino acids which is located on chromosome XII and shows high sequence similarity to the Neurospora crassa NAD-glutamate dehydrogenase. Suppression of the pgi1 deletion by over-expression of GDH2 was abolished in strains with a deletion of the glucose-6-phosphate dehydrogenase gene ZWF1 or gene GDH1 coding for the NADPH-dependent glutamate dehydrogenase. Moreover, this suppression required functional mitochondria. It is proposed that the growth defect of pgi1 deletion mutants on glucose is due to a rapid depletion of NADP which is needed as a cofactor in the oxidative reactions of the pentose phosphate pathway. Over-expression of the NAD-dependent glutamate dehydrogenase leads to a very efficient conversion of glutamate with NADH generation to 2-oxoglutarate which can be converted back to glutamate by the NADPH-dependent glutamate dehydrogenase with the consumption of NADPH. Consequently, over-expression of the NAD-dependent glutamate dehydrogenase causes a substrate cycling between 2-oxoglutarate and glutamate which restores NADP from NADPH through the coupled conversion of NAD to NADH which can be oxidized in the mitochondria. Furthermore, the requirement for an increase in NADPH consumption for the suppression of the phosphoglucose isomerase defect could be met by addition of oxidizing agents which are known to reduce the level of NADPH.

  8. CHANGES IN DISULFIDE BOND CONTENT OF PROTEINS IN A YEAST STRAIN LACKING MAJOR SOURCES OF NADPH

    PubMed Central

    Minard, Karyl I.; Carroll, Christopher A.; Weintraub, Susan T.; Mc-Alister-Henn, Lee

    2006-01-01

    A yeast mutant lacking the two major cytosolic sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1p) and NADP+-specific isocitrate dehydrogenase (Idp2p), has been demonstrated to lose viability when shifted to medium with acetate or oleate as the carbon source. This loss in viability was found to correlate with an accumulation of endogenous oxidative byproducts of respiration and peroxisomal β-oxidation. To assess effects on cellular protein of endogenous versus exogenous oxidative stress, a proteomics approach was used to compare disulfide bond-containing proteins in the idp2Δzwf1Δ strain following shifts to acetate and oleate media with those in the parental strain following similar shifts to media containing hydrogen peroxide. Among prominent disulfide bond-containing proteins were several with known antioxidant functions. These and several other proteins were detected as multiple electrophoretic isoforms, with some isoforms containing disulfide bonds under all conditions and other isoforms exhibiting a redox-sensitive content of disulfide bonds, i.e., in the idp2Δzwf1Δ strain and in the hydrogen peroxide-challenged parental strain. The disulfide bond content of some isoforms of these proteins was also elevated in the parental strain grown on glucose, possibly suggesting a redirection of NADPH reducing equivalents to support rapid growth. Further examination of protein carbonylation in the idp2Δzwf1Δ strain shifted to oleate medium also led to identification of common and unique protein targets of endogenous oxidative stress. PMID:17157197

  9. Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

    SciTech Connect

    Kirkensgaard, Kristine G.; Hägglund, Per; Finnie, Christine; Svensson, Birte; Henriksen, Anette

    2009-09-01

    The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR, HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R{sub cryst} of 19.0% and an R{sub free} of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation.

  10. The NADPH oxidase Nox4 and aging in the heart.

    PubMed

    Ago, Tetsuro; Matsushima, Shouji; Kuroda, Junya; Zablocki, Daniela; Kitazono, Takanari; Sadoshima, Junichi

    2010-12-01

    Oxidative stress in mitochondria is believed to promote aging. Although passive leakage of electron from the mitochondrial electron transport chain has been considered as a major source of oxidative stress in the heart and the cardiomyocytes therein, enzymes actively producing reactive oxygen species may also exist in mitochondria. We have shown recently that Nox4, a member of the NADPH oxidase family, is localized on intracellular membranes, primarily at mitochondria, in cardiomyocytes. Mitochondrial expression of Nox4 is upregulated by cardiac stress and aging in the heart, where Nox4 could become a major source of oxidative stress. This raises an intriguing possibility that Nox4 may play an important role in mediating aging of the heart. Here we discuss the potential involvement of Nox4 in mitochondrial oxidative stress and aging in the heart.

  11. A new regulatory element mediates ethanol repression of KlADH3, a Kluyveromyces lactis gene coding for a mitochondrial alcohol dehydrogenase.

    PubMed

    Saliola, Michele; Getuli, Claudia; Mazzoni, Cristina; Fantozzi, Ivana; Falcone, Claudio

    2007-08-01

    KlADH3 is a Kluyveromyces lactis alcohol dehydrogenase gene induced in the presence of all respiratory carbon sources except ethanol, which specifically represses this gene. Deletion analysis of the KlADH3 promoter revealed the presence of both positive and negative elements. However, by site-directed mutagenesis and gel retardation experiments, we identified a 15-bp element responsible for the transcriptional repression of this gene by ethanol. In particular, this element showed putative sites required for the sequential binding of ethanol-induced factors responsible for the repressed conditions, and the binding of additional factors relieved repression. In addition, we showed that the ethanol element was required for in vivo repression of KlAdh3 activity.

  12. Methylglyoxal-induced modification of arginine residues decreases the activity of NADPH-generating enzymes.

    PubMed

    Morgan, Philip E; Sheahan, Pamela J; Pattison, David I; Davies, Michael J

    2013-08-01

    Inadequate control of plasma and cellular glucose and ketone levels in diabetes is associated with increased generation of reactive aldehydes, including methylglyoxal (MGO). These aldehydes react with protein side chains to form advanced glycation end-products (AGEs). Arg residues are particularly susceptible to MGO glycation and are essential for binding NADP(+) in several enzymes that generate NADPH, a coenzyme for many critical metabolic and antioxidant enzymes. In most animal cells, NADPH is produced predominantly by glucose-6-phosphate dehydrogenase (G6PD) in the oxidative phase of the pentose phosphate pathway and, to a lesser extent, by isocitrate dehydrogenase (IDH) and malic enzyme (ME). In this study, the activities of isolated G6PD, IDH, and ME were inhibited by MGO (0-2.5mM, 2-3h, 37°C), in a dose- and time-dependent manner, with G6PD and IDH more sensitive to modification than ME. Significant inhibition of these two enzymes occurred with MGO levels ≥500μM. Incubation with radiolabeled MGO (0-500µM, 0-3h, 37°C) demonstrated dose- and time-dependent adduction to G6PD and IDH. HPLC analysis provided evidence for AGE formation and particularly the hydroimidazolones MG-H1 and MG-H2 from Arg residues, with corresponding loss of parent Arg residues. Peptide mass mapping studies confirmed hydroimidazolone formation on multiple peptides in G6PD and IDH, including those critical for NADP(+) binding, and substrate binding, in the case of IDH. These results suggest that modification of NADPH-producing enzymes by reactive aldehydes may result in alterations to the cellular redox environment, potentially predisposing cells to further damage by oxidants and reactive aldehydes.

  13. TGL-mediated lipolysis in Manduca sexta fat body: possible roles for lipoamide-dehydrogenase (LipDH) and high-density lipophorin (HDLp)

    PubMed Central

    Wu, Zengying; Soulages, Jose L; Joshi, Bharat D.; Daniel, Stuart M.; Hager, Zachary J.; Arrese, Estela L

    2014-01-01

    Triglyceride-lipase (TGL) is a major fat body lipase in Manduca sexta. The knowledge of how TGL activity is regulated is very limited. A WWE domain, presumably involved in protein-protein interactions, has been previously identified in the N-terminal region of TGL. In this study, we searched for proteins partners that interact with the N-terminal region of TGL. Thirteen proteins were identified by mass spectrometry, and the interaction with four of these proteins was confirmed by immunoblot. The oxidoreductase lipoamide-dehydrogenase (LipDH) and the apolipoprotein components of the lipid transporter, HDLp, were among these proteins. LipDH is the common component of the mitochondrial α-keto acid dehydrogenase complexes whereas HDLp occurs in the hemolymph. However, subcellular fractionation demonstrated that these two proteins are relatively abundant in the soluble fraction of fat body adipocytes. The cofactor lipoate found in typical LipDH substrates was not detected in TGL. However, TGL proved to have critical thiol groups. Additional studies with inhibitors are consistent with the notion that LipDH acting as a diaphorase could preserve the activity of TGL by controlling the redox state of thiol groups. On the other hand, when TG hydrolase activity of TGL was assayed in the presence of HDLp, the production of diacylglycerol (DG) increased. TGL-HDLp interaction could drive the intracellular transport of DG. TGL may be directly involved in the lipoprotein assembly and loading with DG, a process that occurs in the fat body and is essential for insects to mobilize fatty acids. Overall the study suggests that TGL occurs as a multi-protein complex supported by interactions through the WWE domain. PMID:24333838

  14. Depletion of NADP(H) due to CD38 activation triggers endothelial dysfunction in the postischemic heart

    PubMed Central

    Reyes, Levy A.; Boslett, James; Varadharaj, Saradhadevi; De Pascali, Francesco; Hemann, Craig; Druhan, Lawrence J.; Ambrosio, Giuseppe; El-Mahdy, Mohamed; Zweier, Jay L.

    2015-01-01

    In the postischemic heart, coronary vasodilation is impaired due to loss of endothelial nitric oxide synthase (eNOS) function. Although the eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion only partially restores eNOS-mediated coronary vasodilation, indicating that other critical factors trigger endothelial dysfunction. Therefore, studies were performed to characterize the unidentified factor(s) that trigger endothelial dysfunction in the postischemic heart. We observed that depletion of the eNOS substrate NADPH occurs in the postischemic heart with near total depletion from the endothelium, triggering impaired eNOS function and limiting BH4 rescue through NADPH-dependent salvage pathways. In isolated rat hearts subjected to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H) pool occurred and was most marked in the endothelium, with >85% depletion. Repletion of NADPH after I/R increased NOS-dependent coronary flow well above that with BH4 alone. With combined NADPH and BH4 repletion, full restoration of NOS-dependent coronary flow occurred. Profound endothelial NADPH depletion was identified to be due to marked activation of the NAD(P)ase-activity of CD38 and could be prevented by inhibition or specific knockdown of this protein. Depletion of the NADPH precursor, NADP+, coincided with formation of 2’-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibition of CD38 prevented NADP(H) depletion and preserved endothelium-dependent relaxation and NO generation with increased recovery of contractile function and decreased infarction in the postischemic heart. Thus, CD38 activation is an important cause of postischemic endothelial dysfunction and presents a novel therapeutic target for prevention of this dysfunction in unstable coronary syndromes. PMID:26297248

  15. Reversal of coenzyme specificity of 2,3-butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis.

    PubMed

    Ehsani, Maryam; Fernández, Maria R; Biosca, Josep A; Dequin, Sylvie

    2009-10-01

    Saccharomyces cerevisiae NAD(H)-dependent 2,3-butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3-butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)-dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu(221), Ile(222), and Ala(223)) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site-directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration.

  16. Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein.

    PubMed Central

    Huang, Shaohui; Heikal, Ahmed A; Webb, Watt W

    2002-01-01

    Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections (sigma2P) increase rapidly at wavelengths below 800 nm, and the maximum sigma2P of LipDH is approximately 5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., approximately 410-490 nm and approximately 510-650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the sigma2P measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique. PMID:11964266

  17. Natural Compounds as Modulators of NADPH Oxidases

    PubMed Central

    2013-01-01

    Reactive oxygen species (ROS) are cellular signals generated ubiquitously by all mammalian cells, but their relative unbalance triggers also diseases through intracellular damage to DNA, RNA, proteins, and lipids. NADPH oxidases (NOX) are the only known enzyme family with the sole function to produce ROS. The NOX physiological functions concern host defence, cellular signaling, regulation of gene expression, and cell differentiation. On the other hand, increased NOX activity contributes to a wide range of pathological processes, including cardiovascular diseases, neurodegeneration, organ failure, and cancer. Therefore targeting these enzymatic ROS sources by natural compounds, without affecting the physiological redox state, may be an important tool. This review summarizes the current state of knowledge of the role of NOX enzymes in physiology and pathology and provides an overview of the currently available NADPH oxidase inhibitors derived from natural extracts such as polyphenols. PMID:24381714

  18. Mitochondrial alpha-ketoglutarate dehydrogenase complex generates reactive oxygen species.

    PubMed

    Starkov, Anatoly A; Fiskum, Gary; Chinopoulos, Christos; Lorenzo, Beverly J; Browne, Susan E; Patel, Mulchand S; Beal, M Flint

    2004-09-08

    Mitochondria-produced reactive oxygen species (ROS) are thought to contribute to cell death caused by a multitude of pathological conditions. The molecular sites of mitochondrial ROS production are not well established but are generally thought to be located in complex I and complex III of the electron transport chain. We measured H(2)O(2) production, respiration, and NADPH reduction level in rat brain mitochondria oxidizing a variety of respiratory substrates. Under conditions of maximum respiration induced with either ADP or carbonyl cyanide p-trifluoromethoxyphenylhydrazone,alpha-ketoglutarate supported the highest rate of H(2)O(2) production. In the absence of ADP or in the presence of rotenone, H(2)O(2) production rates correlated with the reduction level of mitochondrial NADPH with various substrates, with the exception of alpha-ketoglutarate. Isolated mitochondrial alpha-ketoglutarate dehydrogenase (KGDHC) and pyruvate dehydrogenase (PDHC) complexes produced superoxide and H(2)O(2). NAD(+) inhibited ROS production by the isolated enzymes and by permeabilized mitochondria. We also measured H(2)O(2) production by brain mitochondria isolated from heterozygous knock-out mice deficient in dihydrolipoyl dehydrogenase (Dld). Although this enzyme is a part of both KGDHC and PDHC, there was greater impairment of KGDHC activity in Dld-deficient mitochondria. These mitochondria also produced significantly less H(2)O(2) than mitochondria isolated from their littermate wild-type mice. The data strongly indicate that KGDHC is a primary site of ROS production in normally functioning mitochondria.

  19. Glutamate dehydrogenases: the why and how of coenzyme specificity.

    PubMed

    Engel, Paul C

    2014-01-01

    NAD(+) and NADP(+), chemically similar and with almost identical standard oxidation-reduction potentials, nevertheless have distinct roles, NAD(+) serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD(+)-dependent for glutamate oxidation, NADP(+)-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD(+) reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD(+) but maintains equilibrium using the same pool as accessed by β-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP(+) reduction by NADH, maintaining the coenzyme pools at different oxidation-reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD(+)-dependent, NADP(+)-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD(+) or for NADP(+) has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2'- and 3'-hydroxyls, dictating NAD(+) specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD(+) only, NADP(+) only, or in higher animals both.

  20. Aerobic physiology of redox-engineered Saccharomyces cerevisiae strains modified in the ammonium assimilation for increased NADPH availability.

    PubMed

    Moreira dos Santos, Margarida; Thygesen, Gerda; Kötter, Peter; Olsson, Lisbeth; Nielsen, Jens

    2003-10-01

    Recombinant strains altered in the ammonium assimilation pathways were constructed with the purpose of increasing NADPH availability. The NADPH-dependent glutamate dehydrogenase encoded by GDH1, which accounts for a major fraction of the NADPH consumption during growth on ammonium, was deleted, and alternative pathways for ammonium assimilation were overexpressed: GDH2 (NADH-consuming) or GLN1 and GLT1 (the GS-GOGAT system). The flux through the pentose phosphate pathway during aerobic growth on glucose decreased to about half that of the reference strain Saccharomyces cerevisiae CEN.PK113-7D, indicating a major redox alteration in the strains. The basic growth characteristics of the recombinant strains were not affected to a great extent, but the dilution rate at which the onset of aerobic fermentation occurred decreased, suggesting a relation between the onset of the Crabtree effect and the flux through the Embden-Meyerhof-Parnas pathway downstream of glucose 6-phosphate. No redox effect was observed in a strain containing a deletion of GLR1, encoding glutathione reductase, an enzyme that is NADPH-consuming.

  1. Dehydroepiandrosterone promotes pulmonary artery relaxation by NADPH oxidation-elicited subunit dimerization of protein kinase G 1α

    PubMed Central

    Patel, Dhara; Kandhi, Sharath; Kelly, Melissa; Neo, Boon Hwa

    2013-01-01

    The activity of glucose-6-phosphate dehydrogenase (G6PD) controls a vascular smooth muscle relaxing mechanism promoted by the oxidation of cytosolic NADPH, which has been associated with activation of the 1α form of protein kinase G (PKG-1α) by a thiol oxidation-elicited subunit dimerization. This PKG-1α-activation mechanism appears to contribute to responses of isolated endothelium-removed bovine pulmonary arteries (BPA) elicited by peroxide, cytosolic NADPH oxidation resulting from G6PD inhibition, and hypoxia. Dehydroepiandrosterone (DHEA) is a steroid hormone with pulmonary vasodilator activity, which has beneficial effects in treating pulmonary hypertension. Because multiple mechanisms have been suggested for the vascular effects of DHEA and one of the known actions of DHEA is inhibiting G6PD, we investigated whether it promoted relaxation associated with NADPH oxidation, PKG-1α dimerization, and PKG activation detected by increased vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Relaxation of BPA to DHEA under aerobic or hypoxic conditions was associated with NADPH oxidation, PKG-1α dimerization, and increased VASP phosphorylation. The vasodilator activity of DHEA was markedly attenuated in pulmonary arteries and aorta from a PKG knockin mouse containing a serine in place of a cysteine involved in PKG dimerization. DHEA promoted increased PKG dimerization in lungs from wild-type mice, which was not detected in the PKG knockin mouse model. Thus PKG-1α dimerization is a major contributing factor to the vasodilator actions of DHEA and perhaps its beneficial effects in treating pulmonary hypertension. PMID:24375799

  2. Structural basis for the alteration of coenzyme specificity in a malate dehydrogenase mutant

    SciTech Connect

    Tomita, Takeo; Fushinobu, Shinya; Kuzuyama, Tomohisa; Nishiyama, Makoto . E-mail: umanis@mail.ecc.u-tokyo.ac.jp

    2006-08-25

    To elucidate the structural basis for the alteration of coenzyme specificity from NADH toward NADPH in a malate dehydrogenase mutant EX7 from Thermus flavus, we determined the crystal structures at 2.0 A resolution of EX7 complexed with NADPH and NADH, respectively. In the EX7-NADPH complex, Ser42 and Ser45 form hydrogen bonds with the 2'-phosphate group of the adenine ribose of NADPH, although the adenine moiety is not seen in the electron density map. In contrast, although Ser42 and Ser45 occupy a similar position in the EX7-NADH complex structure, both the adenine and adenine ribose moieties of NADH are missing in the map. These results and kinetic analysis of site-directed mutant enzymes indicate (1) that the preference of EX7 for NADPH over NADH is ascribed to the recognition of the 2'-phosphate group by two Ser and Arg44, and (2) that the adenine moiety of NADPH is not recognized in this mutant.

  3. Protein kinase C-beta contributes to NADPH oxidase activation in neutrophils.

    PubMed Central

    Dekker, L V; Leitges, M; Altschuler, G; Mistry, N; McDermott, A; Roes, J; Segal, A W

    2000-01-01

    We have analysed the involvement of the beta isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils. Using immunofluorescence and cell fractionation, PKC-beta is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcgamma-receptor stimulus). The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli. The PKC-beta specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria. Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-beta, but not other PKC isotypes, is targeted. Neutrophils isolated from a mouse that lacks PKC-beta also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles. The level of inhibition is comparable to that achieved with 379196 in human neutrophils. Thus the PKC-beta isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcgamma receptors in neutrophils. PMID:10727429

  4. NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality

    PubMed Central

    Brothers, Kimberly M.; Gratacap, Remi L.; Barker, Sarah E.; Newman, Zachary R.; Norum, Ashley; Wheeler, Robert T.

    2013-01-01

    Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis. PMID:24098114

  5. Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase.

    PubMed

    Burdette, D; Zeikus, J G

    1994-08-15

    The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling.

  6. Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase.

    PubMed Central

    Burdette, D; Zeikus, J G

    1994-01-01

    The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling. Images Figure 1 PMID:8068002

  7. Lactate dehydrogenase downregulation mediates the inhibitory effect of diallyl trisulfide on proliferation, metastasis, and invasion in triple-negative breast cancer.

    PubMed

    Cheng, Shi-Yann; Yang, Yao-Chih; Ting, Kuan-Lun; Wen, Su-Ying; Viswanadha, Vijaya Padma; Huang, Chih-Yang; Kuo, Wei-Wen

    2017-04-01

    The Warburg effect plays a critical role in tumorigenesis, suggesting that specific agents targeting Warburg effect key proteins may be a promising strategy for cancer therapy. Previous studies have shown that diallyl trisulfide (DATS) inhibits proliferation of breast cancer cells by inducing apoptosis in vitro and in vivo. However, whether the Warburg effect is involved with the apoptosis-promoting action of DATS is unclear. Here, we show that the action of DATS is associated with downregulation of lactate dehydrogenase A (LDHA), an essential protein of the Warburg effect whose upregulation is closely related to tumorigenesis. Interestingly, inhibition of the Warburg effect by DATS in breast cancer cells did not greatly affect normal cells. Furthermore, DATS inhibited growth of breast cancer cells, particularly in MDA-MB-231, a triple-negative breast cancer (TNBC) cell, and reduced proliferation and migration; invasion was reversed by over-expression of LDHA. These data suggest that DATS inhibits breast cancer growth and aggressiveness through a novel pathway targeting the key enzyme of the Warburg effect. Our study shows that LDHA downregulation is involved in the apoptotic effect of DATS on TNBC. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1390-1398, 2017.

  8. 11β-Hydroxysteroid Dehydrogenase Type 1(11β-HSD1) mediates insulin resistance through JNK activation in adipocytes

    PubMed Central

    Peng, Kesong; Pan, Yong; Li, Jieli; Khan, Zia; Fan, Mendi; Yin, Haimin; Tong, Chao; Zhao, Yunjie; Liang, Guang; Zheng, Chao

    2016-01-01

    Glucocorticoids are used to treat a number of human diseases but often lead to insulin resistance and metabolic syndrome. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a key enzyme that catalyzes the intracellular conversion of cortisone to physiologically active cortisol. Despite the known role of 11β-HSD1 and active glucocorticoid in causing insulin resistance, the molecular mechanisms by which insulin resistance is induced remain elusive. The aim of this study is to identify these mechanisms in high fat diet (HFD) experimental models. Mice on a HFD were treated with 11β-HSD1 inhibitor as well as a JNK inhibitor. We then treated 3T3-L1-derived adipocytes with prednisone, a synthetic glucocorticoid, and cells with 11β-HSD1 overexpression to study insulin resistance. Our results show that 11β-HSD1 and JNK inhibition mitigated insulin resistance in HFD mice. Prednisone stimulation or overexpression of 11β-HSD1 also caused JNK activation in cultured adipocytes. Inhibition of 11β-HSD1 blocked the activation of JNK in adipose tissue of HFD mice as well as in cultured adipocytes. Furthermore, prednisone significantly impaired the insulin signaling pathway, and these effects were reversed by 11β-HSD1 and JNK inhibition. Our study demonstrates that glucocorticoid-induced insulin resistance was dependent on 11β-HSD1, resulting in the critical activation of JNK signaling in adipocytes. PMID:27841334

  9. Antisense inhibition of the iron-sulphur subunit of succinate dehydrogenase enhances photosynthesis and growth in tomato via an organic acid-mediated effect on stomatal aperture.

    PubMed

    Araújo, Wagner L; Nunes-Nesi, Adriano; Osorio, Sonia; Usadel, Björn; Fuentes, Daniela; Nagy, Réka; Balbo, Ilse; Lehmann, Martin; Studart-Witkowski, Claudia; Tohge, Takayuki; Martinoia, Enrico; Jordana, Xavier; Damatta, Fábio M; Fernie, Alisdair R

    2011-02-01

    Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the Sl SDH2-2 gene encoding the iron sulfur subunit of the succinate dehydrogenase protein complex in the antisense orientation under the control of the 35S promoter exhibit an enhanced rate of photosynthesis. The rate of the tricarboxylic acid (TCA) cycle was reduced in these transformants, and there were changes in the levels of metabolites associated with the TCA cycle. Furthermore, in comparison to wild-type plants, carbon dioxide assimilation was enhanced by up to 25% in the transgenic plants under ambient conditions, and mature plants were characterized by an increased biomass. Analysis of additional photosynthetic parameters revealed that the rate of transpiration and stomatal conductance were markedly elevated in the transgenic plants. The transformants displayed a strongly enhanced assimilation rate under both ambient and suboptimal environmental conditions, as well as an elevated maximal stomatal aperture. By contrast, when the Sl SDH2-2 gene was repressed by antisense RNA in a guard cell-specific manner, changes in neither stomatal aperture nor photosynthesis were observed. The data obtained are discussed in the context of the role of TCA cycle intermediates both generally with respect to photosynthetic metabolism and specifically with respect to their role in the regulation of stomatal aperture.

  10. Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells

    PubMed Central

    Kim, Da Jung; Kim, Yong Sik

    2015-01-01

    Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells. PMID:26221064

  11. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    SciTech Connect

    Woodward, J.

    1996-10-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based upon the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with continuous cofactor recycle. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value commodity chemical.

  12. Multiple mechanisms of NADPH oxidase inhibition by type A and type B Francisella tularensis

    PubMed Central

    McCaffrey, Ramona L.; Schwartz, Justin T.; Lindemann, Stephen R.; Moreland, Jessica G.; Buchan, Blake W.; Jones, Bradley D.; Allen, Lee-Ann H.

    2010-01-01

    Ft is a facultative intracellular pathogen that infects many cell types, including neutrophils. In previous work, we demonstrated that the type B Ft strain LVS disrupts NADPH oxidase activity throughout human neutrophils, but how this is achieved is incompletely defined. Here, we used several type A and type B strains to demonstrate that Ft-mediated NADPH oxidase inhibition is more complex than appreciated previously. We confirm that phagosomes containing Ft opsonized with AS exclude flavocytochrome b558 and extend previous results to show that soluble phox proteins were also affected, as indicated by diminished phosphorylation of p47phox and other PKC substrates. However, a different mechanism accounts for the ability of Ft to inhibit neutrophil activation by formyl peptides, Staphylococcus aureus, OpZ, and phorbol esters. In this case, enzyme targeting and assembly were normal, and impaired superoxide production was characterized by sustained membrane accumulation of dysfunctional NADPH oxidase complexes. A similar post-assembly inhibition mechanism also diminished the ability of anti-Ft IS to confer neutrophil activation and bacterial killing, consistent with the limited role for antibodies in host defense during tularemia. Studies of mutants that we generated in the type A Ft strain Schu S4 demonstrate that the regulatory factor fevR is essential for NADPH oxidase inhibition, whereas iglI and iglJ, candidate secretion system effectors, and the acid phosphatase acpA are not. As Ft uses multiple mechanisms to block neutrophil NADPH oxidase activity, our data strongly suggest that this is a central aspect of virulence. PMID:20610796

  13. Purification of the enzyme NADPH: protochlorophyllide oxidoreductase.

    PubMed

    Beer, N S; Griffiths, W T

    1981-04-01

    A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

  14. Dual coenzyme activities of high-Km aldehyde dehydrogenase from rat liver mitochondria.

    PubMed

    Tsai, C S; Senior, D J

    1990-04-01

    Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD(+)- and NADP(+)-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate.

  15. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

    The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

  16. Kinetic Characterization of Reduced Pyridine Nucleotide Dehydrogenases (Duroquinone-Dependent) in Cucurbita Microsomes 1

    PubMed Central

    Pupillo, Paolo; Valenti, Vincenzo; De Luca, Letizia; Hertel, Rainer

    1986-01-01

    Some properties of microsomal electron transfer chains, dependent for oxidase activity on addition of NADH or NADPH, duroquinone, and oxygen (L. De Luca et al., 1984, Plant Sci Lett 36: 93-98) are described. Activity is characterized by negatively cooperative kinetics toward reduced pyridine nucleotides, with limiting Km of 10 to 50 micromolar at pH 7.0 (increasing at lower pH), as well as toward duroquinone with limiting Km of 100 to 400 micromolar regardless of pH. Molecular oxygen is reduced by the enzyme complex with S0.5 of about 30 micromolar and production of H2O and H2O2, without superoxide involvement. The ratio NAD(P)H:O2 averages 1.35 in the presence of KCN and 1.85 in its absence. The pyridine nucleotide specificity of the dehydrogenases has been investigated by kinetic competition experiments. Some enzyme heterogeneity was established for all preparations. At least two enzymes are detectable in plasma membrane-enriched fractions: a major NAD(P)H dehydrogenase having an acid pH optimum, and an NADPH dehydrogenase active around neutrality. Addition of Triton X-100 strongly enhances the activity over most of the pH scale, but depresses it increasingly at pH values higher than 8.0, to the effect that pH profile shows, under these conditions, a major peak at about pH 5.8 for both NADH and NADPH oxidase. Results with endoplasmic reticulum preparations are similar, except that they suggest the presence of still more activities at and above pH 7. The results are interpreted in terms of different complexes catalyzing electron transfer from NAD(P)H to O2 without release of intermediates. PMID:16664630

  17. Nebivolol Reduces Proteinuria and Renal NADPH Oxidase-Generated Reactive Oxygen Species in the Transgenic Ren2 Rat

    PubMed Central

    Whaley-Connell, Adam; Habibi, Javad; Johnson, Megan; Tilmon, Roger; Rehmer, Nathan; Rehmer, Jenna; Wiedmeyer, Charles; Ferrario, Carlos M.; Sowers, James R.

    2009-01-01

    Background/Aims Renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system activation are crucial in the pathogenesis of hypertension, cardiovascular and renal disease. NADPH oxidase-mediated increases in reactive oxygen species (ROS) are an important mediator for RAAS-induced cardiovascular and renal injury. Increased levels of ROS can diminish the bioactivity of nitric oxide (NO), a critical modulator of RAAS effects on the kidney. Thereby, we hypothesized that in vivo nebivolol therapy in a rodent model of activated RAAS would attenuate glomerular damage and proteinuria through its actions to reduce NADPH oxidase activity/ROS and increase bioavailable NO. Methods We utilized the transgenic Ren2 rat which displays heightened tissue RAAS, hypertension, and proteinuria. Ren2 rats (6–9 weeks of age) and age-matched Sprague-Dawley littermates were treated with nebivolol 10 mg/kg/day (osmotic mini-pump) for 21 days. Results Ren2 rats exhibited increases in systolic blood pressure, proteinuria, kidney cortical tissue total NADPH oxidase activity and subunits (Rac1, p67phox, and p47phox), ROS and 3-nitrotyrosine, as well as reductions in podocyte protein markers; each of these parameters improved with nebivolol treatment along with increases in renal endothelial NO synthase expression. Conclusions Our data suggest that nebivolol improves proteinuria through reductions in renal RAAS-mediated increases in NADPH oxidase/ROS and increases in bioavailable NO. PMID:19609077

  18. Persistent activation of microglia and NADPH drive hippocampal dysfunction in experimental multiple sclerosis

    PubMed Central

    Di Filippo, Massimiliano; de Iure, Antonio; Giampà, Carmela; Chiasserini, Davide; Tozzi, Alessandro; Orvietani, Pier Luigi; Ghiglieri, Veronica; Tantucci, Michela; Durante, Valentina; Quiroga-Varela, Ana; Mancini, Andrea; Costa, Cinzia; Sarchielli, Paola; Fusco, Francesca Romana; Calabresi, Paolo

    2016-01-01

    Cognitive impairment is common in multiple sclerosis (MS). Unfortunately, the synaptic and molecular mechanisms underlying MS-associated cognitive dysfunction are largely unknown. We explored the presence and the underlying mechanism of cognitive and synaptic hippocampal dysfunction during the remission phase of experimental MS. Experiments were performed in a chronic-relapsing experimental autoimmune encephalomyelitis (EAE) model of MS, after the resolution of motor deficits. Immunohistochemistry and patch-clamp recordings were performed in the CA1 hippocampal area. The hole-board was utilized as cognitive/behavioural test. In the remission phase of experimental MS, hippocampal microglial cells showed signs of activation, CA1 hippocampal synapses presented an impaired long-term potentiation (LTP) and an alteration of spatial tests became evident. The activation of hippocampal microglia mediated synaptic and cognitive/behavioural alterations during EAE. Specifically, LTP blockade was found to be caused by the reactive oxygen species (ROS)-producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We suggest that in the remission phase of experimental MS microglia remains activated, causing synaptic dysfunctions mediated by NADPH oxidase. Inhibition of microglial activation and NADPH oxidase may represent a promising strategy to prevent neuroplasticity impairment associated with active neuro-inflammation, with the aim to improve cognition and counteract MS disease progression. PMID:26887636

  19. Benzene toxicity: emphasis on cytosolic dihydrodiol dehydrogenases

    SciTech Connect

    Bolcsak, L.E.

    1982-01-01

    Blood dyscrasias such as leukopenia and anemia have been clearly identified as consequences of chronic benzene exposure. The metabolites, phenol, catechol, and hydroquinone produced inhibition of /sup 59/Fe uptake in mice which followed the same time course as that produced by benzene. The inhibitor of benzene oxidation, 3-amino-1,2,4-triazole, mitigated the inhibitory effects of benzene and phenol only. These data support the contention that benzene toxicity is mediated by a metabolite and suggest that the toxicity of phenol is a consequence of its metabolism to hydroquinone and that the route of metabolism to catechol may also contribute to the production of toxic metabolite(s). The properties of mouse liver cytosolic dihydrodiol dehydrogenases were examined. These enzymes catalyze the NADP/sup +/-dependent oxidation of trans-1,2-dihydro-1,2-dihydroxybenzene (BDD) to catechol, a possible toxic metabolite of benzene produced via this metabolic route. Four distinct dihydrodiol dehydrogenases (DD1, DD2, DD3, and DD4) were purified to apparent homogeneity as judged by SDS polyacrylamide gel electrophoresis and isoelectric focusing. DD1 appeared to be identical to the major ketone reductase and 17..beta..-hydroxysteroid dehydrogenase activity in the liver. DD2 exhibited aldehyde reductase activity. DD3 and DD4 oxidized 17..beta..-hydroxysteroids, but no carbonyl reductase activity was detected. These relationships between BDD dehydrogenases and carbonyl reductase and/or 17..beta..-hydroxysteroid dehydrogenase activities were supported by several lines of evidence.

  20. NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

    PubMed Central

    Röhm, Marc; Grimm, Melissa J.; D'Auria, Anthony C.; Almyroudis, Nikolaos G.

    2014-01-01

    NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47phox−/−) mice which had resolved in wild-type mice by day 5 but progressed in p47phox−/− mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47phox−/− mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance. PMID:24549323

  1. P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations.

    PubMed

    Beyer, Nina; Kulig, Justyna K; Bartsch, Anette; Hayes, Martin A; Janssen, Dick B; Fraaije, Marco W

    2017-03-01

    To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase. Phosphite-driven conversions of lauric acid at restricted NADPH concentrations confirmed multiple turnovers of the cofactor. Interestingly, both the fusion enzyme and the native P450BM3 displayed enzyme concentration dependent activity and the fused enzyme reached optimal activity at a lower enzyme concentration. This suggests that the fusion enzyme has an improved tendency to form functional oligomers.To explore the constructed phosphite-driven P450BM3 as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450BM3 driven by phosphite was found to be more efficient in terms of total turnover when compared with P450BM3 driven by NADPH. The results suggest that PTDH-P450BM3 is an attractive system for use in biocatalytic and drug metabolism studies.

  2. JWH-018 ω-OH, a shared hydroxy metabolite of the two synthetic cannabinoids JWH-018 and AM-2201, undergoes oxidation by alcohol dehydrogenase and aldehyde dehydrogenase enzymes in vitro forming the carboxylic acid metabolite.

    PubMed

    Holm, Niels Bjerre; Noble, Carolina; Linnet, Kristian

    2016-09-30

    Synthetic cannabinoids are new psychoactive substances (NPS) acting as agonists at the cannabinoid receptors. The aminoalkylindole-type synthetic cannabinoid naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) was among the first to appear on the illicit drug market and its metabolism has been extensively investigated. The N-pentyl side chain is a major site of human cytochrome P450 (CYP)-mediated oxidative metabolism, and the ω-carboxylic acid metabolite appears to be a major in vivo human urinary metabolite. This metabolite is, however, not formed to any significant extent in human liver microsomal (HLM) incubations raising the possibility that the discrepancy is due to involvement of cytosolic enzymes. Here we demonstrate in incubations with human liver cytosol (HLC), that JWH-018 ω-OH, but not the JWH-018 parent compound, is a substrate for nicotinamide adenine dinucleotide (NAD(+))-dependent alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. The sole end-product identified in HLC was the JWH-018 ω-COOH metabolite, while trapping tests with methoxyamine proved the presence of the aldehyde intermediate. ADH/ALDH and UDP-glucuronosyl-transferases (UGT) enzymes may therefore both act on the JWH-018 ω-OH substrate. Finally, we note that for [1-(5-fluoropentyl)indol-3-yl]-naphthalen-1-yl-methanone (AM-2201), the ω-fluorinated analog of JWH-018, a high amount of JWH-018 ω-OH was formed in HLM incubated without NADPH, suggesting that the oxidative defluorination is efficiently catalyzed by non-CYP enzyme(s). The pathway presented here may therefore be especially important for N-(5-fluoropentyl) substituted synthetic cannabinoids, because the oxidative defluorination can occur even if the CYP-mediated metabolism preferentially takes place on other parts of the molecule than the N-alkyl side chain. Controlled clinical studies in humans are ultimately required to demonstrate the in vivo importance of the oxidation pathway presented here.

  3. [Agrobacterium-mediated sunflower transformation (Helianthus annuus L.) in vitro and in Planta using strain of LBA4404 harboring binary vector pBi2E with dsRNA-suppressor proline dehydrogenase gene].

    PubMed

    Tishchenko, E N; Komisarenko, A G; Mikhal'skaia, S I; Sergeeva, L E; Adamenko, N I; Morgun, B V; Kochetov, A V

    2014-01-01

    To estimate the efficiency of proline dehydrogenase gene suppression towards increasing of sunflower (Helianthus annuus L.) tolerance level to water deficit and salinity, we employed strain LBA4404 harboring pBi2E with double-stranded RNA-suppressor, which were prepared on basis arabidopsis ProDH1 gene. The techniques of Agrobacterium-mediated transformation in vitro and in planta during fertilization sunflower have been proposed. There was shown the genotype-depended integration of T-DNA in sunflower genome. PCR-analysis showed that ProDH1 presents in genome of inbred lines transformed in planta, as well as in T1- and T2-generations. In trans-genic regenerants the essential accumulation of free L-proline during early stages of in vitro cultivation under normal conditions was shown. There was established the essential accumulation of free proline in transgenic regenerants during cultivation under lethal stress pressure (0.4 M mannitol and 2.0% sea water salts) and its decline upon the recovery period. These data are declared about effectiveness of suppression of sunflower ProDH and gene participation in processes connected with osmotolerance.

  4. Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes.

    PubMed

    Kasi, Viswanath; Bodiga, Sreedhar; Kommuguri, Upendra Nadh; Sankuru, Suneetha; Bodiga, Vijaya Lakshmi

    2011-07-01

    Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47(phox) phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress.

  5. Contrasting Influence of NADPH and a NADPH-Regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes

    EPA Science Inventory

    Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast ...

  6. Purification, Characterization, and Submitochondrial Localization of the 32-Kilodalton NADH Dehydrogenase from Maize.

    PubMed Central

    Knudten, A. F.; Thelen, J. J.; Luethy, M. H.; Elthon, T. E.

    1994-01-01

    Plant mitochondria have the unique ability to directly oxidize exogenous NAD(P)H. We recently separated two NAD(P)H dehydrogenase activities from maize (Zea mays L.) mitochondria using anion-exchange (Mono Q) chromatography. The first peak of activity oxidized only NADH, whereas the second oxidized both NADH and NADPH. In this paper we describe the purification of the first peak of activity to a 32-kD protein. Polyclonal antibodies to the 32-kD protein were used to show that it was present in mitochondria from several plant species. Two-dimensional gel analysis of the 32-kD NADH dehydrogenase indicated that it consisted of two major and one minor isoelectric forms. Immunoblot analysis of submitochondrial fractions indicated that the 32-kD protein was enriched in the soluble protein fraction after mitochondrial disruption and fractionation; however, some association with the membrane fraction was observed. The membrane-impermeable protein cross-linking agent 3,3[prime] -dithiobis-(sulfosuccinimidylpropionate) was used to further investigate the submitochondrial location of the 32-kD NADH dehydrogenase. The 32-kD protein was localized to the outer surface of the inner mitochondrial membrane or to the intermembrane space. The pH optimum for the enzyme was 7.0. The activity was found to be severely inhibited by p-chloromercuribenzoic acid, mersalyl, and dicumarol, and stimulated somewhat by flavin mononucleotide. PMID:12232393

  7. The complex roles of NADPH oxidases in fungal infection

    PubMed Central

    Hogan, Deborah; Wheeler, Robert T.

    2014-01-01

    Summary NADPH oxidases play key roles in immunity and inflammation that go beyond the production of microbicidal reactive oxygen species (ROS). The past decade has brought a new appreciation for the diversity of roles played by ROS in signaling associated with inflammation and immunity. NADPH oxidase activity affects disease outcome during infections by human pathogenic fungi, an important group of emerging and opportunistic pathogens that includes Candida, Aspergillus and Cryptococcus species. Here we review how alternative roles of NADPH oxidase activity impact fungal infection and how ROS signaling affects fungal physiology. Particular attention is paid to roles for NADPH oxidase in immune migration, immunoregulation in pulmonary infection, neutrophil extracellular trap formation, autophagy and inflammasome activity. These recent advances highlight the power and versatility of spatiotemporally controlled redox regulation in the context of infection, and point to a need to understand the molecular consequences of NADPH oxidase activity in the cell. PMID:24905433

  8. Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity

    PubMed Central

    Walvekar, Adhish S.; Choudhury, Rajarshi; Punekar, Narayan S.

    2014-01-01

    NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol) resulted in preferential attenuation of AnGDH reductive amination (forward) activity but with a negligible effect on oxidative deamination (reverse) activity, when monitored in the described standard assay. Such a disulfide modified AnGDH displaying less than 1.0% forward reaction rate could be isolated after 2-HED treatment. This unique forward inhibited GDH form (FIGDH), resembling a hypothetical ‘one-way’ active enzyme, was characterized. Kinetics of 2-HED mediated inhibition and protein thiol titrations suggested that a single thiol group is modified in FIGDH. Two site-directed cysteine mutants, C141S and C415S, were constructed to identify the relevant thiol in FIGDH. The forward activity of C141S alone was insensitive to 2-HED, implicating Cys141 in FIGDH formation. It was observed that FIGDH displayed maximal reaction rate only after a pre-incubation with 2-oxoglutarate and NADPH. In addition, compared to the native enzyme, FIGDH showed a four fold increase in K0.5 for 2-oxoglutarate and a two fold increase in the Michaelis constants for ammonium and NADPH. With no change in the GDH reaction equilibrium constant, the FIGDH catalyzed rate of approach to equilibrium from reductive amination side was sluggish. Altered kinetic properties of FIGDH at least partly account for the observed apparent loss of forward activity when monitored under defined assay conditions. In sum, although Cys141 is catalytically not essential, its covalent modification provides a striking example of converting the biosynthetic AnGDH into a catabolic enzyme. PMID:24987966

  9. Computational design of short-chain dehydrogenase Gox2181 for altered coenzyme specificity.

    PubMed

    Cui, Dongbing; Zhang, Lujia; Zhang, Lujiang; Yao, Zhiqiang; Liu, Xu; Lin, Jinping; Yuan, Y Adam; Wei, Dongzhi

    2013-09-20

    Short-chain dehydrogenase Gox2181 from Gluconobacter oxydans catalyzes the reduction of 2,3-pentanedione by using NADH as the physiological electron donor. To realize its synthetic biological application for coenzyme recycling use, computational design and site-directed mutagenesis have been used to engineer Gox2181 to utilize not only NADH but also NADPH as the electron donor. Single and double mutations at residues Q20 and D43 were made in a recombinant expression system that corresponded to Gox2181-D43Q and Gox2181-Q20R&D43Q, respectively. The design of mutant Q20R not only resolved the hydrogen bond interaction and electrostatic interaction between R and 2'-phosphate of NADPH, but also could enhance the binding with 2'-phophated of NADPH by combining with D43Q. Molecular dynamics simulation has been carried out to testify the hydrogen bond interactions between mutation sites and 2'-phosphate of NADPH. Steady-state turnover measurement results indicated that Gox2181-D43Q could use both NADH and NADPH as its coenzyme, and so could Gox2181-Q20R&D43Q. Meanwhile, compared to the wild-type enzyme, Gox2181-D43Q exhibited dramatically reduced enzymatic activity while Gox2181-Q20R&D43Q successfully retained the majority of enzymatic activity.

  10. [NADPH oxidases, Nox: new isoenzymes family].

    PubMed

    Chuong Nguyen, Minh Vu; Lardy, Bernard; Paclet, Marie-Hélène; Rousset, Francis; Berthier, Sylvie; Baillet, Athan; Grange, Laurent; Gaudin, Philippe; Morel, Françoise

    2015-01-01

    NADPH oxidases, Nox, are a family of isoenzymes, composed of seven members, whose sole function is to produce reactive oxygen species (ROS). Although Nox catalyze the same enzymatic reaction, they acquired from a common ancestor during evolution, specificities related to their tissue expression, subcellular localization, activation mechanisms and regulation. Their functions could vary depending on the pathophysiological state of the tissues. Indeed, ROS are not only bactericidal weapons in phagocytes but also essential cellular signaling molecules and their overproduction is involved in chronic diseases and diseases of aging. The understanding of the mechanisms involved in the function of Nox and the emergence of Nox inhibitors, require a thorough knowledge of their nature and structure. The objectives of this review are to highlight, in a structure/function approach, the main similar and differentiated properties shared by the human Nox isoenzymes.

  11. NADPH Oxidases in Lung Health and Disease

    PubMed Central

    Bernard, Karen; Hecker, Louise; Luckhardt, Tracy R.; Cheng, Guangjie

    2014-01-01

    Abstract Significance: The evolution of the lungs and circulatory systems in vertebrates ensured the availability of molecular oxygen (O2; dioxygen) for aerobic cellular metabolism of internal organs in large animals. O2 serves as the physiologic terminal acceptor of mitochondrial electron transfer and of the NADPH oxidase (Nox) family of oxidoreductases to generate primarily water and reactive oxygen species (ROS), respectively. Recent advances: The purposeful generation of ROS by Nox family enzymes suggests important roles in normal physiology and adaptation, most notably in host defense against invading pathogens and in cellular signaling. Critical issues: However, there is emerging evidence that, in the context of chronic stress and/or aging, Nox enzymes contribute to the pathogenesis of a number of lung diseases. Future Directions: Here, we review evolving functions of Nox enzymes in normal lung physiology and emerging pathophysiologic roles in lung disease. Antioxid. Redox Signal. 20, 2838–2853. PMID:24093231

  12. NADPH oxidase deficiency underlies dysfunction of aged CD8+ Tregs

    PubMed Central

    Wen, Zhenke; Shimojima, Yasuhiro; Shirai, Tsuyoshi; Li, Yinyin; Ju, Jihang; Yang, Zhen; Tian, Lu; Goronzy, Jörg J.

    2016-01-01

    Immune aging results in progressive loss of both protective immunity and T cell–mediated suppression, thereby conferring susceptibility to a combination of immunodeficiency and chronic inflammatory disease. Here, we determined that older individuals fail to generate immunosuppressive CD8+CCR7+ Tregs, a defect that is even more pronounced in the age-related vasculitic syndrome giant cell arteritis. In young, healthy individuals, CD8+CCR7+ Tregs are localized in T cell zones of secondary lymphoid organs, suppress activation and expansion of CD4 T cells by inhibiting the phosphorylation of membrane-proximal signaling molecules, and effectively inhibit proliferative expansion of CD4 T cells in vitro and in vivo. We identified deficiency of NADPH oxidase 2 (NOX2) as the molecular underpinning of CD8 Treg failure in the older individuals and in patients with giant cell arteritis. CD8 Tregs suppress by releasing exosomes that carry preassembled NOX2 membrane clusters and are taken up by CD4 T cells. Overexpression of NOX2 in aged CD8 Tregs promptly restored suppressive function. Together, our data support NOX2 as a critical component of the suppressive machinery of CD8 Tregs and suggest that repairing NOX2 deficiency in these cells may protect older individuals from tissue-destructive inflammatory disease, such as large-vessel vasculitis. PMID:27088800

  13. Long-chain 3-hydroxy fatty acids accumulating in long-chain 3-hydroxyacyl-CoA dehydrogenase and mitochondrial trifunctional protein deficiencies uncouple oxidative phosphorylation in heart mitochondria.

    PubMed

    Tonin, Anelise M; Amaral, Alexandre U; Busanello, Estela N B; Grings, Mateus; Castilho, Roger F; Wajner, Moacir

    2013-02-01

    Cardiomyopathy is a common clinical feature of some inherited disorders of mitochondrial fatty acid β-oxidation including mitochondrial trifunctional protein (MTP) and isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies. Since individuals affected by these disorders present tissue accumulation of various fatty acids, including long-chain 3-hydroxy fatty acids, in the present study we investigated the effect of 3-hydroxydecanoic (3 HDCA), 3-hydroxydodecanoic (3 HDDA), 3-hydroxytetradecanoic (3 HTA) and 3-hydroxypalmitic (3 HPA) acids on mitochondrial oxidative metabolism, estimated by oximetry, NAD(P)H content, hydrogen peroxide production, membrane potential (ΔΨ) and swelling in rat heart mitochondrial preparations. We observed that 3 HTA and 3 HPA increased resting respiration and diminished the respiratory control and ADP/O ratios using glutamate/malate or succinate as substrates. Furthermore, 3 HDDA, 3 HTA and 3 HPA decreased ΔΨ, the matrix NAD(P)H pool and hydrogen peroxide production. These data indicate that these fatty acids behave as uncouplers of oxidative phosphorylation. We also verified that 3 HTA-induced uncoupling-effect was not mediated by the adenine nucleotide translocator and that this fatty acid induced the mitochondrial permeability transition pore opening in calcium-loaded organelles since cyclosporin A prevented the reduction of mitochondrial ΔΨ and swelling provoked by 3 HTA. The present data indicate that major 3-hydroxylated fatty acids accumulating in MTP and LCHAD deficiencies behave as strong uncouplers of oxidative phosphorylation potentially impairing heart energy homeostasis.

  14. Role of the Rho GTPase Rac in the activation of the phagocyte NADPH oxidase

    PubMed Central

    Pick, Edgar

    2014-01-01

    The superoxide-generating NADPH oxidase of phagocytes consists of the membrane-associated cytochrome b558 (a heterodimer of Nox2 and p22phox) and 4 cytosolic components: p47phox, p67phox, p40phox, and the small GTPase, Rac, in complex with RhoGDI. Superoxide is produced by the NADPH-driven reduction of molecular oxygen, via a redox gradient located in Nox2. Electron flow in Nox2 is initiated by interaction with cytosolic components, which translocate to the membrane, p67phox playing the central role. The participation of Rac is expressed in the following sequence: (1) Translocation of the RacGDP-RhoGDI complex to the membrane; (2) Dissociation of RacGDP from RhoGDI; (3) GDP to GTP exchange on Rac, mediated by a guanine nucleotide exchange factor; (4) Binding of RacGTP to p67phox; (5) Induction of a conformational change in p67phox, promoting interaction with Nox2. The particular involvement of Rac in NADPH oxidase assembly serves as a paradigm for signaling by Rho GTPases, in general. PMID:24598074

  15. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... Elsevier Saunders; 2012:chap 42. Read More Enzyme Glucose-6-phosphate dehydrogenase deficiency Hemoglobin Review Date 2/11/2016 Updated by: ... A.M. Editorial team. Related MedlinePlus Health Topics G6PD Deficiency Browse the Encyclopedia A.D.A.M., Inc. ...

  16. Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging.

    PubMed

    Bremer, Daniel; Leben, Ruth; Mothes, Ronja; Radbruch, Helena; Niesner, Raluca

    2017-04-03

    Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc.

  17. PLATELET-ASSOCIATED NAD(P)H OXIDASE CONTRIBUTES TO THE THROMBOGENIC PHENOTYPE INDUCED BY HYPERCHOLESTEROLEMIA

    PubMed Central

    Stokes, Karen Y.; Russell, Janice M.; Jennings, Merilyn H.; Alexander, J. Steven; Granger., D. Neil

    2007-01-01

    Elevated cholesterol levels promote pro-inflammatory and prothrombogenic responses in venules and impaired endothelium-dependent arteriolar dilation. Although NAD(P)H oxidase-derived superoxide has been implicated in the altered vascular responses to hypercholesterolemia, it remains unclear whether this oxidative pathway mediates the associated arteriolar dysfunction and platelet adhesion in venules. Platelet and leukocyte adhesion in cremasteric postcapillary venules, and arteriolar dilation responses to acetylcholine were monitored in wild-type (WT), Cu,Zn-superoxide dismutase transgenic (SOD-TgN) and NAD(P)H oxidase-knockout (gp91phox-/-) mice placed on normal (ND) or high cholesterol (HC) diet for 2 wk. HC elicited increased platelet and leukocyte adhesion in WT mice, versus ND. Cytosolic subunits of NAD(P)H oxidase (p47phox and p67phox) were expressed in platelets. This was not altered by hypercholesterolemia, however platelets and leukocytes from HC mice exhibited elevated generation of reactive oxygen species when compared to ND mice. Hypercholesterolemia-induced leukocyte recruitment was attenuated in SOD-TgN-HC and gp91phox-/--HC mice. Recruitment of platelets derived from WT-HC mice in venules of SOD-TgN-HC or gp91phox-/--HC recipients was comparable to ND levels. Adhesion of SOD-TgN-HC platelets paralleled the leukocyte response and was attenuated in SOD-TgN-HC recipients, but not in WT-HC recipients. However, gp91phox-/--HC platelets exhibited low levels of adhesion comparable to WT-ND in both hypercholesterolemic gp91phox-/- and WT recipients. Arteriolar dysfunction was evident in WT-HC mice, compared to WT-ND. Overexpression of SOD or, to a lesser extent, gp91phox deficiency, restored arteriolar vasorelaxation responses towards WT-ND levels. These findings reveal a novel role for platelet-associated NAD(P)H oxidase in producing the thrombogenic phenotype in hypercholesterolemia and demonstrate that NAD(P)H oxidase-derived superoxide mediates the HC

  18. Plants Utilize a Highly Conserved System for Repair of NADH and NADPH Hydrates1[W][OPEN

    PubMed Central

    Niehaus, Tom D.; Richardson, Lynn G.L.; Gidda, Satinder K.; ElBadawi-Sidhu, Mona; Meissen, John K.; Mullen, Robert T.; Fiehn, Oliver; Hanson, Andrew D.

    2014-01-01

    NADH and NADPH undergo spontaneous and enzymatic reactions that produce R and S forms of NAD(P)H hydrates [NAD(P)HX], which are not electron donors and inhibit various dehydrogenases. In bacteria, yeast (Saccharomyces cerevisiae), and mammals, these hydrates are repaired by the tandem action of an ADP- or ATP-dependent dehydratase that converts (S)-NAD(P)HX to NAD(P)H and an epimerase that facilitates interconversion of the R and S forms. Plants have homologs of both enzymes, the epimerase homolog being fused to the vitamin B6 salvage enzyme pyridoxine 5′-phosphate oxidase. Recombinant maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) NAD(P)HX dehydratases (GRMZM5G840928, At5g19150) were able to reconvert (S)-NAD(P)HX to NAD(P)H in an ATP-dependent manner. Recombinant maize and Arabidopsis epimerases (GRMZM2G061988, At5g49970) rapidly interconverted (R)- and (S)-NAD(P)HX, as did a truncated form of the Arabidopsis epimerase lacking the pyridoxine 5′-phosphate oxidase domain. All plant NAD(P)HX dehydratase and epimerase sequences examined had predicted organellar targeting peptides with a potential second start codon whose use would eliminate the targeting peptide. In vitro transcription/translation assays confirmed that both start sites were used. Dual import assays with purified pea (Pisum sativum) chloroplasts and mitochondria, and subcellular localization of GFP fusion constructs in tobacco (Nicotiana tabacum) suspension cells, indicated mitochondrial, plastidial, and cytosolic localization of the Arabidopsis epimerase and dehydratase. Ablation of the Arabidopsis dehydratase gene raised seedling levels of all NADHX forms by 20- to 40-fold, and levels of one NADPHX form by 10- to 30-fold. We conclude that plants have a canonical two-enzyme NAD(P)HX repair system that is directed to three subcellular compartments via the use of alternative translation start sites. PMID:24599492

  19. Selected dehydrogenases in Yarrowia lipolytica JMY 861: their role in the synthesis of flavor compounds.

    PubMed

    Aziz, Marya; St-Louis, Richard; Husson, Florence; Kermasha, Selim

    2016-09-01

    The presence of selected dehydrogenases, including alcohol dehydrogenase (ADH-YL) and aldehyde dehydrogenase (ALDH-YL), in Yarrowia lipolytica JMY 861, and their potential role in flavor synthesis were investigated. The experimental findings showed that using reduced form of nicotinamide adenine dinucleotide (NADH) as cofactor, the ADH-YL activity in vitro was 6-fold higher than that with reduced form of nicotinamide adenine dinucleotide phosphate (NADPH); however, under the experimental conditions used in this study, an ALDH-YL activity was not detected. The in situ hexanal reduction reaction was found to be instantaneous; however, when the yeast cells suspension was diluted 150 times, the initial relative hexanal concentration was increased by 84.1%. The chromatographic analyses indicated the conversion, in situ, of linoleic acid hydroperoxides (HPODs) into volatile C6-compounds after 60 min of HPODs addition to the yeast cells suspension.

  20. Thioredoxin f1 and NADPH-Dependent Thioredoxin Reductase C Have Overlapping Functions in Regulating Photosynthetic Metabolism and Plant Growth in Response to Varying Light Conditions1[OPEN

    PubMed Central

    Thormählen, Ina; Meitzel, Tobias; Groysman, Julia; Öchsner, Alexandra Bianca; von Roepenack-Lahaye, Edda; Naranjo, Belén; Cejudo, Francisco J.; Geigenberger, Peter

    2015-01-01

    Two different thiol redox systems exist in plant chloroplasts, the ferredoxin-thioredoxin (Trx) system, which depends on ferredoxin reduced by the photosynthetic electron transport chain and, thus, on light, and the NADPH-dependent Trx reductase C (NTRC) system, which relies on NADPH and thus may be linked to sugar metabolism in the dark. Previous studies suggested, therefore, that the two different systems may have different functions in plants. We now report that there is a previously unrecognized functional redundancy of Trx f1 and NTRC in regulating photosynthetic metabolism and growth. In Arabidopsis (Arabidopsis thaliana) mutants, combined, but not single, deficiencies of Trx f1 and NTRC led to severe growth inhibition and perturbed light acclimation, accompanied by strong impairments of Calvin-Benson cycle activity and starch accumulation. Light activation of key enzymes of these pathways, fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase, was almost completely abolished. The subsequent increase in NADPH-NADP+ and ATP-ADP ratios led to increased nitrogen assimilation, NADP-malate dehydrogenase activation, and light vulnerability of photosystem I core proteins. In an additional approach, reporter studies show that Trx f1 and NTRC proteins are both colocalized in the same chloroplast substructure. Results provide genetic evidence that light- and NADPH-dependent thiol redox systems interact at the level of Trx f1 and NTRC to coordinately participate in the regulation of the Calvin-Benson cycle, starch metabolism, and growth in response to varying light conditions. PMID:26338951

  1. Thioredoxin f1 and NADPH-Dependent Thioredoxin Reductase C Have Overlapping Functions in Regulating Photosynthetic Metabolism and Plant Growth in Response to Varying Light Conditions.

    PubMed

    Thormählen, Ina; Meitzel, Tobias; Groysman, Julia; Öchsner, Alexandra Bianca; von Roepenack-Lahaye, Edda; Naranjo, Belén; Cejudo, Francisco J; Geigenberger, Peter

    2015-11-01

    Two different thiol redox systems exist in plant chloroplasts, the ferredoxin-thioredoxin (Trx) system, which depends on ferredoxin reduced by the photosynthetic electron transport chain and, thus, on light, and the NADPH-dependent Trx reductase C (NTRC) system, which relies on NADPH and thus may be linked to sugar metabolism in the dark. Previous studies suggested, therefore, that the two different systems may have different functions in plants. We now report that there is a previously unrecognized functional redundancy of Trx f1 and NTRC in regulating photosynthetic metabolism and growth. In Arabidopsis (Arabidopsis thaliana) mutants, combined, but not single, deficiencies of Trx f1 and NTRC led to severe growth inhibition and perturbed light acclimation, accompanied by strong impairments of Calvin-Benson cycle activity and starch accumulation. Light activation of key enzymes of these pathways, fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase, was almost completely abolished. The subsequent increase in NADPH-NADP(+) and ATP-ADP ratios led to increased nitrogen assimilation, NADP-malate dehydrogenase activation, and light vulnerability of photosystem I core proteins. In an additional approach, reporter studies show that Trx f1 and NTRC proteins are both colocalized in the same chloroplast substructure. Results provide genetic evidence that light- and NADPH-dependent thiol redox systems interact at the level of Trx f1 and NTRC to coordinately participate in the regulation of the Calvin-Benson cycle, starch metabolism, and growth in response to varying light conditions.

  2. Targeting isocitrate dehydrogenase (IDH) in cancer.

    PubMed

    Fujii, Takeo; Khawaja, Muhammad Rizwan; DiNardo, Courtney D; Atkins, Johnique T; Janku, Filip

    2016-05-01

    Isocitrate dehydrogenase (IDH) is an essential enzyme for cellular respiration in the tricarboxylic acid (TCA) cycle. Recurrent mutations in IDH1 or IDH2 are prevalent in several cancers including glioma, acute myeloid leukemia (AML), cholangiocarcinoma and chondrosarcoma. The mutated IDH1 and IDH2 proteins have a gain-of-function, neomorphic activity, catalyzing the reduction of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG) by NADPH. Cancer-associated IDH mutations block normal cellular differentiation and promote tumorigenesis via the abnormal production of the oncometabolite 2-HG. High levels of 2-HG have been shown to inhibit α-KG dependent dioxygenases, including histone and deoxyribonucleic acid (DNA) demethylases, which play a key role in regulating the epigenetic state of cells. Current targeted inhibitors of IDH1 (AG120, IDH305), IDH2 (AG221), and pan-IDH1/2 (AG881) selectively inhibit mutant IDH protein and induce cell differentiation in in vitro and in vivo models. Preliminary results from phase I clinical trials with IDH inhibitors in patients with advanced hematologic malignancies have demonstrated an objective response rate ranging from 31% to 40% with durable responses (>1 year) observed. Furthermore, the IDH inhibitors have demonstrated early signals of activity in solid tumors with IDH mutations, including cholangiocarcinomas and low grade gliomas.

  3. Purification and characterization of dimeric dihydrodiol dehydrogenase from dog liver.

    PubMed

    Sato, K; Nakanishi, M; Deyashiki, Y; Hara, A; Matsuura, K; Ohya, I

    1994-09-01

    High NADP(+)-linked dihydrodiol dehydrogenase activity was detected in dog liver cytosol, from which a dimeric enzyme composed of M(r) 39,000 subunits was purified to homogeneity. The enzyme oxidized trans-cyclohexanediol, and trans-dihydrodiols of benzene and naphthalene, the [1R,2R]-isomers of which were selectively oxidized. In the reverse reaction in the presence of NADPH as a coenzyme, the enzyme reduced alpha-dicarbonyl compounds, such as methylglyoxal, 3-deoxyglucosone, and diacetyl, and some compounds with a carbonyl group, such as glyceraldehyde, lactaldehyde, and acetoin. 4-Hydroxyphenylketones and ascorbates inhibited the enzyme. The results of steady-state kinetic analyses indicated that the reaction proceeds through an ordered bi bi mechanism with the coenzyme binding to the free enzyme, and suggested that the inhibitors bind to the enzyme-NADP+ binary complex. The dimeric enzyme was detected in liver and kidney of dog, and was immunochemically similar to the dimeric enzymes from monkey kidney, rabbit lens, and pig liver. The sequences (total 127 amino acid residues) of eight peptides derived on enzymatic digestion of the dog liver enzyme did not show significant similarity with the primary structures of members of the aldo-keto reductase and short chain dehydrogenase superfamilies, which include monomeric dihydrodiol dehydrogenases and carbonyl reductase, respectively.

  4. Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase.

    PubMed

    Rosell, Albert; Valencia, Eva; Ochoa, Wendy F; Fita, Ignacio; Parés, Xavier; Farrés, Jaume

    2003-10-17

    Gastric tissues from amphibian Rana perezi express the only vertebrate alcohol dehydrogenase (ADH8) that is specific for NADP(H) instead of NAD(H). In the crystallographic ADH8-NADP+ complex, a binding pocket for the extra phosphate group of coenzyme is formed by ADH8-specific residues Gly223-Thr224-His225, and the highly conserved Leu200 and Lys228. To investigate the minimal structural determinants for coenzyme specificity, several ADH8 mutants involving residues 223 to 225 were engineered and kinetically characterized. Computer-assisted modeling of the docked coenzymes was also performed with the mutant enzymes and compared with the wild-type crystallographic binary complex. The G223D mutant, having a negative charge in the phosphate-binding site, still preferred NADP(H) over NAD(H), as did the T224I and H225N mutants. Catalytic efficiency with NADP(H) dropped dramatically in the double mutants, G223D/T224I and T224I/H225N, and in the triple mutant, G223D/T224I/H225N (kcat/KmNADPH = 760 mm-1 min-1), as compared with the wild-type enzyme (kcat/KmNADPH = 133330 mm-1 min-1). This was associated with a lower binding affinity for NADP+ and a change in the rate-limiting step. Conversely, in the triple mutant, catalytic efficiency with NAD(H) increased, reaching values (kcat/KmNADH = 155000 mm-1 min-1) similar to those of the wild-type enzyme with NADP(H). The complete reversal of ADH8 coenzyme specificity was therefore attained by the substitution of only three consecutive residues in the phosphate-binding site, an unprecedented achievement within the ADH family.

  5. Human dehydrogenase/reductase (SDR family) member 11 is a novel type of 17β-hydroxysteroid dehydrogenase.

    PubMed

    Endo, Satoshi; Miyagi, Namiki; Matsunaga, Toshiyuki; Hara, Akira; Ikari, Akira

    2016-03-25

    We report characterization of a member of the short-chain dehydrogenase/reductase superfamily encoded in a human gene, DHRS11. The recombinant protein (DHRS11) efficiently catalyzed the conversion of the 17-keto group of estrone, 4- and 5-androstenes and 5α-androstanes into their 17β-hydroxyl metabolites with NADPH as a coenzyme. In contrast, it exhibited reductive 3β-hydroxysteroid dehydrogenase activity toward 5β-androstanes, 5β-pregnanes, 4-pregnenes and bile acids. Additionally, DHRS11 reduced α-dicarbonyls (such as diacetyl and methylglyoxal) and alicyclic ketones (such as 1-indanone and loxoprofen). The enzyme activity was inhibited in a mixed-type manner by flavonoids, and competitively by carbenoxolone, glycyrrhetinic acid, zearalenone, curcumin and flufenamic acid. The expression of DHRS11 mRNA was observed widely in human tissues, most abundantly in testis, small intestine, colon, kidney and cancer cell lines. Thus, DHRS11 represents a novel type of 17β-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution.

  6. IDH1 R132H Mutation Enhances Cell Migration by Activating AKT-mTOR Signaling Pathway, but Sensitizes Cells to 5-FU Treatment as NADPH and GSH Are Reduced

    PubMed Central

    Qiu, Jiangdong; Huang, Keting; Wu, Mindan; Xia, Chunlin

    2017-01-01

    Aim of study Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation. Materials and methods Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH. Results We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment. Conclusion Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment. PMID:28052098

  7. Regulation of NADPH Oxidase 5 by Protein Kinase C Isoforms

    PubMed Central

    Chen, Feng; Yu, Yanfang; Haigh, Steven; Johnson, John; Lucas, Rudolf; Stepp, David W.; Fulton, David J. R.

    2014-01-01

    NADPH oxidase5 (Nox5) is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular complications of diabetes

  8. Suppression of NADPH Oxidase Activity May Slow the Expansion of Osteolytic Bone Metastases

    PubMed Central

    McCarty, Mark F.; DiNicolantonio, James

    2016-01-01

    Lysophosphatidic acid (LPA), generated in the microenvironment of cancer cells, can drive the proliferation, invasion, and migration of cancer cells by activating G protein-coupled LPA receptors. Moreover, in cancer cells that have metastasized to bone, LPA signaling can promote osteolysis by inducing cancer cell production of cytokines, such as IL-6 and IL-8, which can stimulate osteoblasts to secrete RANKL, a key promoter of osteoclastogenesis. Indeed, in cancers prone to metastasize to bone, LPA appears to be a major driver of the expansion of osteolytic bone metastases. Activation of NADPH oxidase has been shown to play a mediating role in the signaling pathways by which LPA, as well as RANKL, promote osteolysis. In addition, there is reason to suspect that Nox4 activation is a mediator of the feed-forward mechanism whereby release of TGF-beta from bone matrix by osteolysis promotes expression of PTHrP in cancer cells, and thereby induces further osteolysis. Hence, measures which can down-regulate NADPH oxidase activity may have potential for slowing the expansion of osteolytic bone metastases in cancer patients. Phycocyanin and high-dose statins may have utility in this regard, and could be contemplated as complements to bisphosphonates or denosumab for the prevention and control of osteolytic lesions. Ingestion of omega-3-rich flaxseed or fish oil may also have potential for controlling osteolysis in cancer patients. PMID:27571113

  9. Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein.

    PubMed

    Cacas, Jean-Luc; Gerbeau-Pissot, Patricia; Fromentin, Jérôme; Cantrel, Catherine; Thomas, Dominique; Jeannette, Emmanuelle; Kalachova, Tetiana; Mongrand, Sébastien; Simon-Plas, Françoise; Ruelland, Eric

    2017-04-01

    Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [(33) P]-orthophosphate labelling of tobacco Bright Yellow-2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of DGK5-like was up-regulated in response to cryptogein. Besides DGK5-like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid-mediated events in plant immunity.

  10. Engineering an NADPH/NADP(+) Redox Biosensor in Yeast.

    PubMed

    Zhang, Jie; Sonnenschein, Nikolaus; Pihl, Thomas P B; Pedersen, Kasper R; Jensen, Michael K; Keasling, Jay D

    2016-12-16

    Genetically encoded biosensors have emerged as powerful tools for timely and precise in vivo evaluation of cellular metabolism. In particular, biosensors that can couple intercellular cues with downstream signaling responses are currently attracting major attention within health science and biotechnology. Still, there is a need for bioprospecting and engineering of more biosensors to enable real-time monitoring of specific cellular states and controlling downstream actuation. In this study, we report the engineering and application of a transcription factor-based NADPH/NADP(+) redox biosensor in the budding yeast Saccharomyces cerevisiae. Using the biosensor, we are able to monitor the cause of oxidative stress by chemical induction, and changes in NADPH/NADP(+) ratios caused by genetic manipulations. Because of the regulatory potential of the biosensor, we also show that the biosensor can actuate upon NADPH deficiency by activation of NADPH regeneration. Finally, we couple the biosensor with an expression of dosage-sensitive genes (DSGs) and thereby create a novel tunable sensor-selector useful for synthetic selection of cells with higher NADPH/NADP(+) ratios from mixed cell populations. We show that the combination of exploitation and rational engineering of native signaling components is applicable for diagnosis, regulation, and selection of cellular redox states.

  11. NADPH OXIDASE: STRUCTURE AND ACTIVATION MECHANISMS (REVIEW). NOTE I.

    PubMed

    Filip-Ciubotaru, Florina; Manciuc, Carmen; Stoleriu, Gabriela; Foia, Liliana

    2016-01-01

    NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase), with its generically termed NOX isoforms, is the major source of ROS (reactive oxigen species) in biological systems. ROS are small oxygen-derived molecules with an important role in various biological processes (physiological or pathological). If under physiological conditions some processes are beneficial and necessary for life, under pathophysiological conditions they are noxious, harmful. NADPH oxidases are present in phagocytes and in a wide variety of nonphagocytic cells. The enzyme generates superoxide by transferring electrons from NADPH inside the cell across the membrane and coupling them to molecular oxygen to produce superoxide anion, a reactive free-radical. Structurally, NADPH oxidase is a multicomponent enzyme which includes two integral membrane proteins, glycoprotein gp9 1 Phox and adaptor protein p22(phox), which together form the heterodimeric flavocytochrome b558 that constitutes the core of the enzyme. During the resting state, the multidomain regulatory subunits p40P(phox), p47(phox), p67(Phox) are located in the cytosol organized as a complex. The activation of phagocytic NADPH oxidase occurs through a complex series of protein interactions.

  12. NADPH production, a growth marker, is stimulated by maslinic acid in gilthead sea bream by increased NADP-IDH and ME expression.

    PubMed

    Rufino-Palomares, Eva E; Reyes-Zurita, Fernando J; García-Salguero, Leticia; Peragón, Juan; de la Higuera, Manuel; Lupiáñez, José A

    2016-09-01

    NADPH plays a central role in reductive biosynthesis of membrane lipids, maintenance of cell integrity, protein synthesis and redox balance maintenance. Hence, NADPH is involved in the growth and proliferation processes. In addition, it has been shown that changes in nutritional conditions produced changes in NADPH levels and growth rate. Maslinic acid (MA), a pentacyclic triterpene of natural origin, is able to stimulate NADPH production, through regulation of the two oxidative phase dehydrogenases of the pentose phosphate pathway. Our main objective was to study the effects of MA on the kinetic behaviour and on the molecular expression of two NADPH-generating systems, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and malic enzyme (ME), in the liver and white muscle of gilthead sea bream (Sparus aurata). Four groups of 12g of a mean body mass were fed for 210days in a fish farm, with diets containing 0 (control), and 0.1g of MA per kg of diet. Two groups were fed ad libitum (C-AL and MA-AL) and another's two, with restricted diet of 1% of fish weight (C-R and MA-R). Results showed that MA significantly increased the main kinetic parameter of the NADPH-forming enzymes (NADP-IDH and ME). In this sense, specific activity, maximum velocity, catalytic efficiency and activity ratio values were higher in MA conditions than control groups. Moreover, these changes were observed in both feeding regimen, AL and R. Meanwhile, the Michaelis constant changed mainly in groups fed with the MA and restricted diet, these changes are related to the best substrate affinity by enzyme. Moreover, in the Western-blot result, we found that MA increased both protein levels studied, this behaviour being consistent with the regulation of the number of enzyme molecules. All results, indicate that MA, independently of the fed regimen, could potentially be a nutritional additive for fish as it improved the metabolic state of fish, as consequence of increased activity and expression of NADP

  13. Inhibition effects of some metal ions on the rat liver 6-phosphogluconate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Adem, Şevki; Kayhan, Naciye

    2016-04-01

    6-phosphogluconate dehydrogenase is an enzyme in the pentose phosphate path. The main functions of the pathway are the manufacture of the reduced coenzyme NADPH and the formation of ribose 5-phosphate for nucleic acid synthesis and nucleotide. Both NADPH and ribose 5-phosphate involve a critical biochemical process. Metals have been recognized as important toxic agents for living for a long time. It has been considered that they lead to in the emergence of many diseases. To evaluate whether metals is effect towards rat liver 6PGD, we apply various concentrations of metals and enzyme inhibition was analyzed using enzyme activity assays. The IC50 values of Pb+2, Cr+3, Co+2, Ni+2, Cd+2, and Va+2, metals on rat liver 6PGD were calculated as 138,138, 169, 214, 280, and 350 µM, respectively.

  14. The Reduction of Glyceraldehyde by Human Erythrocytes L-HEXONATE DEHYDROGENASE ACTIVITY

    PubMed Central

    Beutler, E.; Guinto, E.

    1974-01-01

    Incubation of red cell suspensions with D-glyceraldehyde resulted in disappearance of glyceraldehyde and appearance of glycerol. Concomitantly, there was an increase of CO2 formation from glucose. This indicated that the reduction of glyceraldehyde to glycerol occurred through a NADPH-linked system. Studies in hemolysates revealed the presence of an enzyme with the capacity to catalyze the reduction of glyceraldehyde to glycerol by NADPH. This enzyme was partially purified by DEAE chromatography. The elution pattern of the enzyme and its kinetic characteristics indicated that the enzyme was L-hexonate dehydrogenase (L-gulonate: NADP oxidoreductase, EC 1.1.1.19), not aldose reductase (Alditol: NADP oxidoreductase, EC 1.1.1.21), which had previously been thought present in erythrocytes. The reduction of glyceraldehyde to glycerol is one of a number of pathways for the metabolism of glyceraldehyde that have been found in red cells and/or other mammalian tissues. PMID:4825223

  15. Sorbitol production in charged membrane bioreactor with coenzyme regeneration system: II. Theoretical analysis of a continuous reaction with retained and regenerated NADPH.

    PubMed

    Ikemi, M; Ishimatsu, Y; Kise, S

    1990-06-20

    A theoretical model was constructed in order to study charged membrane bioreactors (CMBRs). In this model, it was postulated that a native nicotinamide coenzyme NADP(H) can be partially retained by a charged membrane in continuous operation. A multienzyme system composed of NADPH-dependent aldose reductase (AR) and glucose dehydrogenase (GDH) was used for the production of sorbitol and gluconic acid from glucose and for the conjugated enzymatic regeneration of NADP(H). Both enzymes were studied with respect to their reaction kinetics. AR was determined to obey the Theorell-Chance mechanism. GDH reaction was approximated by the initial velocity equation of the sequential Bi-Bi mechanism since the reverse reaction could be neglected. Significant inhibitions of both enzymes by sorbitol, gluconic acid, and glucose were observed, and the mode of inhibition was estimated to modify the velocity equations. The differential equation system for each component was derived and numerically analyzed according to the model. The theoretical model elucidated several features of the CMBR. (1) When compared at the same productivity, higher retainment was found to bring about a higher coenzyme turnover number, indicating that the feed coenzyme concentration can be reduced. (2) Under constant conversion, a contradictory relationship between turnover number and residence time arises if the feed concentration of a coenzyme varies. The theoretical model predicts that there is a practically optimal concentration for using NADP(H) efficiently. This concentration was consistent with that yielding the estimated minimum total cost. (3) In this system, excess-GDH-to-AR activity was required because of differences in their kinetic constants. The amount of regeneration enzyme required can be reduced by the accumulation of excels NADPH due to coenzyme retainment. (4) Comparison with an ideal repeated batch reaction revealed that the continuously operated CMBR was vastly superior with respect to

  16. NADPH oxidases: key modulators in aging and age-related cardiovascular diseases?

    PubMed Central

    Sahoo, Sanghamitra; Meijles, Daniel N.; Pagano, Patrick J.

    2016-01-01

    Reactive oxygen species (ROS) and oxidative stress have long been linked to aging and diseases prominent in the elderly such as hypertension, atherosclerosis, diabetes and atrial fibrillation (AF). NADPH oxidases (Nox) are a major source of ROS in the vasculature and are key players in mediating redox signalling under physiological and pathophysiological conditions. In this review, we focus on the Nox-mediated ROS signalling pathways involved in the regulation of ‘longevity genes’ and recapitulate their role in age-associated vascular changes and in the development of age-related cardiovascular diseases (CVDs). This review is predicated on burgeoning knowledge that Nox-derived ROS propagate tightly regulated yet varied signalling pathways, which, at the cellular level, may lead to diminished repair, the aging process and predisposition to CVDs. In addition, we briefly describe emerging Nox therapies and their potential in improving the health of the elderly population. PMID:26814203

  17. Characterization of an Arabidopsis thaliana mutant lacking a cytosolic non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Rius, Sebastián P; Casati, Paula; Iglesias, Alberto A; Gomez-Casati, Diego F

    2006-08-01

    Non-phosphorylating glyceraldehyde- 3-phosphate dehydrogenase (NP-GAPDH) is a conserved cytosolic protein found in higher plants. In photosynthetic cells, the enzyme is involved in a shuttle transfer mechanism to export NADPH from the chloroplast to the cytosol. To investigate the role of this enzyme in plant tissues, we characterized a mutant from Arabidopsis thaliana having an insertion at the NP-GAPDH gene locus. The homozygous mutant was determined to be null respect to NP-GAPDH, as it exhibited undetectable levels of both transcription of NP-GAPDH mRNA, protein expression and enzyme activity. Transcriptome analysis demonstrated that the insertion mutant plant shows altered expression of several enzymes involved in carbohydrate metabolism. Significantly, cytosolic phosphorylating (NAD-dependent) glyceraldehyde-3-phosphate dehydrogenase mRNA levels are induced in the mutant, which correlates with an increase in enzyme activity. mRNA levels and enzymatic activity of glucose-6-phosphate dehydrogenase were also elevated, correlating with an increase in NADPH concentration. Moreover, increased ROS levels were measured in the mutant plants. Down-regulation of several glycolytic and photosynthetic genes suggests that NP-GAPDH is important for the efficiency of both metabolic processes. The results presented demonstrate that NP-GAPDH has a relevant role in plant growth and development.

  18. Comparative studies on the cumene hydroperoxide- and NADPH-supported N-oxidation of 4-chloroaniline by cytochrome P-450.

    PubMed

    Hlavica, P; Golly, I; Mietaschk, J

    1983-06-15

    The present study confirms that cytochrome P-450 can act as a catalyst in the cumene hydroperoxide-supported N-oxidation of 4-chloroaniline. Analogous to the NADPH/O2-driven N-oxidation process, product dissociation is likely to limit the overall rate of cytochrome P-450 cycling also in the peroxidatic pathway. The oxy complexes involved in either metabolic route differ with respect to stability, spectral properties and need for thiolate-mediated resonance stabilization. With the organic hydroperoxide, the metabolic profile is shifted from the preponderant production of N-(4-chlorophenyl)hydroxylamine to the formation of 1-chloro-4-nitrobenzene. This finding suggests that the peroxide-sustained N-oxidation mechanism differs in several ways from that functional in the NADPH/O2-dependent oxenoid reaction. Thus one-electron oxidation, triggered by homolytic cleavage of the oxygen donor, is proposed as the mechanism of peroxidatic transformation of 4-chloroaniline.

  19. Biohydrogenation from biomass sugar mediated by in vitro synthetic enzymatic pathways.

    PubMed

    Wang, Yiran; Huang, Weidong; Sathitsuksanoh, Noppadon; Zhu, Zhiguang; Zhang, Y-H Percival

    2011-03-25

    Different from NAD(P)H regeneration approaches mediated by a single enzyme or a whole-cell microorganism, we demonstrate high-yield generation of NAD(P)H from a renewable biomass sugar--cellobiose through in vitro synthetic enzymatic pathways consisting of 12 purified enzymes and coenzymes. When the NAD(P)H generation system was coupled with its consumption reaction mediated by xylose reductase, the NADPH yield was as high as 11.4 mol NADPH per cellobiose (i.e., 95% of theoretical yield--12 NADPH per glucose unit) in a batch reaction. Consolidation of endothermic reactions and exothermic reactions in one pot results in a very high energy-retaining efficiency of 99.6% from xylose and cellobiose to xylitol. The combination of this high-yield and projected low-cost biohydrogenation and aqueous phase reforming may be important for the production of sulfur-free liquid jet fuel in the future.

  20. LACTIC DEHYDROGENASES OF PSEUDOMONAS NATRIEGENS.

    PubMed

    WALKER, H; EAGON, R G

    1964-07-01

    Walker, Hazel (University of Georgia, Athens), and R. G. Eagon. Lactic dehydrogenases of Pseudomonas natriegens. J. Bacteriol. 88:25-30. 1964.-Lactic dehydrogenases specific for d- and l-lactate were demonstrated in Pseudomonas natriegens. The l-lactic dehydrogenase showed considerable heat stability, and 40% of the activity remained in extracts after heating at 60 C for 10 min. An essential thiol group for enzyme activity was noted. The results of these experiments were consistent with the view that lactate was dehydrogenated initially by a flavin cofactor and that electrons were transported through a complete terminal oxidase system to oxygen. The intracellular site of these lactic dehydrogenases was shown to be the cell membrane. It was suggested that the main physiological role of these lactic dehydrogenases is that of lactate utilization.

  1. Engineering of alanine dehydrogenase from Bacillus subtilis for novel cofactor specificity.

    PubMed

    Lerchner, Alexandra; Jarasch, Alexander; Skerra, Arne

    2016-09-01

    The l-alanine dehydrogenase of Bacillus subtilis (BasAlaDH), which is strictly dependent on NADH as redox cofactor, efficiently catalyzes the reductive amination of pyruvate to l-alanine using ammonia as amino group donor. To enable application of BasAlaDH as regenerating enzyme in coupled reactions with NADPH-dependent alcohol dehydrogenases, we alterated its cofactor specificity from NADH to NADPH via protein engineering. By introducing two amino acid exchanges, D196A and L197R, high catalytic efficiency for NADPH was achieved, with kcat /KM  = 54.1 µM(-1)  Min(-1) (KM  = 32 ± 3 µM; kcat  = 1,730 ± 39 Min(-1) ), almost the same as the wild-type enzyme for NADH (kcat /KM  = 59.9 µM(-1)  Min(-1) ; KM  = 14 ± 2 µM; kcat  = 838 ± 21 Min(-1) ). Conversely, recognition of NADH was much diminished in the mutated enzyme (kcat /KM  = 3 µM(-1)  Min(-1) ). BasAlaDH(D196A/L197R) was applied in a coupled oxidation/transamination reaction of the chiral dicyclic dialcohol isosorbide to its diamines, catalyzed by Ralstonia sp. alcohol dehydrogenase and Paracoccus denitrificans ω-aminotransferase, thus allowing recycling of the two cosubstrates NADP(+) and l-Ala. An excellent cofactor regeneration with recycling factors of 33 for NADP(+) and 13 for l-Ala was observed with the engineered BasAlaDH in a small-scale biocatalysis experiment. This opens a biocatalytic route to novel building blocks for industrial high-performance polymers.

  2. [Effects of melaxen and valdoxan on the activity of glutathione antioxidant system and NADPH-producing enzymes in rat heart under experimental hyperthyroidism conditions].

    PubMed

    Gorbenko, M V; Popova, T N; Shul'gin, K K; Popov, S S

    2013-01-01

    The effects of melaxen and valdoxan on the activity of glutathione antioxidant system and some NADPH-producing enzymes have been studied under conditions of experimental hyperthyroidism in rat heart. Under the action of these drugs, reduced glutathione (GSH) content increased as compared to values observed under the conditions of pathology. It has been established that the activities of glutathione reductase (GR), glutathione peroxidase (GP), glucose-6-phosphate dehydrogenase, and NADP isocitrate dehydrogenase (increased under pathological conditions) change toward the intact control values upon the introduction of both drugs. The influence of melaxen and valdoxan, capable of producing antioxidant effect, leads apparently to the inhibition of free-radical oxidation processes and, as a consequence, the reduction of mobilization degree of the glutathione antioxidant system.

  3. Phagocyte NADPH-oxidase. Studies with flavin analogues as active site probes in triton X-100-solubilized preparations.

    PubMed

    Parkinson, J F; Gabig, T G

    1988-06-25

    NADPH-oxidase of stimulated human neutrophil membranes was solubilized in Triton X-100 and activity reconstituted with FAD, 8-F-FAD, 8-phenyl-S-FAD, and 8-S-FAD. The enzyme had similar affinities for all the flavins with Km values in the 60-80 nM range. Vmax was found to increase 4-fold with increasing redox midpoint potential of the flavin. 8-F-FAD reconstituted with the enzyme was reactive toward thiophenol, suggesting exposure of the 8-position to solvent, a finding supported by unsuccessful attempts to label the enzyme with the photoaffinity probe 8-N3-[32P]FAD. Solubilized oxidase stabilized the red thiolate form of 8-S-FAD, a characteristic of flavoproteins of the dehydrogenase/electron transferase classes which stabilize the blue neutral form of the flavin semiquinone radical.

  4. Involvement of activation of NADPH oxidase and extracellular signal-regulated kinase (ERK) in renal cell injury induced by zinc.

    PubMed

    Matsunaga, Yoshiko; Kawai, Yoshiko; Kohda, Yuka; Gemba, Munekazu

    2005-05-01

    Zinc is employed as a supplement; however, zinc-related nephropathy is not generally known. In this study, we investigated zinc-induced renal cell injury using a pig kidney-derived cultured renal epithelial cell line, LLC-PK(1), with proximal kidney tubule-like features, and examined the involvement of free radicals and extracellular signal-regulated kinase (ERK) in the cell injury. The LLC-PK(1) cells showed early uptake of zinc (30 microM), and the release of lactate dehydrogenase (LDH), an index of cell injury, was observed 24 hr after uptake. Three hours after zinc exposure, generation of reactive oxygen species (ROS) was increased. An antioxidant, N, N'-diphenyl-p-phenylenediamine (DPPD), inhibited a zinc-related increase in ROS generation and zinc-induced renal cell injury. An NADPH oxidase inhibitor, diphenyleneiodonium (DPI), inhibited a zinc-related increase in ROS generation and cell injury. We investigated translocation from the cytosol fraction of the p67(phox) subunit, which is involved in the activation of NADPH oxidase, to the membrane fraction, and translocation was induced 3 hr after zinc exposure. We examined the involvement of ERK1/2 in the deterioration of zinc-induced renal cell injury, and the association between ERK1/2 and an increase in ROS generation. Six hours after zinc exposure, the activation (phosphorylation) of ERK1/2 was observed. An antioxidant, DPPD, inhibited the zinc-related activation of ERK1/2. An MAPK/ERK kinase (MEK1/2) inhibitor, U0126, almost completely inhibited zinc-related cell injury (the release of LDH), but did not influence ROS generation. These results suggest that early intracellular uptake of zinc by LLC-PK(1) cells causes the activation of NADPH oxidase, and that ROS generation by the activation of the enzyme leads to the deterioration of renal cell injury via the activation of ERK1/2.

  5. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress.

    PubMed

    McNally, J Scott; Davis, Michael E; Giddens, Don P; Saha, Aniket; Hwang, Jinah; Dikalov, Sergey; Jo, Hanjoong; Harrison, David G

    2003-12-01

    Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

  6. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

    NASA Technical Reports Server (NTRS)

    McNally, J. Scott; Davis, Michael E.; Giddens, Don P.; Saha, Aniket; Hwang, Jinah; Dikalov, Sergey; Jo, Hanjoong; Harrison, David G.

    2003-01-01

    Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

  7. Biochemical and physiological analyses of NADPH-dependent thioredoxin reductase isozymes in Euglena gracilis.

    PubMed

    Tamaki, Shun; Maruta, Takanori; Sawa, Yoshihiro; Shigeoka, Shigeru; Ishikawa, Takahiro

    2015-07-01

    At least four peroxiredoxins that are coupled with the thioredoxin (Trx) system have been shown to play a key role in redox metabolism in the unicellular phytoflagellate Euglena gracilis. In order to clarify Trx-mediated redox regulation in this alga, we herein identified three NADPH-dependent thioredoxin reductases (NTRs) using a homologous search and characterized their enzymatic properties and physiological roles. Each Euglena NTR protein belonged to the small, large, and NTRC types, and were named EgNTR1, EgNTR2, and EgNTRC, respectively. EgNTR2 was phylogenetically different from the known NTRs in eukaryotic algae. EgNTR1 was predicted to be localized in mitochondria, EgNTR2 in the cytosol, and EgNTRC in plastids. The catalytic efficiency of EgNTR2 for NADPH was 30-46-fold higher than those of EgNTR1 and truncated form of EgNTRC, suggested that large type EgNTR2 reduced Trx more efficiently. The silencing of EgNTR2 gene expression resulted in significant growth inhibition and cell hypertrophy in Euglena cells. These results suggest that EgNTRs function in each cellular compartment and are physiologically important, particularly in the cytosol.

  8. Which NADPH Oxidase Isoform Is Relevant for Ischemic Stroke? The Case for Nox 2

    PubMed Central

    Kahles, Timo

    2013-01-01

    Abstract Significance and Recent Advances: Ischemic stroke is the leading cause of disability and third in mortality in industrialized nations. Immediate restoration of cerebral blood flow is crucial to salvage brain tissue, but only few patients are eligible for recanalization therapy. Thus, the need for alternative neuroprotective strategies is huge, and antioxidant interventions have long been studied in this context. Reactive oxygen species (ROS) physiologically serve as signaling molecules, but excessive amounts of ROS, as generated during ischemia/reperfusion (I/R), contribute to tissue injury. Critical Issues: Nevertheless and despite a strong rational of ROS being a pharmacological target, all antioxidant interventions failed to improve functional outcome in human clinical trials. Antioxidants may interfere with physiological functions of ROS or do not reach the crucial target structures of ROS-induced injury effectively. Future Directions: Thus, a potentially more promising approach is the inhibition of the source of disease-promoting ROS. Within recent years, NADPH oxidases (Nox) of the Nox family have been identified as mediators of neuronal pathology. As, however, several Nox homologs are expressed in neuronal tissue, and as many of the pharmacological inhibitors employed are rather unspecific, the concept of Nox as mediators of brain damage is far from being settled. In this review, we will discuss the contribution of Nox homologs to I/R injury at large as well as to neuronal damage in particular. We will illustrate that the current data provide evidence for Nox2 as the most important NADPH oxidase mediating cerebral injury. Antioxid. Redox Signal. 18, 1400–1417. PMID:22746273

  9. The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase

    PubMed Central

    2009-01-01

    Background The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase. Findings We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1) is 68-fold larger than that for the mutant K69A (0.73 s-1). There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 μM; wild-type = 29 μM) and NADPH (K69A = 30 μM; wild-type = 11 μM). The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4) μM and 134 (± 21), respectively. Conclusion Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs. PMID:19917104

  10. The mitochondrial 2-oxoglutarate carrier is part of a metabolic pathway that mediates glucose- and glutamine-stimulated insulin secretion.

    PubMed

    Odegaard, Matthew L; Joseph, Jamie W; Jensen, Mette V; Lu, Danhong; Ilkayeva, Olga; Ronnebaum, Sarah M; Becker, Thomas C; Newgard, Christopher B

    2010-05-28

    Glucose-stimulated insulin secretion from pancreatic islet beta-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic alpha-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic beta-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP(+) ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.

  11. AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: implication of the cell survival in mitochondrial diseases.

    PubMed

    Wu, Shi-Bei; Wei, Yau-Huei

    2012-02-01

    We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H(2)O(2). The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H(2)O(2)-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H(2)O(2) in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H(2)O(2) was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H(2)O(2)-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H(2)O(2)-treated normal skin fibroblasts to oxidative stress.

  12. Human hydroxysteroid dehydrogenases and pre-receptor regulation: Insights into inhibitor design and evaluation

    PubMed Central

    Penning, Trevor M.

    2011-01-01

    Hydroxysteroid dehydrogenases (HSDs) represent a major class of NAD(P)(H) dependent steroid hormone oxidoreductases involved in the pre-receptor regulation of hormone action. This is achieved by HSDs working in pairs so that they can interconvert ketosteroids with hydroxysteroids resulting in a change in ligand potency for nuclear receptors. HSDs belong to two protein superfamilies the aldo-keto reductases and the short-chain dehydrogenase/reductases. In humans, many of the important enzymes have been thoroughly characterized including the elucidation of their three-dimensional structures. Because these enzymes play fundamental roles in steroid hormone action they can be considered to be drug targets for a variety of steroid driven diseases: e.g. metabolic syndrome and obesity, inflammation, and hormone dependent malignancies of the endometrium, prostate and breast. This article will review how fundamental knowledge of these enzymes can be exploited in the development of isoform specific HSD inhibitors from both protein superfamilies. PMID:21272640

  13. NADPH-dependent reductive biotransformation with Escherichia coli and its pfkA deletion mutant: influence on global gene expression and role of oxygen supply.

    PubMed

    Siedler, Solvej; Bringer, Stephanie; Polen, Tino; Bott, Michael

    2014-10-01

    An Escherichia coli ΔpfkA mutant lacking the major phosphofructokinase possesses a partially cyclized pentose phosphate pathway leading to an increased NADPH per glucose ratio. This effect decreases the amount of glucose required for NADPH regeneration in reductive biotransformations, such as the conversion of methyl acetoacetate (MAA) to (R)-methyl 3-hydroxybutyrate (MHB) by an alcohol dehydrogenase from Lactobacillus brevis. Here, global transcriptional analyses were performed to study regulatory responses during reductive biotransformation. DNA microarray analysis revealed amongst other things increased expression of soxS, supporting previous results indicating that a high NADPH demand contributes to the activation of SoxR, the transcriptional activator of soxS. Furthermore, several target genes of the ArcAB two-component system showed a lower mRNA level in the reference strain than in the ΔpfkA mutant, pointing to an increased QH2 /Q ratio in the reference strain. This prompted us to analyze yields and productivities of MAA reduction to MHB under different oxygen regimes in a bioreactor. Under anaerobic conditions, the specific MHB production rates of both strains were comparable (7.4 ± 0.2 mmolMHB  h(-1)  gcdw (-1) ) and lower than under conditions of 15% dissolved oxygen, where those of the reference strain (12.8 mmol h(-1)  gcdw (-1) ) and of the ΔpfkA mutant (11.0 mmol h(-1)  gcdw (-1) ) were 73% and 49% higher. While the oxygen transfer rate (OTR) of the reference strain increased after the addition of MAA, presumably due to the oxidation of the acetate accumulated before MAA addition, the OTR of the ΔpfkA strain strongly decreased, indicating a very low respiration rate despite sufficient oxygen supply. The latter effect can likely be attributed to a restricted conversion of NADPH into NADH via the soluble transhydrogenase SthA, as the enzyme is outcompeted in the presence of MAA by the recombinant NADPH-dependent alcohol

  14. Lactate dehydrogenase-elevating virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This book chapter describes the taxonomic classification of Lactate dehydrogenase-elevating virus (LDV). Included are: host, genome, classification, morphology, physicochemical and physical properties, nucleic acid, proteins, lipids, carbohydrates, geographic range, phylogenetic properties, biologic...

  15. Polyphenols enhance platelet nitric oxide by inhibiting protein kinase C-dependent NADPH oxidase activation: effect on platelet recruitment.

    PubMed

    Pignatelli, P; Di Santo, S; Buchetti, B; Sanguigni, V; Brunelli, A; Violi, F

    2006-06-01

    Several studies demonstrated an inverse association between polyphenol intake and cardiovascular events. Platelet recruitment is an important phase of platelet activation at the site of vascular injury, but it has never been investigated whether polyphenols influence platelet recruitment. The aim of the study was to analyze in vitro whether two polyphenols, quercetin and catechin, were able to affect platelet recruitment. Platelet recruitment was reduced by NO donors and by NADPH oxidase inhibitors and was enhanced by L-NAME, an inhibitor of NO synthase. Quercetin and catechin, but not single polyphenol, significantly inhibited platelet recruitment in a concentration-dependent fashion. The formation of superoxide anion was significantly inhibited in platelets incubated with quercetin and catechin but was unaffected by a single polyphenol. Incubation of platelets with quercetin and catechin resulted in inhibition of PKC and NADPH oxidase activation. Treatment of platelets with quercetin and catechin resulted in an increase of NO and also down-regulated the expression of GpIIb/IIIa glycoprotein. This study shows that the polyphenols quercetin and catechin synergistically act in reducing platelet recruitment via inhibition of PKC-dependent NADPH oxidase activation. This effect, resulting in NO-mediated platelet glycoprotein GpIIb/IIIa down-regulation, could provide a novel mechanism through which polyphenols reduce cardiovascular disease.

  16. 20-HETE increases NADPH oxidase-derived ROS production and stimulates the L-type Ca2+ channel via a PKC-dependent mechanism in cardiomyocytes

    PubMed Central

    Han, Yong; Bao, Yuyan; Li, Wei; Li, Xingting; Shen, Xin; Wang, Xu; Yao, Fanrong; O'Rourke, Stephen T.; Sun, Chengwen

    2010-01-01

    The production of 20-hydroxyeicosatetraenoic acid (20-HETE) is increased during ischemia-reperfusion, and inhibition of 20-HETE production has been shown to reduce infarct size caused by ischemia. This study was aimed to discover the molecular mechanism underlying the action of 20-HETE in cardiac myocytes. The effect of 20-HETE on L-type Ca2+ currents (ICa,L) was examined in rat isolated cardiomyocytes by patch-clamp recording in the whole cell mode. Superfusion of cardiomyocytes with 20-HETE (10–100 nM) resulted in a concentration-dependent increase in ICa,L, and this action of 20-HETE was attenuated by a specific NADPH oxidase inhibitor, gp91ds-tat (5 μM), or a superoxide scavenger, polyethylene glycol-superoxide dismutase (25 U/ml), suggesting that NADPH-oxidase-derived superoxide is involved in the stimulatory action of 20-HETE on ICa,L. Treatment of cardiomyocytes with 20-HETE (100 nM) increased both NADPH oxidase activity and superoxide production by approximately twofold. To study the molecular mechanism mediating the 20-HETE-induced increase in NADPH oxidase activity, PKC activity was measured in cardiomyocytes. Incubation of the cells with 20-HETE (100 nM) significantly increased PKC activity, and pretreatment of cardiomyocytes with a selective PKC inhibitor, GF-109203 (1 μM), attenuated the 20-HETE-induced increases in ICa,L and in NADPH oxidase activity. In summary, 20-HETE stimulates NADPH oxidase-derived superoxide production, which activates L-type Ca2+ channels via a PKC-dependent mechanism in cardiomyocytes. 20-HETE and 20-HETE-producing enzymes could be novel targets for the treatment of cardiac ischemic diseases. PMID:20675568

  17. NADPH oxidase deficient mice develop colitis and bacteremia upon infection with normally avirulent, TTSS-1- and TTSS-2-deficient Salmonella Typhimurium.

    PubMed

    Felmy, Boas; Songhet, Pascal; Slack, Emma Marie Caroline; Müller, Andreas J; Kremer, Marcus; Van Maele, Laurye; Cayet, Delphine; Heikenwalder, Mathias; Sirard, Jean-Claude; Hardt, Wolf-Dietrich

    2013-01-01

    Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn's disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb (-/-)) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm(avir); invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb (-/-) mice are efficiently infected by S.Tm(avir) and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb (-/-) Myd88 (-/-) mice indicated that the S.Tm(avir)-inflicted disease in Cybb (-/-) mice hinges on CD11c(+)CX3CR1(+) monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm(avir) in the mucosal lamina propria. This provides important insights into microbe handling by the large

  18. Alcohol Dehydrogenase from Methylobacterium organophilum

    PubMed Central

    Wolf, H. J.; Hanson, R. S.

    1978-01-01

    The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10−5 M for methanol and 8.2 × 10−5 M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum. Images PMID:80974

  19. Sildenafil Promotes eNOS Activation and Inhibits NADPH Oxidase in the Transgenic Sickle Cell Mouse Penis

    PubMed Central

    Musicki, Biljana; Bivalacqua, Trinity J.; Champion, Hunter C.; Burnett, Arthur L.

    2014-01-01

    Introduction Sickle cell disease (SCD)-associated vasculopathy in the penis is characterized by aberrant nitric oxide and phosphodiesterase (PDE) 5 signaling, and by increased oxidative stress. Preliminary clinical trials show that continuous treatment with PDE5 inhibitor sildenafil unassociated with sexual activity decreases priapic activity in patients with SCD. However, the mechanism of its vasculoprotective effect in the penis remains unclear. Aims We evaluated whether continuous administration of PDE5 inhibitor sildenafil promotes eNOS function at posttranslational levels and decreases superoxide-producing enzyme NADPH oxidase activity in the sickle cell mouse penis. Methods SCD transgenic mice were used as an animal model of SCD. WT mice served as controls. Mice received treatment with the PDE5 inhibitor sildenafil (100 mg/kg/day) or vehicle for 3 weeks. eNOS phosphorylation on Ser-1177 (positive regulatory site), eNOS interactions with heat-shock protein 90 (HSP90) (positive regulator), phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177), an NADPH oxidase catalytic subunit gp91(phox), and a marker of oxidative stress (4-hydroxy-2-nonenal [HNE]) were measured by Western blot. Main Outcome Measures Effect of continuous sildenafil treatment on eNOS posttranslational activation, NADPH oxidase catalytic subunit, and oxidative stress in the penis of the sickle cell mouse. Results Continuous treatment with sildenafil reversed (P < 0.05) the abnormalities in protein expressions of P-eNOS (Ser-1177), eNOS/HSP90 interaction, P-AKT, protein expression of gp91(phox), and 4-HNE, in the sickle cell mouse penis. Sildenafil treatment of WT mice did not affect any of these parameters. Conclusion Our findings that sildenafil enhances eNOS activation and inhibits NADPH oxidase function in the sickle cell mouse penis offers a vasculoprotective molecular basis for the therapeutic effect of sildenafil in the penis in association with SCD. PMID:24251665

  20. Parasitic worms stimulate host NADPH oxidases to produce reactive oxygen species that limit plant cell death and promote infection.

    PubMed

    Siddique, Shahid; Matera, Christiane; Radakovic, Zoran S; Hasan, M Shamim; Gutbrod, Philipp; Rozanska, Elzbieta; Sobczak, Miroslaw; Torres, Miguel Angel; Grundler, Florian M W

    2014-04-08

    Plants and animals produce reactive oxygen species (ROS) in response to infection. In plants, ROS not only activate defense responses and promote cell death to limit the spread of pathogens but also restrict the amount of cell death in response to pathogen recognition. Plants also use hormones, such as salicylic acid, to mediate immune responses to infection. However, there are long-lasting biotrophic plant-pathogen interactions, such as the interaction between parasitic nematodes and plant roots during which defense responses are suppressed and root cells are reorganized to specific nurse cell systems. In plants, ROS are primarily generated by plasma membrane-localized NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidases, and loss of NADPH oxidase activity compromises immune responses and cell death. We found that infection of Arabidopsis thaliana by the parasitic nematode Heterodera schachtii activated the NADPH oxidases RbohD and RbohF to produce ROS, which was necessary to restrict infected plant cell death and promote nurse cell formation. RbohD- and RbohF-deficient plants exhibited larger regions of cell death in response to nematode infection, and nurse cell formation was greatly reduced. Genetic disruption of SID2, which is required for salicylic acid accumulation and immune activation in nematode-infected plants, led to the increased size of nematodes in RbohD- and RbohF-deficient plants, but did not decrease plant cell death. Thus, by stimulating NADPH oxidase-generated ROS, parasitic nematodes fine-tune the pattern of plant cell death during the destructive root invasion and may antagonize salicylic acid-induced defense responses during biotrophic life stages.

  1. Biochemistry, Physiology and Pathophysiology of NADPH Oxidases in the Cardiovascular System

    PubMed Central

    Lassègue, Bernard; San Martín, Alejandra; Griendling, Kathy K.

    2012-01-01

    The NADPH oxidase (Nox) enzymes are critical mediators of cardiovascular physiology and pathophysiology. These proteins are expressed in virtually all cardiovascular cells, and regulate such diverse functions as differentiation, proliferation, apoptosis, senescence, inflammatory responses and oxygen sensing. They target a number of important signaling molecules, including kinases, phosphatases, transcription factors, ion channels and proteins that regulate the cytoskeleton. Nox enzymes have been implicated in many different cardiovascular pathologies: atherosclerosis, hypertension, cardiac hypertrophy and remodeling, angiogenesis and collateral formation, stroke and heart failure. In this review, we discuss in detail the biochemistry of Nox enzymes expressed in the cardiovascular system (Nox1, 2, 4 and 5), their roles in cardiovascular cell biology, and their contributions to disease development. PMID:22581922

  2. NADPH oxidase inhibitor DPI is neuroprotective at femtomolar concentrations through inhibition of microglia over-activation.

    PubMed

    Qian, Li; Gao, Xi; Pei, Zhong; Wu, Xuefei; Block, Michelle; Wilson, Belinda; Hong, Jau-Shyong; Flood, Patrick M

    2007-01-01

    In this paper we report that diphenyliodonium (DPI), a NADPH oxidase inhibitor, shows potent anti-inflammatory and neuroprotective effects at femtomolar concentrations (10(-13) to 10(-14) M) in primary midbrain cultures. Mechanistic studies revealed that DPI-elicited effects were mediated by the inhibition of LPS-induced microglial ROS production and the subsequent release of pro-inflammatory cytokine TNFa, and the production of nitric oxide. Further studies showed that 10(-14) M DPI significantly reduced LPS-induced ERK phosphorylation. Taken together, our results demonstrate that femtomolar concentrations of DPI exert potent anti-inflammatory and neuroprotective effects by inhibiting microglial activation through the inhibition of ERK-regulated PHOX activity.

  3. Purification and characterization of an anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol.

    PubMed

    Meng, Fantao; Xu, Yan

    2010-04-01

    An anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol was purified by 26-fold to homogeneity. The enzyme had a homodimeric structure consisting of 49 kDa subunits, required NADPH, but not NADH, as a cofactor and was a Zn-independent short-chain dehydrogenase. Aliphatic methyl ketones (chain length > or =6 carbon atoms) and aromatic methyl ketones were the preferred substrates for the enzyme, the best being 2-octanone. Maximum enzyme activity with 2-octanone was at 45 degrees C and at pH 8.0.

  4. NADPH oxidase-derived reactive oxygen species in cardiac pathophysiology

    PubMed Central

    Cave, Alison; Grieve, David; Johar, Sofian; Zhang, Min; Shah, Ajay M

    2005-01-01

    Chronic heart failure, secondary to left ventricular hypertrophy or myocardial infarction, is a condition with increasing morbidity and mortality. Although the mechanisms underlying the development and progression of this condition remain a subject of intense interest, there is now growing evidence that redox-sensitive pathways play an important role. This article focuses on the involvement of reactive oxygen species derived from a family of superoxide-generating enzymes, termed NADPH oxidases (NOXs), in the pathophysiology of ventricular hypertrophy, the accompanying interstitial fibrosis and subsequent heart failure. In particular, the apparent ability of the different NADPH oxidase isoforms to define the response of a cell to a range of physiological and pathophysiological stimuli is reviewed. If confirmed, these data would suggest that independently targeting different members of the NOX family may hold the potential for therapeutic intervention in the treatment of cardiac disease. PMID:16321803

  5. Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8).

    PubMed

    Rosell, Albert; Valencia, Eva; Parés, Xavier; Fita, Ignacio; Farrés, Jaume; Ochoa, Wendy F

    2003-06-27

    The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.

  6. Identification of a novel inactivating mutation in Isocitrate Dehydrogenase 1 (IDH1-R314C) in a high grade astrocytoma

    PubMed Central

    van Lith, Sanne A. M.; Navis, Anna C.; Lenting, Krissie; Verrijp, Kiek; Schepens, Jan T. G.; Hendriks, Wiljan J. A. J.; Schubert, Nil A.; Venselaar, Hanka; Wevers, Ron A.; van Rooij, Arno; Wesseling, Pieter; Molenaar, Remco J.; van Noorden, Cornelis J. F.; Pusch, Stefan; Tops, Bastiaan; Leenders, William P. J.

    2016-01-01

    The majority of low-grade and secondary high-grade gliomas carry heterozygous hotspot mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) or the mitochondrial variant IDH2. These mutations mostly involve Arg132 in IDH1, and Arg172 or Arg140 in IDH2. Whereas IDHs convert isocitrate to alpha-ketoglutarate (α-KG) with simultaneous reduction of NADP+ to NADPH, these IDH mutants reduce α-KG to D-2-hydroxyglutarate (D-2-HG) while oxidizing NADPH. D-2-HG is a proposed oncometabolite, acting via competitive inhibition of α-KG-dependent enzymes that are involved in metabolism and epigenetic regulation. However, much less is known about the implications of the metabolic stress, imposed by decreased α-KG and NADPH production, for tumor biology. We here present a novel heterozygous IDH1 mutation, IDH1R314C, which was identified by targeted next generation sequencing of a high grade glioma from which a mouse xenograft model and a cell line were generated. IDH1R314C lacks isocitrate-to-α-KG conversion activity due to reduced affinity for NADP+, and differs from the IDH1R132 mutants in that it does not produce D-2-HG. Because IDH1R314C is defective in producing α-KG and NADPH, without concomitant production of the D-2-HG, it represents a valuable tool to study the effects of IDH1-dysfunction on cellular metabolism in the absence of this oncometabolite. PMID:27460417

  7. The NADPH oxidase Nox4 has anti-atherosclerotic functions

    PubMed Central

    Schürmann, Christoph; Rezende, Flavia; Kruse, Christoph; Yasar, Yakub; Löwe, Oliver; Fork, Christian; van de Sluis, Bart; Bremer, Rolf; Weissmann, Norbert; Shah, Ajay M.; Jo, Hanjoong; Brandes, Ralf P.; Schröder, Katrin

    2015-01-01

    Aims Oxidative stress is thought to be a risk for cardiovascular disease and NADPH oxidases of the Nox family are important producers of reactive oxygen species. Within the Nox family, the NADPH oxidase Nox4 has a unique position as it is constitutively active and produces H2O2 rather than O2− . Nox4 is therefore incapable of scavenging NO and its low constitutive H2O2 production might even be beneficial. We hypothesized that Nox4 acts as an endogenous anti-atherosclerotic enzyme. Methods and results Tamoxifen-induced Nox4-knockout mice were crossed with ApoE−/− mice and spontaneous atherosclerosis under regular chow as well as accelerated atherosclerosis in response to partial carotid artery ligation under high-fat diet were determined. Deletion of Nox4 resulted in increased atherosclerosis formation in both models. Mechanistically, pro-atherosclerotic and pro-inflammatory changes in gene expression were observed prior to plaque development. Moreover, inhibition of Nox4 or deletion of the enzyme in the endothelium but not in macrophages resulted in increased adhesion of macrophages to the endothelial surface. Conclusions The H2O2-producing NADPH oxidase Nox4 is an endogenous anti-atherosclerotic enzyme. Nox4 inhibitors, currently under clinical evaluation, should be carefully monitored for cardiovascular side-effects. PMID:26385958

  8. Mutations that affect coenzyme binding and dimer formation of fungal 17beta-hydroxysteroid dehydrogenase.

    PubMed

    Brunskole, Mojca; Kristan, Katja; Stojan, Jure; Rizner, Tea Lanisnik

    2009-03-25

    The 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is an NADPH-dependent member of the short-chain dehydrogenase/reductase superfamily, and it functions as a dimer that is composed of two identical subunits. By constructing the appropriate mutants, we have examined the M204 residue that is situated in the coenzyme binding pocket, for its role in the binding of the coenzyme NADP(H). We have also studied the importance of hydrophobic interactions through F124, F132, F133 and F177 for 17beta-HSDcl dimer formation. The M204G substitution decreased the catalytic efficiency of 17beta-HSDcl, suggesting that M204 sterically coerces the nicotinamide moiety of the coenzyme into the appropriate position for further hydride transfer. Phenylalanine substitutions introduced at the dimer interface produced inactive aggregates and oligomers with high molecular masses, suggesting that these hydrophobic interactions have important roles in the formation of the active dimer.

  9. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    PubMed Central

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated. PMID:24949265

  10. Redesign of the coenzyme specificity in L-lactate dehydrogenase from bacillus stearothermophilus using site-directed mutagenesis and media engineering.

    PubMed

    Holmberg, N; Ryde, U; Bülow, L

    1999-10-01

    L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox enzyme which has a strong preference for NADH over NADPH as coenzyme. To exclude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspartate residue at position 52. However, this residue is probably not solely responsible for the NADH specificity. In this report we examine the possibilities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, after choosing the mutant with the highest selectivity for NADPH, we also investigated the possibility of further altering the coenzyme specificity by adding an organic solvent to the reaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turnover number of this mutant was increased almost fourfold as compared with wild-type LDH. To explain the altered coenzyme specificity exhibited by the D52SI51K double mutant, molecular dynamics simulations were performed.

  11. Characterization of a Zinc-Containing Alcohol Dehydrogenase with Stereoselectivity from the Hyperthermophilic Archaeon Thermococcus guaymasensis▿

    PubMed Central

    Ying, Xiangxian; Ma, Kesen

    2011-01-01

    An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 ± 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and l-threonine dehydrogenases with binding motifs of catalytic zinc and NADP+. Metal analyses revealed that this NADP+-dependent enzyme contained 0.9 ± 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic (R/S)-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyperthermostable, the enzyme activity increased as the temperature was elevated up to 95°C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent Km values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP+, respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation. PMID:21515780

  12. Proinflammatory adipokine leptin mediates disinfection byproduct bromodichloromethane-induced early steatohepatitic injury in obesity

    SciTech Connect

    Das, Suvarthi; Kumar, Ashutosh; Seth, Ratanesh Kumar; Tokar, Erik J.; Kadiiska, Maria B.; Waalkes, Michael P.; Mason, Ronald P.; Chatterjee, Saurabh

    2013-06-15

    Today's developed world faces a major public health challenge in the rise in the obese population and the increased incidence in fatty liver disease. There is a strong association among diet induced obesity, fatty liver disease and development of nonalcoholic steatohepatitis but the environmental link to disease progression remains unclear. Here we demonstrate that in obesity, early steatohepatitic lesions induced by the water disinfection byproduct bromodichloromethane are mediated by increased oxidative stress and leptin which act in synchrony to potentiate disease progression. Low acute exposure to bromodichloromethane (BDCM), in diet-induced obesity produced oxidative stress as shown by increased lipid peroxidation, protein free radical and nitrotyrosine formation and elevated leptin levels. Exposed obese mice showed histopathological signs of early steatohepatitic injury and necrosis. Spontaneous knockout mice for leptin or systemic leptin receptor knockout mice had significantly decreased oxidative stress and TNF-α levels. Co-incubation of leptin and BDCM caused Kupffer cell activation as shown by increased MCP-1 release and NADPH oxidase membrane assembly, a phenomenon that was decreased in Kupffer cells isolated from leptin receptor knockout mice. In obese mice that were BDCM-exposed, livers showed a significant increase in Kupffer cell activation marker CD68 and, increased necrosis as assessed by levels of isocitrate dehydrogenase, events that were decreased in the absence of leptin or its receptor. In conclusion, our results show that exposure to the disinfection byproduct BDCM in diet-induced obesity augments steatohepatitic injury by potentiating the effects of leptin on oxidative stress, Kupffer cell activation and cell death in the liver. - Highlights: ► BDCM acute exposure sensitizes liver to increased free radical stress in obesity. ► BDCM-induced higher leptin contributes to early steatohepatitic lesions. ► Increased leptin mediates protein

  13. Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase.

    PubMed

    Moon, Hee-Jung; Tiwari, Manish Kumar; Singh, Ranjitha; Kang, Yun Chan; Lee, Jung-Kul

    2012-05-01

    Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces NAD(P)(+) to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD(+) (S156D, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 10.9, where K(m)(,NAD) is the K(m) for NAD(+) and K(m)(,NADP) is the K(m) for NADP(+)). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP(+) as the cofactor (S156H, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.

  14. Alteration of coenzyme specificity of malate dehydrogenase from Streptomyces coelicolor A3(2) by site-directed mutagenesis.

    PubMed

    Ge, Y D; Song, P; Cao, Z Y; Wang, P; Zhu, G P

    2014-07-29

    We describe here for the first time the alteration of coenzyme specificity of malate dehydrogenase (MDH) from Streptomyces coelicolor A3(2) (ScMDH). In the present study, we replaced four amino acid residues in the Rossmann fold (βB-αC) region of NADH-dependent ScMDH by site-directed mutagenesis with those of NADPH-dependent MDH (Glu42Gly, Ile43Ser, Pro45Arg, and Ala46Ser). The coenzyme specificity of the mutant enzyme (ScMDH-T4) was examined. Coenzyme specificity of ScMDH-T4 was shifted 2231.3-fold toward NADPH using kcat/Km(coenzyme) as the measurement of coenzyme specificity. Accordingly, the effect of the replacements on coenzyme specificity is discussed. Our work provides further insight into the coenzyme specificity of ScMDH.

  15. NADH electrochemical sensor coupled with dehydrogenase enzymes

    SciTech Connect

    Yamanaka, Hideko; Mascini, Marco )

    1992-06-01

    A graphite electrode assembled in a flow cell has shown to be a good detector for NADH. Current is linearly dependent on concentration in the range 10{sup {minus}7}-10{sup {minus}3} M without any mediator at the potential applied of 300 mV vs Ag/AgCl. Lactate and alcohol dehydrogenases were immobilized near to the electrode surface or in a reactor to obtain an NADH-based biosensor for lactate or ethanol. With lactate the authors succeeded to obtain a response only if the reactor was used and for alcohol a current proportional to the concentration was obtained either if the enzyme was immobilized in a membrane and placed near the electrode surface or when the enzyme was immobilized in a reactor form. By FIA procedures fast responses and recoveries were obtained, but with a short linear range.

  16. Mapping of functional domains in p47(phox) involved in the activation of NADPH oxidase by "peptide walking".

    PubMed

    Morozov, I; Lotan, O; Joseph, G; Gorzalczany, Y; Pick, E

    1998-06-19

    The superoxide generating NADPH oxidase of phagocytes consists, in resting cells, of a membrane-associated electron transporting flavocytochrome (cytochrome b559) and four cytosolic proteins as follows: p47(phox), p67(phox), p40(phox), and the small GTPase, Rac(1 or 2). Activation of the oxidase is consequent to the assembly of a membrane-localized multimolecular complex consisting of cytochrome b559 and the cytosolic components. We used "peptide walking" (Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082) for mapping domains in the amino acid sequence of p47(phox) participating in the molecular events leading to the activation of NADPH oxidase. Ninety-five overlapping pentadecapeptides, with a four-residue offset between neighboring peptides, spanning the complete p47(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in a cell-free system. This consisted of solubilized macrophage membranes, recombinant p47(phox), p67(phox), and Rac1, and lithium dodecyl sulfate, as the activator. Eight functional domains were identified and labeled a-h. These were (N- and C-terminal residue numbers are given for each domain) as follows: a (21-35); b (105-119); c (149-159); d (193-207); e (253-267); f (305-319); g (325-339), and h (373-387). Four of these domains (c, d, e, and g) correspond to or form parts of regions shown before to participate in NADPH oxidase assembly. Thus, domain c corresponds to a region on the N-terminal boundary of the first src homology 3 (SH3) domain, whereas domains d and e represent more precisely defined sites within the full-length first and second SH3 domains, respectively. Domain g overlaps an extensively investigated arginine-rich region. Domains a and b, in the N-terminal half of p47(phox), and domains f and h, in the C-terminal half, represent newly identified entities, for which there is no earlier experimental evidence of involvement in NADPH oxidase activation. "Peptide walking" was also applied to

  17. Elevated mitochondria-coupled NAD(P)H in endoplasmic reticulum of dopamine neurons

    PubMed Central

    Tucker, Kristal R.; Cavolo, Samantha L.; Levitan, Edwin S.

    2016-01-01

    Pyridine nucleotides are redox coenzymes that are critical in bioenergetics, metabolism, and neurodegeneration. Here we use brain slice multiphoton microscopy to show that substantia nigra dopamine neurons, which are sensitive to stress in mitochondria and the endoplasmic reticulum (ER), display elevated combined NADH and NADPH (i.e., NAD(P)H) autofluorescence. Despite limited mitochondrial mass, organellar NAD(P)H is extensive because much of the signal is derived from the ER. Remarkably, even though pyridine nucleotides cannot cross mitochondrial and ER membranes, inhibiting mitochondrial function with an uncoupler or interrupting the electron transport chain with cyanide (CN−) alters ER NAD(P)H. The ER CN− response can occur without a change in nuclear NAD(P)H, raising the possibility of redox shuttling via the cytoplasm locally between neuronal mitochondria and the ER. We propose that coregulation of NAD(P)H in dopamine neuron mitochondria and ER coordinates cell redox stress signaling by the two organelles. PMID:27582392

  18. Nrf2 promotes reparative angiogenesis through regulation of NADPH oxidase-2 in oxygen-induced retinopathy.

    PubMed

    Wei, Yanhong; Gong, Junsong; Xu, Zhenhua; Duh, Elia J

    2016-10-01

    Revascularization of ischemic tissue is a highly desirable outcome in multiple diseases, including cardiovascular diseases and ischemic retinopathies. Oxidative stress and inflammation are both known to play a role in suppressing reparative angiogenesis in ischemic disease models including oxygen-induced retinopathy (OIR), but the regulatory molecules governing these pathophysiologic processes in retinal ischemia are largely unknown. Nrf2 is a major stress-response transcription factor that has been implicated in regulating ischemic angiogenesis in the retina and other tissue beds. Using Nrf2-deficient mice, we investigated the effects of Nrf2 in regulating revascularization and modulating the retinal tissue milieu during ischemia. Strikingly, Nrf2's beneficial effect on reparative angiogenesis only became manifested in the later phase of ischemia in OIR, from postnatal day 14 (P14) to P17. This was temporally associated with a reduction in both oxidative stress and inflammatory mediators in wild-type compared to Nrf2(-/-) mice. Nrf2(-/-) retinas exhibited an increase in VEGF but also induction of anti-angiogenic Dll4/Notch signaling. NADPH oxidase (NOX), and especially NOX2, is a major pathogenic molecule and a particularly important contributor to oxidative stress in multiple retinal disease processes. Nrf2(-/-) mice exhibited a significant exacerbation of NOX2 induction in OIR that manifested in the later phases of ischemia. Pharmacologic inhibition of NADPH oxidase abrogated the adverse effect of Nrf2 deficiency on reparative angiogenesis. Taken together, this suggests that Nrf2 is an important regulator of the retinal milieu during tissue ischemia, and that the Nrf2/NOX2 balance may play a critical role in determining the fate of ischemic revascularization.

  19. Redox mechanisms in pathological angiogenesis in the retina: roles for NADPH oxidase.

    PubMed

    Chan, Elsa C; Liu, Guei-Sheung; Dusting, Gregory J

    2015-01-01

    Pathological angiogenesis in the retina is a leading cause of serious vision loss in potentially blinding eye diseases, including proliferative diabetic retinopathy, retinopathy of prematurity and the wet form of age-related macular degeneration. Hypoxia is thought to be the driver of pathological angiogenesis, and transcription factors such as hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF) are key mediators in these processes. Current treatments employ either laser photocoagulation or intravitreal injection of therapeutic antibodies for VEGF, in order to arrest the growth of leaky blood vessels in the avascular vitreous cavity and to restore visual acuity. However, all such therapeutic approaches are limited by low or variable efficacy, and the inconvenience, risk and financial burden of such treatments, which need to be given frequently. The lack of noninvasive and efficacious therapy has therefore driven the search for alternative strategies. We have been interested in the roles of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, which when produced intracellularly at low concentration can act as second messengers to regulate physiological and pathological angiogenesis. Accumulating evidence suggests NADPH oxidase-dependent ROS are involved in regulation of the angiogenic signalling pathways of HIF and VEGF. Suppressing pathological neovascularisation in the retina by manipulating such redox mechanisms appears to be an attractive and clinically translatable therapeutic strategy to treat proliferative neovascular eye diseases. Here we provide a brief overview of the roles of NADPH oxidase in the sensing and regulation processes involving HIF and VEGF that contribute to the development of pathological angiogenesis in the retina.

  20. Genetics Home Reference: lactate dehydrogenase deficiency

    MedlinePlus

    ... dehydrogenase-B pieces (subunits) of the lactate dehydrogenase enzyme. This enzyme is found throughout the body and is important ... cells. There are five different forms of this enzyme, each made up of four protein subunits. Various ...

  1. Crystal structure of the MJ0490 gene product of the hyperthermophilic archaebacterium Methanococcus jannaschii, a novel member of the lactate/malate family of dehydrogenases.

    PubMed

    Lee, B I; Chang, C; Cho, S J; Eom, S H; Kim, K K; Yu, Y G; Suh, S W

    2001-04-13

    The MJ0490 gene, one of the only two genes of Methanococcus jannaschii showing sequence similarity to the lactate/malate family of dehydrogenases, was classified initially as coding for a putative l-lactate dehydrogenase (LDH). It has been re-classified as a malate dehydrogenase (MDH) gene, because it shows significant sequence similarity to MT0188, MDH II from Methanobacterium thermoautotrophicum strain DeltaH. The three-dimensional structure of its gene product has been determined in two crystal forms: a "dimeric" structure in the orthorhombic crystal at 1.9 A resolution and a "tetrameric" structure in the tetragonal crystal at 2.8 A. These structures share a similar subunit fold with other LDHs and MDHs. The tetrameric structure resembles typical tetrameric LDHs. The dimeric structure is equivalent to the P-dimer of tetrameric LDHs, unlike dimeric MDHs, which correspond to the Q-dimer. The structure reveals that the cofactor NADP(H) is bound at the active site, despite the fact that it was not intentionally added during protein purification and crystallization. The preference of NADP(H) over NAD(H) has been supported by activity assays. The cofactor preference is explained by the presence of a glycine residue in the cofactor binding pocket (Gly33), which replaces a conserved aspartate (or glutamate) residue in other NAD-dependent LDHs or MDHs. Preference for NADP(H) is contributed by hydrogen bonds between the oxygen atoms of the monophosphate group and the ribose sugar of adenosine in NADP(H) and the side-chains of Ser9, Arg34, His36, and Ser37. The MDH activity of MJ0490 is made possible by Arg86, which is conserved in MDHs but not in LDHs. The enzymatic assay showed that the MJ0490 protein possesses the fructose-1,6-bisphosphate-activated LDH activity (reduction). Thus the MJ0490 gene product appears to be a novel member of the lactate/malate dehydrogenase family, displaying an LDH scaffold and exhibiting a relaxed substrate and cofactor specificities in NADP(H

  2. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    DOE PAGES

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; ...

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important componentmore » in making biofuels production from lignocellulosic biomass feasible.« less

  3. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    SciTech Connect

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; Westpheling, Janet; Elkins, James G.

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important component in making biofuels production from lignocellulosic biomass feasible.

  4. Glucose-6-Phosphate Dehydrogenase Revisited

    PubMed Central

    O'Connell, Jerome T.; Henderson, Alfred R.

    1984-01-01

    Hemolytic diseases associated with drugs have been recognized since antiquity. Many of these anemias have been associated with oxidizing agents and deficiencies in the intraerythrocytic enzyme glucose-6-phosphate dehydrogenase. This paper outlines the discovery, prevalence, and variants of this enzyme. Methods of diagnosis of associated anemias are offered. PMID:6502728

  5. Studies on NADH(NADPH)-cytochrome c reductase (FMN-containing) from yeast: steady-state kinetic properties of the flavoenzyme from top-fermenting ale yeast.

    PubMed

    Johnson, M S; Kuby, S A

    1986-02-15

    A study of the steady-state kinetics of NADH(NADPH)-cytochrome c reductase (FMN-containing) from ale yeast (M. S. Johnson and S. A. Kuby (1985) J. Biol. Chem. 260, 12341-12350) has led to a postulated three-substrate random-ordered hybrid mechanism, where NAD(P)H and FMN add randomly and very likely in a steady-state fashion, followed by an ordered addition of cytochrome c. Kinetic parameters have been derived from this mechanism. Arrhenius plots showed large differences between NADH and NADPH, as the substrate-reductant. Menadione accelerated cytochrome c reduction and also O2 uptake, but vitamin K1 and coenzyme Q10 were ineffective as electron mediators, possibly as a result of their insolubility. With NADPH as the substrate-reductant, the order of the rate of reduction of electron acceptors was ferricyanide greater than DCIP greater than cytochrome c greater than oxygen; with menadione, the specificity sequence was cytochrome c greater than ferricyanide greater than DCIP greater than oxygen. With NADH, the order was ferricyanide greater than cytochrome c greater than oxygen greater than DCIP, which changed to cytochrome c greater than ferricyanide greater than oxygen greater than DCIP on addition of menadione. Cytochrome b5 was also reduced in the absence of oxygen. No transhydrogenase activity was observed, but the reduced thionicotinamide analogs of NADH and NADPH acted as substrates. Superoxide dismutase inhibited cytochrome c reduction in air by 50%, but O2-. was not necessary for cytochrome c reduction, as evidenced by the increase in rate in the absence of O2. The product of the reaction with oxygen appeared to be H2O2.

  6. Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase

    PubMed Central

    Shao, Beili; Bayraktutan, Ulvi

    2014-01-01

    Blood–brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2•- generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2•- by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2•- production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase. PMID:24936444

  7. Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime

    PubMed Central

    Blacker, Thomas S.; Berecz, Tunde; Duchen, Michael R.; Szabadkai, Gyorgy

    2017-01-01

    NADH and NADPH are redox cofactors, primarily involved in catabolic and anabolic metabolic processes respectively. In addition, NADPH plays an important role in cellular antioxidant defence. In live cells and tissues, the intensity of their spectrally-identical autofluorescence, termed NAD(P)H, can be used to probe the mitochondrial redox state, while their distinct enzyme-binding characteristics can be used to separate their relative contributions to the total NAD(P)H intensity using fluorescence lifetime imaging microscopy (FLIM). These protocols allow differences in metabolism to be detected between cell types and altered physiological and pathological states. PMID:28286806

  8. Antisense Inhibition of the Iron-Sulphur Subunit of Succinate Dehydrogenase Enhances Photosynthesis and Growth in Tomato via an Organic Acid–Mediated Effect on Stomatal Aperture[W][OA

    PubMed Central

    Araújo, Wagner L.; Nunes-Nesi, Adriano; Osorio, Sonia; Usadel, Björn; Fuentes, Daniela; Nagy, Réka; Balbo, Ilse; Lehmann, Martin; Studart-Witkowski, Claudia; Tohge, Takayuki; Martinoia, Enrico; Jordana, Xavier; DaMatta, Fábio M.; Fernie, Alisdair R.

    2011-01-01

    Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the Sl SDH2-2 gene encoding the iron sulfur subunit of the succinate dehydrogenase protein complex in the antisense orientation under the control of the 35S promoter exhibit an enhanced rate of photosynthesis. The rate of the tricarboxylic acid (TCA) cycle was reduced in these transformants, and there were changes in the levels of metabolites associated with the TCA cycle. Furthermore, in comparison to wild-type plants, carbon dioxide assimilation was enhanced by up to 25% in the transgenic plants under ambient conditions, and mature plants were characterized by an increased biomass. Analysis of additional photosynthetic parameters revealed that the rate of transpiration and stomatal conductance were markedly elevated in the transgenic plants. The transformants displayed a strongly enhanced assimilation rate under both ambient and suboptimal environmental conditions, as well as an elevated maximal stomatal aperture. By contrast, when the Sl SDH2-2 gene was repressed by antisense RNA in a guard cell–specific manner, changes in neither stomatal aperture nor photosynthesis were observed. The data obtained are discussed in the context of the role of TCA cycle intermediates both generally with respect to photosynthetic metabolism and specifically with respect to their role in the regulation of stomatal aperture. PMID:21307286

  9. Inhibition of arsenic induced-rat liver injury by grape seed exact through suppression of NADPH oxidase and TGF-{beta}/Smad activation

    SciTech Connect

    Pan Xinjuan; Dai Yujie; Li Xing; Niu Nannan; Li Wenjie; Liu Fangli; Zhao Yang; Yu Zengli

    2011-08-01

    fibrogenic genes. > GSE reduced arsenic-mediated Smad2/3 phosphorylation and NADPH oxidase subunits (Nox2, Nox4 and p47phox). > Beneficial effects of GSE on As-induced liver injury was via inhibition of NADPH oxidase and TGF-{beta}/Smad activation.

  10. Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein.

    PubMed

    Gabig, T G; English, D; Akard, L P; Schell, M J

    1987-02-05

    Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.

  11. Short Chain Dehydrogenase/Reductase Rdhe2 Is a Novel Retinol Dehydrogenase Essential for Frog Embryonic Development*

    PubMed Central

    Belyaeva, Olga V.; Lee, Seung-Ah; Adams, Mark K.; Chang, Chenbei; Kedishvili, Natalia Y.

    2012-01-01

    The enzymes responsible for the rate-limiting step in retinoic acid biosynthesis, the oxidation of retinol to retinaldehyde, during embryogenesis and in adulthood have not been fully defined. Here, we report that a novel member of the short chain dehydrogenase/reductase superfamily, frog sdr16c5, acts as a highly active retinol dehydrogenase (rdhe2) that promotes retinoic acid biosynthesis when expressed in mammalian cells. In vivo assays of rdhe2 function show that overexpression of rdhe2 in frog embryos leads to posteriorization and induction of defects resembling those caused by retinoic acid toxicity. Conversely, antisense morpholino-mediated knockdown of endogenous rdhe2 results in phenotypes consistent with retinoic acid deficiency, such as defects in anterior neural tube closure, microcephaly with small eye formation, disruption of somitogenesis, and curved body axis with bent tail. Higher doses of morpholino induce embryonic lethality. Analyses of retinoic acid levels using either endogenous retinoic acid-sensitive gene hoxd4 or retinoic acid reporter cell line both show that the levels of retinoic acid are significantly decreased in rdhe2 morphants. Taken together, these results provide strong evidence that Xenopus rdhe2 functions as a retinol dehydrogenase essential for frog embryonic development in vivo. Importantly, the retinol oxidizing activity of frog rdhe2 is conserved in its mouse homologs, suggesting that rdhe2-related enzymes may represent the previously unrecognized physiologically relevant retinol dehydrogenases that contribute to retinoic acid biosynthesis in higher vertebrates. PMID:22291023

  12. Activation of AMP-activated protein kinase, inhibition of pyruvate dehydrogenase activity, and redistribution of substrate partitioning mediate the acute insulin-sensitizing effects of troglitazone in skeletal muscle cells.

    PubMed

    Fediuc, S; Pimenta, A S; Gaidhu, M P; Ceddia, R B

    2008-05-01

    The aim of this study was to investigate the acute effects of troglitazone on several pathways of glucose and fatty acid (FA) partitioning and the molecular mechanisms involved in these processes in skeletal muscle. Exposure of L6 myotubes to troglitazone for 1 h significantly increased phosphorylation of AMPK and ACC, which was followed by approximately 30% and approximately 60% increases in palmitate oxidation and carnitine palmitoyl transferase-1 (CPT-1) activity, respectively. Troglitazone inhibited basal ( approximately 25%) and insulin-stimulated ( approximately 35%) palmitate uptake but significantly increased basal and insulin-stimulated glucose uptake by approximately 2.2- and 2.7-fold, respectively. Pharmacological inhibition of AMPK completely prevented the effects of troglitazone on palmitate oxidation and glucose uptake. Interestingly, even though troglitazone exerted an insulin sensitizing effect, it reduced basal and insulin-stimulated rates of glycogen synthesis, incorporation of glucose into lipids, and glucose oxidation to values corresponding to approximately 30%, approximately 60%, and 30% of the controls, respectively. These effects were accompanied by an increase in basal and insulin-stimulated phosphorylation of Akt(Thr308), Akt(Ser473), and GSK3alpha/beta. Troglitazone also powerfully suppressed pyruvate decarboxylation, which was followed by a significant increase in basal ( approximately 3.5-fold) and insulin-stimulated ( approximately 5.5-fold) rates of lactate production by muscle cells. In summary, we provide novel evidence that troglitazone exerts acute insulin sensitizing effects by increasing FA oxidation, reducing FA uptake, suppressing pyruvate dehydrogenase activity, and shifting glucose metabolism toward lactate production in muscle cells. These effects seem to be at least partially dependent on AMPK activation and may account for potential acute PPAR-gamma-independent anti-diabetic effects of thiazolidinediones in skeletal

  13. Site-specific bioconjugation of an organometallic electron mediator to an enzyme with retained photocatalytic cofactor regenerating capacity and enzymatic activity.

    PubMed

    Lim, Sung In; Yoon, Sungho; Kim, Yong Hwan; Kwon, Inchan

    2015-04-07

    Photosynthesis consists of a series of reactions catalyzed by redox enzymes to synthesize carbohydrates using solar energy. In order to take the advantage of solar energy, many researchers have investigated artificial photosynthesis systems mimicking the natural photosynthetic enzymatic redox reactions. These redox reactions usually require cofactors, which due to their high cost become a key issue when constructing an artificial photosynthesis system. Combining a photosensitizer and an Rh-based electron mediator (RhM) has been shown to photocatalytically regenerate cofactors. However, maintaining the high concentration of cofactors available for efficient enzymatic reactions requires a high concentration of the expensive RhM; making this process cost prohibitive. We hypothesized that conjugation of an electron mediator to a redox enzyme will reduce the amount of electron mediators necessary for efficient enzymatic reactions. This is due to photocatalytically regenerated NAD(P)H being readily available to a redox enzyme, when the local NAD(P)H concentration near the enzyme becomes higher. However, conventional random conjugation of RhM to a redox enzyme will likely lead to a substantial loss of cofactor regenerating capacity and enzymatic activity. In order to avoid this issue, we investigated whether bioconjugation of RhM to a permissive site of a redox enzyme retains cofactor regenerating capacity and enzymatic activity. As a model system, a RhM was conjugated to a redox enzyme, formate dehydrogenase obtained from Thiobacillus sp. KNK65MA (TsFDH). A RhM-containing azide group was site-specifically conjugated to p-azidophenylalanine introduced to a permissive site of TsFDH via a bioorthogonal strain-promoted azide-alkyne cycloaddition and an appropriate linker. The TsFDH-RhM conjugate exhibited retained cofactor regenerating capacity and enzymatic activity.

  14. NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

    SciTech Connect

    Yang, Chuen-Mao; Lee, I-Ta; Hsu, Ru-Chun; Chi, Pei-Ling; Hsiao, Li-Der

    2013-10-15

    TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47{sup phox}, p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-α induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-α induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF

  15. A core catalytic domain of the TyrA protein family: arogenate dehydrogenase from Synechocystis

    PubMed Central

    2004-01-01

    The TyrA protein family includes prephenate dehydrogenases, cyclohexadienyl dehydrogenases and TyrAas (arogenate dehydrogenases). tyrAa from Synechocystis sp. PCC 6803, encoding a 30 kDa TyrAa protein, was cloned into an overexpression vector in Escherichia coli. TyrAa was then purified to apparent homogeneity and characterized. This protein is a model structure for a catalytic core domain in the TyrA superfamily, uncomplicated by allosteric or fused domains. Competitive inhibitors acting at the catalytic core of TyrA proteins are analogues of any accepted cyclohexadienyl substrate. The homodimeric enzyme was specific for L-arogenate (Km=331 μM) and NADP+ (Km=38 μM), being unable to substitute prephenate or NAD+ respectively. L-Tyrosine was a potent inhibitor of the enzyme (Ki=70 μM). NADPH had no detectable ability to inhibit the reaction. Although the mechanism is probably steady-state random order, properties of 2′,5′-ADP as an inhibitor suggest a high preference for L-arogenate binding first. Comparative enzymology established that both of the arogenate-pathway enzymes, prephenate aminotransferase and TyrAa, were present in many diverse cyanobacteria and in a variety of eukaryotic red and green algae. PMID:15171683

  16. Decavanadate inhibits the cell-free activation of neutrophil NADPH oxidase without affecting tyrosine phosphorylation.

    PubMed

    Okamura, N; Sakai, T; Nishimura, Y; Sakai, M; Araki, S; Yamaguchi, M; Ishibashi, S

    1999-08-01

    NADPH oxidase was activated by arachidonate in a cell-free system consisting of membrane and cytosol fractions prepared from guinea pig neutrophils. Vanadate apparently inhibited the NADPH oxidase activity in the cell-free system (IC50=2 microM) without phosphotyrosine accumulation. The pH dependency and stability of the inhibitory effect observed for vanadate solution indicated that decavanadate, an isopolyanion of vanadate, was responsible for the inhibition. Pervanadate (vanadyl hydroperoxide) also inhibited the oxidase activity but at a higher concentration (IC50=0.2 mM). Decavanadate lowered the Vmax but did not affect the Km value of NADPH oxidase for NADPH. Decavanadate inhibited the activation process of NADPH oxidase but not the oxidase activity itself. Decavanadate-pretreatment of membrane and cytosol fractions irreversibly decreased the abilities of both fractions to activate NADPH oxidase in the cell-free system. Translocation of p47-phox, one of the cytosolic activation factors of NADPH oxidase, from cytosol to membrane, was little affected by decavanadate. These results suggest that decavanadate inhibits the activation of NADPH oxidase in the cell-free system without affecting the phosphotyrosine phosphatase, and that decavanadate can bind to both the membrane and cytosolic activation factors when they are in a dormant state, but not to the active oxidase complex.

  17. Identification of the NAD(P)H binding site of eukaryotic UDP-galactopyranose mutase.

    PubMed

    Dhatwalia, Richa; Singh, Harkewal; Solano, Luis M; Oppenheimer, Michelle; Robinson, Reeder M; Ellerbrock, Jacob F; Sobrado, Pablo; Tanner, John J

    2012-10-31

    UDP-galactopyranose mutase (UGM) plays an essential role in galactofuranose biosynthesis in microorganisms by catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The enzyme has gained attention recently as a promising target for the design of new antifungal, antitrypanosomal, and antileishmanial agents. Here we report the first crystal structure of UGM complexed with its redox partner NAD(P)H. Kinetic protein crystallography was used to obtain structures of oxidized Aspergillus fumigatus UGM (AfUGM) complexed with NADPH and NADH, as well as reduced AfUGM after dissociation of NADP(+). NAD(P)H binds with the nicotinamide near the FAD isoalloxazine and the ADP moiety extending toward the mobile 200s active site flap. The nicotinamide riboside binding site overlaps that of the substrate galactopyranose moiety, and thus NADPH and substrate binding are mutually exclusive. On the other hand, the pockets for the adenine of NADPH and uracil of the substrate are distinct and separated by only 6 Å, which raises the possibility of designing novel inhibitors that bind both sites. All 12 residues that contact NADP(H) are conserved among eukaryotic UGMs. Residues that form the AMP pocket are absent in bacterial UGMs, which suggests that eukaryotic and bacterial UGMs have different NADP(H) binding sites. The structures address the longstanding question of how UGM binds NAD(P)H and provide new opportunities for drug discovery.

  18. Separating NADH and NADPH fluorescence in live cells and tissues using FLIM

    NASA Astrophysics Data System (ADS)

    Blacker, Thomas S.; Mann, Zoe F.; Gale, Jonathan E.; Ziegler, Mathias; Bain, Angus J.; Szabadkai, Gyorgy; Duchen, Michael R.

    2014-05-01

    NAD is a key determinant of cellular energy metabolism. In contrast, its phosphorylated form, NADP, plays a central role in biosynthetic pathways and antioxidant defence. The reduced forms of both pyridine nucleotides are fluorescent in living cells but they cannot be distinguished, as they are spectrally identical. Here, using genetic and pharmacological approaches to perturb NAD(P)H metabolism, we find that fluorescence lifetime imaging (FLIM) differentiates quantitatively between the two cofactors. Systematic manipulations to change the balance between oxidative and glycolytic metabolism suggest that these states do not directly impact NAD(P)H fluorescence decay rates. The lifetime changes observed in cancers thus likely reflect shifts in the NADPH/NADH balance. Using a mathematical model, we use these experimental data to quantify the relative levels of NADH and NADPH in different cell types of a complex tissue, the mammalian cochlea. This reveals NADPH-enriched populations of cells, raising questions about their distinct metabolic roles.

  19. A NADPH-dependent (S)-imine reductase (SIR) from Streptomyces sp. GF3546 for asymmetric synthesis of optically active amines: purification, characterization, gene cloning, and expression.

    PubMed

    Mitsukura, Koichi; Kuramoto, Tatsuya; Yoshida, Toyokazu; Kimoto, Norihiro; Yamamoto, Hiroaki; Nagasawa, Toru

    2013-09-01

    A NADPH-dependent (S)-imine reductase (SIR) was purified to be homogeneous from the cell-free extract of Streptomyces sp. GF3546. SIR appeared to be a homodimer protein with subunits of 30.5 kDa based on SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It also catalyzed the (S)-enantioselective reduction of not only 2-methyl-1-pyrroline (2-MPN) but also 1-methyl-3,4-dihydroisoquinoline and 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline. Specific activities for their imines were 130, 44, and 2.6 nmol min(-1) mg(-1), and their optical purities were 92.7 % ee, 96.4 % ee, and >99 % ee, respectively. Using a NADPH-regenerating system, 10 mM 2-MPN was converted to amine with 100 % conversion and 92 % ee after 24 h. The amino acid sequence analysis revealed that SIR showed about 60 % identity to 6-phosphogluconate dehydrogenase. However, it showed only 37 % identity with Streptomyces sp. GF3587 (R)-imine reductase. Expression of SIR in Escherichia coli was achieved, and specific activity of the cell-free extract was about two times higher than that of the cell-free extract of Streptomyces sp. GF3546.

  20. Antioxidant Protection of NADPH-Depleted Oligodendrocyte Precursor Cells Is Dependent on Supply of Reduced Glutathione

    PubMed Central

    Kilanczyk, Ewa; Saraswat Ohri, Sujata; Whittemore, Scott R.

    2016-01-01

    The pentose phosphate pathway is the main source of NADPH, which by reducing oxidized glutathione, contributes to antioxidant defenses. Although oxidative stress plays a major role in white matter injury, significance of NADPH for oligodendrocyte survival has not been yet investigated. It is reported here that the NADPH antimetabolite 6-amino-NADP (6AN) was cytotoxic to cultured adult rat spinal cord oligodendrocyte precursor cells (OPCs) as well as OPC-derived oligodendrocytes. The 6AN-induced necrosis was preceded by increased production of superoxide, NADPH depletion, and lower supply of reduced glutathione. Moreover, survival of NADPH-depleted OPCs was improved by the antioxidant drug trolox. Such cells were also protected by physiological concentrations of the neurosteroid dehydroepiandrosterone (10−8 M). The protection by dehydroepiandrosterone was associated with restoration of reduced glutathione, but not NADPH, and was sensitive to inhibition of glutathione synthesis. A similar protective mechanism was engaged by the cAMP activator forskolin or the G protein-coupled estrogen receptor (GPER/GPR30) ligand G1. Finally, treatment with the glutathione precursor N-acetyl cysteine reduced cytotoxicity of 6AN. Taken together, NADPH is critical for survival of OPCs by supporting their antioxidant defenses. Consequently, injury-associated inhibition of the pentose phosphate pathway may be detrimental for the myelination or remyelination potential of the white matter. Conversely, steroid hormones and cAMP activators may promote survival of NADPH-deprived OPCs by increasing a NADPH-independent supply of reduced glutathione. Therefore, maintenance of glutathione homeostasis appears as a critical effector mechanism for OPC protection against NADPH depletion and preservation of the regenerative potential of the injured white matter. PMID:27449129

  1. Arctigenin reduces blood pressure by modulation of nitric oxide synthase and NADPH oxidase expression in spontaneously hypertensive rats.

    PubMed

    Liu, Ying; Wang, Guoyuan; Yang, Mingguang; Chen, Haining; zhao, Yan; Yang, Shucai; Sun, Changhao

    2015-12-25

    Arctigenin is a bioactive constituent from dried seeds of Arctium lappa L., which was traditionally used as medicine. Arctigenin exhibits various bioactivities, but its effects on blood pressure regulation are still not widely studied. In this study, we investigated antihypertensive effects of arctigenin by long-term treatment in spontaneously hypertensive rats (SHRs). Arctigenin (50 mg/kg) or vehicle was administered to SHRs or Wistar rats as negative control by oral gavage once a day for total 8 weeks. Nifedipine (3 mg/kg) was used as a positive drug control. After treatment, hemodynamic and physical parameters, vascular reactivity in aorta, the concentration of plasma arctigenin and serum thromboxane B2, NO release and vascular p-eNOS, p-Akt, caveolin-1 protein expression, and vascular superoxide anion generation and p47phox protein expression were detected and analyzed. The results showed that arctigenin significantly reduced systolic blood pressure and ameliorated endothelial dysfunction of SHRs. Arctigenin reduced the levels of thromboxane B2 in plasma and superoxide anion in thoracic aorta of SHRs. Furthermore, arctigenin increased the NO production by enhancing the phosphorylation of Akt and eNOS (Ser 1177), and inhibiting the expression of NADPH oxidase in thoracic aorta of SHRs. Our data suggested that antihypertensive mechanisms of arctigenin were associated with enhanced eNOS phosphorylation and decreased NADPH oxidase-mediated superoxide anion generation.

  2. Involvement of NADPH oxidase in high-dose phenolic acid-induced pro-oxidant activity on rat mesenteric venules.

    PubMed

    Du, Wen-Yuan; Xiao, Ying; Yao, Jian-Jing; Hao, Zhe; Zhao, Yu-Bin

    2017-01-01

    In the present study, we investigated the potential role of phenolic acids in initiating oxidative damage to microvascular endothelial cells and the underlying mechanism mediating the pro-oxidant action. Male Wistar rats received high doses of phenolic acid [caffeic acid (CA), salvianolic acid B (SAB), chlorogenic acid (ChA) or ferulic acid (FA)]. The creation of reactive oxygen species in mesenteric microcirculation endothelial cells and adherent leukocytes along with venules were assessed using intravital microscopy. The expression levels of NADPH oxidase subunits (Nox4 and p22(phox)) in terminal ileum tissues were determined by western blot analysis. Intravenous injection of high-dose ChA or CA (7 mg/kg) markedly increased the peroxide production in the venular walls and upregulated the protein expression levels of Nox4 and p22(phox) in the ileum tissues, while the same dose of CA and SAB made no difference within the observation period. No changes were observed in the number of leukocytes adhering to the venular walls. High-dose ChA and FA led to an imbalance between the oxidant and antioxidant mechanism by boosting the expression levels of NADPH oxidase. Thus, we clarified the rationale behind the adverse effects of a herbal injection containing high levels of phenolic acid compounds.

  3. Hydrogen peroxide produced by NADPH oxidases increases proline accumulation during salt or mannitol stress in Arabidopsis thaliana.

    PubMed

    Ben Rejeb, Kilani; Lefebvre-De Vos, Delphine; Le Disquet, Isabel; Leprince, Anne-Sophie; Bordenave, Marianne; Maldiney, Régis; Jdey, Asma; Abdelly, Chedly; Savouré, Arnould

    2015-12-01

    Many plants accumulate proline, a compatible osmolyte, in response to various environmental stresses such as water deficit and salinity. In some stress responses, plants generate hydrogen peroxide (H2 O2 ) that mediates numerous physiological and biochemical processes. The aim was to study the relationship between stress-induced proline accumulation and H2 O2 production. Using pharmacological and reverse genetic approaches in Arabidopsis thaliana, we investigated the role of NADPH oxidases, Respiratory burst oxidase homologues (Rboh), in the induction of proline accumulation was investigated in response to stress induced by either 200 mM NaCl or 400 mM mannitol. Stress from NaCl or mannitol resulted in a transient increase in H2 O2 content accompanied by accumulation of proline. Dimethylthiourea, a scavenger of H2 O2 , and diphenylene iodonium (DPI), an inhibitor of H2 O2 production by NADPH oxidase, were found to significantly inhibit proline accumulation in these stress conditions. DPI also reduced the expression level of Δ(1) -pyrroline-5-carboxylate synthetase, the key enzyme involved in the biosynthesis of proline. Similarly, less proline accumulated in knockout mutants lacking either AtRbohD or AtRbohF than in wild-type plants in response to the same stresses. Our data demonstrate that AtRbohs (A. thaliana Rbohs) contribute to H2 O2 production in response to NaCl or mannitol stress to increase proline accumulation in this plant.

  4. NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL-1β secretion but not in inflammasome activation.

    PubMed

    Gabelloni, María Laura; Sabbione, Florencia; Jancic, Carolina; Fuxman Bass, Juan; Keitelman, Irene; Iula, Leonardo; Oleastro, Matías; Geffner, Jorge R; Trevani, Analía S

    2013-12-01

    Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin-1 beta (IL-1β) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL-1β processing is still controversial and has not been previously studied in neutrophils. We report here that IL-1β processing in human neutrophils is dependent on caspase-1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase-1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL-1β; whereas ROS negatively controlled caspase-1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL-1β out of the cell, a role never previously described. The complex ROS-mediated regulation of neutrophil IL-1β secretion might constitute a physiological mechanism to control IL-1β-dependent inflammatory processes where neutrophils play a crucial role.

  5. Role of NADPH Oxidase-4 in Human Endothelial Progenitor Cells

    PubMed Central

    Hakami, Nora Y.; Ranjan, Amaresh K.; Hardikar, Anandwardhan A.; Dusting, Greg J.; Peshavariya, Hitesh M.

    2017-01-01

    Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood. Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts. Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs. Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization. PMID:28386230

  6. Nox4 NADPH oxidase-derived reactive oxygen species, via endogenous carbon monoxide, promote survival of brain endothelial cells during TNF-α-induced apoptosis

    PubMed Central

    Basuroy, Shyamali; Tcheranova, Dilyara; Bhattacharya, Sujoy; Leffler, Charles W.

    2011-01-01

    We investigated the role of reactive oxygen species (ROS) in promoting cell survival during oxidative stress induced by the inflammatory mediator tumor necrosis factor-α (TNF-α) in cerebral microvascular endothelial cells (CMVEC) from newborn piglets. Nox4 is the major isoform of NADPH oxidase responsible for TNF-α-induced oxidative stress and apoptosis in CMVEC. We present novel data that Nox4 NADPH oxidase-derived ROS also initiate a cell survival mechanism by increasing production of a gaseous antioxidant mediator carbon monoxide (CO) by constitutive heme oxygenase-2 (HO-2). TNF-α rapidly enhanced endogenous CO production in a superoxide- and NADPH oxidase-dependent manner in CMVEC with innate, but not with small interfering RNA (siRNA)-downregulated Nox4 activity. CORM-A1, a CO-releasing compound, inhibited Nox4-mediated ROS production and enhanced cell survival in TNF-α-challenged CMVEC. The ROS-induced CO-mediated survival mechanism requires functional interactions between the protein kinase B/Akt and extracellular signal-related kinase (ERK)/p38 MAPK signaling pathways activated by TNF-α. In Akt siRNA-transfected CMVEC and in cells with pharmacologically inhibited Akt, Erk1/2, and p38 mitogen-activated protein kinase (MAPK) activities, CORM-A1 was no longer capable of blocking Nox4 activation and apoptosis caused by TNF-α. Overall, Nox4 NADPH oxidase-derived ROS initiate both death and survival pathways in TNF-α-challenged CMVEC. The ROS-dependent cell survival pathway is mediated by an endogenous antioxidant CO, which inhibits Nox4 activation via a mechanism that includes Akt, ERK1/2, and p38 MAPK signaling pathways. The ability of CO to inhibit TNF-α-induced ERK1/2 and p38 MAPK activities in an Akt-dependent manner appears to be the key element in ROS-dependent survival of endothelial cells during TNF-α-mediated brain inflammatory disease. PMID:21123734

  7. Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity.

    PubMed

    Maddock, Danielle J; Patrick, Wayne M; Gerth, Monica L

    2015-08-01

    Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2'-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary-secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2'-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme-cofactor interactions.

  8. Comparative 13C metabolic flux analysis of pyruvate dehydrogenase complex-deficient, L-valine-producing Corynebacterium glutamicum.

    PubMed

    Bartek, Tobias; Blombach, Bastian; Lang, Siegmund; Eikmanns, Bernhard J; Wiechert, Wolfgang; Oldiges, Marco; Nöh, Katharina; Noack, Stephan

    2011-09-01

    L-Valine can be formed successfully using C. glutamicum strains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-type C. glutamicum and four PDHC-deficient strains were compared by (13)C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH for l-valine formation. In accordance, the introduction of the Escherichia coli transhydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into an L-valine-producing C. glutamicum strain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated for L-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP.

  9. NADPH Oxidase Deficient Mice Develop Colitis and Bacteremia upon Infection with Normally Avirulent, TTSS-1- and TTSS-2-Deficient Salmonella Typhimurium

    PubMed Central

    Slack, Emma Marie Caroline; Müller, Andreas J.; Kremer, Marcus; Van Maele, Laurye; Cayet, Delphine; Heikenwalder, Mathias; Sirard, Jean-Claude; Hardt, Wolf-Dietrich

    2013-01-01

    Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn’s disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb−/−) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tmavir; invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb−/− mice are efficiently infected by S.Tmavir and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb−/−Myd88−/− mice indicated that the S.Tmavir-inflicted disease in Cybb−/− mice hinges on CD11c+CX3CR1+ monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tmavir in the mucosal lamina propria. This provides important insights into microbe handling by the large

  10. Characterization of retinaldehyde dehydrogenase 3

    PubMed Central

    Graham, Caroline E.; Brocklehurst, Keith; Pickersgill, Richard W.; Warren, Martin J.

    2005-01-01

    RALDH3 (retinal dehydrogenase 3) was characterized by kinetic and binding studies, protein engineering, homology modelling, ligand docking and electrostatic-potential calculations. The major recognition determinant of an RALDH3 substrate was shown to be an eight-carbon chain bonded to the aldehyde group whose kinetic influence (kcat/Km at pH 8.5) decreases when shortened or lengthened. Surprisingly, the β-ionone ring of all-trans-retinal is not a major recognition site. The dissociation constants (Kd) of the complexes of RALDH3 with octanal, NAD+ and NADH were determined by intrinsic tryptophan fluorescence. The similarity of the Kd values for the complexes with NAD+ and with octanal suggests a random kinetic mechanism for RALDH3, in contrast with the ordered sequential mechanism often associated with aldehyde dehydrogenase enzymes. Inhibition of RALDH3 by tri-iodothyronine binding in competition with NAD+, predicted by the modelling, was established kinetically and by immunoprecipitation. Mechanistic implications of the kinetically influential ionizations with macroscopic pKa values of 5.0 and 7.5 revealed by the pH-dependence of kcat are discussed. Analogies with data for non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans, together with the present modelled structure of the thioacyl RALDH3, suggest (a) that kcat characterizes deacylation of this intermediate for specific substrates and (b) the assignment of the pKa of the major ionization (approximating to 7.5) to the perturbed carboxy group of Glu280 whose conjugate base is envisaged as supplying general base catalysis to attack of a water molecule. The macroscopic pKa of the minor ionization (5.0) is considered to approximate to that of the carboxy group of Glu488. PMID:16241904

  11. High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+

    PubMed Central

    Huang, Rui; Chen, Hui; Zhong, Chao; Kim, Jae Eung; Zhang, Yi-Heng Percival

    2016-01-01

    Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP+ to NAD+. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD+, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT. PMID:27587230

  12. Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase.

    PubMed

    Kobayashi, Michie; Ohura, Ikuko; Kawakita, Kazuhito; Yokota, Naohiko; Fujiwara, Masayuki; Shimamoto, Ko; Doke, Noriyuki; Yoshioka, Hirofumi

    2007-03-01

    Reactive oxygen species (ROS) are implicated in plant innate immunity. NADPH oxidase (RBOH; for Respiratory Burst Oxidase Homolog) plays a central role in the oxidative burst, and EF-hand motifs in the N terminus of this protein suggest possible regulation by Ca(2+). However, regulatory mechanisms are largely unknown. We identified Ser-82 and Ser-97 in the N terminus of potato (Solanum tuberosum) St RBOHB as potential phosphorylation sites. An anti-phosphopeptide antibody (pSer82) indicated that Ser-82 was phosphorylated by pathogen signals in planta. We cloned two potato calcium-dependent protein kinases, St CDPK4 and St CDPK5, and mass spectrometry analyses showed that these CDPKs phosphorylated only Ser-82 and Ser-97 in the N terminus of St RBOHB in a calcium-dependent manner. Ectopic expression of the constitutively active mutant of St CDPK5, St CDPK5VK, provoked ROS production in Nicotiana benthamiana leaves. The CDPK-mediated ROS production was disrupted by knockdown of Nb RBOHB in N. benthamiana. The loss of function was complemented by heterologous expression of wild-type potato St RBOHB but not by a mutant (S82A/S97A). Furthermore, the heterologous expression of St CDPK5VK phosphorylated Ser-82 of St RBOHB in N. benthamiana. These results suggest that St CDPK5 induces the phosphorylation of St RBOHB and regulates the oxidative burst.

  13. Legume NADPH Oxidases Have Crucial Roles at Different Stages of Nodulation

    PubMed Central

    Montiel, Jesús; Arthikala, Manoj-Kumar; Cárdenas, Luis; Quinto, Carmen

    2016-01-01

    Plant NADPH oxidases, formerly known as respiratory burst oxidase homologues (RBOHs), are plasma membrane enzymes dedicated to reactive oxygen species (ROS) production. These oxidases are implicated in a wide variety of processes, ranging from tissue and organ growth and development to signaling pathways in response to abiotic and biotic stimuli. Research on the roles of RBOHs in the plant’s response to biotic stresses has mainly focused on plant-pathogen interactions; nonetheless, recent findings have shown that these oxidases are also involved in the legume-rhizobia symbiosis. The legume-rhizobia symbiosis leads to the formation of the root nodule, where rhizobia reduce atmospheric nitrogen to ammonia. A complex signaling and developmental pathway in the legume root hair and root facilitate rhizobial entrance and nodule organogenesis, respectively. Interestingly, several reports demonstrate that RBOH-mediated ROS production displays versatile roles at different stages of nodulation. The evidence collected to date indicates that ROS act as signaling molecules that regulate rhizobial invasion and also function in nodule senescence. This review summarizes discoveries that support the key and versatile roles of various RBOH members in the legume-rhizobia symbiosis. PMID:27213330

  14. Substance P Exacerbates Dopaminergic Neurodegeneration through Neurokinin-1 Receptor-Independent Activation of Microglial NADPH Oxidase

    PubMed Central

    Chu, Chun-Hsien; Qian, Li; Chen, Shih-Heng; Wilson, Belinda; Oyarzabal, Esteban; Jiang, Lulu; Ali, Syed; Robinson, Bonnie; Kim, Hyoung-Chun

    2014-01-01

    Although dysregulated substance P (SP) has been implicated in the pathophysiology of Parkinson's disease (PD), how SP affects the survival of dopaminergic neurons remains unclear. Here, we found that mice lacking endogenous SP (TAC1−/−), but not those deficient in the SP receptor (neurokinin-1 receptor, NK1R), were more resistant to lipopolysaccharide (LPS)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigral dopaminergic neurodegeneration than wild-type controls, suggesting a NK1R-independent toxic action of SP. In vitro dose–response studies revealed that exogenous SP enhanced LPS- and 1-methyl-4-phenylpyridinium (MPP+)-induced dopaminergic neurodegeneration in a bimodal manner, peaking at submicromolar and subpicomolar concentrations, but was substantially less effective at intermediate concentrations. Mechanistically, the actions of submicromolar levels of SP were NK1R-dependent, whereas subpicomolar SP-elicited actions required microglial NADPH oxidase (NOX2), the key superoxide-producing enzyme, but not NK1R. Subpicomolar concentrations of SP activated NOX2 by binding to the catalytic subunit gp91phox and inducing membrane translocation of the cytosolic subunits p47phox and p67phox. The importance of NOX2 was further corroborated by showing that inhibition or disruption of NOX2 blocked subpicomolar SP-exacerbated neurotoxicity. Together, our findings revealed a critical role of microglial NOX2 in mediating the neuroinflammatory and dopaminergic neurodegenerative effects of SP, which may provide new insights into the pathogenesis of PD. PMID:25209287

  15. Leonurine (SCM-198) attenuates myocardial fibrotic response via inhibition of NADPH oxidase 4.

    PubMed

    Liu, Xin-Hua; Pan, Li-Long; Deng, Hai-Yan; Xiong, Qing-Hui; Wu, Dan; Huang, Guo-Ying; Gong, Qi-Hai; Zhu, Yi-Zhun

    2013-01-01

    In our previous studies, we have reported that leonurine, a plant phenolic alkaloid in Herba leonuri, exerted cardioprotective properties in a number of preclinical experiments. Herein, we investigated the roles and the possible mechanisms of leonurine for reducing fibrotic responses in angiotensin II (Ang II)-stimulated primary neonatal rat cardiac fibroblasts and post-myocardial infarction (MI) rats. In in vitro experiments performed in neonatal rat cardiac fibroblasts, leonurine (10-20 μM) pretreatment attenuated Ang II-induced activation of extracellular signal-regulated kinase 1/2, production of intracellular reactive oxygen species (ROS), expression and activity of matrix metalloproteinase (MMP)-2/9, and expression of α-smooth muscle actin and types I and III collagen. A small interfering RNA-mediated knockdown strategy for NADPH oxidase 4 (Nox4) revealed that Nox4 was required for Ang II-induced activation of cardiac fibroblasts. In vivo studies using a post-MI model in rats indicated that administration of leonurine inhibited myocardial fibrosis while reducing cardiac Nox4 expression, ROS production, NF-κB activation, and plasma MMP-2 activity. In conclusion, our results provide the first evidence that leonurine could prevent cardiac fibrosis and the activation of cardiac fibroblasts partly through modulation of a Nox4-ROS pathway.

  16. Cellobiose dehydrogenase in cellulose degradation

    SciTech Connect

    Eriksson, L.; Igarashi, Kiyohiko; Samejima, Masahiro

    1996-10-01

    Cellobiose dehydrogenase is produced by a variety of fungi. Although it was already discovered during the 70`s, it`s role in cellulose and lignin degradation is yet ambiguous. The enzyme contains both heme and FAD as prosthetic groups, and seems to have a domain specifically designed to bind the enzyme to cellulose. It`s affinity to amorphous cellulose is higher than to crystalline cellulose. We will report on the binding behavior of the enzyme, its usefulness in elucidation of cellulose structures and also, possibilities for applications such as its use in measuring individual and synergistic mechanisms for cellulose degradation by endo- and exo-glucanases.

  17. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

    SciTech Connect

    Brown, Steven D; Guss, Adam M; Karpinets, Tatiana V; Parks, Jerry M; Smolin, Nikolai; Yang, Shihui; Land, Miriam L; Klingeman, Dawn Marie; Bhandiwad, Ashwini; Rodriguez, Jr., Miguel; Raman, Babu; Shao, Xiongjun; Mielenz, Jonathan R; Smith, Jeremy C; Keller, Martin; Lynd, Lee R

    2011-01-01

    Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

  18. The human short-chain dehydrogenase/reductase (SDR) superfamily: a bioinformatics summary.

    PubMed

    Bray, James E; Marsden, Brian D; Oppermann, Udo

    2009-03-16

    The short-chain dehydrogenase/reductase (SDR) superfamily represents one of the largest protein superfamilies known to date. Enzymes of this family usually catalyse NAD(P)(H) dependent reactions with a substrate spectrum ranging from polyols, retinoids, steroids and fatty acid derivatives to xenobiotics. We have currently identified 73 SDR superfamily members within the human genome. A status report of the human SDR superfamily is provided in terms of 3D structure determination, co-factor preferences, subcellular localisation and functional annotation. A simple scoring system for measuring structural and functional information (SFS score) has also been introduced to monitor the status of 5 key metrics. Currently there are 17 SDR members with an SFS score of zero indicating that almost a quarter of the human SDR superfamily lacks substantial functional annotation.

  19. Enantioselective bioconversion using Escherichia coli cells expressing Saccharomyces cerevisiae reductase and Bacillus subtilis glucose dehydrogenase.

    PubMed

    Park, Hyun Joo; Jung, Jihye; Choi, Hyejeong; Uhm, Ki-Nam; Kim, Hyung Kwoun

    2010-09-01

    Ethyl (R, S)-4-chloro-3-hydroxybutanoate (ECHB) is a useful chiral building block for the synthesis of L-carnitine and hypercholesterolemia drugs. The yeast reductase, YOL151W (GenBank locus tag), exhibits an enantioselective reduction activity, converting ethyl-4-chlorooxobutanoate (ECOB) exclusively into (R)-ECHB. YOL151W was generated in Escherichia coli cells and purified via Ni- NTA and desalting column chromatography. It evidenced an optimum temperature of 45 degrees C and an optimum pH of 6.5-7.5. Bacillus subtilis glucose dehydrogenase (GDH) was also expressed in Escherichia coli, and was used for the recycling of NADPH, required for the reduction reaction. Thereafter, Escherichia coli cells co-expressing YOL151W and GDH were constructed. After permeablization treatment, the Escherichia coli whole cells were utilized for ECHB synthesis. Through the use of this system, the 30 mM ECOB substrate could be converted to (R)-ECHB.

  20. Effects of deletion of glycerol-3-phosphate dehydrogenase and glutamate dehydrogenase genes on glycerol and ethanol metabolism in recombinant Saccharomyces cerevisiae.

    PubMed

    Kim, Jin-Woo; Chin, Young-Wook; Park, Yong-Cheol; Seo, Jin-Ho

    2012-01-01

    Bioethanol is currently used as an alternative fuel for gasoline worldwide. For economic production of bioethanol by Saccharomyces cerevisiae, formation of a main by-product, glycerol, should be prevented or minimized in order to reduce a separation cost of ethanol from fermentation broth. In this study, S. cerevisiae was engineered to investigate the effects of the sole and double disruption of NADH-dependent glycerol-3-phosphate dehydrogenase 1 (GPD1) and NADPH-requiring glutamate dehydrogenase 1 (GDH1) on the production of glycerol and ethanol from glucose. Even though sole deletion of GPD1 or GDH1 reduced glycerol production, double deletion of GPD1 and GDH1 resulted in the lowest glycerol concentration of 2.31 g/L, which was 46.4% lower than the wild-type strain. Interestingly, the recombinant S. cerevisiae ∆GPD1∆GDH1 strain showed a slight improvement in ethanol yield (0.414 g/g) compared with the wild-type strain (0.406 g/g). Genetic engineering of the glycerol and glutamate metabolic pathways modified NAD(P)H-requiring metabolic pathways and exerted a positive effect on glycerol reduction without affecting ethanol production.

  1. Nitric Oxide Synthase and Neuronal NADPH Diaphorase are Identical in Brain and Peripheral Tissues

    NASA Astrophysics Data System (ADS)

    Dawson, Ted M.; Bredt, David S.; Fotuhi, Majid; Hwang, Paul M.; Snyder, Solomon H.

    1991-09-01

    NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase-containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity.

  2. Importance of glucose-6-phosphate dehydrogenase (G6PDH) for vanillin tolerance in Saccharomyces cerevisiae.

    PubMed

    Nguyen, Trinh Thi My; Kitajima, Sakihito; Izawa, Shingo

    2014-09-01

    Vanillin is derived from lignocellulosic biomass and, as one of the major biomass conversion inhibitors, inhibits yeast growth and fermentation. Vanillin was recently shown to induce the mitochondrial fragmentation and formation of mRNP granules such as processing bodies and stress granules in Saccharomyces cerevisiae. Furfural, another major biomass conversion inhibitor, also induces oxidative stress and is reduced in an NAD(P)H-dependent manner to its less toxic alcohol derivative. Therefore, the pentose phosphate pathway (PPP), through which most NADPH is generated, plays a role in tolerance to furfural. Although vanillin also induces oxidative stress and is reduced to vanillyl alcohol in a NADPH-dependent manner, the relationship between vanillin and PPP has not yet been investigated. In the present study, we examined the importance of glucose-6-phosphate dehydrogenase (G6PDH), which catalyzes the rate-limiting NADPH-producing step in PPP, for yeast tolerance to vanillin. The growth of the null mutant of G6PDH gene (zwf1Δ) was delayed in the presence of vanillin, and vanillin was efficiently reduced in the culture of wild-type cells but not in the culture of zwf1Δ cells. Furthermore, zwf1Δ cells easily induced the activation of Yap1, an oxidative stress responsive transcription factor, mitochondrial fragmentation, and P-body formation with the vanillin treatment, which indicated that zwf1Δ cells were more susceptible to vanillin than wild type cells. These findings suggest the importance of G6PDH and PPP in the response of yeast to vanillin.

  3. Glioma-derived mutations in isocitrate dehydrogenase 2 beneficial to traditional chemotherapy

    SciTech Connect

    Fu, Yuejun; Huang, Rui; Zheng, Yali; Zhang, Zhiyun; Liang, Aihua

    2011-07-01

    Highlights: {yields} IDH1 and IDH2 mutations are not detected in the rat C6 glioma cell line model. {yields} IDH2 mutations are not required for the tumorigenesis of glioma. {yields} IDH2{sup R172G} can sensitize glioma sensitivity to chemotherapy through NADPH levels. {yields} IDH2{sup R172G} can give a benefit to traditional chemotherapy of glioma. {yields} This finding serves as an important complement to existing research on this topic. -- Abstract: Heterozygous mutations in either the R132 residue of isocitrate dehydrogenase I (IDH1) or the R172 residue of IDH2 in human gliomas were recently highlighted. In the present study, we report that mutations of IDH1 and IDH2 are not detected in the rat C6 glioma cell line model, which suggests that these mutations are not required for the development of glioblastoma induced by N,N'-nitroso-methylurea. The effects of IDH2 and IDH2{sup R172G} on C6 cells proliferation and sensitivity to chemotherapy and the possible mechanism are analyzed at the cellular level. IDH1 and IDH2 mutations lead to simultaneous loss and gain of activities in the production of {alpha}-ketoglutarate ({alpha}-KG) and 2-hydroxyglutarate (2HG), respectively, and result in lowering NADPH levels even further. The low NADPH levels can sensitize tumors to chemotherapy, and account for the prolonged survival of patients harboring the mutations. Our data extrapolate potential importance of the in vitro rat C6 glioma cell model, show that the IDH2{sup R172G} mutation in gliomas may give a benefit to traditional chemotherapy of this cancer and serve as an important complement to existing research on this topic.

  4. Chlorella induces stomatal closure via NADPH oxidase-dependent ROS production and its effects on instantaneous water use efficiency in Vicia faba.

    PubMed

    Li, Yan; Xu, Shan-Shan; Gao, Jing; Pan, Sha; Wang, Gen-Xuan

    2014-01-01

    Reactive oxygen species (ROS) have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs). Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2) scavenger, catalase (CAT), significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi) in Vicia faba via a reduction in leaf transpiration rate (E) without a parallel reduction in net photosynthetic rate (Pn) assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels.

  5. Deficient flavoprotein component of the NADPH-dependent O2-.-generating oxidase in the neutrophils from three male patients with chronic granulomatous disease.

    PubMed Central

    Gabig, T G; Lefker, B A

    1984-01-01

    The NADPH-dependent O2-.-generating oxidase in subcellular fractions from the neutrophils of three male patients with chronic granulomatous disease was compared with the corresponding preparations from normal neutrophils. The oxidase from normal neutrophils contained flavin adenine dinucleotide in an approximately 0.9:1 molar ratio with cytochrome b559. Each of the three chronic granulomatous disease patients had decreased amounts of the flavoprotein component of the oxidase fraction. The oxidase from two chronic granulomatous disease patients had undetectable amounts of cytochrome b559 whereas the third patient had a normal content of cytochrome b559, which was spectrally indistinguishable from the normal. The intrinsic cytochrome b559 in the oxidase fraction from stimulated neutrophils of the latter chronic granulomatous disease patient was not reduced by NADPH under anaerobic conditions, in distinction with the previously reported reduction of the normal cytochrome b559 under identical conditions. We conclude that the flavoprotein component of the oxidase may mediate transfer of electrons from NADPH to the cytochrome b559 in normal neutrophils, and that deficiency of this flavoprotein is associated with the chronic granulomatous disease phenotype in the three patients studied. PMID:6707199

  6. Deficient flavoprotein component of the NADPH-dependent O2-.-generating oxidase in the neutrophils from three male patients with chronic granulomatous disease.

    PubMed

    Gabig, T G; Lefker, B A

    1984-03-01

    The NADPH-dependent O2-.-generating oxidase in subcellular fractions from the neutrophils of three male patients with chronic granulomatous disease was compared with the corresponding preparations from normal neutrophils. The oxidase from normal neutrophils contained flavin adenine dinucleotide in an approximately 0.9:1 molar ratio with cytochrome b559. Each of the three chronic granulomatous disease patients had decreased amounts of the flavoprotein component of the oxidase fraction. The oxidase from two chronic granulomatous disease patients had undetectable amounts of cytochrome b559 whereas the third patient had a normal content of cytochrome b559, which was spectrally indistinguishable from the normal. The intrinsic cytochrome b559 in the oxidase fraction from stimulated neutrophils of the latter chronic granulomatous disease patient was not reduced by NADPH under anaerobic conditions, in distinction with the previously reported reduction of the normal cytochrome b559 under identical conditions. We conclude that the flavoprotein component of the oxidase may mediate transfer of electrons from NADPH to the cytochrome b559 in normal neutrophils, and that deficiency of this flavoprotein is associated with the chronic granulomatous disease phenotype in the three patients studied.

  7. Chlorella Induces Stomatal Closure via NADPH Oxidase-Dependent ROS Production and Its Effects on Instantaneous Water Use Efficiency in Vicia faba

    PubMed Central

    Li, Yan; Xu, Shan-Shan; Gao, Jing; Pan, Sha; Wang, Gen-Xuan

    2014-01-01

    Reactive oxygen species (ROS) have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs). Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2) scavenger, catalase (CAT), significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi) in Vicia faba via a reduction in leaf transpiration rate (E) without a parallel reduction in net photosynthetic rate (Pn) assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels. PMID:24687099

  8. NOX4 NADPH Oxidase-Dependent Mitochondrial Oxidative Stress in Aging-Associated Cardiovascular Disease

    PubMed Central

    Vendrov, Aleksandr E.; Vendrov, Kimberly C.; Smith, Alberto; Yuan, Jinling; Sumida, Arihiro; Robidoux, Jacques; Madamanchi, Nageswara R.

    2015-01-01

    Abstract Aims: Increased oxidative stress and vascular inflammation are implicated in increased cardiovascular disease (CVD) incidence with age. We and others demonstrated that NOX1/2 NADPH oxidase inhibition, by genetic deletion of p47phox, in Apoe−/− mice decreases vascular reactive oxygen species (ROS) generation and atherosclerosis in young age. The present study examined whether NOX1/2 NADPH oxidases are also pivotal to aging-associated CVD. Results: Both aged (16 months) Apoe−/− and Apoe−/−/p47phox−/− mice had increased atherosclerotic lesion area, aortic stiffness, and systolic dysfunction compared with young (4 months) cohorts. Cellular and mitochondrial ROS (mtROS) levels were significantly higher in aortic wall and vascular smooth muscle cells (VSMCs) from aged wild-type and p47phox−/− mice. VSMCs from aged mice had increased mitochondrial protein oxidation and dysfunction and increased vascular cell adhesion molecule 1 expression, which was abrogated with (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) treatment. NOX4 expression was increased in the vasculature and mitochondria of aged mice and its suppression with shRNA in VSMCs from aged mice decreased mtROS levels and improved function. Increased mtROS levels were associated with enhanced mitochondrial NOX4 expression in aortic VSMCs from aged subjects, and NOX4 expression levels in arterial wall correlated with age and atherosclerotic severity. Aged Apoe−/− mice treated with MitoTEMPO and 2-(2-chlorophenyl)-4-methyl-5-(pyridin-2-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione had decreased vascular ROS levels and atherosclerosis and preserved vascular and cardiac function. Innovation and Conclusion: These data suggest that NOX4, but not NOX1/2, and mitochondrial oxidative stress are mediators of CVD in aging under hyperlipidemic conditions. Regulating NOX4 activity/expression and using mitochondrial antioxidants are

  9. Cancer-associated isocitrate dehydrogenase mutations induce mitochondrial DNA instability.

    PubMed

    Kingsbury, Joanne M; Shamaprasad, Nachiketha; Billmyre, R Blake; Heitman, Joseph; Cardenas, Maria E

    2016-08-15

    A major advance in understanding the progression and prognostic outcome of certain cancers, such as low-grade gliomas, acute myeloid leukaemia, and chondrosarcomas, has been the identification of early-occurring mutations in the NADP(+)-dependent isocitrate dehydrogenase genes IDH1 and IDH2 These mutations result in the production of the onco-metabolite D-2-hydroxyglutarate (2HG), thought to contribute to disease progression. To better understand the mechanisms of 2HG pathophysiology, we introduced the analogous glioma-associated mutations into the NADP(+ )isocitrate dehydrogenase genes (IDP1, IDP2, IDP3) in Saccharomyces cerevisiae Intriguingly, expression of the mitochondrial IDP1(R148H) mutant allele results in high levels of 2HG production as well as extensive mtDNA loss and respiration defects. We find no evidence for a reactive oxygen-mediated mechanism mediating this mtDNA loss. Instead, we show that 2HG production perturbs the iron sensing mechanisms as indicated by upregulation of the Aft1-controlled iron regulon and a concomitant increase in iron levels. Accordingly, iron chelation, or overexpression of a truncated AFT1 allele that dampens transcription of the iron regulon, suppresses the loss of respirative capacity. Additional suppressing factors include overexpression of the mitochondrial aldehyde dehydrogenase gene ALD5 or disruption of the retrograde response transcription factor RTG1 Furthermore, elevated α-ketoglutarate levels also suppress 2HG-mediated respiration loss; consistent with a mechanism by which 2HG contributes to mtDNA loss by acting as a toxic α-ketoglutarate analog. Our findings provide insight into the mechanisms that may contribute to 2HG oncogenicity in glioma and acute myeloid leukaemia progression, with the promise for innovative diagnostic and prognostic strategies and novel therapeutic modalities.

  10. Expression and characterization of a cytosolic glucose 6 phosphate dehydrogenase isoform from barley (Hordeum vulgare) roots.

    PubMed

    Castiglia, Daniela; Cardi, Manuela; Landi, Simone; Cafasso, Donata; Esposito, Sergio

    2015-08-01

    In plant cells, glucose 6 phosphate dehydrogenase (G6PDH-EC 1.1.1.49) regulates the oxidative pentose phosphate pathway (OPPP), a metabolic route involved in the production of NADPH for various biosynthetic processes and stress response. In this study, we report the overexpression of a cytosolic G6PDH isoform from barley (Hordeum vulgare) roots in bacteria, and the biochemical characterization of the purified recombinant enzyme (HvCy-G6PDH). A full-length cDNA coding for a cytosolic isoform of G6PDH was isolated, and the sequence was cloned into pET3d vector; the protein was overexpressed in Escherichia coli BL21 (DE3) and purified by anion exchange and affinity chromatography. The kinetic properties were calculated: the recombinant HvCy-G6PDH showed KMs and KINADPH comparable to those observed for the enzyme purified from barley roots; moreover, the analysis of NADPH inhibition suggested a competitive mechanism. Therefore, this enzyme could be utilised for the structural and regulatory characterization of this isoform in higher plants.

  11. Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn-Glycerol-1-phosphate Dehydrogenase*

    PubMed Central

    Carbone, Vincenzo; Schofield, Linley R.; Zhang, Yanli; Sang, Carrie; Dey, Debjit; Hannus, Ingegerd M.; Martin, William F.; Sutherland-Smith, Andrew J.; Ronimus, Ron S.

    2015-01-01

    One of the most critical events in the origins of cellular life was the development of lipid membranes. Archaea use isoprenoid chains linked via ether bonds to sn-glycerol 1-phosphate (G1P), whereas bacteria and eukaryotes use fatty acids attached via ester bonds to enantiomeric sn-glycerol 3-phosphate. NAD(P)H-dependent G1P dehydrogenase (G1PDH) forms G1P and has been proposed to have played a crucial role in the speciation of the Archaea. We present here, to our knowledge, the first structures of archaeal G1PDH from the hyperthermophilic methanogen Methanocaldococcus jannaschii with bound substrate dihydroxyacetone phosphate, product G1P, NADPH, and Zn2+ cofactor. We also biochemically characterized the enzyme with respect to pH optimum, cation specificity, and kinetic parameters for dihydroxyacetone phosphate and NAD(P)H. The structures provide key evidence for the reaction mechanism in the stereospecific addition for the NAD(P)H-based pro-R hydrogen transfer and the coordination of the Zn2+ cofactor during catalysis. Structure-based phylogenetic analyses also provide insight into the origins of G1PDH. PMID:26175150

  12. Glucose-6-phosphate dehydrogenase deficiency (G6PD) as a risk factor of male neonatal sepsis.

    PubMed

    Rostami-Far, Z; Ghadiri, K; Rostami-Far, M; Shaveisi-Zadeh, F; Amiri, A; Rahimian Zarif, B

    2016-01-01

    Introduction.Neonatal sepsis is a disease process, which represents the systemic response of bacteria entering the bloodstream during the first 28 days of life. The prevalence of sepsis is higher in male infants than in females, but the exact cause is unknown. Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme in the pentose phosphate pathway, which leads to the production of NADPH. NADPH is required for the respiratory burst reaction in white blood cells (WBCs) to destroy microorganisms. The purpose of this study was to evaluate the prevalence of G6PD deficiency in neonates with sepsis. Materials and methods.This study was performed on 76 neonates with sepsis and 1214 normal neonates from February 2012 to November 2014 in the west of Iran. The G6PD deficiency status was determined by fluorescent spot test. WBCs number and neutrophils percentages were measured and compared in patients with and without G6PD deficiency. Results.The prevalence of the G6PD deficiency in neonates with sepsis was significantly higher compared to the control group (p=0.03). WBCs number and neutrophils percentages in G6PD deficient patients compared with patients without G6PD deficiency were decreased, but were not statistically significant (p=0.77 and p=0.86 respectively). Conclusions.G6PD deficiency is a risk factor of neonatal sepsis and also a justification for more male involvement in this disease. Therefore, newborn screening for this disorder is recommended.

  13. Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase.

    PubMed

    Madiraju, Anila K; Erion, Derek M; Rahimi, Yasmeen; Zhang, Xian-Man; Braddock, Demetrios T; Albright, Ronald A; Prigaro, Brett J; Wood, John L; Bhanot, Sanjay; MacDonald, Michael J; Jurczak, Michael J; Camporez, Joao-Paulo; Lee, Hui-Young; Cline, Gary W; Samuel, Varman T; Kibbey, Richard G; Shulman, Gerald I

    2014-06-26

    Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide knockdown of hepatic mitochondrial glycerophosphate dehydrogenase in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decreases in plasma glucose concentrations, and inhibition of endogenous glucose production. These findings were replicated in whole-body mitochondrial glycerophosphate dehydrogenase knockout mice. These results have significant implications for understanding the mechanism of metformin's blood glucose lowering effects and provide a new therapeutic target for type 2 diabetes.

  14. NADPH oxidase 4 protects against development of endothelial dysfunction and atherosclerosis in LDL receptor deficient mice

    PubMed Central

    Langbein, Heike; Brunssen, Coy; Hofmann, Anja; Cimalla, Peter; Brux, Melanie; Bornstein, Stefan R.; Deussen, Andreas; Koch, Edmund; Morawietz, Henning

    2016-01-01

    Aims Endothelial dysfunction is an early step in the development of atherosclerosis. Increased formation of superoxide anions by NADPH oxidase Nox1, 2, and 5 reduces nitric oxide availability and can promote endothelial dysfunction. In contrast, recent evidence supports a vasoprotective role of H2O2 produced by main endothelial isoform Nox4. Therefore, we analysed the impact of genetic deletion of Nox4 on endothelial dysfunction and atherosclerosis in the low-density lipoprotein receptor (Ldlr) knockout model. Methods and results Ex vivo analysis of endothelial function by Mulvany myograph showed impaired endothelial function in thoracic aorta of Nox4−/−/Ldlr−/− mice. Further progression of endothelial dysfunction due to high-fat diet increased atherosclerotic plaque burden and galectin-3 staining in Nox4−/−/Ldlr−/− mice compared with Ldlr−/− mice. Under physiological conditions, loss of Nox4 does not influence aortic vascular function. In this setting, loss of Nox4-derived H2O2 production could be partially compensated for by nNOS upregulation. Using an innovative optical coherence tomography approach, we were able to analyse endothelial function by flow-mediated vasodilation in the murine saphenous artery in vivo. This new approach revealed an altered flow-mediated dilation in Nox4−/− mice, indicating a role for Nox4 under physiological conditions in peripheral arteries in vivo. Conclusions Nox4 plays an important role in maintaining endothelial function under physiological and pathological conditions. Loss of Nox4-derived H2O2 could be partially compensated for by nNOS upregulation, but severe endothelial dysfunction is not reversible. This leads to increased atherosclerosis under atherosclerotic prone conditions. PMID:26578199

  15. ROS signaling by NADPH oxidase 5 modulates the proliferation and survival of prostate carcinoma cells

    PubMed Central

    Höll, Monika; Koziel, Rafal; Schäfer, Georg; Pircher, Haymo; Pauck, Alexander; Hermann, Martin; Klocker, Helmut; Jansen‐Dürr, Pidder

    2015-01-01

    Prostate cancer (PCa) is the most commonly diagnosed cancer and second leading cause of male cancer death in Western nations. Thus, new treatment modalities are urgently needed. Elevated production of reactive oxygen species (ROS) by NADPH oxidase (Nox) enzymes is implicated in tumorigenesis of the prostate and other tissues. However, the identity of the Nox enzyme(s) involved in prostate carcinogenesis remains largely unknown. Analysis of radical prostatectomy tissue samples and benign and malignant prostate epithelial cell lines identified Nox5 as an abundantly expressed Nox isoform. Consistently, immunohistochemical staining of a human PCa tissue microarray revealed distinct Nox5 expression in epithelial cells of benign and malignant prostatic glands. shRNA‐mediated knockdown of Nox5 impaired proliferation of Nox5‐expressing (PC‐3, LNCaP) but not Nox5‐negative (DU145) PCa cell lines. Similar effects were observed upon ROS ablation via the antioxidant N‐acetylcysteine confirming ROS as the mediators. In addition, Nox5 silencing increased apoptosis of PC‐3 cells. Concomitantly, protein kinase C zeta (PKCζ) protein levels and c‐Jun N‐terminal kinase (JNK) phosphorylation were reduced. Moreover, the effect of Nox5 knockdown on PC‐3 cell proliferation could be mimicked by pharmacological inhibition of JNK. Collectively, these data indicate that Nox5 is expressed at functionally relevant levels in the human prostate and clinical PCa. Moreover, findings herein suggest that Nox5‐derived ROS and subsequent depletion of PKCζ and JNK inactivation play a critical role in modulating intracellular signaling cascades involved in the proliferation and survival of PCa cells. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc. PMID:25559363

  16. Impaired NADPH oxidase activity in peripheral blood lymphocytes of galactosemia patients.

    PubMed

    Al-Essa, Mazen; Dhaunsi, Gursev S; Al-Qabandi, Wafa'a; Khan, Islam

    2013-07-01

    Galactosemia is an autosomal recessive disorder with a wide range of clinical abnormalities. Cellular oxidative stress is considered as one of the pathogenic mechanisms of galactosemia. In this study, we examined the activity of NADPH oxidase (NOX), a major superoxide-generating enzyme system, in peripheral blood lymphocytes (PBL) from galactosemia patients. PBL were isolated from galactosemia patients and healthy control subjects and used for cell culture studies and biochemical assays. PBL were cultured in the presence or absence of galactose or galactose-1-phosphate (Gal-1-P), and enzyme activities and/or gene expression of NOX, catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in the cell homogenates. PBL isolated from galactosemia patients showed significantly reduced (P < 0.01) activities of catalase and GPx; however SOD activity remained unaltered. Galactosemia patients were found to have significantly (P < 0.01) increased levels of malondialdehyde (MDA) in blood lymphocytes. Enzymatic activity of NOX was significantly (P < 0.001) reduced in galactosemia patients; however, Western blotting revealed that NOX-1 protein was not significantly altered. Interestingly, levels of NOX activity in lymphocytes isolated from galactosemia patients significantly increased but remained subnormal when cultured in galactose-deficient medium for two weeks, indicating a galactose-mediated inhibition of NOX. Lymphocytes isolated from control subjects were found to have significantly (P < 0.01) reduced NOX activity when cultured in the presence of galactose or Gal-1-P for two weeks. These results show that galactose-induced cellular oxidative stress is not NOX mediated. However, impairment of the NOX system might be responsible for some of the clinical complications in galactosemia patients.

  17. Elementary Flux Mode Analysis Revealed Cyclization Pathway as a Powerful Way for NADPH Regeneration of Central Carbon Metabolism.

    PubMed

    Rui, Bin; Yi, Yin; Shen, Tie; Zheng, Meijuan; Zhou, Wenwei; Du, Honglin; Fan, Yadong; Wang, Yongkang; Zhang, Zhengdong; Xu, Shengsheng; Liu, Zhijie; Wen, Han; Xie, Xiaoyao

    2015-01-01

    NADPH regeneration capacity is attracting growing research attention due to its important role in resisting oxidative stress. Besides, NADPH availability has been regarded as a limiting factor in production of industrially valuable compounds. The central carbon metabolism carries the carbon skeleton flux supporting the operation of NADPH-regenerating enzyme and offers flexibility in coping with NADPH demand for varied intracellular environment. To acquire an insightful understanding of its NADPH regeneration capacity, the elementary mode method was employed to compute all elementary flux modes (EFMs) of a network representative of central carbon metabolism. Based on the metabolic flux distributions of these modes, a cluster analysis of EFMs with high NADPH regeneration rate was conducted using the self-organizing map clustering algorithm. The clustering results were used to study the relationship between the flux of total NADPH regeneration and the flux in each NADPH producing enzyme. The results identified several reaction combinations supporting high NADPH regeneration, which are proven to be feasible in cells via thermodynamic analysis and coincident with a great deal of previous experimental report. Meanwhile, the reaction combinations showed some common characteristics: there were one or two decarboxylation oxidation reactions in the combinations that produced NADPH and the combination constitution included certain gluconeogenesis pathways. These findings suggested cyclization pathways as a powerful way for NADPH regeneration capacity of bacterial central carbon metabolism.

  18. Hydroxysteroid dehydrogenases (HSDs) in bacteria: a bioinformatic perspective.

    PubMed

    Kisiela, Michael; Skarka, Adam; Ebert, Bettina; Maser, Edmund

    2012-03-01

    Steroidal compounds including cholesterol, bile acids and steroid hormones play a central role in various physiological processes such as cell signaling, growth, reproduction, and energy homeostasis. Hydroxysteroid dehydrogenases (HSDs), which belong to the superfamily of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR), are important enzymes involved in the steroid hormone metabolism. HSDs function as an enzymatic switch that controls the access of receptor-active steroids to nuclear hormone receptors and thereby mediate a fine-tuning of the steroid response. The aim of this study was the identification of classified functional HSDs and the bioinformatic annotation of these proteins in all complete sequenced bacterial genomes followed by a phylogenetic analysis. For the bioinformatic annotation we constructed specific hidden Markov models in an iterative approach to provide a reliable identification for the specific catalytic groups of HSDs. Here, we show a detailed phylogenetic analysis of 3α-, 7α-, 12α-HSDs and two further functional related enzymes (3-ketosteroid-Δ(1)-dehydrogenase, 3-ketosteroid-Δ(4)(5α)-dehydrogenase) from the superfamily of SDRs. For some bacteria that have been previously reported to posses a specific HSD activity, we could annotate the corresponding HSD protein. The dominating phyla that were identified to express HSDs were that of Actinobacteria, Proteobacteria, and Firmicutes. Moreover, some evolutionarily more ancient microorganisms (e.g., Cyanobacteria and Euryachaeota) were found as well. A large number of HSD-expressing bacteria constitute the normal human gastro-intestinal flora. Another group of bacteria were originally isolated from natural habitats like seawater, soil, marine and permafrost sediments. These bacteria include polycyclic aromatic hydrocarbons-degrading species such as Pseudomonas, Burkholderia and Rhodococcus. In conclusion, HSDs are found in a wide variety of microorganisms including

  19. NADPH oxidases are critical targets for prevention of ethanol-induced bone loss

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular mechanisms through which chronic alcohol consumption induce bone loss and osteoporosis are largely unknown. Ethanol increases expression and activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase enzymes (Nox) in osteoblasts leading to accumulation of reactive oxygen spe...

  20. Histochemical localization of NADPH-diaphorase in neurons of the pheasant ileum.

    PubMed

    Schmidtová, Katarína; Kocisová, Monika; Sirot'áková, Mária

    2002-01-01

    Localization patterns of NADPH-diaphorase-positive neurons in the pheasant ileum were investigated using an enzyme histochemical method. NADPH-diaphorase activity in the pheasant ileum was demonstrated in neuronal cells bodies and nerve fibres. The NADPH-diaphorase-positive nerve cells showed a polygonal shape and were present solitary or arranged in groups in the submucosal and muscular layers. Nerve fibres penetrated the wall of the ileum at its serosal surface, frequently in the vicinity of ileal arterial branches. They were abundantly present in muscular and submucosal layers of the ileum forming thicker nerves. Some nerve fibres traversed the submucosa into the lamina propria mucosae to form dense nerve plexuses. Fine nerve fibres were found to penetrate into intestinal villi encompassing the crypts underneath the epithelium. We conclude that the pheasant ileum is characterized by abundance of NADPH-diaphorase-positive nerve structures which may play a significant functional role in the small intestine of the pheasant.

  1. [The distribution of NADPH-diaphorase and neuronal no synthase in rat medulla oblongata nuclei].

    PubMed

    Chertok, V M; Kotsuba, A E

    2013-01-01

    The distribution of nitroxide ergic neurons in the medulla oblongata nuclei in Wistar rats (n = 8) was studied histochemically (NADPH-diaphorase) and using immunohistochemistry with an antiserum against neuronal form of nitric oxide synthase (nNOS). NADPH-diaphorase activity was found in large and small neurons of the sensory, autonomic and motor nuclei. The latter were especially rich in the cells demonstrating the activity of the enzyme. Unlike NADPH-diaphorase, nNOS in the corresponding nuclei was always detected in the fewer number of neurons, predominantly of small sizes. The sensory nuclei (nucleus of solitary tract, reticular parvocellular and lateral nuclei, spinal nucleus of the trigeminal nerve) contained 1.5-3 times more nNOS neurons than in motor nuclei. In some nuclei (nucleus ambiguus, hypoglossal nerve nucleus), containing numerous NADPH-diaphorase-positive neurons, immunoreactive cells were particularly rare.

  2. Photoprotective role of NADPH:protochlorophyllide oxidoreductase A

    PubMed Central

    Buhr, Frank; El Bakkouri, Majida; Valdez, Oscar; Pollmann, Stephan; Lebedev, Nikolai; Reinbothe, Steffen; Reinbothe, Christiane

    2008-01-01

    A homology model of NADPH:protochlorophyllide (Pchlide) oxidoreductase A (POR; E.C. 1.3.33.1) of barley is developed and verified by site-directed mutagenesis. PORA is considered a globular protein consisting of nine α-helices and seven β-strands. The model predicts the presence of two functionally distinctive Pchlide binding sites where the pigment is coordinated by cystein residues. The pigment bound to the first, high-affinity Pchlide binding site is used for the formation of the photoactive state of the enzyme. The pigment bound to the second, low-affinity Pchlide binding site is involved in the PORA:PORB interaction, allowing for resonance energy transfer between the neighboring PORs in the complex. In the in vitro reconstituted light-harvesting POR:Pchlide complex (LHPP), light absorbed by PORA-bound Pchlide b is transferred to PORB-bound Pchlide a. That induces the conversion of Pchlide a to chlorophyllide (Chlide) a. This energy transfer eliminates the possibility of Pchlide b photoreduction and prevents that excited triplet states of either Pchlides a or b accumulate and provoke singlet oxygen production. Together, our results provide a photoprotective role of PORA during greening. PMID:18723681

  3. Functional Heterogeneity of Nadph Oxidases in Atherosclerotic and Aneurysmal Diseases

    PubMed Central

    Kigawa, Yasuyoshi; Lei, Xiao-Feng; Kim-Kaneyama, Joo-ri; Miyazaki, Akira

    2017-01-01

    NADPH oxidases (NOX) are enzymes that catalyze the production of reactive oxygen species (ROS). Four species of NOX catalytic homologs (NOX1, NOX2, NOX4, and NOX5) are reportedly expressed in vascular tissues. The pro-atherogenic roles of NOX1, NOX2, and their organizer protein p47phox were manifested, and it was noted that the hydrogen peroxide-generating enzyme NOX4 possesses atheroprotective effects. Loss of NOX1 or p47phox appears to ameliorate murine aortic dissection and subsequent aneurysmal diseases; in contrast, the ablation of NOX2 exacerbates the aneurysmal diseases. It is possible that the loss of NOX2 activates inflammatory cascades in macrophages in the lesions. Roles of NOX5 in vascular functions are currently undetermined, owing to the absence of this enzyme in rodents and the limitation of the experimental procedure. Thus, it is possible that the NOX family of enzymes exhibits heterogeneity in the atherosclerotic diseases. In this aspect, subtype-selective NOX inhibitor may be promising when NOX systems serve as a molecular target for atherosclerotic and aneurysmal diseases. PMID:27476665

  4. PKC/NADPH oxidase are involved in the protective effect of pioglitazone in high homocysteine-induced paracrine dyfunction in endothelial progenitor cells

    PubMed Central

    Xu, Shengjie; Zhao, Yanbo; Jin, Chongying; Yu, Lu; Ding, Fang; Fu, Guosheng; Zhu, Junhui

    2017-01-01

    Increasing evidence suggests that EPCs improve neovascularization and endothelial regeneration via the production of paracrine factors. VEGF and IL-8 are major cytokines involved in EPC-mediated angiogenesis and re-endothelialization. In our previous studies, Hcy impaired EPC migratory and adhesive activities. We devised this study to determine whether Hcy could affect the expression and secretion of VEGF and IL-8 from EPCs. We found that high levels of Hcy (100-500 μM) decreased the EPC-mediated protein secretion and mRNA expression of VEGF and IL-8. Moreover, PIO, a PPARγ agonist, has been suggested to regulate EPC adhesion, migration, survival. In this study, PIO normalized the production of these cytokines by EPCs stimulated with Hcy. These effects of Hcy and PIO were primarily mediated by PKC and ROS via NADPH oxidase. We further confirmed this mechanism via knockdown of the NADPH oxidase subunits p67phox and Nox2. Furthermore, the PPARγ inhibitor GW9662 was not observed to abrogate the beneficial effect of PIO, indicating that PIO protected EPC paracrine function against Hcy in a PPARγ-independent manner. PMID:28386331

  5. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  6. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  7. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  8. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  9. Opine dehydrogenases in marine invertebrates.

    PubMed

    Harcet, Matija; Perina, Drago; Pleše, Bruna

    2013-10-01

    It is well known today that opine production anaerobic pathways are analogs to the classical glycolytic pathway (lactate production pathway). These pathways, catalyzed by a group of enzymes called opine dehydrogenases (OpDHs), ensure continuous flux of glycolysis and a constant supply of ATP by maintaining the NADH/NAD(+) ratio during exercise and hypoxia, thus regulating the cytosolic redox balance in glycolysis under anoxia. OpDHs are distributed in a wide range of marine invertebrate phyla, including sponges (Porifera). Phylogenetic analyses supported with enzymatic assays strongly indicate that sponge OpDHs constitute an enzyme class unrelated to other OpDHs. Therefore, OpDHs in marine invertebrates are divided into two groups, a mollusk/annelid type and a sponge type, which belongs to the OCD/mu-crystallin family.

  10. Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

    PubMed Central

    Komati Reddy, Gajendar; Lindner, Steffen N.

    2015-01-01

    Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154–7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH

  11. L-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea.

    PubMed

    Deutch, Charles E

    2013-11-01

    The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. L-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP(+)-dependent enzymes from chloroplasts and was separated from the NAD(+)-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis-Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K m for oxaloacetate was 20 μM; the K m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD(+) increased with pH but there was very little activity with NADP(+). At pH 7.0, the K m for L-malate was 5 mM and the K m for NAD(+) was 24 μM. The reductive activity was quite insensitive to inhibition by L-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10-20 times higher than the oxidative activity. These results indicate that the L-malate dehydrogenase in N. europaea is similar to other NAD(+)-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.

  12. Fluoride-containing bioactive glasses inhibit pentose phosphate oxidative pathway and glucose 6-phosphate dehydrogenase activity in human osteoblasts.

    PubMed

    Bergandi, Loredana; Aina, Valentina; Garetto, Stefano; Malavasi, Gianluca; Aldieri, Elisabetta; Laurenti, Enzo; Matera, Lina; Morterra, Claudio; Ghigo, Dario

    2010-02-12

    Bioactive glasses such as Hench's 45S5 (Bioglass) have applications to tissue engineering as well as bone repair, and the insertion of fluoride in their composition has been proposed to enhance their bioactivity. In view of a potential clinical application, we investigated whether fluoride-containing glasses exert toxic effects on human MG-63 osteoblasts, and whether and how fluoride, which is released in the cell culture medium, might play a role in such cytotoxicity. A 24h incubation with 50 microg/ml (12.5 microg/cm(2)) of fluoride-containing bioactive glasses termed HCaCaF(2) (F content: 5, 10 and 15 mol.%) caused the release of lactate dehydrogenase in the extracellular medium (index of cytotoxicity), the accumulation of intracellular malonyldialdehyde (index of lipoperoxidation), and the increase of glutathione consumption. Furthermore, fluoride-containing glasses inhibited the pentose phosphate oxidative pathway and the glucose 6-phosphate dehydrogenase activity. These effects are ascribable to the fluoride content/release of glass powders, since they were mimicked by NaF solutions and were prevented by dimethyl sulfoxide and tempol (two radical scavengers), by superoxide dismutase (a superoxide scavenger), and by glutathione (the most important intracellular antioxidant molecule), but not by apocynin (an inhibitor of NADPH oxidase). The presence of fluoride-containing glasses and NaF caused also the generation of reactive oxygen species, which was prevented by superoxide dismutase and catalase. The data suggest that fluoride released from glasses is the cause of MG-63 cell oxidative damage and is independent of NADPH oxidase activation. Our data provide a new mechanism to explain F(-) ions toxicity: fluoride could trigger, at least in part, an oxidative stress via inhibition of the pentose phosphate oxidative pathway and, in particular, through the oxidative inhibition of glucose 6-phosphate dehydrogenase.

  13. Involvement of phospholipase D and NADPH-oxidase in salicylic acid signaling cascade.

    PubMed

    Kalachova, Tetiana; Iakovenko, Oksana; Kretinin, Sergii; Kravets, Volodymyr

    2013-05-01

    Salicylic acid is associated with the primary defense responses to biotic stress and formation of systemic acquired resistance. However, molecular mechanisms of early cell reactions to phytohormone application are currently undisclosed. The present study investigates the participation of phospholipase D and NADPH-oxidase in salicylic acid signal transduction cascade. The activation of lipid signaling enzymes within 15 min of salicylic acid application was shown in Arabidopsis thaliana plants by measuring the phosphatidic acid accumulation. Adding of primary alcohol (1-butanol) to the incubation medium led to phosphatidylbutanol accumulation as a result of phospholipase D (PLD) action in wild-type and NADPH-oxidase RbohD deficient plants. Salicylic acid induced rapid increase in NADPH-oxidase activity in histochemical assay with nitroblue tetrazolium but the reaction was not observed in presence of 1-butanol and NADPH-oxidase inhibitor diphenylene iodide (DPI). The further physiological effect of salicylic acid and inhibitory analysis of the signaling cascade were made in the guard cell model. Stomatal closure induced by salicylic acid was inhibited by 1-butanol and DPI treatment. rbohD transgenic plants showed impaired stomatal reaction upon phytohormone effect, while the reaction to H2O2 did not differ from that of wild-type plants. Thus a key role of NADPH-oxidase D-isoform in the process of stomatal closure in response to salicylic acid has been postulated. It has enabled to predict a cascade implication of PLD and NADPH oxidase to salicylic acid signaling pathway.

  14. LMP1 Increases Expression of NADPH Oxidase (NOX) and Its Regulatory Subunit p22 in NP69 Nasopharyngeal Cells and Makes Them Sensitive to a Treatment by a NOX Inhibitor

    PubMed Central

    Zhu, Yinghui; Sun, Rui; Fang, Yujing; Fan, Yuhua; Xu, Fei

    2015-01-01

    Oxidative stress is thought to contribute to cancer development. Epstein–Barr virus (EBV) and its encoded oncoprotein, latent membrane protein 1 (LMP1), are closely associated with the transformation of nasopharyngeal carcinoma (NPC) and Burkitt’s lymphoma (BL). In this study, we used LMP1-transformed NP cells and EBV-related malignant cell lines to assess the effects of LMP1 on reactive oxygen species (ROS) accumulation and glycolytic activity. Using NPC tissue samples and a tissue array to address clinical implications, we report that LMP1 activates NAD(P)H oxidases to generate excessive amount of ROS in EBV-related malignant diseases. By evaluating NAD(P)H oxidase (NOX) subunit expression, we found that the expression of the NAD(P)H oxidase regulatory subunit p22phox was significantly upregulated upon LMP1-induced transformation. Furthermore, this upregulation was mediated by the c-Jun N-terminal kinase (JNK) pathway. In addition, LMP1 markedly stimulated anaerobic glycolytic activity through the PI3K/Akt pathway. Additionally, in both NPC cells and tissue samples, p22phox expression correlated with LMP1 expression. The NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) also exerted a marked cytotoxic effect in LMP1-transformed and malignant cells, providing a novel strategy for anticancer therapy. PMID:26244812

  15. Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus.

    PubMed Central

    Gillooly, D J; Robertson, A G; Fewson, C A

    1998-01-01

    The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases. PMID:9494109

  16. Differing roles of pyruvate dehydrogenase kinases during mouse oocyte maturation

    PubMed Central

    Hou, Xiaojing; Zhang, Liang; Han, Longsen; Ge, Juan; Ma, Rujun; Zhang, Xuesen; Moley, Kelle; Schedl, Tim; Wang, Qiang

    2015-01-01

    ABSTRACT Pyruvate dehydrogenase kinases (PDKs) modulate energy homeostasis in multiple tissues and cell types, under various nutrient conditions, through phosphorylation of the α subunit (PDHE1α, also known as PDHA1) of the pyruvate dehydrogenase (PDH) complex. However, the roles of PDKs in meiotic maturation are currently unknown. Here, by undertaking knockdown and overexpression analysis of PDK paralogs (PDK1–PDK4) in mouse oocytes, we established the site-specificity of PDKs towards the phosphorylation of three serine residues (Ser232, Ser293 and Ser300) on PDHE1α. We found that PDK3-mediated phosphorylation of Ser293-PDHE1α results in disruption of meiotic spindle morphology and chromosome alignment and decreased total ATP levels, probably through inhibition of PDH activity. Unexpectedly, we discovered that PDK1 and PDK2 promote meiotic maturation, as their knockdown disturbs the assembly of the meiotic apparatus, without significantly altering ATP content. Moreover, phosphorylation of Ser232-PDHE1α was demonstrated to mediate PDK1 and PDK2 action in meiotic maturation, possibly through a mechanism that is distinct from PDH inactivation. These findings reveal that there are divergent roles of PDKs during oocyte maturation and indicate a new mechanism controlling meiotic structure. PMID:25991547

  17. Properties and evolution of an alcohol dehydrogenase from the Crenarchaeota Pyrobaculum aerophilum.

    PubMed

    Vitale, Annalisa; Rosso, Francesco; Barbarisi, Alfonso; Labella, Tullio; D'Auria, Sabato

    2010-08-01

    The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the medium chain dehydrogenase/reductase (MDR) superfamily was identified in the hyperthermophilic archaeon, Pyrobaculum aerophilum. The P. aerophilum ADH gene (Pae2687) was over-expressed in Escherichia coli, and the protein (PyAeADHII) was purified to homogeneity and characterized. The PyAeADHII belongs to a medium chain class because its monomer size is 330 residues and even if it is structurally similar to other enzymes belonging to MDR superfamily, it lacks key residues involved in the coordination of the catalytic Zn ion and in the binding of alcoholic substrates typical of other ADHs. Consistently, PyAeADHII does not show activity on a large number of alcohols, aldheydes or ketones. It is active only when alpha-tetralone is used as a substrate. The enzyme has a strict requirement for NADP(H) as the coenzyme and has remarkable thermophilicity, displaying activity at temperatures up to 95 degrees C. The study of the metabolic pathways of P. aerophilum can provide information on the evolution of genes and enzymes and may be crucial for understanding the evolution of eukaryotic cells.

  18. Glucose-6-phosphate dehydrogenase deficiency and Alzheimer's disease: Partners in crime? The hypothesis.

    PubMed

    Ulusu, N Nuray

    2015-08-01

    Alzheimer's disease is a multifaceted brain disorder which involves various coupled irreversible, progressive biochemical reactions that significantly reduce quality of life as well as the actual life expectancy. Aging, genetic predispositions, head trauma, diabetes, cardiovascular disease, deficiencies in insulin signaling, dysfunction of mitochondria-associated membranes, cerebrovascular changes, high cholesterol level, increased oxidative stress and free radical formation, DNA damage, disturbed energy metabolism, and synaptic dysfunction, high blood pressure, obesity, dietary habits, exercise, social engagement, and mental stress are noted among the risk factors of this disease. In this hypothesis review I would like to draw the attention on glucose-6-phosphate dehydrogenase deficiency and its relationship with Alzheimer's disease. This enzymopathy is the most common human congenital defect of metabolism and defined by decrease in NADPH+H(+) and reduced form of glutathione concentration and that might in turn, amplify oxidative stress due to essentiality of the enzyme. This most common enzymopathy may manifest itself in severe forms, however most of the individuals with this deficiency are not essentially symptomatic. To understand the sporadic Alzheimer's disease, the writer of this paper thinks that, looking into a crystal ball might not yield much of a benefit but glucose-6-phosphate dehydrogenase deficiency could effortlessly give some clues.

  19. NADPH oxidase 4 is an oncoprotein localized to mitochondria.

    PubMed

    Graham, Kelly A; Kulawiec, Mariola; Owens, Kjerstin M; Li, Xiurong; Desouki, Mohamed Mokhtar; Chandra, Dhyan; Singh, Keshav K

    2010-08-01

    Reactive oxygen species (ROS) are known to be involved in many physiological and pathological processes. Initially ROS-producing NADPH oxidase (NOX) proteins were thought to be present in phagocytes. However, recent studies have demonstrated that NOX proteins are expressed in many other cell types and tissues. NOX family members' expression and function seems to vary from tissue to tissue. We determined the expression of the NOX family of proteins (NOX1-5) in normal breast tissue and breast tumors. Our study revealed that normal breast tissues express NOX1, 4 and 5 genes. Similar pattern of expression was revealed in a breast epithelial cell line. We found that NOX4 was overexpressed in the majority of breast cancer cell lines and primary breast tumors. NOX4 was also overexpressed in ovarian tumors. Overexpression of NOX4 in normal breast epithelial cells resulted in cellular senescence, resistance to apoptosis, and tumorigenic transformation. Overexpression of NOX4 in already transformed breast tumor cells also showed increased tumorigenicity. Strong evidence suggests that regulation of these processes occurs through NOX4 generation of ROS in the mitochondria. We demonstrate that the NOX4 protein contains a 73 amino acid long mitochondrial localization signal at the N-terminus that is capable of transporting a passenger protein GFP into the mitochondria. Treatment of NOX4 overexpressing cells with catalase resulted in decreased tumorigenic characteristics. Together, this study provides evidence for an oncogenic function for NOX4 protein localized to mitochondria and suggests that NOX4 is a novel source of ROS produced in the mitochondria. This study also identifies a possible treatment of NOX4-induced breast cancer by antioxidant treatment.

  20. Modeling of Anopheles minimus Mosquito NADPH-Cytochrome P450 Oxidoreductase (CYPOR) and Mutagenesis Analysis

    PubMed Central

    Sarapusit, Songklod; Lertkiatmongkol, Panida; Duangkaew, Panida; Rongnoparut, Pornpimol

    2013-01-01

    Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A. minimus CYPOR enzyme (AnCYPOR), which is the redox partner of cytochrome P450s, loses flavin-adenosine di-nucleotide (FAD) and FLAVIN mono-nucleotide (FMN) cofactors that affect its enzyme activity. Replacement of leucine residues at positions 86 and 219 with phenylalanines in FMN binding domain increases FMN binding, enzyme stability, and cytochrome c reduction activity. Membrane-Bound L86F/L219F-AnCYPOR increases A. minimus P450-mediated pyrethroid metabolism in vitro. In this study, we constructed a comparative model structure of AnCYPOR using a rat CYPOR structure as a template. Overall model structure is similar to rat CYPOR, with some prominent differences. Based on primary sequence and structural analysis of rat and A. minimus CYPOR, C427R, W678A, and W678H mutations were generated together with L86F/L219F resulting in three soluble Δ55 triple mutants. The C427R triple AnCYPOR mutant retained a higher amount of FAD binding and increased cytochrome c reduction activity compared to wild-type and L86F/L219F-Δ55AnCYPOR double mutant. However W678A and W678H mutations did not increase FAD and NAD(P)H bindings. The L86F/L219F double and C427R triple membrane-bound AnCYPOR mutants supported benzyloxyresorufin O-deakylation (BROD) mediated by mosquito CYP6AA3 with a two-to three-fold increase in efficiency over wild-type AnCYPOR. The use of rat CYPOR in place of AnCYPOR most efficiently supported CYP6AA3-mediated BROD compared to all AnCYPORs. PMID:23325047

  1. Activation of AMPK protects against hydrogen peroxide-induced osteoblast apoptosis through autophagy induction and NADPH maintenance: new implications for osteonecrosis treatment?

    PubMed

    She, Chang; Zhu, Lun-qing; Zhen, Yun-fang; Wang, Xiao-dong; Dong, Qi-rong

    2014-01-01

    Elevated hydrogen peroxide (H2O2) causes osteoblast dysfunction and apoptosis, serving as an important contributor to the development of osteonecrosis. Here we aimed to understand the role of AMP-activated protein kinase (AMPK) in the process. We observed a high level of AMPK activation in surgery isolated patients' osteonecrosis tissues. In cultured osteoblastoma MG63 cells, H2O2 stimulation induced significant AMPK activation, oxidative stress, cell death and apoptosis. Inhibition of AMPK by its inhibitor (compound C) or by shRNA-mediated knockdown dramatically enhanced H2O2-induced MG63 cell apoptosis, while over-expression of AMPK in HEK-293 cells alleviated H2O2-induced cell damage. These results confirmed that H2O2-activated AMPK is pro-cell survival. We observed that H2O2 induced protective autophagy in MG63 cells, and AMPK-dependent Ulk1 activation and mTORC1 (mTOR complex 1) inactivation might involve autophagy activation. Further, AMPK activation inhibited H2O2-induced oxidative stress, probably through inhibiting NADPH (nicotinamide adenine dinucleotide phosphate) depletion, since more NADPH depletion and oxidative stress were induced by H2O2 in AMPK deficient MG63 cells. Finally, we observed a significant AMPK activation in H2O2-treated primary cultured and transformed (MC3T3-E1) osteoblasts, and AMPK inhibitor compound C enhanced death by H2O2 in these cells. Based on these results, we concluded that H2O2-induced AMPK activation is pro-survival and anti-apoptosis in osteoblasts. Autophagy induction and NADPH maintenance are involved in AMPK-mediated pro-survival effects. AMPK might represent a novel molecular target for osteonecrosis treatment.

  2. NADPH oxidase gp91phox contributes to RANKL-induced osteoclast differentiation by upregulating NFATc1

    PubMed Central

    Kang, In Soon; Kim, Chaekyun

    2016-01-01

    Bone-marrow derived monocyte-macrophages (BMMs) differentiate into osteoclasts by M-CSF along subsequent RANKL stimulation possibly in collaboration with many other unknown cytokines released by pre- or mature osteoblasts. The differentiation process requires receptor activator of nuclear factor kappa-B ligand (RANKL)/RANK signaling and reactive oxygen species (ROS) such as superoxide anion (O2•−). Gp91phox, a plasma membrane subunit of NADPH oxidase (Nox), is constitutively expressed in BMMs and plays a major role in superoxide anion production. In this study, we found that mice deficient in gp91phox (gp91phox−/−) showed defects in osteoclast differentiation. Femurs of these mice produced osteoclasts at about 70% of the levels seen in femurs from wild-type mice, and accordingly exhibited excessive bone density. This abnormal bone growth in the femurs of gp91phox−/− mice resulted from impaired osteoclast differentiation. In addition, gp91phox−/− mice were defective for RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1). However, H2O2 treatment compensated for gp91phox deficiency in BMMs, almost completely rescuing osteoclast differentiation. Treating wild-type BMMs with antioxidants and superoxide inhibitors resulted in a differentiation defect resembling the phenotype of gp91phox−/− BMMs. Therefore, our results demonstrate that gp91phox-derived superoxide is important for promoting efficient osteoclast differentiation by inducing NFATc1 as a downstream signaling mediator of RANK. PMID:27897222

  3. Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

    SciTech Connect

    Xia, Chuanwu; Panda, Satya P.; Marohnic, Christopher C.; Martásek, Pavel; Masters, Bettie Sue; Kim, Jung-Ja P.

    2012-03-15

    NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

  4. The NADPH oxidase inhibitor diphenyleneiodonium activates the human TRPA1 nociceptor.

    PubMed

    Suzuki, Hiroka; Hatano, Noriyuki; Muraki, Yukiko; Itoh, Yuka; Kimura, Satoko; Hayashi, Hidetoshi; Onozaki, Kikuo; Ohi, Yoshiaki; Haji, Akira; Muraki, Katsuhiko

    2014-08-15

    Transient receptor potential ankyrin 1 (TRPA1) is a Ca(2+)-permeable nonselective cation channel expressed in neuronal and nonneuronal cells and plays an important role in acute and inflammatory pain. Here, we show that an NADPH oxidase (NOX) inhibitor, diphenyleneiodonium (DPI), functions as a TRPA1 activator in human embryonic kidney cells expressing human TRPA1 (HEK-TRPA1) and in human fibroblast-like synoviocytes. Application of DPI at 0.03-10 μM induced a Ca(2+) response in HEK-TRPA1 cells in a concentration-dependent manner. The Ca(2+) response was effectively blocked by a selective TRPA1 antagonist, HC-030031 (HC). In contrast, DPI had no effect on HEK cells expressing TRPV1-V4 or TRPM8. Four other NOX inhibitors, apocynin (APO), VAS2870 (VAS), plumbagin, and 2-acetylphenothiazine, also induced a Ca(2+) response in HEK-TRPA1 cells, which was inhibited by pretreatment with HC. In the presence of 5 mM glutathione, the Ca(2+) response to DPI was effectively reduced. Moreover, mutation of cysteine 621 in TRPA1 substantially inhibited the DPI-induced Ca(2+) response, while it did not inhibit the APO- and VAS-induced responses. The channel activity was induced by DPI in excised membrane patches with both outside-out and inside-out configurations. Internal application of neomycin significantly inhibited the DPI-induced inward currents. In inflammatory synoviocytes with TRPA1, DPI evoked a Ca(2+) response that was sensitive to HC. In mice, intraplantar injection of DPI caused a pain-related response which was inhibited by preadministration with HC. Taken together, our findings demonstrate that DPI and other NOX inhibitors activate human TRPA1 without mediating NOX.

  5. NADPH oxidase and aging drive microglial activation, oxidative stress, and dopaminergic neurodegeneration following systemic LPS administration.

    PubMed

    Qin, Liya; Liu, Yuxin; Hong, Jau-Shyong; Crews, Fulton T

    2013-06-01

    Parkinson's disease is characterized by a progressive degeneration of substantia nigra (SN) dopaminergic neurons with age. We previously found that a single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) injection caused a slow progressive loss of tyrosine hydroxylase immunoreactive (TH+IR) neurons in SN associated with increasing motor dysfunction. In this study, we investigated the role of NADPH oxidase (NOX) in inflammation-mediated SN neurotoxicity. A comparison of control (NOX2(+/+) ) mice with NOX subunit gp91(phox) -deficient (NOX2(-/-) ) mice 10 months after LPS administration (5 mg/kg, i.p.) resulted in a 39% (P < 0.01) loss of TH+IR neurons in NOX2(+/+) mice, whereas NOX2(-/-) mice did not show a significant decrease. Microglia (Iba1+IR) showed morphological activation in NOX2(+/+) mice, but not in NOX2(-/-) mice at 1 hr. Treatment of NOX2(+/+) mice with LPS resulted in a 12-fold increase in NOX2 mRNA in midbrain and 5.5-6.5-fold increases in NOX2 protein (+IR) in SN compared with the saline controls. Brain reactive oxygen species (ROS), determined using diphenyliodonium histochemistry, was increased by LPS in SN between 1 hr and 20 months. Diphenyliodonium (DPI), an NOX inhibitor, blocked LPS-induced activation of microglia and production of ROS, TNFα, IL-1β, and MCP-1. Although LPS increased microglial activation and ROS at all ages studied, saline control NOX2(+/+) mice showed age-related increases in microglial activation, NOX, and ROS levels at 12 and 22 months of age. Together, these results suggest that NOX contributes to persistent microglial activation, ROS production, and dopaminergic neurodegeneration that persist and continue to increase with age.

  6. NADPH oxidase and aging drive microglial activation, oxidative stress and dopaminergic neurodegeneration following systemic LPS administration

    PubMed Central

    Qin, Liya; Liu, Yuxin; Hong, Jau-Shyong; Crews, Fulton T.

    2013-01-01

    Parkinson’s disease is characterized by a progressive degeneration of substantia nigra (SN) dopaminergic neurons with age. We previously found that a single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) injection caused a slow progressive loss of tyrosine hydroxylase immunoreactive (TH+IR) neurons in SN associated with increasing motor dysfunction. In this study, we investigated the role of NADPH oxidase (NOX) in inflammation-mediated SN neurotoxicity. A comparison of control (NOX2+/+) mice with NOX subunit gp91phox-deficient (NOX2−/−) mice 10 months after LPS administration (5 mg/kg, i.p.) resulted in a 39% (p<0.01) loss of TH+IR neurons in NOX2+/+ mice, whereas, NOX2−/− mice did not show a significant decrease. Microglia (Iba1+IR) showed morphological activation in NOX2+/+ mice, but not in NOX2−/− mice at 1 hour. Treatment of NOX2+/+ mice with LPS resulted in a 12 fold increase in NOX2 mRNA in midbrain and 5.5–6.5 fold increases in NOX2 protein (+IR) in SN compared to the saline controls. Brain reactive oxygen species (ROS), determined by hydroethidine histochemistry, was increased by LPS in SN between 1 hour and 20 months. Diphenyliodonium (DPI), a NOX inhibitor, blocked LPS-induced activation of microglia and production of ROS, TNFα, IL-1β, and MCP-1. Although LPS increased microglial activation and ROS at all ages studied, saline control NOX2+/+ mice showed age-related increases in microglial activation, NOX and ROS levels at 12 and 22 months of age. Together, these results suggest that NOX contributes to persistent microglial activation, ROS production and dopaminergic neurodegeneration that persist and continue to increase with age. PMID:23536230

  7. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

    ... conversion is essential to begin the series of chemical reactions that produce energy for cells. The pyruvate dehydrogenase ... E3, each of which performs part of the chemical reaction that converts pyruvate to acetyl-CoA. In addition, ...

  8. Genetics Home Reference: dihydropyrimidine dehydrogenase deficiency

    MedlinePlus

    ... of the skin on the palms and soles (hand-foot syndrome); shortness of breath; and hair loss may also ... dehydrogenase deficiency , with its early-onset neurological symptoms, is a rare disorder. Its prevalence is ...

  9. Sinomenine, a natural dextrorotatory morphinan analog, is anti-inflammatory and neuroprotective through inhibition of microglial NADPH oxidase

    PubMed Central

    Qian, Li; Xu, Zongli; Zhang, Wei; Wilson, Belinda; Hong, Jau-Shyong; Flood, Patrick M

    2007-01-01

    Background The mechanisms involved in the induction and regulation of inflammation resulting in dopaminergic (DA) neurotoxicity in Parkinson's disease (PD) are complex and incompletely understood. Microglia-mediated inflammation has recently been implicated as a critical mechanism responsible for progressive neurodegeneration. Methods Mesencephalic neuron-glia cultures and reconstituted cultures were used to investigate the molecular mechanisms of sinomenine (SN)-mediated anti-inflammatory and neuroprotective effects in both the lipopolysaccharide (LPS)- and the 1-methyl-4-phenylpyridinium (MPP+)-mediated models of PD. Results SN showed equivalent efficacy in protecting against DA neuron death in rat midbrain neuron-glial cultures at both micro- and sub-picomolar concentrations, but no protection was seen at nanomolar concentrations. The neuroprotective effect of SN was attributed to inhibition of microglial activation, since SN significantly decreased tumor necrosis factor-α (TNF-α, prostaglandin E2 (PGE2) and reactive oxygen species (ROS) production by microglia. In addition, from the therapeutic point of view, we focused on sub-picomolar concentration of SN for further mechanistic studies. We found that 10-14 M of SN failed to protect DA neurons against MPP+-induced toxicity in the absence of microglia. More importantly, SN failed to show a protective effect in neuron-glia cultures from mice lacking functional NADPH oxidase (PHOX), a key enzyme for extracellular superoxide production in immune cells. Furthermore, we demonstrated that SN reduced LPS-induced extracellular ROS production through the inhibition of the PHOX cytosolic subunit p47phoxtranslocation to the cell membrane. Conclusion Our findings strongly suggest that the protective effects of SN are most likely mediated through the inhibition of microglial PHOX activity. These findings suggest a novel therapy to treat inflammation-mediated neurodegenerative diseases. PMID:17880684

  10. Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

    PubMed

    Pennacchio, Angela; Giordano, Assunta; Pucci, Biagio; Rossi, Mosè; Raia, Carlo A

    2010-03-01

    The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

  11. Isocitrate dehydrogenase mutations in gliomas

    PubMed Central

    Waitkus, Matthew S.; Diplas, Bill H.; Yan, Hai

    2016-01-01

    Over the last decade, extraordinary progress has been made in elucidating the underlying genetic causes of gliomas. In 2008, our understanding of glioma genetics was revolutionized when mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) were identified in the vast majority of progressive gliomas and secondary glioblastomas (GBMs). IDH enzymes normally catalyze the decarboxylation of isocitrate to generate α-ketoglutarate (αKG), but recurrent mutations at Arg132 of IDH1 and Arg172 of IDH2 confer a neomorphic enzyme activity that catalyzes reduction of αKG into the putative oncometabolite D-2-hydroxyglutate (D2HG). D2HG inhibits αKG-dependent dioxygenases and is thought to create a cellular state permissive to malignant transformation by altering cellular epigenetics and blocking normal differentiation processes. Herein, we discuss the relevant literature on mechanistic studies of IDH1/2 mutations in gliomas, and we review the potential impact of IDH1/2 mutations on molecular classification and glioma therapy. PMID:26188014

  12. Biochemical properties of human dehydrogenase/reductase (SDR family) member 7.

    PubMed

    Stambergova, Hana; Skarydova, Lucie; Dunford, James E; Wsol, Vladimir

    2014-01-25

    Dehydrogenase/reductase (SDR family) member 7 (DHRS7, retSDR4, SDR34C1) is a previously uncharacterized member of the short-chain dehydrogenase/reductase (SDR) superfamily. While human SDR members are known to play an important role in various (patho)biochemical pathways including intermediary metabolism and biotransformation of xenobiotics, only 20% of them are considered to be well characterized. Based on phylogenetic tree and SDR sequence clusters analysis DHRS7 is a close relative to well-known SDR member 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) that participates in metabolism of endogenous and xenobiotic substances with carbonyl group. The aim of present study is to determine the basic biochemical properties of DHRS7 and its possible involvement in metabolism of substrates with carbonyl group. For the first time the computational predictions of this membrane protein and membrane topology were experimentally confirmed. DHRS7 has been demonstrated to be an integral protein facing the lumen of the endoplasmic reticulum with lack of posttranscriptional glycosylation modification. Subsequently, NADP(H) cofactor preference and enzymatic reducing activity of DHRS7 was determined towards endogenous substrates with a steroid structure (cortisone, 4-androstene-3,17-dion) and also toward relevant exogenous substances bearing a carbonyl group harmful to human health (1,2-naphtoquinone, 9,10-phenantrenequinone). In addition to 11β-HSD1, DHRS7 is another enzyme from SDR superfamily that have been proved, at least in vitro, to contribute to the metabolism of xenobiotics with carbonyl group.

  13. Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

    NASA Astrophysics Data System (ADS)

    Hamid, Nur Athirah Abd; Ismail, Ismanizan

    2013-11-01

    Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

  14. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

    SciTech Connect

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and {beta}-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from {beta}-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

  15. Reassessment of the transhydrogenase/malate shunt pathway in Clostridium thermocellum ATCC 27405 through kinetic characterization of malic enzyme and malate dehydrogenase.

    PubMed

    Taillefer, M; Rydzak, T; Levin, D B; Oresnik, I J; Sparling, R

    2015-04-01

    Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a kcat of 45.8 s(-1) or 14.9 s(-1), respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a kcat of 520.8 s(-1). The malic enzyme used NADP(+) as a cofactor along with NH4 (+) and Mn(2+) as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a Ki of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH4 (+) based on the characterization of the malate dehydrogenase and malic enzyme.

  16. Globular adiponectin inhibits ethanol-induced reactive oxygen species production through modulation of NADPH oxidase in macrophages: involvement of liver kinase B1/AMP-activated protein kinase pathway.

    PubMed

    Kim, Mi Jin; Nagy, Laura E; Park, Pil-Hoon

    2014-09-01

    Adiponectin, an adipokine predominantly secreted from adipocytes, has been shown to play protective roles against chronic alcohol consumption. Although excessive reactive oxygen species (ROS) production in macrophages is considered one of the critical events for ethanol-induced damage in various target tissues, the effect of adiponectin on ethanol-induced ROS production is not clearly understood. In the present study, we investigated the effect of globular adiponectin (gAcrp) on ethanol-induced ROS production and the potential mechanisms underlying these effects of gAcrp in macrophages. Here we demonstrated that gAcrp prevented ethanol-induced ROS production in both RAW 264.7 macrophages and primary murine peritoneal macrophages. Globular adiponectin also inhibited ethanol-induced activation of NADPH oxidase. In addition, gAcrp suppressed ethanol-induced increase in the expression of NADPH oxidase subunits, including Nox2 and p22(phox), via modulation of nuclear factor-κB pathway. Furthermore, pretreatment with compound C, a selective inhibitor of AMPK, or knockdown of AMPK by small interfering RNA restored suppression of ethanol-induced ROS production and Nox2 expression by gAcrp. Finally, we found that gAcrp treatment induced phosphorylation of liver kinase B1 (LKB1), an upstream signaling molecule mediating AMPK activation. Knockdown of LKB1 restored gAcrp-suppressed Nox2 expression, suggesting that LKB1/AMPK pathway plays a critical role in the suppression of ethanol-induced ROS production and activation of NADPH oxidase by gAcrp. Taken together, these results demonstrate that globular adiponectin prevents ethanol-induced ROS production, at least in part, via modulation of NADPH oxidase in macrophages. Further, LKB1/AMPK axis plays an important role in the suppression of ethanol-induced NADPH oxidase activation by gAcrp in macrophages.

  17. Isolation and Characterization of Anaerobic Ethylbenzene Dehydrogenase, a Novel Mo-Fe-S Enzyme

    PubMed Central

    Johnson, Hope A.; Pelletier, Dale A.; Spormann, Alfred M.

    2001-01-01

    The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus sp. strain EB1 is the oxidation of ethylbenzene to (S)-(−)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (αβγ) with subunit masses of 100 kDa (α), 35 kDa (β), and 25 kDa (γ). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that ebdA encodes the α subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The ebdB gene encodes the β subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The ebdC gene encodes the γ subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes. PMID:11443088

  18. Ascorbate inhibits NADPH oxidase subunit p47phox expression in microvascular endothelial cells.

    PubMed

    Wu, Feng; Schuster, David P; Tyml, Karel; Wilson, John X

    2007-01-01

    The production of reactive oxygen species (ROS) is central to the etiology of endothelial dysfunction in sepsis. Endothelial cells respond to infection by activating NADPH oxidases that are sources of intracellular ROS and potential targets for therapeutic administration of antioxidants. Ascorbate is an antioxidant that accumulates in these cells and improves capillary blood flow, vascular reactivity, arterial blood pressure, and survival in experimental sepsis. Therefore, the present study tested the hypothesis that ascorbate regulates NADPH oxidases in microvascular endothelial cells exposed to septic insult. We observed that incubation with Escherichia coli lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) increased NADPH oxidase activity and expression of the enzyme subunit p47phox in mouse microvascular endothelial cells of skeletal muscle origin. Pretreatment of the cells with ascorbate prevented these increases. Polyethylene glycol-conjugated catalase and selective inhibitors of Jak2 also abrogated induction of p47phox. Exogenous hydrogen peroxide induced p47phox expression that was prevented by pretreatment of the cells with ascorbate. LPS+IFNgamma or hydrogen peroxide activated the Jak2/Stat1/IRF1 pathway and this effect was also inhibited by ascorbate. In conclusion, ascorbate blocks the stimulation by septic insult of redox-sensitive Jak2/Stat1/IRF1 signaling, p47phox expression, and NADPH oxidase activity in microvascular endothelial cells. Because endothelial NADPH oxidases produce ROS that can cause endothelial dysfunction, their inhibition by ascorbate may represent a new strategy for sepsis therapy.

  19. Characterisation of electron currents generated by the human neutrophil NADPH oxidase

    SciTech Connect

    Ahluwalia, Jatinder

    2008-04-11

    Electron transport by the human neutrophil NADPH oxidase is an important microbicidal weapon for phagocytes. The electron current (I{sub e}) generated by the neutrophil NADPH oxidase is poorly characterised due to the lack of appropriate electrophysiological data. In this study, I fully characterise the neutrophil generated I{sub e} when the NADPH oxidase is activated by NADPH and GTP{gamma}S. The neutrophil I{sub e} was markedly voltage-dependent in the entire voltage range in comparison to those electron currents measured after chloride was removed from the external bath solution. The difference in I{sub e} measured in chloride free conditions was not due to a change in the activation kinetics of voltage-gated proton channels. The I{sub e} depolarises the neutrophil plasma membrane at a rate of 2.3 V s{sup -1} and this depolarisation was opposed when voltage-gated proton channels are activated. 3 mM ZnCl{sub 2} depolarised the membrane potential to +97.8 {+-} 2.5 mV (n = 4), and this depolarisation was abolished after NADPH oxidase inhibition.

  20. Effects of NADH kinase on NADPH-dependent biotransformation processes in Escherichia coli.

    PubMed

    Lee, Won-Heong; Kim, Jin-Woo; Park, Eun-Hee; Han, Nam Soo; Kim, Myoung-Dong; Seo, Jin-Ho

    2013-02-01

    Sufficient supply of NADPH is one of the most important factors affecting the productivity of biotransformation processes. In this study, construction of an efficient NADPH-regenerating system was attempted using direct phosphorylation of NADH by NADH kinase (Pos5p) from Saccharomyces cerevisiae for producing guanosine diphosphate (GDP)-L-fucose and ε-caprolactone in recombinant Escherichia coli. Expression of Pos5p in a fed-batch culture of recombinant E. coli producing GDP-L-fucose resulted in a maximum GDP-L-fucose concentration of 291.5 mg/l, which corresponded to a 51 % enhancement compared with the control strain. In a fed-batch Baeyer-Villiger (BV) oxidation of cyclohexanone using recombinant E. coli expressing Pos5p, a maximum ε-caprolactone concentration of 21.6 g/l was obtained, which corresponded to a 96 % enhancement compared with the control strain. Such an increase might be due to the enhanced availability of NADPH in recombinant E. coli expressing Pos5p. These results suggested that efficient regeneration of NADPH was possible by functional expression of Pos5p in recombinant E. coli, which can be applied to other NADPH-dependent biotransformation processes in E. coli.

  1. Dual utilization of NADPH and NADH cofactors enhances xylitol production in engineered Saccharomyces cerevisiae.

    PubMed

    Jo, Jung-Hyun; Oh, Sun-Young; Lee, Hyeun-Soo; Park, Yong-Cheol; Seo, Jin-Ho

    2015-12-01

    Xylitol, a natural sweetener, can be produced by hydrogenation of xylose in hemicelluloses. In microbial processes, utilization of only NADPH cofactor limited commercialization of xylitol biosynthesis. To overcome this drawback, Saccharomyces cerevisiae D452-2 was engineered to express two types of xylose reductase (XR) with either NADPH-dependence or NADH-preference. Engineered S. cerevisiae DWM expressing both the XRs exhibited higher xylitol productivity than the yeast strain expressing NADPH-dependent XR only (DWW) in both batch and glucose-limited fed-batch cultures. Furthermore, the coexpression of S. cerevisiae ZWF1 and ACS1 genes in the DWM strain increased intracellular concentrations of NADPH and NADH and improved maximum xylitol productivity by 17%, relative to that for the DWM strain. Finally, the optimized fed-batch fermentation of S. cerevisiae DWM-ZWF1-ACS1 resulted in 196.2 g/L xylitol concentration, 4.27 g/L h productivity and almost the theoretical yield. Expression of the two types of XR utilizing both NADPH and NADH is a promising strategy to meet the industrial demands for microbial xylitol production.

  2. Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica.

    PubMed Central

    Bruchhaus, I; Richter, S; Tannich, E

    1998-01-01

    The gene encoding a putative NADPH:flavin oxidoreductase of the protozoan parasite Entamoeba histolytica (Eh34) was recombinantly expressed in Escherichia coli. The purified recombinant protein (recEh34) has a molecular mass of about 35 kDa upon SDS/PAGE analysis, exhibits a flavoprotein-like absorption spectrum and contains 1 mol of non-covalently bound FMN per mol of protein. RecEh34 reveals two different enzymic activities. It catalyses the NADPH-dependent reduction of oxygen to hydrogen peroxide (H2O2), as well as of disulphides such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and cystine. The disulphide reductase but not the H2O2-forming NADPH oxidase activity is inhibitable by sulphydryl-active compounds, indicating that a thiol component is part of the active site for the disulphide reductase activity, whereas for the H2O2-forming NADPH oxidase activity only the flavin is required. Compared with the recombinant protein, similar activities are present in amoebic extracts. Native Eh34 is active in a monomeric as well as in a dimeric state. In contrast to recEh34, no flavin was associated with the native protein. However, both NADPH oxidase as well as DTNB reductase activity were found to be dependent on the addition of FAD or FMN. PMID:9494088

  3. NADPH Oxidase as a Therapeutic Target for Oxalate Induced Injury in Kidneys

    PubMed Central

    Peck, Ammon B.; Khan, Saeed R.

    2013-01-01

    A major role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes is to catalyze the production of superoxides and other reactive oxygen species (ROS). These ROS, in turn, play a key role as messengers in cell signal transduction and cell cycling, but when they are produced in excess they can lead to oxidative stress (OS). Oxidative stress in the kidneys is now considered a major cause of renal injury and inflammation, giving rise to a variety of pathological disorders. In this review, we discuss the putative role of oxalate in producing oxidative stress via the production of reactive oxygen species by isoforms of NADPH oxidases expressed in different cellular locations of the kidneys. Most renal cells produce ROS, and recent data indicate a direct correlation between upregulated gene expressions of NADPH oxidase, ROS, and inflammation. Renal tissue expression of multiple NADPH oxidase isoforms most likely will impact the future use of different antioxidants and NADPH oxidase inhibitors to minimize OS and renal tissue injury in hyperoxaluria-induced kidney stone disease. PMID:23840917

  4. Morphometric analysis of NADPH diaphorase reactive neurons in a rat model of focal excitotoxic striatal injury.

    PubMed

    Freire, Marco Aurelio M; Guimaraes, Joanilson S; Santos, Jose Ronaldo; Simplício, Hougelle; Gomes-Leal, Walace

    2016-12-01

    Excitotoxicity is the major component in neuropathological conditions, related to harmful action of imbalanced concentrations of glutamate and its agonists in the nervous tissue, ultimately resulting in cell death. In the present study, we evaluated the effects of an acute striatal lesion induced by a focal N-methyl-D-aspartate (NMDA) microinjection on the morphometry of NADPH diaphorase-reactive neurons (NADPH-d(+) ), a subset of cells which release nitric oxide (NO) in the brain and are known by its resistance in pathological conditions. Two hundred and forty NADPH-d neurons from NMDA-lesioned striatum and contralateral counterpart were tridimensionally reconstructed at 1, 3 and 7 post-lesion days (PLDs). Cell body and dendritic field areas, length of dendrites by order and fractal dimension were analyzed. There were no significant morphometric differences when NADPH-d(+) neurons from lesioned and control striatal regions were compared among PLDs evaluated. Conversely, a conspicuous pallor in striatal neuropil reactivity was evidenced, especially in latter survival time. In addition, we observed a noticeable inflammatory response induced by NMDA. Our results suggest that NADPH-d(+) neurons were spared from deleterious effects of acute NMDA excitotoxic damage in the striatum, reinforcing the notion that this cell group is selectively resistant to injury in the nervous system.

  5. Regulation of L-threonine dehydrogenase in somatic cell reprogramming.

    PubMed

    Han, Chuanchun; Gu, Hao; Wang, Jiaxu; Lu, Weiguang; Mei, Yide; Wu, Mian

    2013-05-01

    Increasing evidence suggests that metabolic remodeling plays an important role in the regulation of somatic cell reprogramming. Threonine catabolism mediated by L-threonine dehydrogenase (TDH) has been recognized as a specific metabolic trait of mouse embryonic stem cells. However, it remains unknown whether TDH-mediated threonine catabolism could regulate reprogramming. Here, we report TDH as a novel regulator of somatic cell reprogramming. Knockdown of TDH inhibits, whereas induction of TDH enhances reprogramming efficiency. Moreover, microRNA-9 post-transcriptionally regulates the expression of TDH and thereby inhibits reprogramming efficiency. Furthermore, protein arginine methyltransferase (PRMT5) interacts with TDH and mediates its post-translational arginine methylation. PRMT5 appears to regulate TDH enzyme activity through both methyltransferase-dependent and -independent mechanisms. Functionally, TDH-facilitated reprogramming efficiency is further enhanced by PRMT5. These results suggest that TDH-mediated threonine catabolism controls somatic cell reprogramming and indicate the importance of post-transcriptional and post-translational regulation of TDH.

  6. Aldehyde dehydrogenase 2 in cardiac protection: a new therapeutic target?

    PubMed Central

    Budas, Grant R; Disatnik, Marie- Hélène; Mochly-Rosen, Daria

    2010-01-01

    Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is emerging as a key enzyme involved in cytoprotection in the heart. ALDH2 mediates both the detoxification of reactive aldehydes such as acetaldehyde and 4-hydroxy-2-nonenal (4-HNE) and the bioactivation of nitroglycerin (GTN) to nitric oxide (NO). In addition, chronic nitrate treatment results in ALDH2 inhibition and contributes to nitrate tolerance. Our lab recently identified ALDH2 to be a key mediator of endogenous cytoprotection. We reported that ALDH2 is phosphorylated and activated by the survival kinase protein kinase C epsilon (PKCε) and found a strong inverse correlation between ALDH2 activity and infarct size. We also identified a small molecule ALDH2 activator (Alda-1) which reduces myocardial infarct size induced by ischemia/reperfusion in vivo. In this review, we discuss evidence that ALDH2 is a key mediator of endogenous survival signaling in the heart, suggest possible cardioprotective mechanisms mediated by ALDH2, and discuss potential clinical implications of these findings. PMID:20005475

  7. Early NADPH oxidase-2 activation is crucial in phenylephrine-induced hypertrophy of H9c2 cells.

    PubMed

    Hahn, Nynke E; Musters, René J P; Fritz, Jan M; Pagano, Patrick J; Vonk, Alexander B A; Paulus, Walter J; van Rossum, Albert C; Meischl, Christof; Niessen, Hans W M; Krijnen, Paul A J

    2014-09-01

    Reactive oxygen species (ROS) produced by different NADPH oxidases (NOX) play a role in cardiomyocyte hypertrophy induced by different stimuli, such as angiotensin II and pressure overload. However, the role of the specific NOX isoforms in phenylephrine (PE)-induced cardiomyocyte hypertrophy is unknown. Therefore we aimed to determine the involvement of the NOX isoforms NOX1, NOX2 and NOX4 in PE-induced cardiomyocyte hypertrophy. Hereto rat neonatal cardiomyoblasts (H9c2 cells) were incubated with 100 μM PE to induce hypertrophy after 24 and 48h as determined via cell and nuclear size measurements using digital imaging microscopy, electron microscopy and an automated cell counter. Digital-imaging microscopy further revealed that in contrast to NOX1 and NOX4, NOX2 expression increased significantly up to 4h after PE stimulation, coinciding and co-localizing with ROS production in the cytoplasm as well as the nucleus. Furthermore, inhibition of NOX-mediated ROS production with apocynin, diphenylene iodonium (DPI) or NOX2 docking sequence (Nox2ds)-tat peptide during these first 4h of PE stimulation significantly inhibited PE-induced hypertrophy of H9c2 cells, both after 24 and 48h of PE stimulation. These data show that early NOX2-mediated ROS production is crucial in PE-induced hypertrophy of H9c2 cells.

  8. Regulation of heart muscle pyruvate dehydrogenase kinase

    PubMed Central

    Cooper, Ronald H.; Randle, Philip J.; Denton, Richard M.

    1974-01-01

    1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [32P]phosphate from [γ-32P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100μm) and cyclic 3′:5′-nucleotides (at 10μm) had no significant effect on kinase activity. 3. The Km for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76μm. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The Km for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9–25.4μm. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The Km for pyruvate in the pyruvate dehydrogenase reaction was 35.5μm. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25–500μm. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate

  9. Characterization of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase in pancreatic islets.

    PubMed

    Lenzen, S; Panten, U

    1983-12-01

    Succinate dehydrogenase activities in homogenates of rat and ob/ob mouse pancreatic islets were only 13% of the activities in homogenates of liver and were also several times lower than in homogenates of pancreatic acinar tissue. This indicates that the content of mitochondria in pancreatic islet cells is very low. The very low activity of succinate dehydrogenase is in agreement with the low mitochondrial volume in the cytoplasmic ground substance of pancreatic islet cells as observed in morphometric studies. This may represent the poor equipment of pancreatic islet cells with electron transport chains and thus provide a regulatory role for the generation of reducing equivalents and chemical energy for the regulation of insulin secretion. The activities of succinate dehydrogenase in tissue homogenates of pancreatic islets, pancreatic acinar tissue, and liver were significantly inhibited by malonate and diazoxide but not by glucose, mannoheptulose, streptozotocin, or verapamil. Tolbutamide inhibited only pancreatic islet succinate dehydrogenase significantly, providing evidence for a different behavior of pancreatic islet cell mitochondria. Therefore diazoxide and tolbutamide may affect pancreatic islet function through their effects on succinate dehydrogenase activity. The activities of alpha-glycerophosphate dehydrogenase in homogenates of pancreatic islets and liver from rats and ob/ob mice were in the same range, while activities in homogenates of pancreatic acinar tissue were lower. None of the test agents affected alpha-glycerophosphate dehydrogenase activity. Thus the results provide no support for the recent contention that alpha-glycerophosphate dehydrogenase activity may be critical for the regulation of insulin secretion.

  10. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    SciTech Connect

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; Snow, Christopher D.; Brustad, Eric M.; McIntosh, John A.; Meinhold, Peter; Zhang, Liang; Arnold, Frances H.

    2013-06-17

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.

  11. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    DOE PAGES

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; ...

    2013-06-17

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymesmore » having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.« less

  12. C-6 proton of tetrahydrobiopterin is acquired from water, not NADPH, during de novo biosynthesis

    SciTech Connect

    Smith, G.K.; Cichetti, J.A.; Chandrasurin, P.; Nichol, C.A.

    1985-05-10

    Tetrahydrobiopterin, the cofactor for the aromatic amino acid hydroxylases, is synthesized in mammals from GTP via a pathway involving both dihydropterin and tetrahydropterin intermediates. In this work, the authors have investigated the mechanism of conversion of the product formed from GTP, 7,8-dihydroneopterin triphosphate, into the tetrahydropterin intermediates. Tetrahydrobiopterin can be oxidized under conditions which yield pterin or pterin 6-carboxylate without exchange of the C-6 and C-7 protons. Using these techniques, a gas chromatography/mass spectrometry method was developed to determine that in the biosynthesis of tetrahydrobiopterin de novo, in preparations of bovine adrenal medulla, the C-6 proton of tetrahydrobiopterin is derived from water and not from NADPH. In contrast, the C-6 proton of tetrahydrobiopterin produced from sepiapterin (6-lactoyl-7,8-dihydropterin) comes from NADPH. The results are consistent with evidence for the formation of the first tetrahydropterin intermediate by a tautomerization without any requirement for NADPH.

  13. NADPH oxidases, reactive oxygen species, and hypertension: clinical implications and therapeutic possibilities.

    PubMed

    Paravicini, Tamara M; Touyz, Rhian M

    2008-02-01

    Reactive oxygen species (ROS) influence many physiological processes including host defense, hormone biosynthesis, fertilization, and cellular signaling. Increased ROS production (termed "oxidative stress") has been implicated in various pathologies, including hypertension, atherosclerosis, diabetes, and chronic kidney disease. A major source for vascular and renal ROS is a family of nonphagocytic NAD(P)H oxidases, including the prototypic Nox2 homolog-based NAD(P)H oxidase, as well as other NAD(P)H oxidases, such as Nox1 and Nox4. Other possible sources include mitochondrial electron transport enzymes, xanthine oxidase, cyclooxygenase, lipoxygenase, and uncoupled nitric oxide synthase. NAD(P)H oxidase-derived ROS plays a physiological role in the regulation of endothelial function and vascular tone and a pathophysiological role in endothelial dysfunction, inflammation, hypertrophy, apoptosis, migration, fibrosis, angiogenesis, and rarefaction, important processes underlying cardiovascular and renal remodeling in hypertension and diabetes. These findings have evoked considerable interest because of the possibilities that therapies against nonphagocytic NAD(P)H oxidase to decrease ROS generation and/or strategies to increase nitric oxide (NO) availability and antioxidants may be useful in minimizing vascular injury and renal dysfunction and thereby prevent or regress target organ damage associated with hypertension and diabetes. Here we highlight current developments in the field of reactive oxygen species and cardiovascular disease, focusing specifically on the recently identified novel Nox family of NAD(P)H oxidases in hypertension. We also discuss the potential role of targeting ROS as a therapeutic possibility in the management of hypertension and cardiovascular disease.

  14. [Increasing reductant NADPH content via metabolic engineering of PHB synthesis pathway in Synechocystis sp. PCC 6803].

    PubMed

    Xie, Juan; Zhou, Jie; Zhang, Haifeng; Li, Yin

    2011-07-01

    Cyanobacteria have become attractive hosts for renewable chemicals production. The low productivity, however, prevents it from industrial application. Reductant NAD(P)H availability is a chief hurdle for the production of reductive metabolites in microbes. To increase NADPH content in Synechocystis sp. PCC 6803, PHB synthase encoding gene phaC and phaE in Synechocystis was inactivated by replacing phaC&E genes with chloromycetin resistance cassette via homologous recombination. PCR analysis showed that mutant S.delta phaC&E with complete genome segregation was generated. The comparison between growth curves of S.wt and S.delta phaC&E indicated the knockout of phaC & phaE genes did not affect obviously the cell growth. Gas chromatography analysis showed that the accumulation of PHB in wild type was about 2.3% of the dry cell weight, whereas no PHB was detected in the mutant S.delta phaC&E. The data indicated that inactivation of PHB synthase gene phaC and phaE interrupted the synthesis of PHB. Further comparative study of wild type and mutant demonstrated that NADPH content in S.delta phaC&E was obviously increased. On the third day, the NADPH content in S.delta phaC&E was up to 1.85 fold higher than that in wild type. These results indicated that deleting PHB synthase gene phaC and phaE not only can block the synthesis of PHB, but also can save NADPH to contribute reductant sink in cyanobacteria. Hence, the engineered cyanobacterial strain S.delta phaC&E, in which carbon flux was redirected and NADPH was increased, will be a potential host strain for chemicals production in cyanobacteria.

  15. Stereoselective synthesis of (R)-phenylephrine using recombinant Escherichia coli cells expressing a novel short-chain dehydrogenase/reductase gene from Serratia marcescens BCRC 10948.

    PubMed

    Peng, Guan-Jhih; Kuan, Yi-Chia; Chou, Hsiao-Yi; Fu, Tze-Kai; Lin, Jia-Shin; Hsu, Wen-Hwei; Yang, Ming-Te

    2014-01-20

    (R)-Phenylephrine [(R)-PE] is an α1-adrenergic receptor agonist and is widely used as a nasal decongestant to treat the common cold without the side effects of other ephedrine adrenergic drugs. We identified a short-chain dehydrogenase/reductase (SM_SDR) from Serratia marcescens BCRC 10948 that was able to convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into (R)-PE. The SM_SDR used NADPH and NADH as cofactors with specific activities of 17.35±0.71 and 5.57±0.07mU/mg protein, respectively, at 30°C and pH 7.0, thereby indicating that this enzyme could be categorized as an NADPH-preferring short-chain dehydrogenase/reductase. Escherichia coli strain BL21 (DE3) expressing SM_SDR could convert HPMAE into (R)-PE with more than 99% enantiomeric excess. The productivity and conversion yield were 0.57mmolPE/lh and 51.06%, respectively, using 10mM HPMAE. Fructose was the most effective carbon source for the conversion of HPMAE to (R)-PE.

  16. Targeting NADPH Oxidase Decreases Oxidative Stress in the Transgenic Sickle Cell Mouse Penis

    PubMed Central

    Musicki, Biljana; Liu, Tongyun; Sezen, Sena F.; Burnett, Arthur L.

    2012-01-01

    Introduction Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. Aims We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. Methods SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67phox, p47phox, and gp91phox), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Main Outcome Measures Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Results Relative to hemi mice, SCD increased (P < 0.05) protein expression of NADPH oxidase subunits p67phox, p47phox, and gp91phox, 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P < 0.05) the abnormalities in protein expressions of p47phox, gp91phox (but not p67phox) and 4-HNE, but only slightly (P > 0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. Conclusion NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a

  17. Role of NADPH Oxidase in Metabolic Disease-Related Renal Injury: An Update

    PubMed Central

    Su, Hua

    2016-01-01

    Metabolic syndrome has been linked to an increased risk of chronic kidney disease. The underlying pathogenesis of metabolic disease-related renal injury remains obscure. Accumulating evidence has shown that NADPH oxidase is a major source of intrarenal oxidative stress and is upregulated by metabolic factors leading to overproduction of ROS in podocytes, endothelial cells, and mesangial cells in glomeruli, which is closely associated with the initiation and progression of glomerular diseases. This review focuses on the role of NADPH oxidase-induced oxidative stress in the pathogenesis of metabolic disease-related renal injury. Understanding of the mechanism may help find potential therapeutic strategies. PMID:27597884

  18. Sorbitol dehydrogenase: structure, function and ligand design.

    PubMed

    El-Kabbani, O; Darmanin, C; Chung, R P-T

    2004-02-01

    Sorbitol dehydrogenase (SDH), a member of the medium-chain dehydrogenase/reductase protein family and the second enzyme of the polyol pathway of glucose metabolism, converts sorbitol to fructose strictly using NAD(+) as coenzyme. SDH is expressed almost ubiquitously in all mammalian tissues. The enzyme has attracted considerable interest due to its implication in the development of diabetic complications and thus its tertiary structure may facilitate the development of drugs for the treatment of diabetes sufferers. Modelling studies suggest that SDH is structurally homologous to mammalian alcohol dehydrogenase with respect to conserved zinc binding motif and a hydrophobic substrate-binding pocket. Recently, the three-dimensional (3-D) structure of a mammalian SDH was solved, and it was found that while the overall 3-D structures of SDH and alcohol dehydrogenase are similar, the zinc coordination in the active sites of the two enzymes is different. The available structural and biochemical information of SDH are currently being utilized in a structure-based approach to develop drugs for the treatment or prevention of the complications of diabetes. This review provides an overview of the recent advances in the structure, function and drug development fields of sorbitol dehydrogenase.

  19. Towards a systematic analysis of human short-chain dehydrogenases/reductases (SDR): Ligand identification and structure-activity relationships.

    PubMed

    Bhatia, Chitra; Oerum, Stephanie; Bray, James; Kavanagh, Kathryn L; Shafqat, Naeem; Yue, Wyatt; Oppermann, Udo

    2015-06-05

    Short-chain dehydrogenases/reductases (SDRs) constitute a large, functionally diverse branch of enzymes within the class of NAD(P)(H) dependent oxidoreductases. In humans, over 80 genes have been identified with distinct metabolic roles in carbohydrate, amino acid, lipid, retinoid and steroid hormone metabolism, frequently associated with inherited genetic defects. Besides metabolic functions, a subset of atypical SDR proteins appears to play critical roles in adapting to redox status or RNA processing, and thereby controlling metabolic pathways. Here we present an update on the human SDR superfamily and a ligand identification strategy using differential scanning fluorimetry (DSF) with a focused library of oxidoreductase and metabolic ligands to identify substrate classes and inhibitor chemotypes. This method is applicable to investigate structure-activity relationships of oxidoreductases and ultimately to better understand their physiological roles.

  20. Purification and properties of three NAD(P)+ isozymes of L-glutamate dehydrogenase of Chlamydomonas reinhardtii.

    PubMed

    Moyano, E; Cárdenas, J; Muñoz-Blanco, J

    1992-02-13

    Three isozymes of glutamate dehydrogenase (GDH) of Chlamydomonas reinhardtii, induced under different trophic and stress conditions, have been purified about 800-1000-fold to electrophoretic homogeneity. They are hexamers of Mr 266,000-269,000 as deduced from gel filtration and sedimentation coefficient data. GDH1 consisted of six identical subunits of 44 kDa each, whereas both GDH2 and GDH3 consisted of six similar-sized monomers (4 of 44 kDa and 2 of 46 kDa). Optimum pH for the three activities with each pyridine nucleotide was identical (8.5 with NADH; 7.7 with NADPH; and 9.0 with NAD+). The isozymes exhibited similar high optimum temperature values (60-62 degrees C) and isoelectric points (7.9-8.1). Activity was enhanced in vitro by Ca2+ ions and strongly inhibited by pyridoxal 5'-phosphate, KCN, o-phenanthroline and EDTA, and to a lesser extent by pHMB and methylacetimidate. In the aminating reaction the three isozymes were inhibited in a concentration-dependent process by both NADH and NADPH, with apparent Km values for NH4+ ranging from 13-53 mM; 0.36-1.85 mM for 2-oxoglutarate and 0.07-0.78 mM for NADH and NADPH. In the deaminating reaction apparent Km values ranged from 0.64-3.52 mM for L-glutamate and 0.20-0.32 for NAD+. In addition, the three isozymes exhibited a non-hyperbolic kinetics for NAD+ with negative cooperativity (n = 0.8).

  1. Nitro-oleic acid ameliorates oxygen and glucose deprivation/re-oxygenation triggered oxidative stress in renal tubular cells via activation of Nrf2 and suppression of NADPH oxidase.

    PubMed

    Nie, Huibin; Xue, Xia; Liu, Gang; Guan, Guangju; Liu, Haiying; Sun, Lina; Zhao, Long; Wang, Xueling; Chen, Zhixin

    2016-01-01

    Nitroalkene derivative of oleic acid (OA-NO2), due to its ability to mediate revisable Michael addition, has been demonstrated to have various biological properties and become a therapeutic agent in various diseases. Though its antioxidant properties have been reported in different models of acute kidney injury (AKI), the mechanism by which OA-NO2 attenuates intracellular oxidative stress is not well investigated. Here, we elucidated the anti-oxidative mechanism of OA-NO2 in an in vitro model of renal ischemia/reperfusion (I/R) injury. Human tubular epithelial cells were subjected to oxygen and glucose deprivation/re-oxygenation (OGD/R) injury. Pretreatment with OA-NO2 (1.25 μM, 45 min) attenuated OGD/R triggered reactive oxygen species (ROS) generation and subsequent mitochondrial membrane potential disruption. This action was mediated via up-regulating endogenous antioxidant defense components including superoxide dismutase (SOD1), heme oxygenase 1 (HO-1), and γ-glutamyl cysteine ligase modulatory subunits (GCLM). Moreover, subcellular fractionation analyses demonstrated that OA-NO2 promoted nuclear translocation of nuclear factor-E2- related factor-2 (Nrf2) and Nrf2 siRNA partially abrogated these protective effects. In addition, OA-NO2 inhibited NADPH oxidase activation and NADPH oxidase 4 (NOX4), NADPH oxidase 2 (NOX2) and p22(phox) up-regulation after OGD/R injury, which was not relevant to Nrf2. These results contribute to clarify that the mechanism of OA-NO2 reno-protection involves both inhibition of NADPH oxidase activity and induction of SOD1, Nrf2-dependent HO-1, and GCLM.

  2. Biochemical, Cellular, and Biophysical Characterization of a Potent Inhibitor of Mutant Isocitrate Dehydrogenase IDH1*

    PubMed Central

    Davis, Mindy I.; Gross, Stefan; Shen, Min; Straley, Kimberly S.; Pragani, Rajan; Lea, Wendy A.; Popovici-Muller, Janeta; DeLaBarre, Byron; Artin, Erin; Thorne, Natasha; Auld, Douglas S.; Li, Zhuyin; Dang, Lenny; Boxer, Matthew B.; Simeonov, Anton

    2014-01-01

    Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC50 = 68 nm), whereas the (−) isomer is over 400-fold less active (IC50 = 29 μm) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)-ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC50 >36 μm). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α-ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC50 = 250 nm) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered. PMID:24668804

  3. Tobacco Nectaries Express a Novel NADPH Oxidase Implicated in the Defense of Floral Reproductive Tissues against Microorganisms1[OA

    PubMed Central

    Carter, Clay; Healy, Rosanne; O'Tool, Nicole M.; Naqvi, S.M. Saqlan; Ren, Gang; Park, Sanggyu; Beattie, Gwyn A.; Horner, Harry T.; Thornburg, Robert W.

    2007-01-01

    Hydrogen peroxide produced from the nectar redox cycle was shown to be a major factor contributing to inhibition of most microbial growth in floral nectar; however, this obstacle can be overcome by the floral pathogen Erwinia amylovora. To identify the source of superoxide that leads to hydrogen peroxide accumulation in nectary tissues, nectaries were stained with nitroblue tetrazolium. Superoxide production was localized near nectary pores and inhibited by diphenylene iodonium but not by cyanide or azide, suggesting that NAD(P)H oxidase is the source of superoxide. Native PAGE assays demonstrated that NADPH (not NADH) was capable of driving the production of superoxide, diphenyleneiodonium chloride was an efficient inhibitor of this activity, but cyanide and azide did not inhibit. These results confirm that the production of superoxide was due to an NADPH oxidase. The nectary enzyme complex was distinct by migration on gels from the leaf enzyme complex. Temporal expression patterns demonstrated that the superoxide production (NADPH oxidase activity) was coordinated with nectar secretion, the expression of Nectarin I (a superoxide dismutase in nectar), and the expression of NOX1, a putative gene for a nectary NADPH oxidase that was cloned from nectaries and identified as an rbohD-like NADPH oxidase. Further, in situ hybridization studies indicated that the NADPH oxidase was expressed in the early stages of flower development although superoxide was generated at later stages (after Stage 10), implicating posttranslational regulation of the NADPH oxidase in the nectary. PMID:17114277

  4. Tobacco nectaries express a novel NADPH oxidase implicated in the defense of floral reproductive tissues against microorganisms.

    PubMed

    Carter, Clay; Healy, Rosanne; O'Tool, Nicole M; Naqvi, S M Saqlan; Ren, Gang; Park, Sanggyu; Beattie, Gwyn A; Horner, Harry T; Thornburg, Robert W

    2007-01-01

    Hydrogen peroxide produced from the nectar redox cycle was shown to be a major factor contributing to inhibition of most microbial growth in floral nectar; however, this obstacle can be overcome by the floral pathogen Erwinia amylovora. To identify the source of superoxide that leads to hydrogen peroxide accumulation in nectary tissues, nectaries were stained with nitroblue tetrazolium. Superoxide production was localized near nectary pores and inhibited by diphenylene iodonium but not by cyanide or azide, suggesting that NAD(P)H oxidase is the source of superoxide. Native PAGE assays demonstrated that NADPH (not NADH) was capable of driving the production of superoxide, diphenyleneiodonium chloride was an efficient inhibitor of this activity, but cyanide and azide did not inhibit. These results confirm that the production of superoxide was due to an NADPH oxidase. The nectary enzyme complex was distinct by migration on gels from the leaf enzyme complex. Temporal expression patterns demonstrated that the superoxide production (NADPH oxidase activity) was coordinated with nectar secretion, the expression of Nectarin I (a superoxide dismutase in nectar), and the expression of NOX1, a putative gene for a nectary NADPH oxidase that was cloned from nectaries and identified as an rbohD-like NADPH oxidase. Further, in situ hybridization studies indicated that the NADPH oxidase was expressed in the early stages of flower development although superoxide was generated at later stages (after Stage 10), implicating posttranslational regulation of the NADPH oxidase in the nectary.

  5. Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N(6)-monooxygenase.

    PubMed

    Abdelwahab, Heba; Robinson, Reeder; Rodriguez, Pedro; Adly, Camelia; El-Sohaimy, Sohby; Sobrado, Pablo

    2016-09-15

    l-lysine (l-Lys) N(6)-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)(+) or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding.

  6. The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I*

    PubMed Central

    Quinlan, Casey L.; Goncalves, Renata L. S.; Hey-Mogensen, Martin; Yadava, Nagendra; Bunik, Victoria I.; Brand, Martin D.

    2014-01-01

    Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD+ or NADH at about the same operating redox potential as the NADH/NAD+ pool and comprise the NADH/NAD+ isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)+ ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I. PMID:24515115

  7. Engineering of 2,3-butanediol dehydrogenase to reduce acetoin formation by glycerol-overproducing, low-alcohol Saccharomyces cerevisiae.

    PubMed

    Ehsani, Maryam; Fernández, Maria R; Biosca, Josep A; Julien, Anne; Dequin, Sylvie

    2009-05-01

    Engineered Saccharomyces cerevisiae strains overexpressing GPD1, which codes for glycerol-3-phosphate dehydrogenase, and lacking the acetaldehyde dehydrogenase Ald6 display large-scale diversion of the carbon flux from ethanol toward glycerol without accumulating acetate. Although GPD1 ald6 strains have great potential for reducing the ethanol contents in wines, one major side effect is the accumulation of acetoin, having a negative sensory impact on wine. Acetoin is reduced to 2,3-butanediol by the NADH-dependent 2,3-butanediol dehydrogenase Bdh1. In order to investigate the influence of potential factors limiting this reaction, we overexpressed BDH1, coding for native NADH-dependent Bdh1, and the engineered gene BDH1(221,222,223), coding for an NADPH-dependent Bdh1 enzyme with the amino acid changes 221 EIA 223 to 221 SRS 223, in a glycerol-overproducing wine yeast. We have shown that both the amount of Bdh1 and the NADH availability limit the 2,3-butanediol dehydrogenase reaction. During wine fermentation, however, the major limiting factor was the level of synthesis of Bdh1. Consistent with this finding, the overproduction of native or engineered Bdh1 made it possible to redirect 85 to 90% of the accumulated acetoin into 2,3-butanediol, a compound with neutral sensory characteristics. In addition, the production of diacetyl, a compound causing off-flavor in alcoholic beverages, whose production is increased in glycerol-overproducing yeast cells, was decreased by half. The production of higher alcohols and esters, which was slightly decreased or unchanged in GPD1 ald6 cells compared to that in the control cells, was not further modified in BDH1 cells. Overall, rerouting carbons toward glycerol and 2,3-butanediol represents a new milestone in the engineering of a low-alcohol yeast with desirable organoleptic features, permitting the decrease of the ethanol contents in wines by up to 3 degrees.

  8. Decreased Glutathione S-transferase Level and Neonatal Hyperbilirubinemia Associated with Glucose-6-phosphate Dehydrogenase Deficiency: A Perspective Review.

    PubMed

    Al-Abdi, Sameer Yaseen

    2017-02-01

    Classically, genetically decreased bilirubin conjugation and/or hemolysis account for the mechanisms contributing to neonatal hyperbilirubinemia associated with glucose-6-phosphate dehydrogenase (G6PD) deficiency. However, these mechanisms are not involved in most cases of this hyperbilirubinemia. Additional plausible mechanisms for G6PD deficiency-associated hyperbilirubinemia need to be considered. Glutathione S-transferases (GST) activity depends on a steady quantity of reduced form of glutathione (GSH). If GSH is oxidized, it is reduced back by glutathione reductase, which requires the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). The main source of NADPH is the pentose phosphate pathway, in which G6PD is the first enzyme. Rat kidney GSH, rat liver GST, and human red blood cell GST levels have been found to positively correlate with G6PD levels in their respective tissues. As G6PD is expressed in hepatocytes, it is expected that GST levels would be significantly decreased in hepatocytes of G6PD-deficient neonates. As hepatic GST binds bilirubin and prevents their reflux into circulation, hypothesis that decreased GST levels in hepatocytes is an additional mechanism contributing to G6PD deficiency-associated hyperbilirubinemia seems plausible. Evidence for and against this hypothesis are discussed in this article hoping to stimulate further research on the role of GST in G6PD deficiency-associated hyperbilirubinemia.

  9. Involvement of glucose-6-phosphate dehydrogenase in reduced glutathione maintenance and hydrogen peroxide signal under salt stress.

    PubMed

    Wang, Xiaomin; Ma, Yuanyuan; Huang, Chenghong; Li, Jisheng; Wan, Qi; Bi, Yurong

    2008-06-01

    Cellular redox homeostasis is essential for plant growth, development as well as for the resistance to biotic and abiotic stresses, which is governed by the complex network of prooxidant and antioxidant systems. Recently, new evidence has been published that NADPH, produced by glucose-6-phosephate dehydrogenase enzyme (G6PDH), not only acted as the reducing potential for the output of reduced glutathione (GSH), but was involved in the activity of plasma membrane (PM) NADPH oxidase under salt stress, which resulted in hydrogen peroxide (H(2)O(2)) accumulation. H(2)O(2) acts as a signal in regulating G6PDH activity and expression, and the activities of the enzymes in the glutathione cycle as well, through which the ability of GSH regeneration was increased under salt stress. Thus, G6PDH plays a critical role in maintaining cellular GSH levels under long-term salt stress. In this addendum, a hypothetical model for the roles of G6PDH in modulating the intracellular redox homeostasis under salt stress is presented.

  10. Mitochondrial NADP(+)-Dependent Isocitrate Dehydrogenase Deficiency Exacerbates Mitochondrial and Cell Damage after Kidney Ischemia-Reperfusion Injury.

    PubMed

    Han, Sang Jun; Jang, Hee-Seong; Noh, Mi Ra; Kim, Jinu; Kong, Min Jung; Kim, Jee In; Park, Jeen-Woo; Park, Kwon Moo

    2017-04-01

    Mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate, synthesizing NADPH, which is essential for mitochondrial redox balance. Ischemia-reperfusion (I/R) is one of most common causes of AKI. I/R disrupts the mitochondrial redox balance, resulting in oxidative damage to mitochondria and cells. Here, we investigated the role of IDH2 in I/R-induced AKI. I/R injury in mice led to the inactivation of IDH2 in kidney tubule cells. Idh2 gene deletion exacerbated the I/R-induced increase in plasma creatinine and BUN levels and the histologic evidence of tubule injury, and augmented the reduction of NADPH levels and the increase in oxidative stress observed in the kidney after I/R. Furthermore, Idh2 gene deletion exacerbated I/R-induced mitochondrial dysfunction and morphologic fragmentation, resulting in severe apoptosis in kidney tubule cells. In cultured mouse kidney proximal tubule cells, Idh2 gene downregulation enhanced the mitochondrial damage and apoptosis induced by treatment with hydrogen peroxide. This study demonstrates that Idh2 gene deletion exacerbates mitochondrial damage and tubular cell death via increased oxidative stress, suggesting that IDH2 is an important mitochondrial antioxidant enzyme that protects cells from I/R insult.

  11. Conserved catalytic residues of the ALDH1L1 aldehyde dehydrogenase domain control binding and discharging of the coenzyme.

    PubMed

    Tsybovsky, Yaroslav; Krupenko, Sergey A

    2011-07-01

    The C-terminal domain (C(t)-FDH) of 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an NADP(+)-dependent oxidoreductase and a structural and functional homolog of aldehyde dehydrogenases. Here we report the crystal structures of several C(t)-FDH mutants in which two essential catalytic residues adjacent to the nicotinamide ring of bound NADP(+), Cys-707 and Glu-673, were replaced separately or simultaneously. The replacement of the glutamate with an alanine causes irreversible binding of the coenzyme without any noticeable conformational changes in the vicinity of the nicotinamide ring. Additional replacement of cysteine 707 with an alanine (E673A/C707A double mutant) did not affect this irreversible binding indicating that the lack of the glutamate is solely responsible for the enhanced interaction between the enzyme and the coenzyme. The substitution of the cysteine with an alanine did not affect binding of NADP(+) but resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme: unlike the wild-type C(t)-FDH/NADPH complex, in the C707A mutant the position of NADPH is identical to the position of NADP(+) with the nicotinamide ring well ordered within the catalytic center. Thus, whereas the glutamate restricts the affinity for the coenzyme, the cysteine is the sensor of the coenzyme redox state. These conclusions were confirmed by coenzyme binding experiments. Our study further suggests that the binding of the coenzyme is additionally controlled by a long-range communication between the catalytic center and the coenzyme-binding domain and points toward an α-helix involved in the adenine moiety binding as a participant of this communication.

  12. Aldehyde dehydrogenase is used by cancer cells for energy metabolism

    PubMed Central

    Kang, Joon Hee; Lee, Seon-Hyeong; Hong, Dongwan; Lee, Jae-Seon; Ahn, Hee-Sung; Ahn, Ju-Hyun; Seong, Tae Wha; Lee, Chang-Hun; Jang, Hyonchol; Hong, Kyeong Man; Lee, Cheolju; Lee, Jae-Ho; Kim, Soo-Youl

    2016-01-01

    We found that non-small-cell lung cancer (NSCLC) cells express high levels of multiple aldehyde dehydrogenase (ALDH) isoforms via an informatics analysis of metabolic enzymes in NSCLC and immunohistochemical staining of NSCLC clinical tumor samples. Using a multiple reaction-monitoring mass spectrometry analysis, we found that multiple ALDH isozymes were generally abundant in NSCLC cells compared with their levels in normal IMR-90 human lung cells. As a result of the catalytic reaction mediated by ALDH, NADH is produced as a by-product from the conversion of aldehyde to carboxylic acid. We hypothesized that the NADH produced by ALDH may be a reliable energy source for ATP production in NSCLC. This study revealed that NADH production by ALDH contributes significantly to ATP production in NSCLC. Furthermore, gossypol, a pan-ALDH inhibitor, markedly reduced the level of ATP. Gossypol combined with phenformin synergistically reduced the ATP levels, which efficiently induced cell death following cell cycle arrest. PMID:27885254

  13. Biofuel cell anode: NAD +/glucose dehydrogenase-coimmobilized ketjenblack electrode

    NASA Astrophysics Data System (ADS)

    Miyake, T.; Oike, M.; Yoshino, S.; Yatagawa, Y.; Haneda, K.; Kaji, H.; Nishizawa, M.

    2009-09-01

    We have studied the coimmobilization of glucose dehydrogenase (GDH) and its cofactor, oxidized nicotinamide adenine dinucleotide (NAD +), on a ketjenblack (KB) electrode as a step toward a biofuel cell anode that works without mediators. A KB electrode was first treated with a sulfuric acid/nitric acid/water mixture to lower the overvoltage for NADH oxidation, and was next chemically modified with NAD + and GDH. The improved GDH/NAD +/KB electrode is found to oxidize glucose around 0 V vs. Ag/AgCl. A biofuel cell constructed with a bilirubin oxidase-immobilized KB cathode showed a maximum power density of 52 μW/cm 2 at 0.3 V.

  14. Differential response of NADP-dehydrogenases and carbon metabolism in leaves and roots of two durum wheat (Triticum durum Desf.) cultivars (Karim and Azizi) with different sensitivities to salt stress.

    PubMed

    Bouthour, Donia; Kalai, Tawba; Chaffei, Haouari C; Gouia, Houda; Corpas, Francisco J

    2015-05-01

    Wheat (Triticum durum Desf.) is a common Mediterranean species of considerable agronomic importance. Salinity is one of the major threats to sustainable agricultural production mainly because it limits plant productivity. After exposing the Karim and Azizi durum wheat cultivars, which are of agronomic significance in Tunisia, to 100mM NaCl salinity, growth parameters (dry weight and length), proline content and chlorophylls were evaluated in their leaves and roots. In addition, we analyzed glutathione content and key enzymatic activities, including phosphoenolpyruvate carboxylase (PEPC), NADP-isocitrate dehydrogenase (NADP-ICDH), NADP-malic enzyme (NADP-ME), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), involved in the carbon metabolism and NADPH-generating system. The sensitivity index indicates that cv Karim was more tolerant to salinity than cv Azizi. This higher tolerance was corroborated at the biochemical level, as cv Karim showed a greater capacity to accumulate proline, especially in leaves, while the enzyme activities studied were differentially regulated in both organs, with NADP-ICDH being the only activity to be unaffected in all organs. In summary, the data indicate that higher levels of proline accumulation and the differential responses of some key enzymes involved in the carbon metabolism and NADPH regeneration contribute to the salinity tolerance mechanism and lead to increased biomass accumulation in cv Karim.

  15. Fundamental molecular differences between alcohol dehydrogenase classes.

    PubMed Central

    Danielsson, O; Atrian, S; Luque, T; Hjelmqvist, L; Gonzàlez-Duarte, R; Jörnvall, H

    1994-01-01

    Two types of alcohol dehydrogenase in separate protein families are the "medium-chain" zinc enzymes (including the classical liver and yeast forms) and the "short-chain" enzymes (including the insect form). Although the medium-chain family has been characterized in prokaryotes and many eukaryotes (fungi, plants, cephalopods, and vertebrates), insects have seemed to possess only the short-chain enzyme. We have now also characterized a medium-chain alcohol dehydrogenase in Drosophila. The enzyme is identical to insect octanol dehydrogenase. It is a typical class III alcohol dehydrogenase, similar to the corresponding human form (70% residue identity), with mostly the same residues involved in substrate and coenzyme interactions. Changes that do occur are conservative, but Phe-51 is of functional interest in relation to decreased coenzyme binding and increased overall activity. Extra residues versus the human enzyme near position 250 affect the coenzyme-binding domain. Enzymatic properties are similar--i.e., very low activity toward ethanol (Km beyond measurement) and high selectivity for formaldehyde/glutathione (S-hydroxymethylglutathione; kcat/Km = 160,000 min-1.mM-1). Between the present class III and the ethanol-active class I enzymes, however, patterns of variability differ greatly, highlighting fundamentally separate molecular properties of these two alcohol dehydrogenases, with class III resembling enzymes in general and class I showing high variation. The gene coding for the Drosophila class III enzyme produces an mRNA of about 1.36 kb that is present at all developmental stages of the fly, compatible with the constitutive nature of the vertebrate enzyme. Taken together, the results bridge a previously apparent gap in the distribution of medium-chain alcohol dehydrogenases and establish a strictly conserved class III enzyme, consistent with an important role for this enzyme in cellular metabolism. Images PMID:8197167

  16. The involvement of superoxide and iron ions in the NADPH-dependent lipid peroxidation in human placental mitochondria.

    PubMed

    Klimek, J

    1988-01-19

    Incubation of human term placental mitochondria with Fe2+ and a NADPH-generating system initiated high levels of lipid peroxidation, as measured by the production of malondialdehyde. Malondialdehyde formation was accompanied by a corresponding decrease of the unsaturated fatty acid content. This NADPH-dependent lipid peroxidation was strongly inhibited by superoxide dismutase and singlet oxygen scavengers, markedly stimulated by paraquat, but was not affected by hydroxyl radical scavengers. Catalase enhanced the production of malondialdehyde by placental mitochondria. The effects of catalase and hydroxyl radical scavengers suggest that the initiation of NADPH-dependent lipid peroxidation is not dependent upon the hydroxyl radical produced via an iron-catalyzed Fenton reaction. These studies provide evidence that hydrogen peroxide strongly inhibits NADPH-dependent mitochondrial lipid peroxidation. The inhibitory effect of superoxide dismutase and stimulatory effect of paraquat, which was abolished by the addition of superoxide dismutase, suggests that superoxide may promote NADPH-dependent lipid peroxidation in human placental mitochondria.

  17. Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum.

    PubMed

    Kleijn, Roelco J; Liu, Feng; van Winden, Wouter A; van Gulik, Walter M; Ras, Cor; Heijnen, Joseph J

    2007-01-01

    This study addresses the relation between NADPH supply and penicillin synthesis, by comparing the flux through the oxidative branch of the pentose phosphate pathway (PPP; the main source of cytosolic NADPH) in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. The fluxes through the oxidative part of the PPP were determined using the recently introduced gluconate-tracer method. Significantly higher oxidative PPP fluxes were observed in penicillin-G producing chemostat cultures, indicating that penicillin production puts a major burden on the supply of cytosolic NADPH. To our knowledge this is the first time direct experimental proof is presented for the causal relationship between penicillin production and NADPH supply. Additional insight in the metabolism of P. chrysogenum was obtained by comparing the PPP fluxes from the gluconate-tracer experiment to oxidative PPP fluxes derived via metabolic flux analysis, using different assumptions for the stoichiometry of NADPH consumption and production.

  18. Analysis and prediction of the physiological effects of altered coenzyme specificity in xylose reductase and xylitol dehydrogenase during xylose fermentation by Saccharomyces cerevisiae.

    PubMed

    Krahulec, Stefan; Klimacek, Mario; Nidetzky, Bernd

    2012-04-30

    An advanced strategy of Saccharomyces cerevisiae strain development for fermentation of xylose applies tailored enzymes in the process of metabolic engineering. The coenzyme specificities of the NADPH-preferring xylose reductase (XR) and the NAD⁺-dependent xylitol dehydrogenase (XDH) have been targeted in previous studies by protein design or evolution with the aim of improving the recycling of NADH or NADPH in their two-step pathway, converting xylose to xylulose. Yeast strains expressing variant pairs of XR and XDH that according to in vitro kinetic data were suggested to be much better matched in coenzyme usage than the corresponding pair of wild-type enzymes, exhibit widely varying capabilities for xylose fermentation. To achieve coherence between enzyme properties and the observed strain performance during fermentation, we explored the published kinetic parameters for wild-type and engineered forms of XR and XDH as possible predictors of xylitol by-product formation (Y(xylitol)) in yeast physiology. We found that the ratio of enzymatic reaction rates using NADP(H) and NAD(H) that was calculated by applying intracellular reactant concentrations to rate equations derived from bi-substrate kinetic analysis, succeeded in giving a statistically reliable forecast of the trend effect on Y(xylitol). Prediction based solely on catalytic efficiencies with or without binding affinities for NADP(H) and NAD(H) were not dependable, and we define a minimum demand on the enzyme kinetic characterization to be performed for this purpose. An immediate explanation is provided for the typically lower Y(xylitol) in the current strains harboring XR engineered for utilization of NADH as compared to strains harboring XDH engineered for utilization of NADP⁺. The known XDH enzymes all exhibit a relatively high K(m) for NADP⁺ so that physiological boundary conditions are somewhat unfavorable for xylitol oxidation by NADP⁺. A criterion of physiological fitness is developed for

  19. Analysis and prediction of the physiological effects of altered coenzyme specificity in xylose reductase and xylitol dehydrogenase during xylose fermentation by Saccharomyces cerevisiae

    PubMed Central

    Krahulec, Stefan; Klimacek, Mario; Nidetzky, Bernd

    2012-01-01

    An advanced strategy of Saccharomyces cerevisiae strain development for fermentation of xylose applies tailored enzymes in the process of metabolic engineering. The coenzyme specificities of the NADPH-preferring xylose reductase (XR) and the NAD+-dependent xylitol dehydrogenase (XDH) have been targeted in previous studies by protein design or evolution with the aim of improving the recycling of NADH or NADPH in their two-step pathway, converting xylose to xylulose. Yeast strains expressing variant pairs of XR and XDH that according to in vitro kinetic data were suggested to be much better matched in coenzyme usage than the corresponding pair of wild-type enzymes, exhibit widely varying capabilities for xylose fermentation. To achieve coherence between enzyme properties and the observed strain performance during fermentation, we explored the published kinetic parameters for wild-type and engineered forms of XR and XDH as possible predictors of xylitol by-product formation (Yxylitol) in yeast physiology. We found that the ratio of enzymatic reaction rates using NADP(H) and NAD(H) that was calculated by applying intracellular reactant concentrations to rate equations derived from bi-substrate kinetic analysis, succeeded in giving a statistically reliable forecast of the trend effect on Yxylitol. Prediction based solely on catalytic efficiencies with or without binding affinities for NADP(H) and NAD(H) were not dependable, and we define a minimum demand on the enzyme kinetic characterization to be performed for this purpose. An immediate explanation is provided for the typically lower Yxylitol in the current strains harboring XR engineered for utilization of NADH as compared to strains harboring XDH engineered for utilization of NADP+. The known XDH enzymes all exhibit a relatively high Km for NADP+ so that physiological boundary conditions are somewhat unfavorable for xylitol oxidation by NADP+. A criterion of physiological fitness is developed for engineered XR

  20. NADPH- and linoleic acid hydroperoxide-induced lipid peroxidation and destruction of cytochrome P-450 in hepatic microsomes.

    PubMed

    Iba, M M; Mannering, G J

    1987-05-01

    Temporal aspects of the effects of inhibitors on hepatic cytochrome P-450 destruction and lipid peroxidation induced by NADPH and linoleic acid hydroperoxide (LAHP) were compared. In the absence of added Fe2+, NADPH-induced lipid peroxidation in hepatic microsomes exhibited a slow phase followed by a fast phase. The addition of Fe2+ eliminated the slow phase, thus demonstrating that iron is a rate-limiting component in the reaction. EDTA, which complexes iron, and p-chloromercurobenzoate (pCMB), which inhibits NADPH-cytochrome P-450 reductase, inhibited both phases of the reaction. Catalase as well as scavengers of hydroxyl radical, inhibited NADPH-induced lipid peroxidation almost completely. GSH also inhibited the NADPH-dependent reaction but only when added at the beginning of the reaction. In contrast with NADPH-dependent lipid peroxidation, the autocatalytic reaction induced by LAHP was not biphasic, NADPH-dependent or iron-dependent, nor was it inhibited by hydroxyl radical scavengers, catalase or GSH. A synergistic effect on lipid peroxidation was observed when both NADPH and LAHP were added to microsomes. It is concluded that both the fast and slow phases of NADPH-dependent microsomal lipid peroxidation are catalyzed enzymatically and are dependent upon Fe2+, whereas LAHP-dependent lipid peroxidation is autocatalytic. Since the fast phase of enzymatic lipid peroxidation occurred during the fast phase of destruction of cytochrome P-450, it is postulated that iron made available from cytochrome P-450 is sufficient to promote optimal lipid peroxidation. Since catalase and hydroxyl radical scavengers inhibited NADPH-dependent but not LAHP-dependent lipid peroxidation, it is concluded that the hydroxyl radical derived from H2O2 is the initiating active-oxygen species in the enzymatic reaction but not in the autocatalytic reaction.

  1. The involvement of P2Y12 receptors, NADPH oxidase, and lipid rafts in the action of extracellular ATP on synaptic transmission at the frog neuromuscular junction.

    PubMed

    Giniatullin, A; Petrov, A; Giniatullin, R

    2015-01-29

    Adenosine 5'-triphosphate (ATP) is the main co-transmitter accompanying the release of acetylcholine from motor nerve terminals. Previously, we revealed the direct inhibitory action of extracellular ATP on transmitter release via redox-dependent mechanism. However, the receptor mechanism of ATP action and ATP-induced sources of reactive oxygen sources (ROS) remained not fully understood. In the current study, using microelectrode recordings of synaptic currents from the frog neuromuscular junction, we analyzed the receptor subtype involved in synaptic action of ATP, receptor coupling to NADPH oxidase and potential location of ATP receptors within the lipid rafts. Using subtype-specific antagonists, we found that the P2Y13 blocker 2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde did not prevent the depressant action of ATP. In contrast, the P2Y12 antagonist 2-methylthioadenosine 5'-monophosphate abolished the inhibitory action of ATP, suggesting the key role of P2Y12 receptors in ATP action. As the action of ATP is redox-dependent, we also tested potential involvement of the NADPH oxidase, known as a common inducer of ROS. The depressant action of extracellular ATP was significantly reduced by diphenyleneiodonium chloride and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, two structurally different inhibitors of NADPH oxidase, indicating that this enzyme indeed mediates the action of ATP. Since the location and activity of various receptors are often associated with lipid rafts, we next tested whether ATP-driven inhibition depends on lipid rafts. We found that the disruption of lipid rafts with methyl-beta-cyclodextrin reduced and largely delayed the action of ATP. Taken together, these data revealed key steps in the purinergic control of synaptic transmission via P2Y12 receptors associated with lipid rafts, and identified NADPH oxidase as the main source of ATP-induced inhibitory ROS at the neuromuscular

  2. Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation.

    PubMed

    Gabig, T G; Crean, C D; Mantel, P L; Rosli, R

    1995-02-01

    Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1-166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47-phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates

  3. Directed evolution and structural analysis of NADPH-dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics.

    PubMed

    Matsumoto, Ken'ichiro; Tanaka, Yoshikazu; Watanabe, Tsuyoshi; Motohashi, Ren; Ikeda, Koji; Tobitani, Kota; Yao, Min; Tanaka, Isao; Taguchi, Seiichi

    2013-10-01

    NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.

  4. Chronic intake of red wine polyphenols by young rats prevents aging-induced endothelial dysfunction and decline in physical performance: role of NADPH oxidase.

    PubMed

    Dal-Ros, Stéphanie; Zoll, Joffrey; Lang, Anne-Laure; Auger, Cyril; Keller, Nathalie; Bronner, Christian; Geny, Bernard; Schini-Kerth, Valérie B

    2011-01-14

    Aging is associated with oxidative stress-mediated endothelial dysfunction and decline in physical performance, which promote cardiovascular diseases. This study examined whether chronic intake of red wine polyphenols (RWPs), a rich source of natural antioxidants, prevents aging-related impairment of vascular function and physical exercise capacity. Vascular reactivity from 12, 20 and 40 week-old rats was assessed in organ chambers. Rats received from week 16 to 40 either solvent, RWPs or the antioxidant and NADPH oxidase inhibitor, apocynin. Aging was associated with blunted endothelium-dependent relaxations, oxidative stress (dihydroethidine staining), and an upregulation of eNOS, arginase I, NADPH oxidase p22phox and nox1 subunits, and AT1 and AT2 receptors (assessed by immunohistochemistry) in the mesenteric artery. RWPs and apocynin improved the endothelial dysfunction, normalized oxidative stress and the expression of the different proteins. RWPs also improved aging-related decline in physical exercise. Thus, intake of RWPs protects against aging-induced endothelial dysfunction and decline in physical performance. These effects likely involve the ability of RWPs to normalize oxidative stress and the expression of proteins involved in the formation of NO and the angiotensin II pathway.

  5. Growth arrest of lung carcinoma cells (A549) by polyacrylate-anchored peroxovanadate by activating Rac1-NADPH oxidase signalling axis.

    PubMed

    Chatterjee, Nirupama; Anwar, Tarique; Islam, Nashreen S; Ramasarma, T; Ramakrishna, Gayatri

    2016-09-01

    Hydrogen peroxide is often required in sublethal, millimolar concentrations to show its oxidant effects on cells in culture as it is easily destroyed by cellular catalase. Previously, we had shown that diperoxovanadate, a physiologically stable peroxovanadium compound, can substitute H2O2 effectively in peroxidation reactions. We report here that peroxovanadate when anchored to polyacrylic acid (PAPV) becomes a highly potent inhibitor of growth of lung carcinoma cells (A549). The early events associated with PAPV treatment included cytoskeletal modifications, increase in GTPase activity of Rac1, accumulation of the reactive oxygen species, and also increase in phosphorylation of H2AX (γH2AX), a marker of DNA damage. These effects persisted even at 24 h after removal of the compound and culminated in increased levels of p53 and p21 together with growth arrest. The PAPV-mediated growth arrest was significantly abrogated in cells pre-treated with the N-acetylcysteine, Rac1 knocked down by siRNA and DPI an inhibitor of NADPH oxidase. In conclusion, our results show that polyacrylate derivative of peroxovanadate efficiently arrests growth of A549 cancerous cells by activating the axis of Rac1-NADPH oxidase leading to oxidative stress and DNA damage.

  6. Role of the NADPH Oxidases DUOX and NOX4 in Thyroid Oxidative Stress.

    PubMed

    Carvalho, Denise P; Dupuy, Corinne

    2013-09-01

    Somatic mutations are present at high levels in the rat thyroid gland, indicating that the thyrocyte is under oxidative stress, a state in which cellular oxidant levels are high. The most important class of free radicals, or reactive metabolites, is reactive oxygen species (ROS), such as superoxide anion (O2 (-)), hydroxyl radical (OH) and hydrogen peroxide (H2O2). The main source of ROS in every cell type seems to be mitochondrial respiration; however, recent data support the idea that NADPH:O(2) oxidoreductase flavoproteins or simply NADPH oxidases (NOX) are enzymes specialized in controlled ROS generation at the subcellular level. Several decades ago, high concentrations of H2O2 were detected at the apical surface of thyrocytes, where thyroid hormone biosynthesis takes place. Only in the last decade has the enzymatic source of H2O2 involved in thyroid hormone biosynthesis been well characterized. The cloning of two thyroid genes encoding NADPH oxidases dual oxidases 1 and 2 (DUOX1 and DUOX2) revealed that DUOX2 mutations lead to hereditary hypothyroidism in humans. Recent reports have also described the presence of NOX4 in the thyroid gland and have suggested a pathophysiological role of this member of the NOX family. In the present review, we describe the participation of NADPH oxidases not only in thyroid physiology but also in gland pathophysiology, particularly the involvement of these enzymes in the regulation of thyroid oxidative stress.

  7. Role of the NADPH Oxidases DUOX and NOX4 in Thyroid Oxidative Stress

    PubMed Central

    Carvalho, Denise P.; Dupuy, Corinne

    2013-01-01

    Somatic mutations are present at high levels in the rat thyroid gland, indicating that the thyrocyte is under oxidative stress, a state in which cellular oxidant levels are high. The most important class of free radicals, or reactive metabolites, is reactive oxygen species (ROS), such as superoxide anion (O2-), hydroxyl radical (OH) and hydrogen peroxide (H2O2). The main source of ROS in every cell type seems to be mitochondrial respiration; however, recent data support the idea that NADPH:O(2) oxidoreductase flavoproteins or simply NADPH oxidases (NOX) are enzymes specialized in controlled ROS generation at the subcellular level. Several decades ago, high concentrations of H2O2 were detected at the apical surface of thyrocytes, where thyroid hormone biosynthesis takes place. Only in the last decade has the enzymatic source of H2O2 involved in thyroid hormone biosynthesis been well characterized. The cloning of two thyroid genes encoding NADPH oxidases dual oxidases 1 and 2 (DUOX1 and DUOX2) revealed that DUOX2 mutations lead to hereditary hypothyroidism in humans. Recent reports have also described the presence of NOX4 in the thyroid gland and have suggested a pathophysiological role of this member of the NOX family. In the present review, we describe the participation of NADPH oxidases not only in thyroid physiology but also in gland pathophysiology, particularly the involvement of these enzymes in the regulation of thyroid oxidative stress. PMID:24847449

  8. Nonhematopoietic NADPH oxidase regulation of lung eosinophilia and airway hyperresponsiveness in experimentally induced asthma

    PubMed Central

    Abdala-Valencia, Hiam; Earwood, Julie; Bansal, Shelly; Jansen, Michael; Babcock, George; Garvy, Beth; Wills-Karp, Marsha; Cook-Mills, Joan M.

    2009-01-01

    Pulmonary eosinophilia is one of the most consistent hallmarks of asthma. Infiltration of eosinophils into the lung in experimental asthma is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Ligation of VCAM-1 activates endothelial cell NADPH oxidase, which is required for VCAM-1-dependent leukocyte migration in vitro. To examine whether endothelial-derived NADPH oxidase modulates eosinophil recruitment in vivo, mice deficient in NADPH oxidase (CYBB mice) were irradiated and received wild-type hematopoietic cells to generate chimeric CYBB mice. In response to ovalbumin (OVA) challenge, the chimeric CYBB mice had increased numbers of eosinophils bound to the endothelium as well as reduced eosinophilia in the lung tissue and bronchoalveolar lavage. This occurred independent of changes in VCAM-1 expression, cytokine/chemokine levels (IL-5, IL-10, IL-13, IFNγ, or eotaxin), or numbers of T cells, neutrophils, or mononuclear cells in the lavage fluids or lung tissue of OVA-challenged mice. Importantly, the OVA-challenged chimeric CYBB mice had reduced airway hyperresponsiveness (AHR). The AHR in OVA-challenged chimeric CYBB mice was restored by bypassing the endothelium with intratracheal administration of eosinophils. These data suggest that VCAM-1 induction of NADPH oxidase in the endothelium is necessary for the eosinophil recruitment during allergic inflammation. Moreover, these studies provide a basis for targeting VCAM-1-dependent signaling pathways in asthma therapies. PMID:17293377

  9. Inhibition of NADPH oxidases prevents chronic ethanol-induced bone loss in female rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous in vitro data suggest that ethanol (EtOH) activates NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox) in osteoblasts leading to accumulation of reactive oxygen species (ROS). This might be a mechanism underlying inhibition of bone formation and increased bone resorption obse...

  10. Titanium Dioxide Nanoparticles Increase Superoxide Anion Production by Acting on NADPH Oxidase.

    PubMed

    Masoud, Rawand; Bizouarn, Tania; Trepout, Sylvain; Wien, Frank; Baciou, Laura; Marco, Sergio; Houée Levin, Chantal

    2015-01-01

    Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development.

  11. Alcohol-induced bone loss is blocked in p47phox -/- mice lacking functional nadph oxidases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic ethanol (EtOH) consumption produces bone loss. Previous data suggest a role for NADPH oxidase enzymes (Nox) since the pan-Nox inhibitor diphenylene iodonium (DPI) blocks EtOH-induced bone loss in rats. The current study utilized mice in which Nox enzymes 1,2,3 and 5 are inactivated as a resu...

  12. A pathway for repair of NAD(P)H in plants.

    PubMed

    Colinas, Maite; Shaw, Holly V; Loubéry, Sylvain; Kaufmann, Markus; Moulin, Michael; Fitzpatrick, Teresa B

    2014-05-23

    Unwanted enzyme side reactions and spontaneous decomposition of metabolites can lead to a build-up of compounds that compete with natural enzyme substrates and must be dealt with for efficient metabolism. It has recently been realized that there are enzymes that process such compounds, formulating the concept of metabolite repair. NADH and NADPH are vital cellular redox cofactors but can form non-functional hydrates (named NAD(P)HX) spontaneously or enzymatically that compete with enzymes dependent on NAD(P)H, impairing normal enzyme function. Here we report on the functional characterization of components of a potential NAD(P)H repair pathway in plants comprising a stereospecific dehydratase (NNRD) and an epimerase (NNRE), the latter being fused to a vitamin B6 salvage enzyme. Through the use of the recombinant proteins, we show that the ATP-dependent NNRD and NNRE act concomitantly to restore NAD(P)HX to NAD(P)H. NNRD behaves as a tetramer and NNRE as a dimer, but the proteins do not physically interact. In vivo fluorescence analysis demonstrates that the proteins are localized to mitochondria and/or plastids, implicating these as the key organelles where this repair is required. Expression analysis indicates that whereas NNRE is present ubiquitously, NNRD is restricted to seeds but appears to be dispensable during the normal Arabidopsis life cycle.

  13. 21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...-phosphogluconate dehydrogenase (6 PGD) in serum and erythrocytes. Measurements of 6-phosphogluconate dehydrogenase are used in the diagnosis and treatment of certain liver diseases (such as hepatitis) and anemias....

  14. NADPH diaphorase-positive neurons in the lizard hippocampus: a distinct subpopulation of GABAergic interneurons.

    PubMed

    Dávila, J C; Megías, M; Andreu, M J; Real, M A; Guirado, S

    1995-01-01

    We analyzed the distribution and light-microscopic features of the NADPH diaphorase-containing structures in the lizard hippocampus, likely to correspond to nitric oxide synthase-containing cells and fibers, and thus likely to release nitric oxide. We also studied co-localization of NADPH diaphorase with the neurotransmitter GABA, the calcium-binding protein parvalbumin, and the neuropeptide somatostatin, in order to examine whether putative nitric oxide-synthesizing neurons represent a different subpopulation of GABA cells, on which the authors recently reported in lizards. We also studied co-localization of NADPH diaphorase with parvalbumin or somatostatin in mice to ascertain whether the characteristics of this population in reptiles parallel the situation in mammals. Most of the positive NADPH diaphorase neurons were stained in a Golgi-like manner and were in the plexiform layers of the lizard hippocampus with morphologies ranging from bipolar to multipolar. Co-localization with GABA was 100%, and NADPH diaphorase-positive neurons in the lizard hippocampus did not contain parvalbumin or somatostatin. The results indicate that putative nitric oxide-synthesizing neurons represent a distinct subpopulation of GABA interneurons in the lizard hippocampus. Two different types of fibers were described in the plexiform layers: one type bearing thick varicosities, and the other thinner ones. We discuss the possibility that at least part of the positive fibers arise from a hypothalamic aminergic nucleus contacting the third ventricle, the periventricular hypothalamic organ. Most radial glia were stained almost completely and formed typical end-feet both at the pia and around capillaries. The results of this study confirm that the capacity for synthesizing nitric oxide is linked to a determined set of neuronal markers depending on the specific brain region, and they provide new resemblances between hippocampal regions in different classes of vertebrates.

  15. Decline in NAD(P)H autofluorescence precedes apoptotic cell death from chemotherapy

    NASA Astrophysics Data System (ADS)

    Toms, Steven A.; Muhammad, Osman; Jackson, Heather; Lin, Wei-Chiang

    2005-11-01

    OBJECTIVE: Optical spectroscopic tools exist that allow open surgical and minimally invasive assays of intrinsic tissue optics. Optical detection of cellular and tissue viability may offer a minimally invasive way to assess tumor responsiveness to chemotherapies. We report on an optical spectroscopic change that precedes apoptotic cell death and appears related to NAD(P)H autofluorescence. METHODS: The cell lines SW 480 and U87-MG were grown in culture and treated with cisplatin 100 μg/ml and tamoxifen 10 μM, respectively. Fluorescence spectroscopy at 355 nm excitation and 460 nm emission were collected. MTS assays were used to determine cell viability. Cell lysates were analyzed for NAD(P)H concentrations by mass spectroscopy. RESULTS: Autoflourescence at 355 nm excitation and 460 nm emission declines markedly despite normalization for cell number and total protein concentration after treatment with tamoxifen or cisplatin. The autofluorescence drop precedes the loss of cell viability as measured by MTS assay. For example, the relative viability of the U87-MG cell treated with tamoxifen at hours 0, 8, 12 and 24 of treatment was 100 +/- 6, 85 +/- 6, 53 +/- 9 and 0 +/- 3. The relative fluorescence at the same time points were 100 +/- 2, 57 +/- 6, 47 +/- 3, and 0 +/- 1. TUNNEL assays confirm that cell death is via apoptosis. The key cellular fluorophore at these wavelengths is NAD(P)H. Mass spectroscopic analysis of cell lysates at these time points reveals a drop in NAD(P)H concentrations that is parallel to the loss of fluorescence signal. CONCLUSIONS: NAD(P)H autofluoresence decline precedes apoptotic cell death. This may allow the design of minimally invasive spectroscopic tools to monitor chemotherapeutic response.

  16. NADPH oxidase 4 regulates homocysteine metabolism and protects against acetaminophen-induced liver damage in mice.

    PubMed

    Murray, Thomas V A; Dong, Xuebin; Sawyer, Greta J; Caldwell, Anna; Halket, John; Sherwood, Roy; Quaglia, Alberto; Dew, Tracy; Anilkumar, Narayana; Burr, Simon; Mistry, Rajesh K; Martin, Daniel; Schröder, Katrin; Brandes, Ralf P; Hughes, Robin D; Shah, Ajay M; Brewer, Alison C

    2015-12-01

    Glutathione is the major intracellular redox buffer in the liver and is critical for hepatic detoxification of xenobiotics and other environmental toxins. Hepatic glutathione is also a major systemic store for other organs and thus impacts on pathologies such as Alzheimer's disease, Sickle Cell Anaemia and chronic diseases associated with aging. Glutathione levels are determined in part by the availability of cysteine, generated from homocysteine through the transsulfuration pathway. The partitioning of homocysteine between remethylation and transsulfuration pathways is known to be subject to redox-dependent regulation, but the underlying mechanisms are not known. An association between plasma Hcy and a single nucleotide polymorphism within the NADPH oxidase 4 locus led us to investigate the involvement of this reactive oxygen species- generating enzyme in homocysteine metabolism. Here we demonstrate that NADPH oxidase 4 ablation in mice results in increased flux of homocysteine through the betaine-dependent remethylation pathway to methionine, catalysed by betaine-homocysteine-methyltransferase within the liver. As a consequence NADPH oxidase 4-null mice display significantly lowered plasma homocysteine and the flux of homocysteine through the transsulfuration pathway is reduced, resulting in lower hepatic cysteine and glutathione levels. Mice deficient in NADPH oxidase 4 had markedly increased susceptibility to acetaminophen-induced hepatic injury which could be corrected by administration of N-acetyl cysteine. We thus conclude that under physiological conditions, NADPH oxidase 4-derived reactive oxygen species is a regulator of the partitioning of the metabolic flux of homocysteine, which impacts upon hepatic cysteine and glutathione levels and thereby upon defence against environmental toxins.

  17. [Interaction of succinate dehydrogenase and oxaloacetate].

    PubMed

    Kotliar, A B; Vinogradov, A D

    1984-04-01

    The equilibrium and rate constants for interaction of the reduced and oxidized membrane-bound succinate dehydrogenase (EC 1.3.99.1) with oxaloacetate were determined. The 10-fold decrease in the oxaloacetate affinity for the reduced enzyme was shown to be due to the 10-fold increase of the enzyme-inhibitor complex dissociation rate, which occurs upon its reduction. The rate of dissociation induced by succinate is 10 times higher than that induced by malonate in the submitochondrial particles, being equal in the soluble enzyme preparations. The rates of dissociation induced by malonate excess, or by the enzyme irreversibly utilizing oxaloacetate (transaminase in the presence of glutamate) are also equal. The data obtained suggest that succinate dehydrogenase interaction with succinate and oxaloacetate results from the competition for a single dicarboxylate-specific site. In submitochondrial particles all succinate dehydrogenase molecules are in redox equilibrium provided for by endogenous ubiquinone. No electronic equilibrium between the individual enzyme molecules exists, when succinate dehydrogenase is solubilized.

  18. Type 2 NADH Dehydrogenases in the Cyanobacterium Synechocystis sp. Strain PCC 6803 Are Involved in Regulation Rather Than Respiration

    PubMed Central

    Howitt, Crispin A.; Udall, Pacer K.; Vermaas, Wim F. J.

    1999-01-01

    Analysis of the genome of Synechocystis sp. strain PCC 6803 reveals three open reading frames (slr0851, slr1743, and sll1484) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in Synechocystis sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames, slr1743, was able to functionally complement an Escherichia coli mutant lacking both NDH-1s and NDH-2s. Therefore, slr0851, slr1743, and sll1484 have been designated ndbA, ndbB, and ndbC, respectively. Strains that lacked one or more of the ndb genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion of ndb genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in Synechocystis sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool. PMID:10383967

  19. Development of an amine dehydrogenase for synthesis of chiral amines.

    PubMed

    Abrahamson, Michael J; Vázquez-Figueroa, Eduardo; Woodall, Nicholas B; Moore, Jeffrey C; Bommarius, Andreas S

    2012-04-16

    A leucine dehydrogenase has been successfully altered through several rounds of protein engineering to an enantioselective amine dehydrogenase. Instead of the wild-type α-keto acid, the new amine dehydrogenase now accepts the analogous ketone, methyl isobutyl ketone (MIBK), which corresponds to exchange of the carboxy group by a methyl group to produce chiral (R)-1,3-dimethylbutylamine.

  20. WRKY Transcription Factors Phosphorylated by MAPK Regulate a Plant Immune NADPH Oxidase in Nicotiana benthamiana[OPEN

    PubMed Central

    Adachi, Hiroaki; Nakano, Takaaki; Miyagawa, Noriko; Ishihama, Nobuaki; Yoshioka, Miki; Katou, Yuri; Yaeno, Takashi

    2015-01-01

    Pathogen attack sequentially confers pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) after sensing of pathogen patterns and effectors by plant immune receptors, respectively. Reactive oxygen species (ROS) play pivotal roles in PTI and ETI as signaling molecules. Nicotiana benthamiana RBOHB, an NADPH oxidase, is responsible for both the transient PTI ROS burst and the robust ETI ROS burst. Here, we show that RBOHB transactivation mediated by MAPK contributes to R3a/AVR3a-triggered ETI (AVR3a-ETI) ROS burst. RBOHB is markedly induced during the ETI and INF1-triggered PTI (INF1-PTI), but not flg22-tiggered PTI (flg22-PTI). We found that the RBOHB promoter contains a functional W-box in the R3a/AVR3a and INF1 signal-responsive cis-element. Ectopic expression of four phospho-mimicking mutants of WRKY transcription factors, which are MAPK substrates, induced RBOHB, and yeast one-hybrid analysis indicated that these mutants bind to the cis-element. Chromatin immunoprecipitation assays indicated direct binding of the WRKY to the cis-element in plants. Silencing of multiple WRKY genes compromised the upregulation of RBOHB, resulting in impairment of AVR3a-ETI and INF1-PTI ROS bursts, but not the flg22-PTI ROS burst. These results suggest that the MAPK-WRKY pathway is required for AVR3a-ETI and INF1-PTI ROS bursts by activation of RBOHB. PMID:26373453

  1. Potential role of the NADPH oxidase NOX1 in the pathogenesis of 5-fluorouracil-induced intestinal mucositis in mice.

    PubMed

    Yasuda, Masashi; Kato, Shinichi; Yamanaka, Naoki; Iimori, Maho; Utsumi, Daichi; Kitahara, Yumeno; Iwata, Kazumi; Matsuno, Kuniharu; Amagase, Kikuko; Yabe-Nishimura, Chihiro; Takeuchi, Koji

    2012-05-15

    Although NADPH oxidase 1 (NOX1) has been shown to be highly expressed in the gastrointestinal tract, the physiological and pathophysiological roles of this enzyme are not yet fully understood. In the present study, we investigated the role of NOX1 in the pathogenesis of intestinal mucositis induced by the cancer chemotherapeutic agent 5-fluorouracil (5-FU) in mice. Intestinal mucositis was induced in Nox1 knockout (Nox1KO) and littermate wild-type (WT) mice via single, daily administration of 5-FU for 5 days. In WT mice, 5-FU caused severe intestinal mucositis characterized by a shortening of villus height, a disruption of crypts, a loss of body weight, and diarrhea. In Nox1KO mice, however, the severity of mucositis was significantly reduced, particularly with respect to crypt disruption. The numbers of apoptotic caspase-3- and caspase-8-activated cells in the intestinal crypt increased 24 h after the first 5-FU administration but were overall significantly lower in Nox1KO than in WT mice. Furthermore, the 5-FU-mediated upregulation of TNF-α, IL-1β, and NOX1 and the production of reactive oxygen species were significantly attenuated in Nox1KO mice compared with that in WT mice. These findings suggest that NOX1 plays an important role in the pathogenesis of 5-FU-induced intestinal mucositis. NOX1-derived ROS production following administration of 5-FU may promote the apoptotic response through upregulation of inflammatory cytokines.

  2. Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity.

    PubMed

    Kadota, Yasuhiro; Sklenar, Jan; Derbyshire, Paul; Stransfeld, Lena; Asai, Shuta; Ntoukakis, Vardis; Jones, Jonathan Dg; Shirasu, Ken; Menke, Frank; Jones, Alexandra; Zipfel, Cyril

    2014-04-10

    The rapid production of reactive oxygen species (ROS) burst is a conserved signaling output in immunity across kingdoms. In plants, perception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) activates the NADPH oxidase RBOHD by hitherto unknown mechanisms. Here, we show that RBOHD exists in complex with the receptor kinases EFR and FLS2, which are the PRRs for bacterial EF-Tu and flagellin, respectively. The plasma-membrane-associated kinase BIK1, which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. BIK1 phosphorylates different residues than calcium-dependent protein kinases, and both PAMP-induced BIK1 activation and BIK1-mediated phosphorylation of RBOHD are calcium independent. Importantly, phosphorylation of these residues is critical for the PAMP-induced ROS burst and antibacterial immunity. Our study reveals a rapid regulatory mechanism of a plant RBOH, which occurs in parallel with and is essential for its paradigmatic calcium-based regulation.

  3. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity

    PubMed Central

    Deep, Gagan; Kumar, Rahul; Jain, Anil K.; Dhar, Deepanshi; Panigrahi, Gati K.; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P.; Oberlies, Nicholas H.; Agarwal, Rajesh

    2016-01-01

    Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1–5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47phox). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity. PMID:26979487

  4. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity.

    PubMed

    Deep, Gagan; Kumar, Rahul; Jain, Anil K; Dhar, Deepanshi; Panigrahi, Gati K; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P; Oberlies, Nicholas H; Agarwal, Rajesh

    2016-03-16

    Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1-5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47(phox)). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity.

  5. [Thermal stability of lactate dehydrogenase and alcohol dehydrogenase incorporated into highly concentrated gels].

    PubMed

    Kulis, Iu Iu

    1979-03-01

    The rate constants for inactivation of lactate dehydrogenase and alcohol dehydrogenase in solution at 65 degrees C (pH 7,5) are 0,72 and 0,013 min-1, respectively. The enzyme incorporation into acrylamide gels results in immobilized enzymes, whose residual activity is 18--25% of the original one. In 6,7% gels the rate of thermal inactivation for lactate dehydrogenase is decreased nearly 10-fold, whereas the inactivation rate for alcohol dehydrogenase is increased 4,6-fold as compared to the soluble enzymes. In 14% and 40% gels the inactivation constants for lactate dehydrogenase are 6,3.10(-3) and 5,9.10(-4) min-1, respectively. In 60% gels the thermal inactivation of lactate dehydrogenase is decelerated 3600-fold as compared to the native enzyme. The enthalpy and enthropy for the inactivation of the native enzyme are equal to 62,8 kcal/mole and 116,9 cal/(mole.grad.) for the native enzyme and those of gel-incorporated (6,7%) enzyme -- 38,7 kcal/mole and 42 cal/(mole.grad.), respectively. The thermal stability of alcohol dehydrogenase in 60% gels is increased 12-fold. To prevent gel swelling, methacrylic acid and allylamine were added to the matrix, with subsequent treatment by dicyclohexylcarbodiimide. The enzyme activity of the modified gels is 2,7--3% of that for the 6,7% gels. The stability of lactate dehydrogenase in such gels is significantly increased. A mechanism of stabilization of the subunit enzymes in highly concentrated gels is discussed.

  6. Dehydrogenase and Oxoreductase Activities of Porcine Placental 11Beta-Hydroxysteroid Dehydrogenase

    DTIC Science & Technology

    2016-06-07

    dehydrogenase (IIB-HSD) were measured in tissue fragment cultures on day 75 of gestation. Dehydrogenase activity was over fivefold greater than oxoreductase...oxoreductase activities in porcine placentae under physiological conditions using placental explant culture and endogenous concentrations of coenzymes and...f!M range). In human placental tissue fragments at midterm and late pregnancy ( 12, 18) and in trophoblast cell cultures from term placentae ( 41

  7. Characterization of xylitol dehydrogenase from Debaryomyces hansenii

    SciTech Connect

    Girio, F.M.; Amaral-Collaco, M.T.; Pelica, F.

    1996-01-01

    The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells of Debaryomyces hansenii was partially purified in two chromatographic steps, and characterization studies were carried out in order to investigate the role of the xylitol dehydrogenase-catalyzed step in the regulation of D-xylose metabolism. The enzyme was most active at pH 9.0-9.5, and exhibited a broad polyol specificity. The Michaelis constants for xylitol and NAD{sup +} were 16.5 and 0.55 mM, respectively. Ca{sup 2+}, Mg{sup 2+}, and Mn{sup 2+} did not affect the enzyme activity. Conversely, Zn{sup 2+}, Cd{sup 2+}, and Co{sup 2+} strongly inhibited the enzyme activity. It was concluded that NAD{sup +}-xylitol dehydrogenase from D. hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and K{sub m} value for xylitol, and therefore should be named L-iditol:NAD{sup +}-5-oxidoreductase (EC 1.1.1.14). The reason D. hansenii is a good xylitol producer is not because of its value of K for xylitol, which is low enough to assure its fast oxidation by NAD{sup +}-xylitol dehydrogenase. However, a higher K{sub m} value of xylitol dehydrogenase for NAD{sup +} compared to the K{sub m} values of other xylose-fermenting yeasts may be responsible for the higher xylitol yields. 22 refs., 4 figs., 2 tabs.

  8. Properties of formate dehydrogenase in Methanobacterium formicicum

    SciTech Connect

    Schauer, N.L.; Ferry, J.G.

    1982-04-01

    Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50..mu..mol of methyl viologen per min per mg of protein and 8.2 ..mu..mol of coenzyme F/sub 420/ per min per mg of protein. The apparent K/sub m/ for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F/sub 420/, was 10-fold greater (63 ..mu..M) than for coenzyme F/sub 420/ (6 ..mu..M). The purified enzyme also reduced flavin mononucleotide (K/sub m/ = 13 ..mu..M) and flavin adenine dinucleotide (K/sub m/ = 25 ..mu..M) with formate, but did not reduce NAD/sup +/ or NADP/sup +/. The reduction of NADP/sup +/ with formate required formate dehydrogenase, coenzyme F/sub 420/, and coenzyme F/sub 420/:NADP/sup +/ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F/sub 420/. The optimal reaction rate occurred at 55/sup 0/C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (K/sub i/ = 6 ..mu..M), azide (K/sub i/ = 39 ..mu..M),..cap alpha..,..cap alpha..-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.

  9. D-2-Hydroxyglutarate does not mimic all the IDH mutation effects, in particular the reduced etoposide-triggered apoptosis mediated by an alteration in mitochondrial NADH.

    PubMed

    Oizel, K; Gratas, C; Nadaradjane, A; Oliver, L; Vallette, F M; Pecqueur, C

    2015-03-26

    Somatic mutations in isocitrate dehydrogenase (IDH)-1 and -2 have recently been described in glioma. This mutation leads to a neomorphic enzymatic activity as the conversion of isocitrate to alpha ketoglutarate (αKG) is replaced by the conversion of αKG to D-2-hydroxyglutarate (D-2HG) with NADPH oxidation. It has been suggested that this oncometabolite D-2HG via inhibition of αKG-dioxygenases is involved in multiple functions such as epigenetic modifications or hypoxia responses. The present study is aimed at deciphering how the mutant IDH can affect cancer pathogenesis, in particular with respect to its associated oncometabolite D-2HG. We show that the overexpression of mutant IDH in glioma cells or treatment with D-2HG triggered an increase in cell proliferation. However, although mutant IDH reduced cell sensitivity to the apoptotic inducer etoposide, D-2HG exhibited no effect on apoptosis. Instead, we found that the apoptotic effect was mediated through the mitochondrial NADH pool reduction and could be inhibited by oxamate. These data show that besides D-2HG production, mutant IDH affects other crucial metabolite pools. These observations lead to a better understanding of the biology of IDH mutations in gliomas and their response to therapy.

  10. Induction of reactive oxygen species and the potential role of NADPH oxidase in hyperhydricity of garlic plantlets in vitro.

    PubMed

    Tian, Jie; Cheng, Yaqi; Kong, Xiangyu; Liu, Min; Jiang, Fangling; Wu, Zhen

    2017-01-01

    Hyperhydricity is a physiological disorder associated with oxidative stress. Reactive oxygen species (ROS) generation in plants is initiated by various enzymatic sources, including plasma membrane-localized nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, cell wall-bound peroxidase (POD), and apoplastic polyamine oxidase (PAO). The origin of the oxidative burst associated with hyperhydricity remains unknown. To investigate the role of NADPH oxidases, POD, and PAO in ROS production and hyperhydricity, exogenous hydrogen peroxide (H2O2) and inhibitors of each ROS-producing enzyme were applied to explore the mechanism of oxidative stress induction in garlic plantlets in vitro. A concentration of 1.5 mM H2O2 increased endogenous ROS production and hyperhydricity occurrence and enhanced the activities of NADPH oxidases, POD, and PAO. During the entire treatment period, NADPH oxidase activity increased continuously, whereas POD and PAO activities exhibited a transient increase and subsequently declined. Histochemical and cytochemical visualization demonstrated that specific inhibitors of each enzyme effectively suppressed ROS accumulation. Moreover, superoxide anion generation, H2O2 content, and hyperhydric shoot frequency in H2O2-stressed plantlets decreased significantly. The NADPH oxidase inhibitor was the most effective at suppressing superoxide anion production. The results suggested that NADPH oxidases, POD, and PAO were responsible for endogenous ROS induction. NADPH oxidase activation might play a pivotal role in the oxidative burst in garlic plantlets in vitro during hyperhydricity.

  11. Neuropil and neuronal changes in hippocampal NADPH-diaphorase histochemistry in the ME7 model of murine prion disease.

    PubMed

    Picanço-Diniz, C W; Boche, D; Gomes-Leal, W; Perry, V H; Cunningham, C

    2004-06-01

    Nitric oxide (NO) has been implicated in neurotoxicity and cerebral blood flow changes in chronic neurodegeneration, but its activity in the mammalian prion diseases has not been studied in detail. Nicotine adenine dinucleotide phosphate (NADPH)-diaphorase (NADPH-d) histochemistry is a simple and robust histochemical procedure that allows localization of the tissue distribution of NO synthases. The aim of the present study is to assess whether NADPH-d histochemical activity is altered in the hippocampus in the ME7 model of prion disease in C57BL/6J mice. At early and late stages after the initiation of the disease we assessed features of the NADPH-d positive cells and the neuropil histochemical activity in CA1 and dentate gyrus using densitometric analysis. In C57BL/6J mice 13 weeks postinjection of the prion agent ME7, when behavioural changes first become apparent, neuropil NADPH-d histochemical staining increases, whereas at late stages it decreases dramatically. Both type I and type II NADPH-d positive cells were found to survive throughout the hippocampal formation into the late stages of the disease, but diaphorase activity was reduced in dendritic branches and abnormal varicosities were present in both dendritic and axonal processes of NADPH-d positive type I cells. The pathophysiological implications of the results remain to be investigated but both blood flow alteration and NO neurotoxicity may be features of the disease.

  12. NADPH oxidase activation played a critical role in the oxidative stress process in stable coronary artery disease

    PubMed Central

    Zhang, Jiefang; Wang, Meihui; Li, Zhengwei; Bi, Xukun; Song, Jiale; Weng, Shaoxiang; Fu, Guosheng

    2016-01-01

    Objectives: The study was designed to investigate the oxidative stress levels of endothelial progenitor cells (EPCs) in stable coronary artery disease (CAD) and to explore the underlying mechanisms of NADPH oxidase activation and subsequent EPCs dysfunction. Methods: EPCs were isolated from patients with stable CAD (n=50) and matched healthy volunteers (n=50). NADPH oxidase activation was detected by measuring the expression of each subunit using western blotting and qPCR analyses and the membrane translocation of p47phox using immunofluorescence. The in vivo angiogenesis capacity was evaluated using immunofluorescence by transplanting EPCs into a rat hind limb ischemia model. The PKC inhibitor GÖ-6983 was used to determine the role of PKC in NADPH oxidase activation. Results: Oxidative stress level was increased and the in vivo angiogenesis capacity was impaired in EPCs obtained from CAD subjects with the activation of NADPH oxidase. P47phox membrane translocation increased in CAD group vs controls. These effects were resolved by NADPH oxidase inhibition. Up-regulation of PKCα/β2 was found in EPCs from CAD subjects, PKC inhibition GÖ-6983 could reduce the expression and activity of NADPH oxidation. Conclusions: NADPH oxidase activation via p47phox membrane translocation played a critical role in the initiation and progression of CAD, and the PKCα/β2 signaling pathway might be involved. PMID:28077995

  13. Deletion of hexose-6-phosphate dehydrogenase activates the unfolded protein response pathway and induces skeletal myopathy.

    PubMed

    Lavery, Gareth G; Walker, Elizabeth A; Turan, Nil; Rogoff, Daniela; Ryder, Jeffery W; Shelton, John M; Richardson, James A; Falciani, Francesco; White, Perrin C; Stewart, Paul M; Parker, Keith L; McMillan, Daniel R

    2008-03-28

    Hexose-6-phosphate dehydrogenase (H6PD) is the initial component of a pentose phosphate pathway inside the endoplasmic reticulum (ER) that generates NADPH for ER enzymes. In liver H6PD is required for the 11-oxoreductase activity of 11beta-hydroxysteroid dehydrogenase type 1, which converts inactive 11-oxo-glucocorticoids to their active 11-hydroxyl counterparts; consequently, H6PD null mice are relatively insensitive to glucocorticoids, exhibiting fasting hypoglycemia, increased insulin sensitivity despite elevated circulating levels of corticosterone, and increased basal and insulin-stimulated glucose uptake in muscles normally enriched in type II (fast) fibers, which have increased glycogen content. Here, we show that H6PD null mice develop a severe skeletal myopathy characterized by switching of type II to type I (slow) fibers. Running wheel activity and electrically stimulated force generation in isolated skeletal muscle are both markedly reduced. Affected muscles have normal sarcomeric structure at the electron microscopy level but contain large intrafibrillar membranous vacuoles and abnormal triads indicative of defects in structure and function of the sarcoplasmic reticulum (SR). SR proteins involved in calcium metabolism, including the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), calreticulin, and calsequestrin, show dysregulated expression. Microarray analysis and real-time PCR demonstrate overexpression of genes encoding proteins in the unfolded protein response pathway. We propose that the absence of H6PD induces a progressive myopathy by altering the SR redox state, thereby impairing protein folding and activating the unfolded protein response pathway. These studies thus define a novel metabolic pathway that links ER stress to skeletal muscle integrity and function.

  14. Prenatal Hypoxic-Ischemic Insult Changes the Distribution and Number of NADPH-Diaphorase Cells in the Cerebellum

    PubMed Central

    Savignon, Tiago; Costa, Everton; Tenorio, Frank; Manhães, Alex C.; Barradas, Penha C.

    2012-01-01

    Astrogliosis, oligodendroglial death and motor deficits have been observed in the offspring of female rats that had their uterine arteries clamped at the 18th gestational day. Since nitric oxide has important roles in several inflammatory and developmental events, here we evaluated NADPH-diaphorase (NADPH-d) distribution in the cerebellum of rats submitted to this hypoxia-ischemia (HI) model. At postnatal (P) day 9, Purkinje cells of SHAM and non-manipulated (NM) animals showed NADPH-d+ labeling both in the cell body and dendritic arborization in folia 1 to 8, while HI animals presented a weaker labeling in both cellular structures. NADPH-d+ labeling in the molecular (ML), and in both the external and internal granular layer, was unaffected by HI at this age. At P23, labeling in Purkinje cells was absent in all three groups. Ectopic NADPH-d+ cells in the ML of folia 1 to 4 and folium 10 were present exclusively in HI animals. This labeling pattern was maintained up to P90 in folium 10. In the cerebellar white matter (WM), at P9 and P23, microglial (ED1+) NADPH-d+ cells, were observed in all groups. At P23, only HI animals presented NADPH-d labeling in the cell body and processes of reactive astrocytes (GFAP+). At P9 and P23, the number of NADPH-d+ cells in the WM was higher in HI animals than in SHAM and NM ones. At P45 and at P90 no NADPH-d+ cells were observed in the WM of the three groups. Our results indicate that HI insults lead to long-lasting alterations in nitric oxide synthase expression in the cerebellum. Such alterations in cerebellar differentiation might explain, at least in part, the motor deficits that are commonly observed in this model. PMID:22540005

  15. Sexual dimorphism of vocal control nuclei in budgerigars (Melopsittacus undulatus) revealed with Nissl and NADPH-d staining.

    PubMed

    Brauth, Steven E; Liang, Wenru; Amateau, Stuart K; Roberts, Todd F; Robert, Todd F

    2005-03-28

    Nissl staining and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry were used to explore the existence of sexual dimorphism in vocal control nuclei of adult budgerigars (Melopsittacus undulatus), a parrot species capable of lifelong vocal learning. Behavioral studies indicate that adult males possess larger vocal repertoires than adult females and learn new calls more quickly. The results of the present study show that the volumes of all vocal nuclei, as measured using both Nissl-stained and NADPH-d-stained material, as well as the total numbers of NADPH-d neurons, were 35-110% greater in males. Furthermore, all vocal nuclei exhibit conspicuous NADPH-d staining compared to surrounding fields in both adult males and females. Nevertheless, there were no significant gender differences in either the intensity of neuropil staining or the densities of NADPH-d neurons in vocal nuclei. Moreover NADPH-d neuron somal shapes were similar in males and females. Diameters of NADPH-d neurons in vocal nuclei were 8.5-32% larger in males than in females. Greater size of NADPH-d neuronal somata in males may be a general property of this cell type in budgerigars because a similar gender difference was found in a visual nucleus, the entopallium, which is not directly associated with the vocal control system and does not exhibit sexual dimorphism in total volume or total NADPH-d neuron numbers. Taken together, the results of the present study favor the hypothesis that superior lifelong vocal learning ability in male budgerigars rests largely on larger volumes of vocal control nuclei in males rather than on sexual dimorphism in the internal composition of vocal nuclei.

  16. Use of NAD(P)H and Flavoprotein Autofluorescence Transients to Probe Neuron and Astrocyte Responses to Synaptic Activation

    PubMed Central

    Shuttleworth, C. William

    2010-01-01

    Synaptic stimulation in brain slices is accompanied by changes in tissue autofluorescence, which are a consequence of changes in tissue metabolism. Autofluorescence excited by ultraviolet light has been most extensively studied, and is due to reduced pyridine nucleotides (NADH and NADPH, collectively termed NAD(P)H). Stimulation generates a characteristic compound NAD(P)H response, comprising an initial fluorescence decrease and then an overshooting increase that slowly recovers to baseline levels. Evoked NAD(P)H transients are relatively easy to record, do not require the addition of exogenous indicators and have good signal-noise ratios. These characteristics make NAD(P)H imaging methods very useful for tracking the spread of neuronal activity in complex brain tissues, however the cellular basis of synaptically-evoked autofluorescence transients has been the subject of recent debate. Of particular importance is the question of whether signals are due primarily to changes in neuronal mitochondrial function, and/or whether astrocyte metabolism triggered by glutamate uptake may be a significant contributor to the overshooting NAD(P)H fluorescence increases. This mini-review addresses the subcellular origins of NAD(P)H autofluorescence and the evidence for mitochondrial and glycolytic contributions to compound transients. It is concluded that there is no direct evidence for a contribution to NAD(P)H signals from glycolysis in astrocytes following synaptic glutamate uptake. In contrast, multiple lines of evidence, including from complimentary flavoprotein autofluorescence signals, imply that mitochondrial NADH dynamics in neurons dominate compound evoked NAD(P)H transients. These signals are thus appropriate for studies of mitochondrial function and dysfunction in brain slices, in addition to providing robust maps of postsynaptic neuronal activation following physiological activation. PMID:20036704

  17. Regulation of NAD+- and NADP+-linked isocitrate dehydrogenase in the obligate methylotrophic bacterium Pseudomonas W6.

    PubMed

    Hofmann, K H; Babel, W

    1980-01-01

    Cell-free extracts of the obligate methanol-utilizing bacterium Pseudomonas W6 catalyze the oxydation of isocitrate to alpha-ketoglutarate in the presence of NAD+ and NADP+. After electro-focusing of the crude extract of Pseudomonas W6 actually two distinct bands each of NAD+-linked isocitrate dehydrogenase (NAD+-IDH) and of NADP+-linked isocitrate dehydrogenase (NADP+-IDH) could be observed. The NAD+-IDH was completely separated from the NADP+-IDH by employing DEAE ion exchange chromatography and further purified by affinity chromatography using Cibacron blue F 3G-A. The NAD+-IDH was inhibited by a high energy charge, whereas the NADP+-IDH was found to be independent of energy charge. Consequently the NAD+-IDH showed the control behaviour of an enzyme of an energy-generating sequence which, however, equally fulfils a catabolic and an anabolic function. With respect to the inhibition by reduced pyridine nucleotides and alpha-ketoglutarate differences between NAD+-IDH and NADP+-IDH were also found. Only the NADP+-linked enzyme exhibited a feedback inhibition by its reaction products alpha-ketoglutarate and NADPH. This control behaviour gives evidence for the biosynthetic function of the NADP+-IDH. These results confer an amphibolic character to the sequence from citrate to alpha-ketoglutarate in the incomplete citric-acid cycle of Pseudomonas W6.

  18. Group III alcohol dehydrogenase from Pectobacterium atrosepticum: insights into enzymatic activity and organization of the metal ion-containing region.

    PubMed

    Elleuche, Skander; Fodor, Krisztian; von der Heyde, Amélie; Klippel, Barbara; Wilmanns, Matthias; Antranikian, Garabed

    2014-05-01

    NAD(P)(+)-dependent alcohol dehydrogenases (ADH) are widely distributed in all phyla. These proteins can be assigned to three nonhomologous groups of isozymes, with group III being highly diverse with regards to catalytic activity and primary structure. Members of group III ADHs share a conserved stretch of amino acid residues important for cofactor binding and metal ion coordination, while sequence identities for complete proteins are highly diverse (<20 to >90 %). A putative group III ADH PaYqhD has been identified in BLAST analysis from the plant pathogenic enterobacterium Pectobacterium atrosepticum. The PaYqhD gene was expressed in the heterologous host Escherichia coli, and the recombinant protein was purified in a two-step purification procedure to homogeneity indicating an obligate dimerization of monomers. Four conserved amino acid residues involved in metal ion coordination were substituted with alanine, and their importance for catalytic activity was confirmed by circular dichroism spectrum determination, in vitro, and growth experiments. PaYqhD exhibits optimal activity at 40 °C with short carbon chain aldehyde compounds and NADPH as cofactor indicating the enzyme to be an aldehyde reductase. No oxidative activities towards alcoholic compounds were detectable. EDTA completely inhibited catalytic activity and was fully restored by the addition of Co(2+). Activity measurements together with sequence alignments and structure analysis confirmed that PaYqhD belongs to the butanol dehydrogenase-like enzymes within group III of ADHs.

  19. NADPH oxidase 4 regulates cardiomyocyte differentiation via redox activation of c-Jun protein and the cis-regulation of GATA-4 gene transcription.

    PubMed

    Murray, Thomas V A; Smyrnias, Ioannis; Shah, Ajay M; Brewer, Alison C

    2013-05-31

    NADPH oxidase 4 (Nox4) generates reactive oxygen species (ROS) that can modulate cellular phenotype and function in part through the redox modulation of the activity of transcription factors. We demonstrate here the potential of Nox4 to drive cardiomyocyte differentiation in pluripotent embryonal carcinoma cells, and we show that this involves the redox activation of c-Jun. This in turn acts to up-regulate GATA-4 expression, one of the earliest markers of cardiotypic differentiation, through a defined and highly conserved cis-acting motif within the GATA-4 promoter. These data therefore suggest a mechanism whereby ROS act in pluripotential cells in vivo to regulate the initial transcription of critical tissue-restricted determinant(s) of the cardiomyocyte phenotype, including GATA-4. The ROS-dependent activation, mediated by Nox4, of widely expressed redox-regulated transcription factors, such as c-Jun, is fundamental to this process.