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Sample records for nitrite reductase activity

  1. Nitrite Reductase Activity in Engineered Azurin Variants.

    PubMed

    Berry, Steven M; Strange, Jacob N; Bladholm, Erika L; Khatiwada, Balabhadra; Hedstrom, Christine G; Sauer, Alexandra M

    2016-05-01

    Nitrite reductase (NiR) activity was examined in a series of dicopper P.a. azurin variants in which a surface binding copper site was added through site-directed mutagenesis. Four variants were synthesized with copper binding motifs inspired by the catalytic type 2 copper binding sites found in the native noncoupled dinuclear copper enzymes nitrite reductase and peptidylglycine α-hydroxylating monooxygenase. The four azurin variants, denoted Az-NiR, Az-NiR3His, Az-PHM, and Az-PHM3His, maintained the azurin electron transfer copper center, with the second designed copper site located over 13 Å away and consisting of mutations Asn10His,Gln14Asp,Asn16His-azurin, Asn10His,Gln14His,Asn16His-azurin, Gln8Met,Gln14His,Asn16His-azurin, and Gln8His,Gln14His,Asn16His-azurin, respectively. UV-visible absorption spectroscopy, EPR spectroscopy, and electrochemistry of the sites demonstrate copper binding as well as interaction with small exogenous ligands. The nitrite reduction activity of the variants was determined, including the catalytic Michaelis-Menten parameters. The variants showed activity (0.34-0.59 min(-1)) that was slower than that of native NiRs but comparable to that of other model systems. There were small variations in activity of the four variants that correlated with the number of histidines in the added copper site. Catalysis was found to be reversible, with nitrite produced from NO. Reactions starting with reduced azurin variants demonstrated that electrons from both copper centers were used to reduce nitrite, although steady-state catalysis required the T2 copper center and did not require the T1 center. Finally, experiments separating rates of enzyme reduction from rates of reoxidation by nitrite demonstrated that the reaction with nitrite was rate limiting during catalysis. PMID:27055058

  2. The functional nitrite reductase activity of the heme-globins

    PubMed Central

    2008-01-01

    Hemoglobin and myoglobin are among the most extensively studied proteins, and nitrite is one of the most studied small molecules. Recently, multiple physiologic studies have surprisingly revealed that nitrite represents a biologic reservoir of NO that can regulate hypoxic vasodilation, cellular respiration, and signaling. These studies suggest a vital role for deoxyhemoglobin- and deoxymyoglobin-dependent nitrite reduction. Biophysical and chemical analysis of the nitrite-deoxyhemoglobin reaction has revealed unexpected chemistries between nitrite and deoxyhemoglobin that may contribute to and facilitate hypoxic NO generation and signaling. The first is that hemoglobin is an allosterically regulated nitrite reductase, such that oxygen binding increases the rate of nitrite conversion to NO, a process termed R-state catalysis. The second chemical property is oxidative denitrosylation, a process by which the NO formed in the deoxyhemoglobin-nitrite reaction that binds to other deoxyhemes can be released due to heme oxidation, releasing free NO. Third, the reaction undergoes a nitrite reductase/anhydrase redox cycle that catalyzes the anaerobic conversion of 2 molecules of nitrite into dinitrogen trioxide (N2O3), an uncharged molecule that may be exported from the erythrocyte. We will review these reactions in the biologic framework of hypoxic signaling in blood and the heart. PMID:18596228

  3. A high-throughput assay format for determination of nitrate reductase and nitrite reductase enzyme activities

    SciTech Connect

    McNally, N.; Liu, Xiang Yang; Choudary, P.V.

    1997-01-01

    The authors describe a microplate-based high-throughput procedure for rapid assay of the enzyme activities of nitrate reductase and nitrite reductase, using extremely small volumes of reagents. The new procedure offers the advantages of rapidity, small sample size-nanoliter volumes, low cost, and a dramatic increase in the throughput sample number that can be analyzed simultaneously. Additional advantages can be accessed by using microplate reader application software packages that permit assigning a group type to the wells, recording of the data on exportable data files and exercising the option of using the kinetic or endpoint reading modes. The assay can also be used independently for detecting nitrite residues/contamination in environmental/food samples. 10 refs., 2 figs.

  4. A dissimilatory nitrite reductase in Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

    Grant, M. A.; Hochstein, L. I.

    1984-01-01

    Paracoccus halodenitrificans produced a membrane-associated nitrite reductase. Spectrophotometric analysis showed it to be associated with a cd-cytochrome and located on the inner side of the cytoplasmic membrane. When supplied with nitrite, membrane preparations produced nitrous oxide and nitric oxide in different ratios depending on the electron donor employed. The nitrite reductase was maximally active at relatively low concentrations of sodium chloride and remained attached to the membranes at 100 mM sodium chloride.

  5. A genetic screen reveals a periplasmic copper chaperone required for nitrite reductase activity in pathogenic Neisseria.

    PubMed

    Jen, Freda E-C; Djoko, Karrera Y; Bent, Stephen J; Day, Christopher J; McEwan, Alastair G; Jennings, Michael P

    2015-09-01

    Under conditions of low oxygen availability, Neisseria meningitidis and Neisseria gonorrhoeae are able to respire via a partial denitrification pathway in which nitrite is converted to nitrous oxide. In this process, nitrite reductase (AniA), a copper (Cu)-containing protein converts nitrite to NO, and this product is converted to nitrous oxide by nitric oxide reductase (NorB). NorB also confers protection against toxic NO, and so we devised a conditional lethal screen, using a norB mutant, to identify mutants that were resistant to nitrite-dependent killing. After random-deletion mutagenesis of N. meningitidis, this genetic screen identified a gene encoding a Cu chaperone that is essential for AniA function, AccA. Purified AccA binds one Cu (I) ion and also possesses a second binding site for Cu (II). This novel periplasmic Cu chaperone (AccA) appears to be essential for provision of Cu ions to AniA of pathogenic Neisseria to generate an active nitrite reductase. Apart from the Neisseria genus, AccA is distributed across a wide range of environmental Proteobacteria species. PMID:26031293

  6. Regulating the nitrite reductase activity of myoglobin by redesigning the heme active center.

    PubMed

    Wu, Lei-Bin; Yuan, Hong; Gao, Shu-Qin; You, Yong; Nie, Chang-Ming; Wen, Ge-Bo; Lin, Ying-Wu; Tan, Xiangshi

    2016-07-01

    Heme proteins perform diverse functions in living systems, of which nitrite reductase (NIR) activity receives much attention recently. In this study, to better understand the structural elements responsible for the NIR activity, we used myoglobin (Mb) as a model heme protein and redesigned the heme active center, by introducing one or two distal histidines, and by creating a channel to the heme center with removal of the native distal His64 gate (His to Ala mutation). UV-Vis kinetic studies, combined with EPR studies, showed that a single distal histidine with a suitable position to the heme iron, i.e., His43, is crucial for nitrite (NO2(-)) to nitric oxide (NO) reduction. Moreover, creation of a water channel to the heme center significantly enhanced the NIR activity compared to the corresponding mutant without the channel. In addition, X-ray crystallographic studies of F43H/H64A Mb and its complexes with NO2(-) or NO revealed a unique hydrogen-bonding network in the heme active center, as well as unique substrate and product binding models, providing valuable structural information for the enhanced NIR activity. These findings enriched our understanding of the structure and NIR activity relationship of heme proteins. The approach of creating a channel in this study is also useful for rational design of other functional heme proteins. PMID:27108710

  7. Enhanced nitrite reductase activity associated with the haptoglobin complexed hemoglobin dimer: Functional and antioxidative implications

    PubMed Central

    Roche, Camille J.; Dantsker, David; Alayash, Abdu I.; Friedman, Joel M.

    2012-01-01

    The presence of acellular hemoglobin (Hb) within the circulation is generally viewed as a pathological state that can result in toxic consequences. Haptoglobin (Hp), a globular protein found in the plasma, binds with high avidity the αβ dimers derived from the dissociation of Hb tetramer and thus helps clear free Hb. More recently there have been compelling indications that the redox properties of the Hp bound dimer (Hb–Hp) may play a more active role in controlling toxicity by limiting the potential tissue damage caused by propagation of the free-radicals generated within the heme containing globin chains. The present study further examines the potential protective effect of Hp through its impact on the production of nitric oxide (NO) from nitrite through nitrite reductase activity of the Hp bound αβ Hb dimer. The presented results show that the Hb dimer in the Hb–Hp complex has oxygen binding, CO recombination and spectroscopic properties consistent with an Hb species having properties similar to but not exactly the same as the R quaternary state of the Hb tetramer. Consistent with these observations is the finding that the initial nitrite reductase rate for Hb–Hp is approximately ten times that of HbA under the same conditions. These results in conjunction with the earlier redox properties of the Hb–Hp are discussed in terms of limiting the pathophysiological consequences of acellular Hb in the circulation. PMID:22521791

  8. Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity.

    PubMed

    Kumar, Nitin; Astegno, Alessandra; Chen, Jian; Giorgetti, Alejandro; Dominici, Paola

    2016-01-01

    It is well-established that plant hemoglobins (Hbs) are involved in nitric oxide (NO) metabolism via NO dioxygenase and/or nitrite reductase activity. The ferrous-deoxy Arabidopsis Hb1 and Hb2 (AHb1 and AHb2) have been shown to reduce nitrite to NO under hypoxia. Here, to test the hypothesis that a six- to five-coordinate heme iron transition might mediate the control of the nitrite reduction rate, we examined distal pocket mutants of AHb1 and AHb2 for nitrite reductase activity, NO production and spectroscopic features. Absorption spectra of AHbs distal histidine mutants showed that AHb1 mutant (H69L) is a stable pentacoordinate high-spin species in both ferrous and ferric states, whereas heme iron in AHb2 mutant (H66L) is hexacoordinated low-spin with Lys69 as the sixth ligand. The bimolecular rate constants for nitrite reduction to NO were 13.3 ± 0.40, 7.3 ± 0.5, 10.6 ± 0.8 and 171.90 ± 9.00 M(-1)·s(-1) for AHb1, AHb2, AHb1 H69L and AHb2 H66L, respectively, at pH 7.4 and 25 °C. Consistent with the reductase activity, the amount of NO detected by chemiluminescence was significantly higher in the AHb2 H66L mutant. Our data indicate that nitrite reductase activity is determined not only by heme coordination, but also by a unique distal heme pocket in each AHb. PMID:27136534

  9. Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity

    PubMed Central

    Kumar, Nitin; Astegno, Alessandra; Chen, Jian; Giorgetti, Alejandro; Dominici, Paola

    2016-01-01

    It is well-established that plant hemoglobins (Hbs) are involved in nitric oxide (NO) metabolism via NO dioxygenase and/or nitrite reductase activity. The ferrous-deoxy Arabidopsis Hb1 and Hb2 (AHb1 and AHb2) have been shown to reduce nitrite to NO under hypoxia. Here, to test the hypothesis that a six- to five-coordinate heme iron transition might mediate the control of the nitrite reduction rate, we examined distal pocket mutants of AHb1 and AHb2 for nitrite reductase activity, NO production and spectroscopic features. Absorption spectra of AHbs distal histidine mutants showed that AHb1 mutant (H69L) is a stable pentacoordinate high-spin species in both ferrous and ferric states, whereas heme iron in AHb2 mutant (H66L) is hexacoordinated low-spin with Lys69 as the sixth ligand. The bimolecular rate constants for nitrite reduction to NO were 13.3 ± 0.40, 7.3 ± 0.5, 10.6 ± 0.8 and 171.90 ± 9.00 M−1·s−1 for AHb1, AHb2, AHb1 H69L and AHb2 H66L, respectively, at pH 7.4 and 25 °C. Consistent with the reductase activity, the amount of NO detected by chemiluminescence was significantly higher in the AHb2 H66L mutant. Our data indicate that nitrite reductase activity is determined not only by heme coordination, but also by a unique distal heme pocket in each AHb. PMID:27136534

  10. Nitrite-Reductase and Peroxynitrite Isomerization Activities of Methanosarcina acetivorans Protoglobin

    PubMed Central

    Ascenzi, Paolo; Leboffe, Loris; Pesce, Alessandra; Ciaccio, Chiara; Sbardella, Diego; Bolognesi, Martino; Coletta, Massimo

    2014-01-01

    Within the globin superfamily, protoglobins (Pgb) belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb), since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies) to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb*) are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II)) is biphasic and values of the second-order rate constant for the reduction of NO2– to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II) (Ma-Pgb*-Fe(II)-NO) are kapp1 = 9.6±0.2 M–1 s–1 and kapp2 = 1.2±0.1 M–1 s–1 (at pH 7.4 and 20°C). The kapp1 and kapp2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III)) is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III) and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are happ = 3.8×104 M–1 s–1 and h0 = 2.8×10–1 s–1 (at pH 7.4 and 20°C). The pH-dependence of hon and h0 values reflects the acid-base equilibrium of peroxynitrite (pKa = 6.7 and 6.9, respectively; at 20°C), indicating that HOONO is the species that reacts preferentially with the heme-Fe(III) atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species. PMID:24827820

  11. The nitrite reductase activity of horse heart carboxymethylated-cytochrome c is modulated by cardiolipin.

    PubMed

    Ascenzi, Paolo; Sbardella, Diego; Sinibaldi, Federica; Santucci, Roberto; Coletta, Massimo

    2016-06-01

    Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO2 (-)-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k on = (7.3 ± 0.7) × 10(-2) M(-1) s(-1); at pH 7.4], whereas the value of k on for NO2 (-) reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M(-1) s(-1) (at pH 7.4). CL facilitates the NO2 (-)-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k on for the NO2 (-)-mediated conversion of CL-CM-cytc-Fe(II) to CL-CM-cytc-Fe(II)-NO (5.6 ± 0.6 M(-1) s(-1); at pH 7.4) being slightly higher than that for the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO (2.6 ± 0.3 M(-1) s(-1); at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10(-6) M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k on versus pH are -1.05 ± 0.07 and -1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH(-). These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL-CM-cytc. PMID:27010463

  12. Solubilization and Resolution of the Membrane-Bound Nitrite Reductase from Paracoccus Halodenitrificans into Nitrite and Nitric Oxide Reductases

    NASA Technical Reports Server (NTRS)

    Grant, Michael A.; Cronin, Sonja E.; Hochstein, Lawrence I.

    1984-01-01

    Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-Chlolamidoporopyldimethylammonio)-1-(2- hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.

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

    PubMed Central

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

    1996-01-01

    Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems. PMID:8694757

  14. Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils

    PubMed Central

    Tegoni, Matteo; Yu, Fangting; Bersellini, Manuela; Penner-Hahn, James E.; Pecoraro, Vincent L.

    2012-01-01

    One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)3+/2+, where Cu(I/II) is embeded in α-helical coiled coils, as a model for the CuT2 center of copper nitrite reductase. In Cu(I/II)(TRIL23H)3+/2+, Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His)3 active site is characterized in both oxidation states, revealing similarities to the CuT2 site in the natural enzyme. The species Cu(II)(TRIL23H)32+ in aqueous solution can be reduced to Cu(I)(TRIL23H)3+ using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)32+ acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed. PMID:23236170

  15. Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils.

    PubMed

    Tegoni, Matteo; Yu, Fangting; Bersellini, Manuela; Penner-Hahn, James E; Pecoraro, Vincent L

    2012-12-26

    One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)(3)(+/2+), where Cu(I/II) is embeded in α-helical coiled coils, as a model for the Cu(T2) center of copper nitrite reductase. In Cu(I/II)(TRIL23H)(3)(+/2+), Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His)(3) active site is characterized in both oxidation states, revealing similarities to the Cu(T2) site in the natural enzyme. The species Cu(II)(TRIL23H)(3)(2+) in aqueous solution can be reduced to Cu(I)(TRIL23H)(3)(+) using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)(3)(2+) acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed. PMID:23236170

  16. Boletus edulis Nitrite Reductase Reduces Nitrite Content of Pickles and Mitigates Intoxication in Nitrite-intoxicated Mice

    PubMed Central

    Zhang, Weiwei; Tian, Guoting; Feng, Shanshan; Wong, Jack Ho; Zhao, Yongchang; Chen, Xiao; Wang, Hexiang; Ng, Tzi Bun

    2015-01-01

    Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45 °C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite, and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120 minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications. PMID:26446494

  17. Boletus edulis Nitrite Reductase Reduces Nitrite Content of Pickles and Mitigates Intoxication in Nitrite-intoxicated Mice.

    PubMed

    Zhang, Weiwei; Tian, Guoting; Feng, Shanshan; Wong, Jack Ho; Zhao, Yongchang; Chen, Xiao; Wang, Hexiang; Ng, Tzi Bun

    2015-01-01

    Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45 °C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite, and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120 minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications. PMID:26446494

  18. De novo designed metallopeptides with type 2 copper centers: modulation of reduction potentials and nitrite reductase activities

    PubMed Central

    Yu, Fangting; Penner-Hahn, James E.; Pecoraro, Vincent L.

    2014-01-01

    Enzymatic reactions involving redox processes are highly sensitive to the local electrostatic environment. Despite considerable effort, the complex interactions between different influential factors in native proteins impede progress towards complete understanding of the structure-function relationship. Of particular interest is the type 2 copper center Cu(His)3, which may act as an electron transfer center in peptidylglycine α-hydroxylating monooxygenase (PHM) or a catalytic center in copper nitrite reductase (CuNiR). A de novo design strategy is used to probe the effect of modifying charged amino acid residues around, but not directly bound to, a Cu(His)3 center embedded in three-stranded coiled coils (TRI-H)3 [TRI-H = Ac-G WKALEEK LKALEEK LKALEEK HKALEEK G-NH2]. Specifically, the peptide TRI-EH [TRI-EH = TRI-HK22E] alters an important lysine to glutamate just above the copper binding center. With a series of TRI-EH peptides mutated below the metal center, we use a variety of spectroscopies (EPR, UV-Vis, XAS) to show a direct impact on the protonation equilibria, copper binding affinities, reduction potentials and nitrite reductase activities of these copper-peptide complexes. The potentials at a specific pH vary by 100 mV and nitrite reductase activity ranges over a factor of four in rates. We also observe that affinities, potentials and catalytic activities are strongly influenced by pH conditions (pH 5.8 ~ 7.4). In general, Cu(II) affinities for the peptides are diminished at low pH values. The interplay between these factors can lead to a 200 mV shift in reduction potentials across these peptides, which is determined by the pH-dependent affinities of copper in both oxidation states. This study illustrates the strength of de novo protein design in elucidating the influence of ionizable residues on a particular redox system, an important step towards understanding the factors that govern the properties of this metalloenzyme with a goal of eventually improving

  19. Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase expression and activity in response to different nitrogen sources in nitrogen-starved wheat seedlings.

    PubMed

    Balotf, Sadegh; Kavoosi, Gholamreza; Kholdebarin, Bahman

    2016-03-01

    The objective of this study was to examine the expression and activity of nitrate reductase (NR, EC 1.7.1.1), nitrite reductase (NiR, EC 1.7.2.2), glutamine synthetase (GS, EC 6.3.1.2), and glutamate synthase (GOGAT, EC 1.4.7.1) in response to potassium nitrate, ammonium chloride, and ammonium nitrate in nitrogen-starved wheat seedlings. Plants were grown in standard nutrient solution for 17 days and then subjected to nitrogen starvation for 7 days. The starved plants were supplied with potassium nitrate ammonium nitrate and ammonium chloride (50 mM) for 4 days and the leaves were harvested. The relative expression of NR, NiR, GS, and GOGAT as well as the enzyme activities were investigated. Nitrogen starvation caused a significant decrease both in transcript levels and in NR, NiR, GS, and GOGAT activities. Potassium nitrate and ammonium nitrate treatments restored NR, NiR, GS, and GOGAT expressions and activities. Ammonium chloride increased only the expressions and activities of GS and GOGAT in a dose-dependent manner. The results of our study highlight the differential effects between the type and the amount of nitrogen salts on NR, NiR, GS, and GOGAT activities in wheat seedlings while potassium nitrate being more effective. PMID:25676153

  20. Role of nitrite in the induction of nitrate reductase activity in barley leaves

    SciTech Connect

    Aslam, M.; Huffaker, R.C.

    1986-04-01

    High levels of nitrate reductase activity (NRA) were induced in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings when supplied with NO/sub 2//sup -/ in the induction solutions. At similar N flux, the level of the enzyme activity induced by NO/sub 2//sup -/ was about one-half of that induced by NO/sub 3//sup -/. Significant levels of NO/sub 3//sup -/ accumulated in NO/sub 2//sup -/-fed leaves. Traces of NO/sub 3//sup -/ (0.6%) were detected in solutions of reagent grade KNO/sub 2/. However, the amount of NO/sub 3//sup -/ absorbed from the NO/sub 2//sup -/ solutions was only one-tenth of that accumulated in the leaves during the induction period, showing the actual conversion of NO/sub 2//sup -/ to NO/sub 3//sup -/ within the leaf. When the NO/sub 3//sup -/ concentrations in the NO/sub 2//sup -/-fed leaves were plotted against NRA, a highly positive correlation was obtained. The results suggest that NO/sub 2//sup -/ induces NRA indirectly after being oxidized to NO/sub 3//sup -/ within the leaf.

  1. Effects of Nitrite, Chlorate, and Chlorite on Nitrate Uptake and Nitrate Reductase Activity 1

    PubMed Central

    Siddiqi, M. Yaeesh; King, Bryan J.; Glass, Anthony D. M.

    1992-01-01

    Effects of NO2−, ClO3−, and ClO2− on the induction of nitrate transport and nitrate reductase activity (NRA) as well as their effects on NO3− influx into roots of intact barley (Hordeum vulgare cv Klondike) seedlings were investigated. A 24-h pretreatment with 0.1 mol m−3 NO2− fully induced NO3− transport but failed to induce NRA. Similar pretreatments with ClO3− and ClO2− induced neither NO3− transport nor NRA. Net ClO3− uptake was induced by NO3− but not by ClO3− itself, indicating that NO3− and ClO3− transport occur via the NO3− carrier. At the uptake step, NO2− and ClO2− strongly inhibited NO3− influx; the former exhibited classical competitive kinetics, whereas the latter exhibited complex mixed-type kinetics. ClO3− proved to be a weak inhibitor of NO3− influx (Ki = 16 mol m−3) in a noncompetitive manner. The implications of these findings are discussed in the context of the suitability of these NO3− analogs as screening agents for the isolation of mutants defective in NO3− transport. PMID:16653041

  2. Nitrite and nitrite reductases: from molecular mechanisms to significance in human health and disease.

    PubMed

    Castiglione, Nicoletta; Rinaldo, Serena; Giardina, Giorgio; Stelitano, Valentina; Cutruzzolà, Francesca

    2012-08-15

    Nitrite, previously considered physiologically irrelevant and a simple end product of endogenous nitric oxide (NO) metabolism, is now envisaged as a reservoir of NO to be activated in response to oxygen (O(2)) depletion. In the first part of this review, we summarize and compare the mechanisms of nitrite-dependent production of NO in selected bacteria and in eukaryotes. Bacterial nitrite reductases, which are copper or heme-containing enzymes, play an important role in the adaptation of pathogens to O(2) limitation and enable microrganisms to survive in the human body. In mammals, reduction of nitrite to NO under hypoxic conditions is carried out in tissues and blood by an array of metalloproteins, including heme-containing proteins and molybdenum enzymes. In humans, tissues play a more important role in nitrite reduction, not only because most tissues produce more NO than blood, but also because deoxyhemoglobin efficiently scavenges NO in blood. In the second part of the review, we outline the significance of nitrite in human health and disease and describe the recent advances and pitfalls of nitrite-based therapy, with special attention to its application in cardiovascular disorders, inflammation, and anti-bacterial defence. It can be concluded that nitrite (as well as nitrate-rich diet for long-term applications) may hold promise as therapeutic agent in vascular dysfunction and ischemic injury, as well as an effective compound able to promote angiogenesis. PMID:22304560

  3. Community structures and activity of denitrifying microbes in a forested catchment in central Japan: survey using nitrite reductase genes

    NASA Astrophysics Data System (ADS)

    Ohte, N.; Aoki, M.; Katsuyama, C.; Suwa, Y.; Tange, T.

    2012-12-01

    To elucidate the mechanisms of denitrification processes in the forested catchment, microbial ecological approaches have been applied in an experimental watershed that has previously investigated its hydrological processes. The study catchment is located in the Chiba prefecture in central Japan under the temperate Asian monsoon climate. Potential activities of denitrification of soil samples were measured by incubation experiments under anoxic condition associated with Na15NO3 addition. Existence and variety of microbes having nitrite reductase genes were investigated by PCR amplification, cloning and sequencings of nirK and nirS fragments after DNA extraction. Contrary to our early expectation that the potential denitrification activity was higher at deeper soil horizon with consistent groundwater residence than that in the surface soil, denitrification potential was higher in shallower soil horizons than deeper soils. This suggested that the deficiency of NO3- as a respiratory substrate for denitrifier occurred in deeper soils especially in the summer. However, high denitrification activity and presence of microbes having nirK and nirS in surface soils usually under aerobic condition was explainable by the fact that the majority of denitrifying bacteria have been recognized as a facultative anaerobic bacterium. This also suggests the possibility of that denitrification occurs even in the surface soils if the wet condition is provided by rainwater during and after a storm event. Community structures of microbes having nirK were different between near surface and deeper soil horizons, and ones having nirS was different between saturated zone (under groundwater table) and unsaturated soil horizons. These imply that microbial communities with nisK are sensitive to the concentration of soil organic matters and ones with nirS is sensitive to soil moisture contents.

  4. Nitrate metabolism in tobacco leaves overexpressing Arabidopsis nitrite reductase.

    PubMed

    Davenport, Susie; Le Lay, Pascaline; Sanchez-Tamburrrino, Juan Pablo

    2015-12-01

    Primary nitrogen assimilation in plants includes the reduction of nitrite to ammonium in the chloroplasts by the enzyme nitrite reductase (NiR EC:1.7.7.1) or in the plastids of non-photosynthetic organs. Here we report on a study overexpressing the Arabidopsis thaliana NiR (AtNiR) gene in tobacco plants under the control of a constitutive promoter (CERV - Carnation Etched Ring Virus). The aim was to overexpress AtNiR in an attempt to alter the level of residual nitrite in the leaf which can act as precursor to the formation of nitrosamines. The impact of increasing the activity of AtNiR produced an increase in leaf protein and a stay-green phenotype in the primary transformed AtNiR population. Investigation of the T1 homozygous population demonstrated elevated nitrate reductase (NR) activity, reductions in leaf nitrite and nitrate and the amino acids proline, glutamine and glutamate. Chlorophyl content of the transgenic lines was increased, as evidenced by the stay-green phenotype. This reveals the importance of NiR in primary nitrogen assimilation and how modification of this key enzyme affects both the nitrogen and carbon metabolism of tobacco plants. PMID:26447683

  5. Nitrite Reductase and Nitric-oxide Synthase Activity of the Mitochondrial Molybdopterin Enzymes mARC1 and mARC2*

    PubMed Central

    Sparacino-Watkins, Courtney E.; Tejero, Jesús; Sun, Bin; Gauthier, Marc C.; Thomas, John; Ragireddy, Venkata; Merchant, Bonnie A.; Wang, Jun; Azarov, Ivan; Basu, Partha; Gladwin, Mark T.

    2014-01-01

    Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production. PMID:24500710

  6. Cardiac contractility in Antarctic teleost is modulated by nitrite through xanthine oxidase and cytochrome p-450 nitrite reductase.

    PubMed

    Garofalo, Filippo; Amelio, Daniela; Gattuso, Alfonsina; Cerra, Maria Carmela; Pellegrino, Daniela

    2015-09-15

    In mammalian and non-mammalian vertebrates, nitrite anion, the largest pool of intravascular and tissue nitric oxide storage, represents a key player of many biological processes, including cardiac modulation. As shown by our studies on Antarctic teleosts, nitrite-dependent cardiac regulation is of great relevance also in cold-blooded vertebrates. This study analysed the influence elicited by nitrite on the performance of the perfused beating heart of two Antarctic stenotherm teleosts, the haemoglobinless Chionodraco hamatus (icefish) and the red-blooded Trematomus bernacchii. Since haemoglobin is crucial in nitric oxide homeostasis, the icefish, a naturally occurring genetic knockout for this protein, provides exclusive opportunities to investigate nitric oxide/nitrite signaling. In vivo, nitrite conversion to nitric oxide requires the nitrite reductase activity of xanthine oxidase and cytochrome P-450, thus the involvement of these enzymes was also evaluated. We showed that, in C. hamatus and T. bernacchii, nitrite influenced cardiac performance by inducing a concentration-dependent positive inotropic effect which was unaffected by nitric oxide scavenging by PTIO in C. hamatus, while it was abolished in T. bernacchii. Specific inhibition of xanthine oxidase and cytochrome P-450 revealed, in the two teleosts, that the nitrite-dependent inotropism required the nitrite reductase activity of both enzymes. We also found that xanthine oxidase is more expressed in C. hamatus than in T. bernacchii, while the opposite was observed concerning cytochrome P-450. Results suggested that in the heart of C. hamatus and T. bernacchii, nitrite is an integral physiological source of nitric oxide with important signaling properties, which require the nitrite reductase activity of xanthine oxidase and cytochrome P-450. PMID:26045289

  7. Biochemical predetermination of the NO synthase and nitrite reductase components of the nitric oxide cycle.

    PubMed

    Reutov, V P

    1999-05-01

    This review presents some aspects of a concept of cellular evolution bearing a relationship to nitrate--nitrite respiration, the endosymbiosis theory, and the origin of NO synthase and nitrite reductase activity in heme-containing proteins. Analysis of structural and functional unity of the NO synthase and nitrite reductase systems suggests that these systems did not arise without any relation to evolutionarily ancient energetic systems of cells. The use of symmetry principles reveals commonalities among many electron transport chains which in the language of physics is called "invariance". This work also comparatively analyzes the nitric oxide cycle and the known nitrogen cycle. The ideas about evolution of the NO synthase and nitrite reductase systems developed here are clearly compatible with the endosymbiotic theory and the hypothesis that nitrate--nitrite respiration was a precursor of oxygen-dependent respiration. PMID:10381613

  8. The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase

    SciTech Connect

    Liu, M.-C.; Peck, H.D., Jr.

    1981-12-01

    Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal M/sub r/=66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced..cap alpha..-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase,however, is able to transfer electrons from H/sub 2/ to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially reoxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57/sup o/C. Purified nitrite reductase also has hydroxylamine reductase activity, and the K/sub m/ for nitrite was determined to be 1.14 mM.

  9. Nitrite controls the release of nitric oxide in Pseudomonas aeruginosa cd{sub 1} nitrite reductase

    SciTech Connect

    Rinaldo, Serena; Brunori, Maurizio; Cutruzzola, Francesca

    2007-11-23

    Nitrite reductase (cd{sub 1}NIR) from Pseudomonas aeruginosa, which catalyses the reduction of nitrite to nitric oxide (NO), contains a c-heme as the electron acceptor and a d{sub 1}-heme where catalysis occurs. Reduction involves binding of nitrite to the reduced d{sub 1}-heme, followed by dehydration to yield NO; release of NO and re-reduction of the enzyme close the cycle. Since NO is a powerful inhibitor of ferrous hemeproteins, enzymatic turnover demands the release of NO. We recently discovered that NO dissociation from the ferrous d{sub 1}-heme is fast, showing that cd{sub 1}NIR behaves differently from other hemeproteins. Here we demonstrate for the first time that the physiological substrate nitrite displaces NO from the ferrous enzyme, which enters a new catalytic cycle; this reaction depends on the conserved His369 whose role in substrate stabilization is crucial for catalysis. Thus we suggest that also in vivo the activity of cd{sub 1}NIR is controlled by nitrite.

  10. Direct electrochemistry of Shewanella oneidensis cytochrome c nitrite reductase: evidence for interactions across the dimeric interface

    PubMed Central

    Judd, Evan T.; Youngblut, Matthew; Pacheco, A. Andrew; Elliott, Sean J.

    2013-01-01

    Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, carries out the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by ‘boosts’ and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed, e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA (Angove, H. C., Cole, J. A., Richardson, D. J., and Butt, J. N. (2002) Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome C nitrite reductase, J Biol Chem 277, 23374-23381). Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which have previously been observed in any cytochrome c nitrite reductase. Collectively these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer. PMID:23210513

  11. Expression and purification of spinach nitrite reductase in E. coli

    SciTech Connect

    Bellissimo, D.; Privalle, L. )

    1991-03-11

    The study of structure-function relationships in nitrite reductase (NiR) by site-directed mutagenesis requires an expression system from which suitable quantities of active enzyme can be purified. Spinach NiR cDNA was cloned into pUC18 and expressed in E.coli JM109 as a beta-galactosidase fusion protein. The IPTG-induced fusion protein contains five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive indicating the presence of inclusion bodies. By altering growth conditions, active NiR could represent 0.5-1.0% of the total E.coli protein, Effects of the addition of delta-aminolevulinic acid, a heme precursor, and anaerobic growth were also examined. Spinach NiR was purified approximately 200 fold to homogeneity. When subjected to electrophoresis on SDS polyacrylamide gels, the NiR migrated as a single band with similar mobility to pure spinach enzyme. The expressed enzyme also reacted with rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of the E.coli-expressed enzyme was identical to spinach enzyme with a Soret and alpha band a 386 and 573 nm, respectively, and an A{sub 278}/A{sub 386} = 1.9. The addition of nitrite produced the characteristic shifts in the spectrum. The E. coli-expressed NiR catalyzed the methylviologen-dependent reduction of nitrite. The specific activity was 100 U/mg. The K{sub m} determined for nitrite was 0.3 mM which is in agreement with values reported for the enzyme. These results indicate that the E.coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity and physical state. Site-directed mutants have been made using PCR to examine structure-function relationships in this enzyme.

  12. Quantum mechanical interpretation of nitrite reduction by cytochrome cd1 nitrite reductase from Paracoccus pantotrophus.

    PubMed

    Ranghino, G; Scorza, E; Sjögren, T; Williams, P A; Ricci, M; Hajdu, J

    2000-09-12

    The reduction of nitrite to nitric oxide in respiratory denitrification is catalyzed by a cytochrome cd(1) nitrite reductase in Paracoccus pantotrophus (formerly known as Thiosphaera pantotropha LMD 92.63). High-resolution structures are available for the fully oxidized [Fülöp, V., Moir, J. W., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Baker, S. C., Saunders, N. F., Willis, A. C., Ferguson, S. J., Hajdu, J., and Fülöp, V. (1997) J. Mol. Biol. 269, 440-455] and fully reduced forms of this enzyme, as well as for various intermediates in its catalytic cycle [Williams, P. A., Fülöp, V., Garman, E. F., Saunders, N. F., Ferguson, S. J., and Hajdu, J. (1997) Nature 389, 406-412]. On the basis of these structures, quantum mechanical techniques (QM), including density functional methods (DFT), were combined with simulated annealing (SA) and molecular mechanics techniques (MM) to calculate the electronic distribution of molecular orbitals in the active site during catalysis. The results show likely trajectories for electrons, protons, substrates, and products in the process of nitrite reduction, and offer an interpretation of the reaction mechanism. The calculations indicate that the redox state of the d(1) heme and charges on two histidines in the active site orchestrate catalysis locally. Binding of nitrite to the reduced iron is followed by proton transfer from His345 and His388 to one of the oxygens of nitrite, creating a water molecule and an [Fe(II)-NO(+)] complex. Valence isomerization within this complex gives [Fe(III)-NO]. The release of NO from the ferric iron is influenced by the protonation state of His345 and His388, and by the orientation of NO on the d(1) heme. Return of Tyr25 to a hydrogen-bonding position between His345 and His388 facilitates product release, but a rebinding of Tyr25 to the oxidized iron may be bypassed in steady-state catalysis. PMID:10998232

  13. The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

    Mancinelli, R. L.; Cronin, S.; Hochstein, L. I.

    1986-01-01

    Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membrane-bound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membrane-bound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.

  14. Characterization of the gene encoding nitrite reductase and the physiological consequences of its expression in the nondenitrifying Rhizobium {open_quotes}hedysari{close_quotes} strain HCNT1

    SciTech Connect

    Toffanin, A.; Shapleigh, J.P.; Maskus, M.

    1996-11-01

    Rhizobium {open_quotes}hedysari{close_quotes} HCNT1 is an unclassified rhizobium which contains a nitric oxide-producing nitrite reductase but is apparently incapable of coupling the reduction of nitrite to energy conservation. The gene encoding the nitrite reductase, nirK, has been cloned and sequenced and was found to encode a protein closely related to the copper-containing family of nitrite reductases. Unlike other members of this family, nirK expression in HCNT1 is not dependent on the presence of nitrogen oxides, being dependent only on oxygen concentration. Oxygen respiration of microaerobically grown Nir-deficient cells is not affected by concentrations of nitrite that completely inhibit oxygen respiration in wild-type cells. This loss of sensitivity suggests that the product of nitrite reductase, nitric oxide, is responsible for inhibition of oxygen respiration. By using a newly developed chemically modified electrode to detect nitric oxide, it was found that nitrite reduction by HCNT1 produces significantly higher nitric oxide concentrations than are observed in true denitrifiers. This indicates that nitrite reductase is the only nitrogen oxide reductase active in HCNT1. The capacity to generate such large concentrations of freely diffusible nitric oxide as a consequence of nitrite respiration makes HCNT1 unique among bacteria. 33 refs., 6 figs., 1 tab.

  15. Molecular Cloning of Complementary DNA Encoding Maize Nitrite Reductase

    PubMed Central

    Lahners, Kristine; Kramer, Vance; Back, Eduard; Privalle, Laura; Rothstein, Steven

    1988-01-01

    Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20-26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize. Images Fig. 3 Fig. 4 Fig. 5 PMID:16666376

  16. A novel nitrite biosensor based on conductometric electrode modified with cytochrome c nitrite reductase composite membrane.

    PubMed

    Zhang, Zhiqiang; Xia, Siqing; Leonard, Didier; Jaffrezic-Renault, Nicole; Zhang, Jiao; Bessueille, François; Goepfert, Yves; Wang, Xuejiang; Chen, Ling; Zhu, Zhiliang; Zhao, Jianfu; Almeida, M Gabriela; Silveira, Célia M

    2009-02-15

    A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 degrees C), the conductometric biosensor showed a fast response to nitrite (about 10s) with a linear range of 0.2-120 microM, a sensitivity of 0.194 microS/microM [NO(2)(-)], and a detection limit of 0.05 microM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n=5). The apparent Michaelis-Menten constant (K(M,app)) was 338 microM. When stored in potassium phosphate buffer (100mM, pH 7.6) at 4 degrees C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples. PMID:18804367

  17. Role of nitrate and nitrite in the induction of nitrite reductase in leaves of barley seedlings

    NASA Technical Reports Server (NTRS)

    Aslam, M.; Huffaker, R. C.

    1989-01-01

    The role of NO3- and NO2- in the induction of nitrite reductase (NiR) activity in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings was investigated. Barley leaves contained 6 to 8 micromoles NO2-/gram fresh weight x hour of endogenous NiR activity when grown in N-free solutions. Supply of both NO2- and NO3- induced the enzyme activity above the endogenous levels (5 and 10 times, respectively at 10 millimolar NO2- and NO3- over a 24 hour period). In NO3(-)-supplied leaves, NiR induction occurred at an ambient NO3- concentration of as low as 0.05 millimolar; however, no NiR induction was found in leaves supplied with NO2- until the ambient NO2- concentration was 0.5 millimolar. Nitrate accumulated in NO2(-)-fed leaves. The amount of NO3- accumulating in NO2(-)-fed leaves induced similar levels of NiR as did equivalent amounts of NO3- accumulating in NO3(-)-fed leaves. Induction of NiR in NO2(-)-fed leaves was not seen until NO3- was detectable (30 nanomoles/gram fresh weight) in the leaves. The internal concentrations of NO3-, irrespective of N source, were highly correlated with the levels of NiR induced. When the reduction of NO3- to NO2- was inhibited by WO4(2-), the induction of NiR was inhibited only partially. The results indicate that in barley leaves in NiR is induced by NO3- directly, i.e. without being reduced to NO2-, and that absorbed NO2- induces the enzyme activity indirectly after being oxidized to NO3- within the leaf.

  18. Evaluation of nitrate reductase activity in Rhizobium japonicum

    SciTech Connect

    Streeter, J.G.; DeVine, P.J.

    1983-08-01

    Nitrate reductase activity was evaluated by four approaches, using four strains of Rhizobium japonicum and 11 chlorate-resistant mutants of the four strains. It was concluded that in vitro assays with bacteria or bacteroids provide the most simple and reliable assessment of the presence or absence of nitrate reductase. Nitrite reductase activity with methyl viologen and dithionite was found, but the enzyme activity does not confound the assay of nitrate reductase. 18 references

  19. Nitrogen and oxygen regulation of Bacillus subtilis nasDEF encoding NADH-dependent nitrite reductase by TnrA and ResDE.

    PubMed

    Nakano, M M; Hoffmann, T; Zhu, Y; Jahn, D

    1998-10-01

    The nitrate and nitrite reductases of Bacillus subtilis have two different physiological functions. Under conditions of nitrogen limitation, these enzymes catalyze the reduction of nitrate via nitrite to ammonia for the anabolic incorporation of nitrogen into biomolecules. They also function catabolically in anaerobic respiration, which involves the use of nitrate and nitrite as terminal electron acceptors. Two distinct nitrate reductases, encoded by narGHI and nasBC, function in anabolic and catabolic nitrogen metabolism, respectively. However, as reported herein, a single NADH-dependent, soluble nitrite reductase encoded by the nasDE genes is required for both catabolic and anabolic processes. The nasDE genes, together with nasBC (encoding assimilatory nitrate reductase) and nasF (required for nitrite reductase siroheme cofactor formation), constitute the nas operon. Data presented show that transcription of nasDEF is driven not only by the previously characterized nas operon promoter but also from an internal promoter residing between the nasC and nasD genes. Transcription from both promoters is activated by nitrogen limitation during aerobic growth by the nitrogen regulator, TnrA. However, under conditions of oxygen limitation, nasDEF expression and nitrite reductase activity were significantly induced. Anaerobic induction of nasDEF required the ResDE two-component regulatory system and the presence of nitrite, indicating partial coregulation of NasDEF with the respiratory nitrate reductase NarGHI during nitrate respiration. PMID:9765565

  20. Nitrogen and Oxygen Regulation of Bacillus subtilis nasDEF Encoding NADH-Dependent Nitrite Reductase by TnrA and ResDE

    PubMed Central

    Nakano, Michiko M.; Hoffmann, Tamara; Zhu, Yi; Jahn, Dieter

    1998-01-01

    The nitrate and nitrite reductases of Bacillus subtilis have two different physiological functions. Under conditions of nitrogen limitation, these enzymes catalyze the reduction of nitrate via nitrite to ammonia for the anabolic incorporation of nitrogen into biomolecules. They also function catabolically in anaerobic respiration, which involves the use of nitrate and nitrite as terminal electron acceptors. Two distinct nitrate reductases, encoded by narGHI and nasBC, function in anabolic and catabolic nitrogen metabolism, respectively. However, as reported herein, a single NADH-dependent, soluble nitrite reductase encoded by the nasDE genes is required for both catabolic and anabolic processes. The nasDE genes, together with nasBC (encoding assimilatory nitrate reductase) and nasF (required for nitrite reductase siroheme cofactor formation), constitute the nas operon. Data presented show that transcription of nasDEF is driven not only by the previously characterized nas operon promoter but also from an internal promoter residing between the nasC and nasD genes. Transcription from both promoters is activated by nitrogen limitation during aerobic growth by the nitrogen regulator, TnrA. However, under conditions of oxygen limitation, nasDEF expression and nitrite reductase activity were significantly induced. Anaerobic induction of nasDEF required the ResDE two-component regulatory system and the presence of nitrite, indicating partial coregulation of NasDEF with the respiratory nitrate reductase NarGHI during nitrate respiration. PMID:9765565

  1. Structures of complexes of octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens with sulfite and cyanide.

    PubMed

    Trofimov, Anton A; Polyakov, Konstantin M; Boyko, Konstantin M; Tikhonova, Tamara V; Safonova, Tatyana N; Tikhonov, Alexey V; Popov, Alexandre N; Popov, Vladimir O

    2010-10-01

    The structures of complexes of octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens (TvNiR) with the substrate sulfite (1.4 Å resolution; R(cryst) = 0.126) and the inhibitor cyanide (1.55 Å resolution; R(cryst) = 0.148) have been established. The complex with sulfite was prepared by the reduction of the protein crystal with sodium dithionite. The sulfite ion is bound to the iron ion of the catalytic haem through the S atom. The Fe-S distance is 2.24 Å. The structure of the cyanide complex with full occupancy of the ligand site was established for the first time for cytochrome c nitrite reductases. The cyanide ion is bound to the catalytic haem iron through the C atom. The Fe-C distance is 1.91 Å and the Fe-C-N angle is 171°. The sulfite reductase activity of TvNiR was measured at different pH values. The activity is 0.02 µmol of HS(-) per minute per milligram at pH 7.0; it decreases with increasing pH and is absent at pH 9.0. PMID:20944237

  2. Theoretical study on reaction mechanisms of nitrite reduction by copper nitrite complexes: toward understanding and controlling possible mechanisms of copper nitrite reductase.

    PubMed

    Maekawa, Shintaro; Matsui, Toru; Hirao, Kimihiko; Shigeta, Yasuteru

    2015-04-30

    Using density functional theory, we studied denitrification reaction mechanisms of copper adducts of tris(pyrazolyl)methane and hydrotris(pyrazolyl)borate models of a copper nitrite reductase (Cu-NiR), and herein propose several possible reaction pathways, including some parts that have never been examined previously. Because electron and proton transfer reactions participate in the enzymatic cycles of Cu-NiR, the Gibbs energy of a proton in solution, G(H(+)), and the redox potential, Eredox, of the model Cu-NiR are also evaluated. Although the pathway where a nitrite is provided as HNO2 is energetically preferable, a well-known reaction pathway passing through the resting state with an active site occupied by a water molecule where nitrite is provided as NO2(-) is the main recognized pathway under normal conditions. These features do not change whether the electron transfer occurs before production of NO or not. However, our results suggest that the pathway involving HNO2 might become dominant under low pH conditions in conjunction with experimental results. PMID:25845517

  3. Measuring the Cytochrome c Nitrite Reductase Activity—Practical Considerations on the Enzyme Assays

    PubMed Central

    Silveira, Célia M.; Besson, Stéphane; Moura, Isabel; Moura, José J. G.; Almeida, M. Gabriela

    2010-01-01

    The cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 is able to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensively characterized from the spectroscopic and structural points-of-view, some of its kinetic aspects are still under explored. In this work the kinetic behaviour of ccNiR has been evaluated in a systematic manner using two different spectrophotometric assays carried out in the presence of different redox mediators and a direct electrochemical approach. Solution assays have proved that the specific activity of ccNiR decreases with the reduction potential of the electronic carriers and ammonium is always the main product of nitrite reduction. The catalytic parameters were discussed on the basis of the mediator reducing power and also taking into account the location of their putative docking sites with ccNiR. Due to the fast kinetics of ccNiR, electron delivering from reduced electron donors is rate-limiting in all spectrophotometric assays, so the estimated kinetic constants are apparent only. Nevertheless, this limitation could be overcome by using a direct electrochemical approach which shows that the binding affinity for nitrite decreases whilst turnover increases with the reductive driving force. PMID:20689707

  4. Six-electron reduction of nitrite to ammonia by cytochrome c nitrite reductase: insights from density functional theory studies.

    PubMed

    Bykov, Dmytro; Neese, Frank

    2015-10-01

    In this Forum Article, an extensive discussion of the mechanism of six-electron, seven-proton nitrite reduction by the cytochrome c nitrite reductase enzyme is presented. On the basis of previous studies, the entire mechanism is summarized and a unified picture of the most plausible sequence of elementary steps is presented. According to this scheme, the mechanism can be divided into five functional stages. The first phase of the reaction consists of substrate binding and N-O bond cleavage. Here His277 plays a crucial role as a proton donor. In this step, the N-O bond is cleaved heterolytically through double protonation of the substrate. The second phase of the mechanism consists of two proton-coupled electron-transfer events, leading to an HNO intermediate. The third phase involves the formation of hydroxylamine, where Arg114 provides the necessary proton for the reaction. The second N-O bond is cleaved in the fourth phase of the mechanism, again triggered by proton transfer from His277. The Tyr218 side chain governs the fifth and last phase of the mechanism. It consists of radical transfer and ammonia formation. Thus, this mechanism implies that all conserved active-site side chains work in a concerted way in order to achieve this complex chemical transformation from nitrite to ammonia. The Forum Article also provides a detailed discussion of the density functional theory based cluster model approach to bioinorganic reactivity. A variety of questions are considered: the resting state of enzyme and substrate binding modes, interaction with the metal site and with active-site side chains, electron- and proton-transfer events, substrate dissociation, etc. PMID:26237518

  5. Stable Copper-Nitrosyl Formation By Nitrite Reductase in Either Oxidation State

    SciTech Connect

    Tocheva, E.I.; Rosell, F.I.; Mauk, A.G.; Murphy, M.E.P.

    2009-06-04

    Nitrite reductase (NiR) is an enzyme that uses type 1 and type 2 copper sites to reduce nitrite to nitric oxide during bacterial denitrification. A copper-nitrosyl intermediate is a proposed, yet poorly characterized feature of the NiR catalytic cycle. This intermediate is formally described as Cu(I)-NO{sup +} and is proposed to be formed at the type 2 copper site after nitrite binding and electron transfer from the type 1 copper site. In this study, copper-nitrosyl complexes were formed by prolonged exposure of exogenous NO to crystals of wild-type and two variant forms of NiR from Alcaligenes faecalis (AfNiR), and the structures were determined to 1.8 {angstrom} or better resolution. Exposing oxidized wild-type crystals to NO results in the reverse reaction and formation of nitrite that remains bound at the active site. In a type 1 copper site mutant (H145A) that is incapable of electron transfer to the type 2 site, the reverse reaction is not observed. Instead, in both oxidized and reduced H145A crystals, NO is observed bound in a side-on manner to the type 2 copper. In AfNiR, Asp98 forms hydrogen bonds to both substrate and product bound to the type 2 Cu. In the D98N variant, NO is bound side-on but is more disordered when observed for the wild-type enzyme. The solution EPR spectra of the crystallographically characterized NiR-NO complexes indicate the presence of an oxidized type 2 copper site and thus are interpreted as resulting from stable copper-nitrosyls and formally assigned as Cu(II)-NO{sup -}. A reaction scheme in which a second NO molecule is oxidized to nitrite can account for the formation of a CuD-NO{sup -} species after exposure of the oxidized H145A variant to NO gas.

  6. Structure of octaheme cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens in a complex with phosphate

    SciTech Connect

    Trofimov, A. A.; Polyakov, K. M.; Boiko, K. M.; Filimonenkov, A. A.; Dorovatovskii, P. V.; Tikhonova, T. V.; Popov, V. O.; Koval'chuk, M. V.

    2010-01-15

    Octaheme cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR) catalyzes the reduction of nitrite and hydroxylamine to ammonia. The structures of the free enzyme and of the enzyme in complexes with the substrate (nitrite ion) and the inhibitor (azide ion) have been solved previously. In this study we report the structures of the oxidized complex of TvNiR with phosphate and of this complex reduced by europium(II) chloride (1.8- and 2.0-A resolution, the R factors are 15.9 and 16.7%, respectively) and the structure of the enzyme in the complex with cyanide (1.76-A resolution, the R factor is 16.5%), which was prepared by soaking a crystal of the oxidized phosphate complex of TvNiR. In the active site of the enzyme, the phosphate ion binds to the iron ion of the catalytic heme and to the side chains of the catalytic residues Arg131, Tyr303, and His361. The cyanide ion is coordinated to the heme-iron ion and is hydrogen bonded to the residue His361. In the structure of reduced TvNiR, the phosphate ion is bound in the same manner as in the structure of oxidized TvNiR, and the nine{sub c}oordinated europium ion is located on the surface of one of the crystallographically independent monomers of the enzyme.

  7. Metabolic Interactions between Spinach Leaf Nitrite Reductase and Ferredoxin-NADP Reductase

    PubMed Central

    Baysdorfer, Chris; Robinson, J. Michael

    1985-01-01

    Steady state rates of NADP reduction decline upon commencement of nitrite reduction in reconstituted chloroplast preparations. Similarly, steady state rates of nitrite reduction are lower, but not zero, during concurrent NADP reduction. These results imply that competition for substrate occurs and suggest that nitrite reduction can successfully compete for reduced ferredoxin, even at high rates of NADP reduction. PMID:16664050

  8. Nitrite and Nitrous Oxide Reductase Regulation by Nitrogen Oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106

    PubMed Central

    Sabaty, Monique; Schwintner, Carole; Cahors, Sandrine; Richaud, Pierre; Verméglio, Andre

    1999-01-01

    We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N2O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N2O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase. PMID:10498715

  9. Nitrite and nitrous oxide reductase regulation by nitrogen oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106.

    PubMed

    Sabaty, M; Schwintner, C; Cahors, S; Richaud, P; Verméglio, A

    1999-10-01

    We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase. PMID:10498715

  10. Nitrite reductase function of deoxymyoglobin: oxygen sensor and regulator of cardiac energetics and function.

    PubMed

    Rassaf, Tienush; Flögel, Ulrich; Drexhage, Christine; Hendgen-Cotta, Ulrike; Kelm, Malte; Schrader, Jürgen

    2007-06-22

    Although the primary function of myoglobin (Mb) has been considered to be cellular oxygen storage and supply, recent studies have suggested to classify Mb as a multifunctional allosteric enzyme. In the heart, Mb acts as a potent scavenger of nitric oxide (NO) and contributes to the attenuation of oxidative damage. Here we report that a dynamic cycle exists in which a decrease in tissue oxygen tension drives the conversion of Mb from being an NO scavenger in normoxia to an NO producer in hypoxia. The NO generated by reaction of deoxygenated Mb with nitrite is functionally relevant and leads to a downregulation of cardiac energy status, which was not observed in mice lacking Mb. As a consequence, myocardial oxygen consumption is reduced and cardiac contractility is dampened in wild-type mice. We propose that this pathway represents a novel homeostatic mechanism by which a mismatch between oxygen supply and demand in muscle is translated into the fractional increase of deoxygenated Mb exhibiting enhanced nitrite reductase activity. Thus, Mb may act as an oxygen sensor which through NO can adjust muscle energetics to limited oxygen supply. PMID:17495223

  11. Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal.

    PubMed

    Horrell, Sam; Antonyuk, Svetlana V; Eady, Robert R; Hasnain, S Samar; Hough, Michael A; Strange, Richard W

    2016-07-01

    Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to study in crystallo enzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07-1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a 'catalytic reaction movie' highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines. PMID:27437114

  12. Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal

    PubMed Central

    Horrell, Sam; Antonyuk, Svetlana V.; Eady, Robert R.; Hasnain, S. Samar; Hough, Michael A.; Strange, Richard W.

    2016-01-01

    Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to study in crystallo enzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07–1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a ‘catalytic reaction movie’ highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines. PMID:27437114

  13. A nitrite biosensor based on co-immobilization of nitrite reductase and viologen-modified chitosan on a glassy carbon electrode.

    PubMed

    Quan, De; Shin, Woonsup

    2010-01-01

    An electrochemical nitrite biosensor based on co-immobilization of copper-containing nitrite reductase (Cu-NiR, from Rhodopseudomonas sphaeroides forma sp. denitrificans) and viologen-modified chitosan (CHIT-V) on a glassy carbon electrode (GCE) is presented. Electron transfer (ET) between a conventional GCE and immobilized Cu-NiR was mediated by the co-immobilized CHIT-V. Redox-active viologen was covalently linked to a chitosan backbone, and the thus produced CHIT-V was co-immobilized with Cu-NiR on the GCE surface by drop-coating of hydrophilic polyurethane (HPU). The electrode responded to nitrite with a limit of detection (LOD) of 40 nM (S/N = 3). The sensitivity, linear response range, and response time (t(90%)) were 14.9 nA/μM, 0.04-11 μM (r(2) = 0.999) and 15 s, respectively. The corresponding Lineweaver-Burk plot showed that the apparent Michaelis-Menten constant (K(M) (app)) was 65 μM. Storage stability of the biosensor (retaining 80% of initial activity) was 65 days under ambient air and room temperature storage conditions. Reproducibility of the sensor showed a relative standard deviation (RSD) of 2.8% (n = 5) for detection of 1 μM of nitrite. An interference study showed that anions commonly found in water samples such as chlorate, chloride, sulfate and sulfite did not interfere with the nitrite detection. However, nitrate interfered with a relative sensitivity of 64% and this interference effect was due to the intrinsic character of the NiR employed in this study. PMID:22219710

  14. Spectroelectrochemical investigation of intramolecular and interfacial electron-transfer rates reveals differences between nitrite reductase at rest and during turnover.

    PubMed

    Krzemiński, Łukasz; Ndamba, Lionel; Canters, Gerard W; Aartsma, Thijs J; Evans, Stephen D; Jeuken, Lars J C

    2011-09-28

    A combined fluorescence and electrochemical method is described that is used to simultaneously monitor the type-1 copper oxidation state and the nitrite turnover rate of a nitrite reductase (NiR) from Alcaligenes faecalis S-6. The catalytic activity of NiR is measured electrochemically by exploiting a direct electron transfer to fluorescently labeled enzyme molecules immobilized on modified gold electrodes, whereas the redox state of the type-1 copper site is determined from fluorescence intensity changes caused by Förster resonance energy transfer (FRET) between a fluorophore attached to NiR and its type-1 copper site. The homotrimeric structure of the enzyme is reflected in heterogeneous interfacial electron-transfer kinetics with two monomers having a 25-fold slower kinetics than the third monomer. The intramolecular electron-transfer rate between the type-1 and type-2 copper site changes at high nitrite concentration (≥520 μM), resulting in an inhibition effect at low pH and a catalytic gain in enzyme activity at high pH. We propose that the intramolecular rate is significantly reduced in turnover conditions compared to the enzyme at rest, with an exception at low pH/nitrite conditions. This effect is attributed to slower reduction rate of type-2 copper center due to a rate-limiting protonation step of residues in the enzyme's active site, gating the intramolecular electron transfer. PMID:21863850

  15. A cytochrome cd1-type nitrite reductase isolated from the marine denitrifier Pseudomonas nautica 617: purification and characterization.

    PubMed

    Besson, S; Carneiro, C; Moura, J J; Moura, I; Fauque, G

    1995-08-01

    Nitrite reductase (cytochrome cd1) was purified to electrophoretic homogeneity from the soluble extract of the marine denitrifying bacterium Pseudomonas nautica strain 617. Cells were anaerobically grown with 10 mM nitrate as final electron acceptor. The soluble fraction was purified by four successive chromatographic steps and the purest cytochrome cd1 exhibited an A280 nm(oxidized)/A410nm(oxidized) coefficient of 0.90. In the course of purification, cytochrome cd1 specific activity presented a maximum value of 0.048 units/mg of protein. This periplasmic enzyme is a homodimer and each 60 kDa subunit contains one heme c and one heme d1 as prosthetic moieties, both in a low spin state. Redox potentials of hemes c and d1 were determined at three different pH values (6.6, 7.6 and 8.6) and did not show any pH dependence. The first 20 amino acids of the NH2-terminal region of the protein were identified and the sequence showed 45% identity with the corresponding region of Pseudomonas aeruginosa nitrite reductase but no homology to Pseudomonas stutzeri and Paracoccus denitrificans enzymes. Spectroscopic properties of Pseudomonas nautica 617 cytochrome cd1 in the ultraviolet-visible range and in electron paramagnetic resonance are described. The formation of a heme d1 -nitric-oxide complex as an intermediate of nitrite reduction was demonstrated by electron paramagnetic resonance experiments. PMID:16887530

  16. DFT Study on Enzyme Turnover Including Proton and Electron Transfers of Copper-Containing Nitrite Reductase.

    PubMed

    Lintuluoto, Masami; Lintuluoto, Juha M

    2016-08-23

    The reaction mechanism of copper-containing nitrite reductase (CuNiR) has been proposed to include two important events, an intramolecular electron transfer and a proton transfer. The two events have been suggested to be coupled, but the order of these events is currently under debate. We investigated the entire enzyme reaction mechanism of nitrite reduction at the T2 Cu site in thermophilic Geobacillus CuNiR from Geobacillus thermodenitrificans NG80-2 (GtNiR) using density functional theory calculations. We found significant conformational changes of His ligands coordinated to the T2 Cu site upon nitrite binding during the catalytic reaction. The reduction potentials and pKa values calculated for the relevant protonation and reduction states show two possible routes, A and B. Reduction of the T2 Cu site in the resting state is followed by endothermic nitrite binding in route A, while exothermic nitrite binding occurs prior to reduction of the T2 Cu site in route B. We concluded that our results support the random-sequential mechanism rather than the ordered mechanism. PMID:27455866

  17. Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120

    PubMed Central

    Frías, José E.

    2015-01-01

    ABSTRACT Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively. IMPORTANCE Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many

  18. Nitrite reductase is critical for Pseudomonas aeruginosa survival during co-infection with the oral commensal Streptococcus parasanguinis.

    PubMed

    Scoffield, Jessica A; Wu, Hui

    2016-02-01

    Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in cystic fibrosis (CF) patients. However, recent evidence suggests that the polymicrobial community of the CF lung may also harbour oral streptococci, and colonization by these micro-organisms may have a negative impact on P. aeruginosa within the CF lung. Our previous studies demonstrated that nitrite abundance plays an important role in P. aeruginosa survival during co-infection with oral streptococci. Nitrite reductase is a key enzyme involved in nitrite metabolism. Therefore, the objective of this study was to examine the role nitrite reductase (gene nirS) plays in P. aeruginosa survival during co-infection with an oral streptococcus, Streptococcus parasanguinis. Inactivation of nirS in both the chronic CF isolate FRD1 and acute wound isolate PAO1 reduced the survival rate of P. aeruginosa when co-cultured with S. parasanguinis. Growth of both mutants was restored when co-cultured with S. parasanguinis that was defective for H2O2 production. Furthermore, the nitrite reductase mutant was unable to kill Drosophila melanogaster during co-infection with S. parasanguinis. Taken together, these results suggest that nitrite reductase plays an important role for survival of P. aeruginosa during co-infection with S. parasanguinis. PMID:26673783

  19. Inhibition of xanthine oxidase by the aldehyde oxidase inhibitor raloxifene: implications for identifying molybdopterin nitrite reductases.

    PubMed

    Weidert, E R; Schoenborn, S O; Cantu-Medellin, N; Choughule, K V; Jones, J P; Kelley, E E

    2014-02-15

    when choosing inhibition strategies as well as inhibitor concentrations when assigning relative NO2- reductase activity of AO and XOR. PMID:24406683

  20. Nitrite reductase gene upregulated during conidiation is involved in macroconidium formation in Fusarium oxysporum.

    PubMed

    Iida, Y; Kurata, T; Harimoto, Y; Tsuge, T

    2008-10-01

    Fusarium oxysporum produces three kinds of asexual spores, microconidia, macroconidia, and chlamydospores. We previously found that the transcript level of the nitrite reductase gene of F. oxysporum, named FoNIIA, was markedly upregulated during conidiation compared with during vegetative growth. FoNIIA was also found to be positively regulated by Ren1 that is a transcription regulator controlling development of microconidia and macroconidia. In this study, we analyzed the function of FoNIIA in conidiation of F. oxysporum. Conidiation cultures showed markedly higher level of accumulation of FoNiiA protein as well as FoNIIA mRNA than vegetative growth cultures. FoNIIA protein was significantly decreased in cultures of the REN1 disruption mutant compared with that of the wild type. These results confirmed that FoNIIA expression is upregulated during conidiation and is positively regulated by REN1. The FoNIIA disruption mutants produced microconidia, macroconidia, and chlamydospores, which were morphologically indistinguishable from those of the wild type. The mutants, however, produced significantly fewer macroconidia than the wild type, although the wild type and mutant strains produced similar numbers of microconidia and chlamydospores. These results demonstrate that nitrite reductase is involved in quantitative control of macroconidium formation as well as nitrate utilization in F. oxysporum. PMID:18943456

  1. Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography

    PubMed Central

    Fukuda, Yohta; Tse, Ka Man; Nakane, Takanori; Nakatsu, Toru; Suzuki, Mamoru; Sugahara, Michihiro; Inoue, Shigeyuki; Masuda, Tetsuya; Yumoto, Fumiaki; Matsugaki, Naohiro; Nango, Eriko; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Song, Changyong; Hatsui, Takaki; Nureki, Osamu; Murphy, Michael E. P.; Inoue, Tsuyoshi; Iwata, So; Mizohata, Eiichi

    2016-01-01

    Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme–substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes. PMID:26929369

  2. Redox-coupled structural changes in nitrite reductase revealed by serial femtosecond and microfocus crystallography.

    PubMed

    Fukuda, Yohta; Tse, Ka Man; Suzuki, Mamoru; Diederichs, Kay; Hirata, Kunio; Nakane, Takanori; Sugahara, Michihiro; Nango, Eriko; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Song, Changyong; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Matsumura, Hiroyoshi; Inoue, Tsuyoshi; Iwata, So; Mizohata, Eiichi

    2016-05-01

    Serial femtosecond crystallography (SFX) has enabled the damage-free structural determination of metalloenzymes and filled the gaps of our knowledge between crystallographic and spectroscopic data. Crystallographers, however, scarcely know whether the rising technique provides truly new structural insights into mechanisms of metalloenzymes partly because of limited resolutions. Copper nitrite reductase (CuNiR), which converts nitrite to nitric oxide in denitrification, has been extensively studied by synchrotron radiation crystallography (SRX). Although catalytic Cu (Type 2 copper (T2Cu)) of CuNiR had been suspected to tolerate X-ray photoreduction, we here showed that T2Cu in the form free of nitrite is reduced and changes its coordination structure in SRX. Moreover, we determined the completely oxidized CuNiR structure at 1.43 Å resolution with SFX. Comparison between the high-resolution SFX and SRX data revealed the subtle structural change of a catalytic His residue by X-ray photoreduction. This finding, which SRX has failed to uncover, provides new insight into the reaction mechanism of CuNiR. PMID:26769972

  3. Redox-coupled structural changes in nitrite reductase revealed by serial femtosecond and microfocus crystallography

    PubMed Central

    Fukuda, Yohta; Suzuki, Mamoru; Matsumura, Hiroyoshi; Inoue, Tsuyoshi; Iwata, So; Mizohata, Eiichi

    2016-01-01

    Serial femtosecond crystallography (SFX) has enabled the damage-free structural determination of metalloenzymes and filled the gaps of our knowledge between crystallographic and spectroscopic data. Crystallographers, however, scarcely know whether the rising technique provides truly new structural insights into mechanisms of metalloenzymes partly because of limited resolutions. Copper nitrite reductase (CuNiR), which converts nitrite to nitric oxide in denitrification, has been extensively studied by synchrotron radiation crystallography (SRX). Although catalytic Cu (Type 2 copper (T2Cu)) of CuNiR had been suspected to tolerate X-ray photoreduction, we here showed that T2Cu in the form free of nitrite is reduced and changes its coordination structure in SRX. Moreover, we determined the completely oxidized CuNiR structure at 1.43 Å resolution with SFX. Comparison between the high-resolution SFX and SRX data revealed the subtle structural change of a catalytic His residue by X-ray photoreduction. This finding, which SRX has failed to uncover, provides new insight into the reaction mechanism of CuNiR. PMID:26769972

  4. Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography.

    PubMed

    Fukuda, Yohta; Tse, Ka Man; Nakane, Takanori; Nakatsu, Toru; Suzuki, Mamoru; Sugahara, Michihiro; Inoue, Shigeyuki; Masuda, Tetsuya; Yumoto, Fumiaki; Matsugaki, Naohiro; Nango, Eriko; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Song, Changyong; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Murphy, Michael E P; Inoue, Tsuyoshi; Iwata, So; Mizohata, Eiichi

    2016-03-15

    Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme-substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes. PMID:26929369

  5. The energy-conserving nitric-oxide-reductase system in Paracoccus denitrificans. Distinction from the nitrite reductase that catalyses synthesis of nitric oxide and evidence from trapping experiments for nitric oxide as a free intermediate during denitrification.

    PubMed

    Carr, G J; Page, M D; Ferguson, S J

    1989-02-15

    1. A Clark-type electrode that responds to nitric oxide has been used to show that cytoplasmic membrane vesicles of Paracoccus denitrificans have a nitric-oxide reductase activity. Nitrous oxide is the reaction product. NADH, succinate or isoascorbate plus 2,3,5,6-tetramethyl-1,4-phenylene diamine can act as reductants. The NADH-dependent activity is resistant to freezing of the vesicles and thus the NADH:nitric-oxide oxidoreductase activity of stored frozen vesicles provides a method for calibrating the electrode by titration of dissolved nitric oxide with NADH. The periplasmic nitrite reductase and nitrous-oxide reductase enzymes are absent from the vesicles which indicates that nitric-oxide reductase is a discrete enzyme associated with the denitrification process. This conclusion was supported by the finding that nitric-oxide reductase activity was absent from both membranes prepared from aerobically grown P. denitrificans and bovine heart submitochondrial particles. 2. The NADH: nitric-oxide oxidoreductase activity was inhibited by concentrations of antimycin or myxothiazol that were just sufficient to inhibit the cytochrome bc1 complex of the ubiquinol--cytochrome-c oxidoreductase. The activity was deduced to be proton translocating by the observations of: (a) up to 3.5-fold stimulation upon addition of an uncoupler; and (b) ATP synthesis with a P:2e ratio of 0.75. 3. Nitrite reductase of cytochrome cd1 type was highly purified from P. denitrificans in a new, high-yield, rapid two- or three-step procedure. This enzyme catalysed stoichiometric synthesis of nitric oxide. This observation, taken together with the finding that the maximum rate of NADH:nitric-oxide oxidoreductase activity catalysed by the vesicles was comparable with that of NADH:nitrate-oxidoreductase, is consistent with a role for nitric-oxide reductase in the physiological conversion of nitrate or nitrite to dinitrogen gas. 4. Intact cells of P. denitrificans also reduced nitric oxide in an

  6. Laue Crystal Structure of Shewanella oneidensis Cytochrome c Nitrite Reductase from a High-yield Expression System

    PubMed Central

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-01-01

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “Small Tetra-heme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated ~20 mg crude ccNiR/L culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for E. coli ccNiR, and is stable for over two weeks in pH 7 solution at 4° C. UV/Vis spectropotentiometric titrations and protein film voltammetry identified 5 independent 1-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the 5 reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed amongst the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good quality crystals, with which the 2.59 Å resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein). PMID:22382353

  7. Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system

    SciTech Connect

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-09-11

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein 'small tetraheme c' replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-{angstrom}-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).

  8. Electron transfer and docking between cytochrome cd1 nitrite reductase and different redox partners - A comparative study.

    PubMed

    Pedroso, Humberto A; Silveira, Célia M; Almeida, Rui M; Almeida, Ana; Besson, Stéphane; Moura, Isabel; Moura, José J G; Almeida, M Gabriela

    2016-09-01

    Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the reduction of nitrite to nitric oxide in denitrifying bacteria, such as Marinobacter hydrocarbonoclasticus. Previous work demonstrated that the enzymatic activity depends on a structural pre-activation triggered by the entry of electrons through the electron transfer (ET) domain, which houses a heme c center. The catalytic activity of M. hydrocarbonoclasticus cd1NiR (Mhcd1NiR) was tested by mediated electrochemistry, using small ET proteins and chemical redox mediators. The rate of enzymatic reaction depends on the nature of the redox partner, with cytochrome (cyt) c552 providing the highest value. In situations where cyt c552 is replaced by either a biological (cyt c from horse heart) or a chemical mediator the catalytic response was only observed at very low scan rates, suggesting that the intermolecular ET rate is much slower. Molecular docking simulations with the 3D model structure of Mhcd1NiR and cyt c552 or cyt c showed that hydrophobic interactions favor the formation of complexes where the heme c domain of the enzyme is the principal docking site. However, only in the case of cyt c552 the preferential areas of contact and Fe-Fe distances between heme c groups of the redox partners allow establishing competent ET pathways. The coupling of the enzyme with chemical redox mediators was also found not to be energetically favorable. These results indicate that although low activity functional complexes can be formed between Mhcd1NiR and different types of redox mediators, efficient ET is only observed with the putative physiological electron donor cyt c552. PMID:27133504

  9. nasST, two genes involved in the induction of the assimilatory nitrite-nitrate reductase operon (nasAB) of Azotobacter vinelandii.

    PubMed

    Gutierrez, J C; Ramos, F; Ortner, L; Tortolero, M

    1995-11-01

    An operon including two new genes (nasS and nasT) has been defined, cloned and sequenced. The deduced NASS protein is homologous to NRTA from Synechococcus sp. and to NASF from Klebsiella pneumoniae, two proteins involved in nitrate uptake. The predicted NAST polypeptide is homologous to the regulator proteins of the two-component regulatory systems. NASS plays a negative regulatory role in the synthesis of the nitrate and nitrite reductase. NAST is required for the expression of the nitrite-nitrate reductase operon (nasAB). Expression of the nasST operon is not under the control of the NTR system and is not regulated by the nitrogen source. A Phi(nasA-lacZ) fusion has been used to analyse expression of the nasAB operon in three different genetic backgrounds with altered nitrate reductase activity. Beta-galactosidase activity in two of them was independent of nitrate but in a mutant unable to reduce nitrate, nas-4, it was normally induced by nitrate. PMID:8748040

  10. Electron transfer in zinc-reconstituted nitrite reductase from Pseudomonas aeruginosa.

    PubMed Central

    Bellelli, A; Brzezinski, P; Arese, M; Cutruzzola, F; Silvestrini, M C; Brunori, M

    1996-01-01

    1. The catalytic cycle of the haem-containing nitrite reductase (NIR) from Pseudomonas aeruginosa involves electron transfer between the two prosthetic groups of the enzyme, the c-haem and the d1-haem; this reaction was shown to be slow by stopped-flow analysis. The recombinant enzyme, expressed in Pseudomonas putida, contains the c-haem but no d1-haem; we have reconstituted this protein with Zn-protoporphyrin IX in the place of the d1-haem. 2. Photoexcitation of Zn-NIR is followed by electron transfer from the triplet excited state of the Zn-porphyrin to the oxidized c-haem, with a rate constant of 7 x 10(5) s-1; since the intermediate with reduced c-haem is not significantly populated, we conclude that the back reaction is probably as fast. 3. Even taking into account that in the native NIR the driving force is close to zero, the rate constant for the c-->d1 electron transfer, estimated from our experiments, is still much higher than that observed by stopped flow (k = 0.3 s-1) using reduced azurin as the electron donor. This finding may be a direct kinetic indication that reduction of the d1-haem is associated with a substantial reorganization of the co-ordination of the metal, as shown by spectroscopy of the oxidized and reduced NIR. PMID:8912674

  11. Nitrite

    Integrated Risk Information System (IRIS)

    Nitrite ; CASRN 14797 - 65 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effects

  12. Nitrate, nitrite and nitric oxide reductases: from the last universal common ancestor to modern bacterial pathogens.

    PubMed

    Vázquez-Torres, Andrés; Bäumler, Andreas J

    2016-02-01

    The electrochemical gradient that ensues from the enzymatic activity of cytochromes such as nitrate reductase, nitric oxide reductase, and quinol oxidase contributes to the bioenergetics of the bacterial cell. Reduction of nitrogen oxides by bacterial pathogens can, however, be uncoupled from proton translocation and biosynthesis of ATP or NH4(+), but still linked to quinol and NADH oxidation. Ancestral nitric oxide reductases, as well as cytochrome c oxidases and quinol bo oxidases evolved from the former, are capable of binding and detoxifying nitric oxide to nitrous oxide. The NO-metabolizing activity associated with these cytochromes can be a sizable source of antinitrosative defense in bacteria during their associations with host cells. Nitrosylation of terminal cytochromes arrests respiration, reprograms bacterial metabolism, stimulates antioxidant defenses and alters antibiotic cytotoxicity. Collectively, the bioenergetics and regulation of redox homeostasis that accompanies the utilization of nitrogen oxides and detoxification of nitric oxide by cytochromes of the electron transport chain increases fitness of many Gram-positive and -negative pathogens during their associations with invertebrate and vertebrate hosts. PMID:26426528

  13. SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes

    PubMed Central

    Silveira, Célia M.; Quintas, Pedro O.; Moura, Isabel; Moura, José J. G.; Hildebrandt, Peter; Almeida, M. Gabriela; Todorovic, Smilja

    2015-01-01

    Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd1NiR from Marinobacter hydrocarbonoclasticus (Mhcd1) depends on the presence of its physiological redox partner, cytochrome c552 (cyt c552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd1 and cyt c552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd1 // cyt c552 and Ag // SAM // cyt c552 // Mhcd1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c552 retains its native properties, while the redox potential of apparently intact Mhcd1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd1, reinforcing the idea that subtle and very specific interactions between Mhcd1 and cyt c552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd1. PMID:26091174

  14. SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes.

    PubMed

    Silveira, Célia M; Quintas, Pedro O; Moura, Isabel; Moura, José J G; Hildebrandt, Peter; Almeida, M Gabriela; Todorovic, Smilja

    2015-01-01

    Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd1NiR from Marinobacter hydrocarbonoclasticus (Mhcd1) depends on the presence of its physiological redox partner, cytochrome c552 (cyt c552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd1 and cyt c552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd1 // cyt c552 and Ag // SAM // cyt c552 // Mhcd1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c552 retains its native properties, while the redox potential of apparently intact Mhcd1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd1, reinforcing the idea that subtle and very specific interactions between Mhcd1 and cyt c552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd1. PMID:26091174

  15. Probing the nitrite and nitric oxide reductase activity of cbb3 oxidase: resonance Raman detection of a six-coordinate ferrous heme-nitrosyl species in the binuclear b3/CuB center.

    PubMed

    Loullis, Andreas; Pinakoulaki, Eftychia

    2015-12-21

    In this work we report the first spectroscopic evidence demonstrating that cbb3 oxidase catalyzes the reduction of nitrite to nitrous oxide under reducing anaerobic conditions. The reaction proceeds through the formation of a ferrous six-coordinate heme b3-nitrosyl species that has been characterized by resonance Raman spectroscopy. PMID:26465875

  16. Inhibition of denitrification activity but not of mRNA induction in Paracoccus denitrificans by nitrite at a suboptimal pH.

    PubMed

    Baumann, B; van der Meer, J R; Snozzi, M; Zehnder, A J

    1997-10-01

    The influence of pH on the denitrification activity of a continuous culture of Paracoccus denitrificans was studied in relation to the presence of nitrite. After a transition from aerobic to anaerobic conditions at the suboptimal pH of 6.8, P. denitrificans was not able to build up a functional denitrification pathway. Nitrite accumulated in the medium as the predominant denitrification product. Although the nitrite reductase gene was induced properly, the enzyme could not be detected at sufficient amounts in the culture. These observations was somehow inhibited, or once synthesized nitrite reductase was inactivated, possibly by the high concentrations of nitrous acid (HNO2). Interestingly, when a P. denitrificans culture which was grown to steady-state under anaerobic conditions was then exposed to suboptimal pHs, cells exhibited a reduced overall denitrification activity, but neither nitrite nor any other denitrification intermediate accumulated. PMID:9403103

  17. Gene cluster for dissimilatory nitrite reductase (nir) from Pseudomonas aeruginosa: sequencing and identification of a locus for heme d1 biosynthesis.

    PubMed Central

    Kawasaki, S; Arai, H; Kodama, T; Igarashi, Y

    1997-01-01

    The primary structure of an nir gene cluster necessary for production of active dissimilatory nitrite reductase was determined from Pseudomonas aeruginosa. Seven open reading frames, designated nirDLGHJEN, were identified downstream of the previously reported nirSMCF genes. From nirS through nirN, the stop codon of one gene and the start codon of the next gene were closely linked, suggesting that nirSMCFDLGHJEN are expressed from a promoter which regulates the transcription of nirSM. The amino acid sequences deduced from the nirDLGH genes were homologous to each other. A gene, designated nirJ, which encodes a protein of 387 amino acids, showed partial identity with each of the nirDLGH genes. The nirE gene encodes a protein of 279 amino acids homologous to S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase from other bacterial strains. In addition, NirE shows 21.0% identity with NirF in the N-terminal 100-amino-acid residues. A gene, designated nirN, encodes a protein of 493 amino acids with a conserved binding motif for heme c (CXXCH) and a typical N-terminal signal sequence for membrane translocation. The derived NirN protein shows 23.9% identity with nitrite reductase (NirS). Insertional mutation and complementation analyses showed that all of the nirFDLGHJE genes were necessary for the biosynthesis of heme d1. PMID:8982003

  18. Crystallization and preliminary structure determination of the membrane-bound complex cytochrome c nitrite reductase from Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    Rodrigues, M. L.; Oliveira, T.; Matias, P. M.; Martins, I. C.; Valente, F. M. A.; Pereira, I. A. C.; Archer, M.

    2006-06-01

    The cytochrome c nitrite reductase complex from D. vulgaris Hildenborough has been crystallized. The preliminary crystallographic structure reveals a 2:1 NrfA:NrfH complex stoichiometry. The cytochrome c nitrite reductase (cNiR) isolated from Desulfovibrio vulgaris Hildenborough is a membrane-bound complex formed of NrfA and NrfH subunits. The catalytic subunit NrfA is a soluble pentahaem cytochrome c that forms a physiological dimer of about 120 kDa. The electron-donor subunit NrfH is a membrane-anchored tetrahaem cytochrome c of about 18 kDa molecular weight and belongs to the NapC/NirT family of quinol dehydrogenases, for which no structures are known. Crystals of the native cNiR membrane complex, solubilized with dodecylmaltoside detergent (DDM), were obtained using PEG 4K as precipitant. Anomalous diffraction data were measured at the Swiss Light Source to 2.3 Å resolution. Crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 79.5, b = 256.7, c = 578.2 Å. Molecular-replacement and MAD methods were combined to solve the structure. The data presented reveal that D. vulgaris cNiR contains one NrfH subunit per NrfA dimer.

  19. Orientational control over nitrite reductase on modified gold electrode and its effects on the interfacial electron transfer.

    PubMed

    Krzemiński, Lukasz; Cronin, Samuel; Ndamba, Lionel; Canters, Gerard W; Aartsma, Thijs J; Evans, Stephen D; Jeuken, Lars J C

    2011-11-01

    Recently, studies have been reported in which fluorescently labeled redox proteins have been studied with a combination of spectroscopy and electrochemistry. In order to understand the effect of the dye on the protein-electrode interaction, voltammetry and surface analysis have been performed on protein films of dye-labeled and unlabeled forms of a cysteine-surface variant (L93C) and the wild type (wt) of the copper containing nitrite reductase (NiR) from Alcaligenes faecalis S6. The protein has been adsorbed onto gold electrodes modified with self-assembled monolayers (SAMs) made up of 6-mercaptohexanol (6-OH) and mixtures of various octanethiols. Electrochemical and surface-analytical techniques were utilized to explore the influence of the SAM composition on wt and L93C NiR enzyme activity and the orientation of the enzyme molecules with respect to the electrode/SAM. The unlabeled L93C NiR enzyme is only electroactive on mixed SAMs composed of positive 8-aminooctanethiol (8-NH(2)) and 8-mercaptooctanol (8-OH). No enzymatic activity is observed on SAMs consisting of pure 6-OH, 8-OH, or pure 8-NH(2). Modification of L93C NiR with the ATTO 565 dye resulted in enzymatic activity on SAMs of 6-OH, but not on SAMs of 8-OH. Quartz crystal microbalance with dissipation measurements show that well-ordered and rigid protein films (single orientation of the protein) are formed when NiR is electroactive. By contrast, electrode-NiR combinations for which no electrochemical activity is observed still have NiR adsorbed on the surfaces, but a less-structured and water-rich film is formed. For the unlabeled L93C NiR, bilayer formation is observed, suggesting that the Cys93 residue is orientated away from the surface and able to form disulfide bridges to a second layer of L93C NiR. The results indicate that interfacial electron transfer is only possible if the negatively charged surface patch surrounding the electron-entry site of NiR is directed toward the electrode. This can be

  20. The possible involvement of copper-containing nitrite reductase (NirK) and flavohemoglobin in denitrification by the fungus Cylindrocarpon tonkinense.

    PubMed

    Kim, Sang-Wan; Fushinobu, Shinya; Zhou, Shengmin; Wakagi, Takayoshi; Shoun, Hirofumi

    2010-01-01

    The occurrence of denitrification and nitrate respiration among eukaryotes has been established during the last few decades. However, denitrification-related eukaryotic genes have been isolated from only a few fungi, and eukaryotic denitrification (or nitrate respiration) is still inadequately understood. In this study, we identified genes that were up-regulated under denitrifying conditions in the fungus Cylindrocarpon tonkinense using the suppression subtraction hybridization technique, and the expression patterns of these genes were characterized by Northern analysis. We identified copper-containing nitrite reductase, cytochrome P450 nitric oxide reductase, flavohemoglobin (Fhb), and formate/nitrite transporter homolog genes as possibly involved in fungal denitrification. Our results concerning the involvement of Fhb and formate/nitrite transporter perhaps provide new insight into the fungal denitrification system. PMID:20622453

  1. The napF and narG Nitrate Reductase Operons in Escherichia coli Are Differentially Expressed in Response to Submicromolar Concentrations of Nitrate but Not Nitrite

    PubMed Central

    Wang, Henian; Tseng, Ching-Ping; Gunsalus, Robert P.

    1999-01-01

    Escherichia coli synthesizes two biochemically distinct nitrate reductase enzymes, a membrane-bound enzyme encoded by the narGHJI operon and a periplasmic cytochrome c-linked nitrate reductase encoded by the napFDAGHBC operon. To address why the cell makes these two enzymes, continuous cell culture techniques were used to examine napF and narG gene expression in response to different concentrations of nitrate and/or nitrite. Expression of the napF-lacZ and narG-lacZ reporter fusions in strains grown at different steady-state levels of nitrate revealed that the two nitrate reductase operons are differentially expressed in a complementary pattern. The napF operon apparently encodes a “low-substrate-induced” reductase that is maximally expressed only at low levels of nitrate. Expression is suppressed under high-nitrate conditions. In contrast, the narGHJI operon is only weakly expressed at low nitrate levels but is maximally expressed when nitrate is elevated. The narGHJI operon is therefore a “high-substrate-induced” operon that somehow provides a second and distinct role in nitrate metabolism by the cell. Interestingly, nitrite, the end product of each enzyme, had only a minor effect on the expression of either operon. Finally, nitrate, but not nitrite, was essential for repression of napF gene expression. These studies reveal that nitrate rather than nitrite is the primary signal that controls the expression of these two nitrate reductase operons in a differential and complementary fashion. In light of these findings, prior models for the roles of nitrate and nitrite in control of narG and napF expression must be reconsidered. PMID:10464201

  2. Nitrite Reductase from Pseudomonas aeruginosa Released by Antimicrobial Agents and Complement Induces Interleukin-8 Production in Bronchial Epithelial Cells

    PubMed Central

    Sar, Borann; Oishi, Kazunori; Wada, Akihiro; Hirayama, Toshiya; Matsushima, Kouji; Nagatake, Tsuyoshi

    1999-01-01

    We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of Pseudomonas aeruginosa, could induce interleukin-8 (IL-8) generation in a variety of respiratory cells, including bronchial epithelial cells (K. Oishi et al. Infect. Immun. 65:2648–2655, 1997). In this report, we examined the mode of nitrite reductase (PNR) release from a serum-sensitive strain of live P. aeruginosa cells during in vitro treatment with four different antimicrobial agents or human complement. Bacterial killing of P. aeruginosa by antimicrobial agents induced PNR release and mediated IL-8 production in human bronchial epithelial (BET-1A) cells. Among these agents, imipenem demonstrated rapid killing of P. aeruginosa as well as rapid release of PNR and resulted in the highest IL-8 production. Complement-mediated killing of P. aeruginosa was also associated with PNR release and enhanced IL-8 production. The immunoprecipitates of the aliquots of bacterial culture containing imipenem or complement with anti-PNR immunoglobulin G (IgG) induced a twofold-higher IL-8 production than did the immunoprecipitates of the aliquots of bacterial culture with a control IgG. These pieces of evidence confirmed that PNR released in the aliquots of bacterial culture was responsible for IL-8 production in the BET-1A cells. Furthermore, the culture supernatants of the BET-1A cells stimulated with aliquots of bacterial culture containing antimicrobial agents or complement similarly mediated neutrophil migration in vitro. These data support the possibility that a potent inducer of IL-8, PNR, could be released from P. aeruginosa after exposure to antimicrobial agents or complement and contributes to neutrophil migration in the airways during bronchopulmonary infections with P. aeruginosa. PMID:10103183

  3. Mechanisms of human erythrocytic bioactivation of nitrite.

    PubMed

    Liu, Chen; Wajih, Nadeem; Liu, Xiaohua; Basu, Swati; Janes, John; Marvel, Madison; Keggi, Christian; Helms, Christine C; Lee, Amber N; Belanger, Andrea M; Diz, Debra I; Laurienti, Paul J; Caudell, David L; Wang, Jun; Gladwin, Mark T; Kim-Shapiro, Daniel B

    2015-01-01

    Nitrite signaling likely occurs through its reduction to nitric oxide (NO). Several reports support a role of erythrocytes and hemoglobin in nitrite reduction, but this remains controversial, and alternative reductive pathways have been proposed. In this work we determined whether the primary human erythrocytic nitrite reductase is hemoglobin as opposed to other erythrocytic proteins that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. Our studies show that NO is formed by red blood cells and inhibits platelet activation. Nitric oxide formation and signaling can be recapitulated with isolated deoxyhemoglobin. Importantly, there is limited NO production from erythrocytic xanthine oxidoreductase and nitric-oxide synthase. Under certain conditions we find dorzolamide (an inhibitor of carbonic anhydrase) results in diminished nitrite bioactivation, but the role of carbonic anhydrase is abrogated when physiological concentrations of CO2 are present. Importantly, carbon monoxide, which inhibits hemoglobin function as a nitrite reductase, abolishes nitrite bioactivation. Overall our data suggest that deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions and accounts for nitrite-mediated NO signaling in blood. PMID:25471374

  4. Ferrisiderophore reductase activity in Agrobacterium tumefaciens.

    PubMed Central

    Lodge, J S; Gaines, C G; Arceneaux, J E; Byers, B R

    1982-01-01

    Reduction of the iron in ferriagrobactin by the cytoplasmic fraction of Agrobacterium tumefaciens strictly required NaDH as the reductant. Addition of flavin mononucleotide and anaerobic conditions were necessary for the reaction; when added with flavin mononucleotide, magnesium was stimulatory. This ferrisiderophore reductase activity may be a part of the iron assimilation process in A. tumefaciens. PMID:7056702

  5. Comparative induction of nitrate reductase by nitrate and nitrite in barley leaves

    NASA Technical Reports Server (NTRS)

    Aslam, M.; Rosichan, J. L.; Huffaker, R. C.

    1987-01-01

    The comparative induction of nitrate reductase (NR) by ambient NO3- and NO2- as a function of influx, reduction (as NR was induced) and accumulation in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings was determined. The dynamic interaction of NO3- influx, reduction and accumulation on NR induction was shown. The activity of NR, as it was induced, influenced its further induction by affecting the internal concentration of NO3-. As the ambient concentration of NO3- increased, the relative influences imposed by influx and reduction on NO3- accumulation changed with influx becoming a more predominant regulant. Significant levels of NO3- accumulated in NO2(-)-fed leaves. When the leaves were supplied cycloheximide or tungstate along with NO2-, about 60% more NO3- accumulated in the leaves than in the absence of the inhibitors. In NO3(-)-supplied leaves NR induction was observed at an ambient concentration of as low as 0.02 mM. No NR induction occurred in leaves supplied with NO2- until the ambient NO2- concentration was 0.5 mM. In fact, NR induction from NO2- solutions was not seen until NO3- was detected in the leaves. The amount of NO3- accumulating in NO2(-)-fed leaves induced similar levels of NR as did equivalent amounts of NO3- accumulating from NO3(-)-fed leaves. In all cases the internal concentration of NO3-, but not NO2-, was highly correlated with the amount of NR induced. The evidence indicated that NO3- was a more likely inducer of NR than was NO2-.

  6. Effect of self-alkalization on nitrite accumulation in a high-rate denitrification system: Performance, microflora and enzymatic activities.

    PubMed

    Li, Wei; Shan, Xiao-Yu; Wang, Zhi-Yao; Lin, Xiao-Yu; Li, Chen-Xu; Cai, Chao-Yang; Abbas, Ghulam; Zhang, Meng; Shen, Li-Dong; Hu, Zhi-Qiang; Zhao, He-Ping; Zheng, Ping

    2016-01-01

    The self-alkalization of denitrifying automatic circulation (DAC) reactor resulted in a large increase of pH up to 9.20 and caused a tremendous accumulation of nitrite up to 451.1 ± 49.0 mgN L(-1) at nitrate loading rate (NLR) from 35 kgN m(-3) d(-1) to 55 kgN m(-3) d(-1). The nitrite accumulation was greatly relieved even at the same NLR once the pH was maintained at 7.6 ± 0.2 in the system. Enzymatic assays indicated that the long-term bacterial exposure to high pH significantly inhibited the activity of copper type nitrite reductase (NirK) rather than the cytochrome cd1 type nitrite reductase (NirS). The terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the dominant denitrifying bacteria shifted from the NirS-containing Thauear sp. 27 to the NirK-containing Hyphomicrobium nitrativorans strain NL23 during the self-alkalization. The significant nitrite accumulation in the high-rate denitrification system could be therefore, due to the inhibition of Cu-containing NirK by high pH from the self-alkalization. The results suggest that the NirK-containing H. nitrativorans strain NL23 could be an ideal functional bacterium for the conversion of nitrate to nitrite, i.e. denitritation, which could be combined with anaerobic ammonium oxidation (Anammox) to develop a new process for nitrogen removal from wastewater. PMID:26595097

  7. Detection and diversity of copper containing nitrite reductase genes (nirK) in prokaryotic and fungal communities of agricultural soils.

    PubMed

    Long, Andrew; Song, Bongkeun; Fridey, Kelly; Silva, Amy

    2015-02-01

    Microorganisms are capable of producing N2 and N2O gases as the end products of denitrification. Copper-containing nitrite reductase (NirK), a key enzyme in the microbial N-cycle, has been found in bacteria, archaea and fungi. This study seeks to assess the diversity of nirK genes in the prokaryotic and fungal communities of agricultural soils in the United States. New primers targeting the nirK genes in fungi were developed, while nirK genes in archaea and bacteria were detected using previously published methods. The new primers were able to detect fungal nirK genes as well as bacterial nirK genes from a group that could not be observed with previously published primers. Based on the sequence analyses from three different primer sets, five clades of nirK genes were identified, which were associated with soil archaea, ammonium-oxidizing bacteria, denitrifying bacteria and fungi. The diversity of nirK genes in the two denitrifying bacteria clades was higher than the diversity found in other clades. Using a newly designed primer set, this study showed the detection of fungal nirK genes from environmental samples. The newly designed PCR primers in this study enhance the ability to detect the diversity of nirK-encoding microorganisms in soils. PMID:25764542

  8. NADPH-cytochrome P450 reductase-mediated denitration reaction of 2,4,6-trinitrotoluene to yield nitrite in mammals.

    PubMed

    Shinkai, Yasuhiro; Nishihara, Yuya; Amamiya, Masahiro; Wakayama, Toshihiko; Li, Song; Kikuchi, Tomohiro; Nakai, Yumi; Shimojo, Nobuhiro; Kumagai, Yoshito

    2016-02-01

    While the biodegradation of 2,4,6-trinitrotoluene (TNT) via the release of nitrite is well established, mechanistic details of the reaction in mammals are unknown. To address this issue, we attempted to identify the enzyme from rat liver responsible for the production of nitrite from TNT. A NADPH-cytochrome P450 reductase (P450R) was isolated and identified from rat liver microsomes as the enzyme responsible for not only the release of nitrite from TNT but also formation of superoxide and 4-hydroxyamino-2,6-dinitrotoluene (4-HADNT) under aerobic conditions. In this context, reactive oxygen species generated during P450R-catalyzed TNT reduction were found to be, at least in part, a mediator for the production of 4-HADNT from TNT via formation of 4-nitroso-2,6-dinitrotoluene. P450R did not catalyze the formation of the hydride-Meisenheimer complex (H(-)-TNT) that is thought to be an intermediate for nitrite release from TNT. Furthermore, in a time-course experiment, 4-HADNT formation reached a plateau level and then declined during the reaction between TNT and P450R with NADPH, while the release of nitrite was subjected to a lag period. Notably, the produced 4-HADNT can react with the parent compound TNT to produce nitrite and dimerized products via formation of a Janovsky complex. Our results demonstrate for the first time that P450R-mediated release of nitrite from TNT results from the process of chemical interaction of TNT and its 4-electron reduction metabolite 4-HADNT. PMID:26454083

  9. HY5 regulates nitrite reductase 1 (NIR1) and ammonium transporter1;2 (AMT1;2) in Arabidopsis seedlings.

    PubMed

    Huang, Lifen; Zhang, Hongcheng; Zhang, Huiyong; Deng, Xing Wang; Wei, Ning

    2015-09-01

    HY5 (Long Hypocotyles 5) is a key transcription factor in Arabidopsis thaliana that has a pivotal role in seedling development. Soil nitrogen is an essential macronutrient, and its uptake, assimilation and metabolism are influenced by nutrient availability and by lights. To understand the role of HY5 in nitrogen assimilation pathways, we examined the phenotype as well as the expression of selected nitrogen assimilation-related genes in hy5 mutant grown under various nitrogen limiting and nitrogen sufficient conditions, or different light conditions. We report that HY5 positively regulates nitrite reductase gene NIR1 and negatively regulates the ammonium transporter gene AMT1;2 under all nitrogen and light conditions tested, while it affects several other genes in a nitrogen supply-dependent manner. HY5 is not required for light induction of NIR1, AMT1;2 and NIA genes, but it is necessary for high level expression of NIR1 and NIA under optimal nutrient and light conditions. In addition, nitrogen deficiency exacerbates the abnormal root system of hy5. Together, our results suggest that HY5 exhibits the growth-promoting activity only when sufficient nutrients, including lights, are provided, and that HY5 has a complex involvement in nitrogen acquisition and metabolism in Arabidopsis seedlings. PMID:26259199

  10. Relationship between Nitrite Reduction and Active Phosphate Uptake in the Phosphate-Accumulating Denitrifier Pseudomonas sp. Strain JR 12

    PubMed Central

    Barak, Yoram; van Rijn, Jaap

    2000-01-01

    Phosphate uptake by the phosphate-accumulating denitrifier Pseudomonas sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H+-ATPase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a c-type cytochrome. Based on these findings, we propose that this c-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this c-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed. PMID

  11. Conserved Active Site Residues Limit Inhibition of a Copper-Containing Nitrite By Small Molecules

    SciTech Connect

    Tocheva, E.I.; Eltis, L.D.; Murphy, M.E.P.

    2009-05-26

    The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme ( K i >50 mM), the Cu-O distances in these AfNiR-inhibitor complexes were approximately 0.15 A longer than that observed in the AfNiR-nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR-acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly ( K ic = 2.0 +/- 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu-N c distance of approximately 2 A, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.

  12. Aldose reductase mediates retinal microglia activation.

    PubMed

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

    2016-04-29

    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy. PMID:27033597

  13. Regulation of nap Gene Expression and Periplasmic Nitrate Reductase Activity in the Phototrophic Bacterium Rhodobacter sphaeroides DSM158

    PubMed Central

    Gavira, Mónica; Roldán, M. Dolores; Castillo, Francisco; Moreno-Vivián, Conrado

    2002-01-01

    Bacterial periplasmic nitrate reductases (Nap) can play different physiological roles and are expressed under different conditions depending on the organism. Rhodobacter sphaeroides DSM158 has a Nap system, encoded by the napKEFDABC gene cluster, but nitrite formed is not further reduced because this strain lacks nitrite reductase. Nap activity increases in the presence of nitrate and oxygen but is unaffected by ammonium. Reverse transcription-PCR and Northern blots demonstrated that the napKEFDABC genes constitute an operon transcribed as a single 5.5-kb product. Northern blots and nap-lacZ fusions revealed that nap expression is threefold higher under aerobic conditions but is regulated by neither nitrate nor ammonium, although it is weakly induced by nitrite. On the other hand, nitrate but not nitrite causes a rapid enzyme activation, explaining the higher Nap activity found in nitrate-grown cells. Translational nap′-′lacZ fusions reveal that the napK and napD genes are not efficiently translated, probably due to mRNA secondary structures occluding the translation initiation sites of these genes. Neither butyrate nor caproate increases nap expression, although cells growing phototrophically on these reduced substrates show a very high Nap activity in vivo (nitrite accumulation is sevenfold higher than in medium with malate). Phototrophic growth on butyrate or caproate medium is severely reduced in the NapA− mutants. Taken together, these results indicate that nitrate reduction in R. sphaeroides is mainly regulated at the level of enzyme activity by both nitrate and electron supply and confirm that the Nap system is involved in redox balancing using nitrate as an ancillary oxidant to dissipate excess reductant. PMID:11872721

  14. Diversity and Abundance of Ammonia-Oxidizing Archaeal Nitrite Reductase (nirK) Genes in Estuarine Sediments of San Francisco Bay

    NASA Astrophysics Data System (ADS)

    Reji, L.; Lee, J. A.; Damashek, J.; Francis, C. A.

    2013-12-01

    Nitrification, the microbially-mediated aerobic oxidation of ammonia to nitrate via nitrite, is an integral component of the global biogeochemical nitrogen cycle. The first and rate-limiting step of nitrification, ammonia oxidation, is carried out by two distinct microbial groups: ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Molecular ecological studies targeting the amoA gene have revealed the abundance and ubiquity of AOA in terrestrial as well as aquatic environments. In addition to the ammonia oxidation machinery that includes the amoA gene, AOA also encode a gene for copper-containing nitrite reductase (nirK). The distribution patterns and functional role of nirK in AOA remain mostly unknown; proposed functions include the indirect involvement in ammonia oxidation through the production of nitric oxide during nitrite reduction, and (2) nitrite detoxification. In the present study, the diversity and abundance of archaeal nirK genes in estuarine sediments were investigated using quantitative polymerase chain reaction, cloning and sequencing approaches. In sediment samples collected from the San Francisco Bay estuary, two archaeal nirK variants (AnirKa and AnirKb) were amplified using specific primer sets. Overall, AnirKa was observed to be significantly more abundant than AnirKb in the sediment samples, with variation in relative abundance spanning two to three orders of magnitude between sampling sites. Phylogenetic analysis revealed a number of unique archaeal nirK sequence types, as well as many that clustered with sequences from previous estuarine studies and cultured AOA isolates, such as Nitrosopumilus maritimus. This study yielded new insights into the diversity and abundance of archaeal nirK genes in estuarine sediments, and highlights the importance of further investigating the physiological role of this gene in AOA, as well as its suitability as a marker gene for studying AOA in the environment.

  15. Sodium Nitrite Blocks the Activity of Aminoglycosides against Pseudomonas aeruginosa Biofilms

    PubMed Central

    Zemke, Anna C.; Gladwin, Mark T.

    2015-01-01

    Sodium nitrite has broad antimicrobial activity at pH 6.5, including the ability to prevent biofilm growth by Pseudomonas aeruginosa on the surfaces of airway epithelial cells. Because of its antimicrobial activity, nitrite is being investigated as an inhaled agent for chronic P. aeruginosa airway infections in cystic fibrosis patients. However, the interaction between nitrite and commonly used aminoglycosides is unknown. This paper investigates the interaction between nitrite and tobramycin in liquid culture, abiotic biofilms, and a biotic biofilm model simulating the conditions in the cystic fibrosis airway. The addition of nitrite prevented killing by aminoglycosides in liquid culture, with dose dependence between 1.5 and 15 mM. The effect was not blocked by the nitric oxide scavenger CPTIO or dependent on efflux pump activity. Nitrite shifted the biofilm minimal bactericidal concentration (MBC-biofilm) from 256 μg/ml to >1,024 μg/ml in an abiotic biofilm model. In a biotic biofilm model, the addition of 50 mM nitrite decreased the antibiofilm activity of tobramycin by up to 1.2 log. Respiratory chain inhibition recapitulated the inhibition of aminoglycoside activity by nitrite, suggesting a potential mechanism of inhibition of energy-dependent aminoglycoside uptake. In summary, sodium nitrite induces resistance to both gentamicin and tobramycin in P. aeruginosa grown in liquid culture, as an abiotic biofilm, or as a biotic biofilm. PMID:25801569

  16. Low apparent aldose reductase activity produced by monosaccharide autoxidation.

    PubMed Central

    Wolff, S P; Crabbe, M J

    1985-01-01

    Low apparent aldose reductase activity, as measured by NADPH oxidation, can be produced by the spontaneous autoxidation of monosaccharides. NADPH is oxidized to metabolically active NADP+ in a solution of autoxidizing DL-glyceraldehyde at rates of up to 15 X 10(-4) A340/min. The close parallelism between the effects of buffer salt type and concentration, monosaccharide structure and temperature activation on autoxidation and NADPH oxidation imply that autoxidation is a prerequisite for the NADPH oxidation, probably via the hydroperoxy radical. Nucleotide-binding proteins enhanced NADPH oxidation induced by DL-glyceraldehyde, up to 10.6-fold with glucose-6-phosphate dehydrogenase. Glutathione reductase-catalysed NADPH oxidation in the presence of autoxidizing monosaccharide showed many characteristics of the aldose reductase reaction. Aldose reductase inhibitors acted as antioxidants in inhibiting this NADPH oxidation. These results indicate that low apparent aldose reductase activities may be due to artifacts of monosaccharide autoxidation, and could provide an explanation for the non-linear steady-state kinetics observed with DL-glyceraldehyde and aldose reductase. PMID:2985042

  17. Sulfite Oxidase Catalyzes Single-Electron Transfer at Molybdenum Domain to Reduce Nitrite to Nitric Oxide

    PubMed Central

    Wang, Jun; Krizowski, Sabina; Fischer-Schrader, Katrin; Niks, Dimitri; Tejero, Jesús; Sparacino-Watkins, Courtney; Wang, Ling; Ragireddy, Venkata; Frizzell, Sheila; Kelley, Eric E.; Zhang, Yingze; Basu, Partha; Hille, Russ

    2015-01-01

    Abstract Aims: Recent studies suggest that the molybdenum enzymes xanthine oxidase, aldehyde oxidase, and mARC exhibit nitrite reductase activity at low oxygen pressures. However, inhibition studies of xanthine oxidase in humans have failed to block nitrite-dependent changes in blood flow, leading to continued exploration for other candidate nitrite reductases. Another physiologically important molybdenum enzyme—sulfite oxidase (SO)—has not been extensively studied. Results: Using gas-phase nitric oxide (NO) detection and physiological concentrations of nitrite, SO functions as nitrite reductase in the presence of a one-electron donor, exhibiting redox coupling of substrate oxidation and nitrite reduction to form NO. With sulfite, the physiological substrate, SO only facilitates one turnover of nitrite reduction. Studies with recombinant heme and molybdenum domains of SO indicate that nitrite reduction occurs at the molybdenum center via coupled oxidation of Mo(IV) to Mo(V). Reaction rates of nitrite to NO decreased in the presence of a functional heme domain, mediated by steric and redox effects of this domain. Using knockdown of all molybdopterin enzymes and SO in fibroblasts isolated from patients with genetic deficiencies of molybdenum cofactor and SO, respectively, SO was found to significantly contribute to hypoxic nitrite signaling as demonstrated by activation of the canonical NO-sGC-cGMP pathway. Innovation: Nitrite binds to and is reduced at the molybdenum site of mammalian SO, which may be allosterically regulated by heme and molybdenum domain interactions, and contributes to the mammalian nitrate-nitrite-NO signaling pathway in human fibroblasts. Conclusion: SO is a putative mammalian nitrite reductase, catalyzing nitrite reduction at the Mo(IV) center. Antioxid. Redox Signal. 23, 283–294. PMID:25314640

  18. Nitrite attenuated peroxynitrite and hypochlorite generation in activated neutrophils.

    PubMed

    Ren, Xiaoming; Ding, Yun; Lu, Naihao

    2016-03-15

    Oxidative stress is usually considered as an important factor to the pathogenesis of various diseases. Peroxynitrite (ONOO(-)) and hypochlorite (OCl(-)) are formed in immune cells as a part of the innate host defense system, but excessive reactive oxygen species generation can cause progressive inflammation and tissue damage. It has been proven that through mediating nitric oxide (NO) homeostasis, inorganic nitrite (NO2(-)) shows organ-protective effects on oxidative stress and inflammation. However, the effects of NO2(-) on the function of immune cells were still not clear. The potential role of NO2(-) in modulating ONOO(-) and OCl(-) generation in neutrophil cells was investigated in this study. As an immune cell activator, lipopolysaccharide (LPS) increased both ONOO(-) and OCl(-) production in neutrophils, which was significantly attenuated by NO2(-). NO2(-) reduced superoxide (O2(·-)) generation via a NO-dependent mechanism and increased NO formation in activated neutrophils, suggesting a crucial role of O2(·-) in NO2(-)-mediated reduction of ONOO(-). Moreover, the reduced effect of NO2(-) on OCl(-) production was attributed to that NO2(-) reduced H2O2 production in activated neutrophils without influencing the release of myeloperoxidase (MPO), thus limiting OCl(-) production by MPO/H2O2 system. Therefore, NO2(-) attenuates ONOO(-) and OCl(-) formation in activated neutrophils, opening a new direction to modulate the inflammatory response. PMID:26854590

  19. Equine 5α-reductase activity and expression in epididymis.

    PubMed

    Corbin, C J; Legacki, E L; Ball, B A; Scoggin, K E; Stanley, S D; Conley, A J

    2016-10-01

    The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of 5α-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and DHT, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine 5α-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies. PMID:27466384

  20. Volatile fatty acid impacts on nitrite oxidation and carbon dioxide fixation in activated sludge.

    PubMed

    Oguz, Merve T; Robinson, Kevin G; Layton, Alice C; Sayler, Gary S

    2006-02-01

    Batch test were performed to assess nitrite removal, nitrate formation, CO2 fixation, gaseous nitrogen production and microbial density in activated sludge exposed to volatile fatty acid (VFA) mixtures. Nitrite removal and nitrate formation were both affected by the presence of VFAs, but to different degrees. Nitrate formation rates were reduced to a greater extent (79%) than nitrite removal rates (36%) resulting in an apparent unbalanced nitrite oxidation reaction. Since the total bacterial density and the nitrite oxidizing bacteria (NOB, Nitrospira) concentration remained essentially constant under all test conditions, the reduction in rates was not due to heterotrophic uptake of nitrogen or to a decrease in the NOB population. In contrast to the nitrogen results, VFAs were not found to impact CO2 fixation efficiency. It appeared that nitrite oxidation occurred when VFAs were present since the oxidation of nitrite provides energy for CO2 fixation. However, nitrate produced from the oxidation of nitrite was reduced to gaseous nitrogen products. N2O gas was detected in the presence of VFAs which was a clear indication that VFAs stimulated an alternative pathway, such as aerobic denitrification, during biotransformation of nitrogen in activated sludge. PMID:16436292

  1. Compensatory periplasmic nitrate reductase activity supports anaerobic growth of Pseudomonas aeruginosa PAO1 in the absence of membrane nitrate reductase.

    PubMed

    Van Alst, Nadine E; Sherrill, Lani A; Iglewski, Barbara H; Haidaris, Constantine G

    2009-10-01

    Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa. Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. The inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon, and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The roles of the 2 dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expressions, were examined by use of a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 h. In contrast, the nitrate sensor-response regulator mutant DeltanarXL displayed growth arrest initially, but resumed growth after 72 h and reached the early stationary phase in liquid culture after 120 h. Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild-type P. aeruginosa PAO1, the nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase. PMID:19935885

  2. 5. cap alpha. -reductase activity in rat adipose tissue

    SciTech Connect

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

    1987-11-01

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

  3. Activated and unactivated forms of human erythrocyte aldose reductase.

    PubMed Central

    Srivastava, S K; Hair, G A; Das, B

    1985-01-01

    Aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) has been partially purified from human erythrocytes by DEAE-cellulose (DE-52) column chromatography. This enzyme is activated severalfold upon incubation with 10 microM each glucose 6-phosphate, NADPH, and glucose. The activation of the enzyme was confirmed by following the oxidation of NADPH as well as the formation of sorbitol with glucose as substrate. The activated form of aldose reductase exhibited monophasic kinetics with both glyceraldehyde and glucose (Km of glucose = 0.68 mM and Km of glyceraldehyde = 0.096 mM), whereas the native (unactivated) enzyme exhibited biphasic kinetics (Km of glucose = 9.0 and 0.9 mM and Km of glyceraldehyde = 1.1 and 0.14 mM). The unactivated enzyme was strongly inhibited by aldose reductase inhibitors such as sorbinil, alrestatin, and quercetrin, and by phosphorylated intermediates such as ADP, glycerate 3-phosphate, glycerate 1,3-bisphosphate, and glycerate 2,3-trisphosphate. The activated form of the enzyme was less susceptible to inhibition by aldose reductase inhibitors and phosphorylated intermediates. PMID:3933003

  4. A composite biochemical system for bacterial nitrate and nitrite assimilation as exemplified by Paracoccus denitrificans.

    PubMed

    Gates, Andrew J; Luque-Almagro, Victor M; Goddard, Alan D; Ferguson, Stuart J; Roldán, M Dolores; Richardson, David J

    2011-05-01

    The denitrifying bacterium Paracoccus denitrificans can grow aerobically or anaerobically using nitrate or nitrite as the sole nitrogen source. The biochemical pathway responsible is expressed from a gene cluster comprising a nitrate/nitrite transporter (NasA), nitrite transporter (NasH), nitrite reductase (NasB), ferredoxin (NasG) and nitrate reductase (NasC). NasB and NasG are essential for growth with nitrate or nitrite as the nitrogen source. NADH serves as the electron donor for nitrate and nitrite reduction, but only NasB has a NADH-oxidizing domain. Nitrate and nitrite reductase activities show the same Km for NADH and can be separated by anion-exchange chromatography, but only fractions containing NasB retain the ability to oxidize NADH. This implies that NasG mediates electron flux from the NADH-oxidizing site in NasB to the sites of nitrate and nitrite reduction in NasC and NasB respectively. Delivery of extracellular nitrate to NasBGC is mediated by NasA, but both NasA and NasH contribute to nitrite uptake. The roles of NasA and NasC can be substituted during anaerobic growth by the biochemically distinct membrane-bound respiratory nitrate reductase (Nar), demonstrating functional overlap. nasG is highly conserved in nitrate/nitrite assimilation gene clusters, which is consistent with a key role for the NasG ferredoxin, as part of a phylogenetically widespread composite nitrate and nitrite reductase system. PMID:21348864

  5. Measurement of nitrous oxide reductase activity in aquatic sediments

    SciTech Connect

    Miller, L.G.; Oremland, R.S.; Paulsen, S.

    1986-01-01

    Denitrification in aquatic sediments was measured by an N/sub 2/O reductase assay. Sediments consumed small added quantities of N/sub 2/O over short periods (a few hours). In experiments with sediment slurries, N/sub 2/O reductase activity was inhibited by 0/sub 2/, C/sub 2/H/sub 2/, heat treatment, and by high levels of nitrate (1 mM) or sulfide (10 mM). However, ambient levels of nitrate (<100 ..mu..M) did not influence activity, and moderate levels (about 150 ..mu..M) induced only a short lag before reductase activity began. Moderate levels of sulfide (<1 mM) had no effect on N/sub 2/O reductase activity. Nitrous oxide reductase displayed Michaelis-Menten kinetics in sediments from freshwater, estuarine, and alkaline-saline environments. An in situ assay was devised in which a solution of N/sub 2/O was injected into sealed glass cores containing intact sediment. Two estimates of net rates of denitrification in San Francisco Bay under approximated in situ conditions were 0.009 and 0.041 mmol of N/sub 2/O per m/sup 2/ per h. Addition of chlorate to inhibit denitrification in these intact-core experiments (to estimate gross rates of N/sub 2/O consumption) resulted in approximately a 14% upward revision of estimates of net rates. These results were comparable to an in situ estimate of 0.022 mmol of N/sub 2/O per m/sup 2/ per h made with the acetylene block assay.

  6. Changes in the mutagenic and estrogenic activities of bisphenol A upon treatment with nitrite.

    PubMed

    Masuda, Shuichi; Terashima, Yumeko; Sano, Ayako; Kuruto, Ryoko; Sugiyama, Yasumasa; Shimoi, Kayoko; Tanji, Kenichi; Yoshioka, Hisashi; Terao, Yoshiyasu; Kinae, Naohide

    2005-08-01

    Bisphenol A (4,4'isopropylidenediphenol: BPA), an endocrine-disrupting chemical, is contained in food-packaging and can-coating agents as well as in dental sealants. Nitrite is present in vegetables, fish and tap water as an ingredient or contaminant, and also in human saliva. Here, we explored the possible generation of genotoxicity from the reactions of BPA and nitrite under acidic conditions, a situation simulating the stomach. We determined the changes in the mutagenic and estrogenic activities of BPA before and after nitrite treatment. Untreated BPA did not exhibit any mutagenicity. However, the mixture of BPA and sodium nitrite after incubation at pH 3.0 showed strong mutagenic activity toward Salmonella typhimurium strains TA 100 and TA 98 either with or without a metabolic activation system (S9 mix). The clastogenic properties of nitrite-treated and untreated BPA were analyzed by a micronucleus test with male ICR mice. A single gastric intubation of nitrite-treated BPA induced a significantly higher frequency of micronucleated reticulocytes (MNRETs) in mice. The results of analysis of electron spin resonance (ESR) suggest that the expression of the mutagenic activity of nitrite-treated BPA is related to the generation of radicals in the reaction mixture. By applying 1H and 13C NMR, AB-MS and APCI/LC/MS, we identified two compounds 3-nitrobisphenol A and 3,3'-dinitro-bisphenol A. These compounds were synthesized by the reaction of BPA with nitric acid. 3,3'-Dinitro-bisphenol induced a significantly greater frequency of MNRETs in male ICR mice. By applying a green fluorescent protein (GFP)-reporter expression system and an estrogen R(alpha) competitor screening kit, we found that nitrite-treated BPA and 3,3'-dinitro-bisphenol A showed weak estrogenic activity compared to that of untreated BPA. PMID:15936980

  7. Regulation of Nitrite Stress Response in Desulfovibrio vulgaris Hildenborough, a Model Sulfate-Reducing Bacterium

    PubMed Central

    Rajeev, Lara; Chen, Amy; Kazakov, Alexey E.; Luning, Eric G.; Zane, Grant M.; Novichkov, Pavel S.; Wall, Judy D.

    2015-01-01

    ABSTRACT Sulfate-reducing bacteria (SRB) are sensitive to low concentrations of nitrite, and nitrite has been used to control SRB-related biofouling in oil fields. Desulfovibrio vulgaris Hildenborough, a model SRB, carries a cytochrome c-type nitrite reductase (nrfHA) that confers resistance to low concentrations of nitrite. The regulation of this nitrite reductase has not been directly examined to date. In this study, we show that DVU0621 (NrfR), a sigma54-dependent two-component system response regulator, is the positive regulator for this operon. NrfR activates the expression of the nrfHA operon in response to nitrite stress. We also show that nrfR is needed for fitness at low cell densities in the presence of nitrite because inactivation of nrfR affects the rate of nitrite reduction. We also predict and validate the binding sites for NrfR upstream of the nrfHA operon using purified NrfR in gel shift assays. We discuss possible roles for NrfR in regulating nitrate reductase genes in nitrate-utilizing Desulfovibrio spp. IMPORTANCE The NrfA nitrite reductase is prevalent across several bacterial phyla and required for dissimilatory nitrite reduction. However, regulation of the nrfA gene has been studied in only a few nitrate-utilizing bacteria. Here, we show that in D. vulgaris, a bacterium that does not respire nitrate, the expression of nrfHA is induced by NrfR upon nitrite stress. This is the first report of regulation of nrfA by a sigma54-dependent two-component system. Our study increases our knowledge of nitrite stress responses and possibly of the regulation of nitrate reduction in SRB. PMID:26283774

  8. Structural and functional characterization of the Geobacillus copper nitrite reductase: involvement of the unique N-terminal region in the interprotein electron transfer with its redox partner.

    PubMed

    Fukuda, Yohta; Koteishi, Hiroyasu; Yoneda, Ryohei; Tamada, Taro; Takami, Hideto; Inoue, Tsuyoshi; Nojiri, Masaki

    2014-03-01

    The crystal structures of copper-containing nitrite reductase (CuNiR) from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426 and the amino (N)-terminal 68 residue-deleted mutant were determined at resolutions of 1.3Å and 1.8Å, respectively. Both structures show a striking resemblance with the overall structure of the well-known CuNiRs composed of two Greek key β-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one β-strand and one α-helix extended to the northern surface of the type-1 copper site. The superposition of the Geobacillus CuNiR model on the electron-transfer complex structure of CuNiR with the redox partner cytochrome c551 in other denitrifier system led us to infer that this region contributes to the transient binding with the partner protein during the interprotein electron transfer reaction in the Geobacillus system. Furthermore, electron-transfer kinetics experiments using N-terminal residue-deleted mutant and the redox partner, Geobacillus cytochrome c551, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition. PMID:24440558

  9. Molecular Components of Nitrate and Nitrite Efflux in Yeast

    PubMed Central

    Cabrera, Elisa; González-Montelongo, Rafaela; Giraldez, Teresa; de la Rosa, Diego Alvarez

    2014-01-01

    Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation. PMID:24363367

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

    PubMed

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

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth. PMID:26940877

  11. Sequence homology requirements for transcriptional silencing of 35S transgenes and post-transcriptional silencing of nitrite reductase (trans)genes by the tobacco 271 locus.

    PubMed

    Thierry, D; Vaucheret, H

    1996-12-01

    The transgene locus of the tobacco plant 271 (271 locus) is located on a telomere and consists of multiple copies of a plasmid carrying an NptII marker gene driven by the cauliflower mosaic virus (CaMV) 19S promoter and the leaf-specific nitrite reductase Nii1 cDNA cloned in the antisense orientation under the control of the CaMV 35S promoter. Previous analysis of gene expression in leaves has shown that this locus triggers both post-transcriptional silencing of the host leaf-specific Nii genes and transcriptional silencing of transgenes driven by the 19S or 35S promoter irrespective of their coding sequence and of their location in the genome. In this paper we show that silencing of transgenes carrying Nii1 sequences occurs irrespective of the promoter driving their expression and of their location within the genome. This phenomenon occurs in roots as well as in leaves although root Nii genes share only 84% identity with leaf-specific Nii1 sequences carried by the 271 locus. Conversely, transgenes carrying the bean Nii gene (which shares 76% identity with the tobacco Nii1 gene) escape silencing by the 271 locus. We also show that transgenes driven by the figwort mosaic virus 34S promoter (which shares 63% identity with the 35S promoter) also escape silencing by the 271 locus. Taken together, these results indicate that a high degree of sequence similarity is required between the sequences of the silencing locus and of the target (trans)genes for both transcriptional and post-transcriptional silencing. PMID:9002606

  12. Impact of long-term fertilization on the composition of denitrifier communities based on nitrite reductase analyses in a paddy soil.

    PubMed

    Chen, Zhe; Luo, Xiqian; Hu, Ronggui; Wu, Minna; Wu, Jinshui; Wei, Wenxue

    2010-11-01

    The effect of long-term fertilization on soil-denitrifying communities was determined by measuring the abundance and diversity of the nitrite reductase genes nirK and nirS. Soil samples were collected from plots of a long-term fertilization experiment started in 1990, located in Taoyuan (110°72″ E, 28°52″ N), China. The treatments were no fertilizer (NF), urea (UR), balanced mineral fertilizers (BM), and BM combined with rice straw (BMR). The abundance, diversity, and composition of the soil-denitrifying bacteria were determined by using real-time quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of nirK and nirS genes. There was a pronounced difference in the community composition and diversity of nirK-containing denitrifiers responding to the long-term fertilization regimes; however, less variation was observed in communities of nirS-containing denitrifiers, indicating that denitrifiers possessing nirK were more sensitive to the fertilization practices than those with nirS. In contrast, fertilization regimes had similar effects on the copy numbers of nirK and nirS genes. The BMR treatment had the highest copy numbers of nirK and nirS, followed by the two mineral fertilization regimes (UR and BM), and the lowest was in the NF treatment. Of the measured soil parameters, the differences in the community composition of nirK and the abundance of nir denitrifiers were highly correlated with the soil carbon content. Therefore, long-term fertilization resulted in a strong impact on the community structure of nirK populations only, and total organic carbon was the dominant factor in relation to the variations of nir community sizes. PMID:20563573

  13. Aldose reductase inhibitory activity of compounds from Zea mays L.

    PubMed

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

    2013-01-01

    Aldose reductase (AR) inhibitors have a considerable therapeutic potential against diabetes complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of an EtOH extract of the kernel from purple corn (Zea mays L.), 7 nonanthocyanin phenolic compounds (compound 1-7) and 5 anthocyanins (compound 8-12) were isolated. These compounds were investigated by rat lens aldose reductase (RLAR) inhibitory assays. Kinetic analyses of recombinant human aldose reductase (rhAR) were performed, and intracellular galactitol levels were measured. Hirsutrin, one of 12 isolated compounds, showed the most potent RLAR inhibitory activity (IC(50), 4.78 μ M). In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate concentration, hirsutrin showed competitive inhibition against rhAR. Furthermore, hirsutrin inhibited galactitol formation in rat lens and erythrocytes sample incubated with a high concentration of galactose; this finding indicates that hirsutrin may effectively prevent osmotic stress in hyperglycemia. Therefore, hirsutrin derived from Zea mays L. may be a potential therapeutic agent against diabetes complications. PMID:23586057

  14. Oral Nitrate Reductase Activity Is Not Associated with Development of Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH): A Pilot Study

    PubMed Central

    Barzin, Gilda; Merat, Shahin; Nokhbeh-Zaeem, Habibeh; Saniee, Parastoo; Pedramnia, Shahrzad; Mostashfi Habibabadi, Ali; Nasseri-Moghaddam, Siavosh

    2014-01-01

    BACKGROUND NAFLD/NASH is a manifestation of metabolic syndrome and is associated with obesity/overweight. Not all obese/overweight individuals develop NASH. Gastro-esophageal reflux disease (GERD) is considered a gastrointestinal manifestation of the metabolic syndrome and is associated with obesity/overweight. Again not all obese/overweight individuals develop GERD. Recent data show association of dietary nitrate content and oral nitrate reductase activity (NRA) with GERD. Nitrates need to be converted to nitrite (done in human beings by nitrate reductase of oral bacteria exclusively) to be active in metabolic pathways. OBJECTIVE To assess the relation between NASH/NAFLD and oral NRA. METHODS Oral NRA was measured in individuals with NASH (compatible abdominal ultrasound and two elevated ALT/AST levels over six months) and was compared with that of those without NASH. Oral NRA was measured according to a previously reported protocol. RESULTS Eleven NASH patients and twelve controls were enrolled. Mean oral NRA activity were 2.82 vs. 3.51 μg nitrite-N formed per person per minute for cases and controls respectively (p=0.46). CONCLUSION According to our data, oral nitrite production is not different between individual swith and without NASH. PMID:24829701

  15. Ultrasonic Treatment Enhanced Ammonia-Oxidizing Bacterial (AOB) Activity for Nitritation Process.

    PubMed

    Zheng, Min; Liu, Yan-Chen; Xin, Jia; Zuo, Hao; Wang, Cheng-Wen; Wu, Wei-Min

    2016-01-19

    Oxidation of ammonia to nitrite rather than nitrate is critical for nitritation process for wastewater treatment. We proposed a promising approach by using controlled ultrasonic treatment to enhance the activity of ammonia-oxidizing bacteria (AOB) and suppress that of nitrite-oxidizing bacteria (NOB). Batch activity assays indicated that when ultrasound was applied, AOB activity reached a peak level and then declined but NOB activity deteriorated continuously as the power intensity of ultrasound increased. Kinetic analysis of relative microbial activity versus ultrasonic energy density was performed to investigate the effect of operational factors (power, sludge concentration, and aeration) on AOB and NOB activities and the test parameters were selected for reactor tests. Laboratory sequential batch reactor (SBR) was further used to test the ultrasonic stimulus with 8 h per day operational cycle and synthetic waste urine as influent. With specific ultrasonic energy density of 0.09 kJ/mg VSS and continuously fed influent containing above 200 mg NH3-N/L, high AOB reproductive activity was achieved and nearly complete conversion of ammonia-N to nitrite was maintained. Microbial structure analysis confirmed that the treatment changed community of AOB, NOB, and heterotrophs. Known AOB Nitrosomonas genus remained at similar level in the biomass while typical NOB Nitrospira genus disappeared in the SBR under ultrasonic treatment and after the treatment was off for 30 days. PMID:26678011

  16. Hypoxia induces NO-dependent release of heparan sulfate in fibroblasts from the Alzheimer mouse Tg2576 by activation of nitrite reduction.

    PubMed

    Cheng, Fang; Bourseau-Guilmain, Erika; Belting, Mattias; Fransson, Lars-Åke; Mani, Katrin

    2016-06-01

    There is a functional relationship between the heparan sulfate proteoglycan glypican-1 and the amyloid precursor protein (APP) of Alzheimer disease. In wild-type mouse embryonic fibroblasts, expression and processing of the APP is required for endosome-to-nucleus translocation of anhydromannose-containing heparan sulfate released from S-nitrosylated glypican-1 by ascorbate-induced, nitrosothiol-catalyzed deaminative cleavage. In fibroblasts from the transgenic Alzheimer mouse Tg2576, there is increased processing of the APP to amyloid-β peptides. Simultaneously, there is spontaneous formation of anhydromannose-containing heparan sulfate by an unknown mechanism. We have explored the effect of hypoxia on anhydromannose-containing heparan sulfate formation in wild-type and Tg2576 fibroblasts by deconvolution immunofluorescence microscopy and flow cytometry using an anhydromannose-specific monoclonal antibody and by (35)SO4-labeling experiments. Hypoxia prevented ascorbate-induced heparan sulfate release in wild-type fibroblasts, but induced an increased formation of anhydromannose-positive and (35)S-labeled heparan sulfate in Tg2576 fibroblasts. This appeared to be independent of glypican-1 S-nitrosylation as demonstrated by using a monoclonal antibody specific for S-nitrosylated glypican-1. In hypoxic wild-type fibroblasts, addition of nitrite to the medium restored anhydromannose-containing heparan sulfate formation. The increased release of anhydromannose-containing heparan sulfate in hypoxic Tg2576 fibroblasts did not require addition of nitrite. However, it was suppressed by inhibition of the nitrite reductase activity of xanthine oxidoreductase/aldehyde oxidase or by inhibition of p38 mitogen-activated protein kinase or by chelation of iron. We propose that normoxic Tg2576 fibroblasts maintain a high level of anhydromannose-containing heparan sulfate production by a stress-activated generation of nitric oxide from endogenous nitrite. This activation is enhanced

  17. Nitrate and periplasmic nitrate reductases

    PubMed Central

    Sparacino-Watkins, Courtney; Stolz, John F.; Basu, Partha

    2014-01-01

    The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed. PMID:24141308

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

    PubMed

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

    2016-03-01

    Two new ent-prenylaromadendrane-type diterpenoids, diplotaxifols A (1) and B (2), a new ent-eudesmol, ent-eudesma-4(15),11(13)-dien-6α,12-diol (3), eight new eudesmanolides enantiomers (4-11) of the corresponding compounds from higher plants along with four known ent-eudesmanolides (12-15) were isolated from the 95% EtOH extract of Chinese liverwort Diplophyllum taxifolium. Their structures were elucidated on the basis of MS, NMR and IR spectral data, and confirmed by single-crystal X-ray diffraction analysis. The quinone reductase-inducing activity of the compounds was evaluated. PMID:26656409

  19. Aggregate size and architecture determine microbial activity balance for one-stage partial nitritation and anammox.

    PubMed

    Vlaeminck, Siegfried E; Terada, Akihiko; Smets, Barth F; De Clippeleir, Haydée; Schaubroeck, Thomas; Bolca, Selin; Demeestere, Lien; Mast, Jan; Boon, Nico; Carballa, Marta; Verstraete, Willy

    2010-02-01

    Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing. PMID:19948857

  20. Kinetics of inter- and intramolecular electron transfer of Pseudomonas nautica cytochrome cd1 nitrite reductase: regulation of the NO-bound end product.

    PubMed

    Lopes, H; Besson, S; Moura, I; Moura, J J

    2001-01-01

    The intermolecular electron transfer kinetics between nitrite reductase (NiR, cytochrome cd1) isolated from Pseudomonas nautica and three cytochromes c isolated from the same strain, as well as the intramolecular electron transfer between NiR heme c and NiR heme d1, were investigated by cyclic voltammetry. All cytochromes (cytochrome c552, cytochrome c553 and cytochrome C553(548)) exhibited well-behaved electrochemistry. The individual diffusion coefficients and mid-point redox potentials were determined. Under the experimental conditions, only cytochrome c552 established a rapid electron transfer with NiR. At acidic pH, the intermolecular electron transfer (cytochrome c(552red)-->NiR heme cox) is a second-order reaction with a rate constant (k2) of 4.1+/-0.1x10(5) M(-1) s(-1) (pH=6.3 and 100 mM NaCl). Under these conditions, the intermolecular reaction represents the rate-limiting step. A minimum estimate of 33 s(-1) could be determined for the first-order rate constant (k1) of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox. The pH dependence of k2 values was investigated at pH values ranging from 5.8 to 8.0. When the pH is progressively shifted towards basic values, the rate constant of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox decreases gradually to a point where it becomes rate limiting. At pH 8.0 we determined a value of 1.4+/-0.7 s(-1), corresponding to a k2 value of 2.2+/-1.1x10(4) M(-1) s(-1) for the intermolecular step. The physiological relevance of these results is discussed with a particular emphasis on the proposed mechanism of "dead-end product" formation. PMID:11191223

  1. Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in Pacific northwest marine sediment communities

    SciTech Connect

    Braker, G.; Zhou, J.; Wu, L.; Devol, A.H.; Tiedje, J.M.

    2000-05-01

    Genetic heterogeneity of denitrifying bacteria in sediment samples from Puget Sound and two sites on the Washington continental margin was studied by PCR approaches amplifying nirK and nirS genes. These structurally different but functionally equivalent single-copy genes coding for nitrite reductases, a key enzyme of the denitrification process, were used as a molecular marker for denitrifying bacteria. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially for nirS clones from Puget Sound and a slightly lower level of diversity for nirK and nirS clones from the Washington margin. One group dominated within nirK clones, but no dominance and only a few redundant clones were seen between sediment samples for nirS clones in both habitats. Hybridization and sequencing confirmed that all but one of the 228 putative nirS clones were nirS with levels of nucleotide identities as low as 45.3%. Phylogenetic analysis grouped nirS clones into three distinct subclusters within the nirS gene tree which corresponded to the two habitats from which they were obtained. These sequences had little relationship to any strain with known nirS sequences or to isolates (mostly close relatives of Pseudomonas stutzeri) from the Washington margin sediment samples. nirK clones were more closely related to each other than were the nirS clones, with 78.6% and higher nucleotide identities; clones showing only weak hybridization signals were not related to known nirK sequences. All nirK clones were also grouped into a distinct cluster which could not be placed with any strain with known nirK sequences. These findings show a very high diversity of nir sequences within small samples and that

  2. Changes in Benthic Denitrification, Nitrate Ammonification, and Anammox Process Rates and Nitrate and Nitrite Reductase Gene Abundances along an Estuarine Nutrient Gradient (the Colne Estuary, United Kingdom)▿ †

    PubMed Central

    Dong, Liang F.; Smith, Cindy J.; Papaspyrou, Sokratis; Stott, Andrew; Osborn, A. Mark; Nedwell, David B.

    2009-01-01

    Estuarine sediments are the location for significant bacterial removal of anthropogenically derived inorganic nitrogen, in particular nitrate, from the aquatic environment. In this study, rates of benthic denitrification (DN), dissimilatory nitrate reduction to ammonium (DNRA), and anammox (AN) at three sites along a nitrate concentration gradient in the Colne estuary, United Kingdom, were determined, and the numbers of functional genes (narG, napA, nirS, and nrfA) and corresponding transcripts encoding enzymes mediating nitrate reduction were determined by reverse transcription-quantitative PCR. In situ rates of DN and DNRA decreased toward the estuary mouth, with the findings from slurry experiments suggesting that the potential for DNRA increased while the DN potential decreased as nitrate concentrations declined. AN was detected only at the estuary head, accounting for ∼30% of N2 formation, with 16S rRNA genes from anammox-related bacteria also detected only at this site. Numbers of narG genes declined along the estuary, while napA gene numbers were stable, suggesting that NAP-mediated nitrate reduction remained important at low nitrate concentrations. nirS gene numbers (as indicators of DN) also decreased along the estuary, whereas nrfA (an indicator for DNRA) was detected only at the two uppermost sites. Similarly, nitrate and nitrite reductase gene transcripts were detected only at the top two sites. A regression analysis of log(n + 1) process rate data and log(n + 1) mean gene abundances showed significant relationships between DN and nirS and between DNRA and nrfA. Although these log-log relationships indicate an underlying relationship between the genetic potential for nitrate reduction and the corresponding process activity, fine-scale environmentally induced changes in rates of nitrate reduction are likely to be controlled at cellular and protein levels. PMID:19304834

  3. Optimisation of nitrate reductase enzyme activity to synthesise silver nanoparticles.

    PubMed

    Khodashenas, Bahareh; Ghorbani, Hamid Reza

    2016-06-01

    Today, the synthesis of silver nanoparticles (Ag NPs) is very common since it has many applications in different areas. The synthesis of these nanoparticles is done by means of physical, chemical, or biological methods. However, due to its inexpensive and environmentally friendly features, the biological method is more preferable. In the present study, using nitrate reductase enzyme available in the Escherichia coli (E. coli) bacterium, the biosynthesis of Ag NPs was investigated. In addition, the activity of the nitrate reductase enzyme was optimised by changing its cultural conditions, and the effects of silver nitrate (AgNO(3)) concentration and enzyme amount on nanoparticles synthesis were studied. Finally, the produced nanoparticles were studied using ultraviolet -visible (UV-Vis) spectrophotometer, dynamic light scattering technique, and transmission electron microscopy. UV-Visible spectrophotometric study showed the characteristic peak for Ag NPs at wavelength 405-420 nm for 1 mM metal precursor solution (AgNO(3)) with 1, 5, 10, and 20 cc supernatant and 435 nm for 0.01M AgNO(3) with 20 cc supernatant. In this study, it was found that there is a direct relationship between the AgNO(3) concentration and the size of produced Ag NPs. PMID:27256897

  4. Periplasmic nitrate reductase and formate dehydrogenase: similar molecular architectures with very different enzymatic activities.

    PubMed

    Cerqueira, Nuno M F S A; Gonzalez, Pablo J; Fernandes, Pedro A; Moura, José J G; Ramos, Maria João

    2015-11-17

    It is remarkable how nature has been able to construct enzymes that, despite sharing many similarities, have simple but key differences that tune them for completely different functions in living cells. Periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) from the DMSOr family are representative examples of this. Both enzymes share almost identical three-dimensional protein foldings and active sites, in terms of coordination number, geometry and nature of the ligands. The substrates of both enzymes (nitrate and formate) are polyatomic anions that also share similar charge and stereochemistry. In terms of the catalytic mechanism, both enzymes have a common activation mechanism (the sulfur-shift mechanism) that ensures a constant coordination number around the metal ion during the catalytic cycle. In spite of these similarities, they catalyze very different reactions: Nap abstracts an oxygen atom from nitrate releasing nitrite, whereas FdH catalyzes a hydrogen atom transfer from formate and releases carbon dioxide. In this Account, a critical analysis of structure, function, and catalytic mechanism of the molybdenum enzymes periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) is presented. We conclude that the main structural driving force that dictates the type of reaction, catalyzed by each enzyme, is a key difference on one active site residue that is located in the top region of the active sites of both enzymes. In both enzymes, the active site is centered on the metal ion of the cofactor (Mo in Nap and Mo or W in Fdh) that is coordinated by four sulfur atoms from two pyranopterin guanosine dinucleotide (PGD) molecules and by a sulfido. However, while in Nap there is a Cys directly coordinated to the Mo ion, in FdH there is a SeCys instead. In Fdh there is also an important His that interacts very closely with the SeCys, whereas in Nap the same position is occupied by a Met. The role of Cys in Nap and SeCys in FdH is similar in both

  5. Response of denitrifying genes coding for nitrite (nirK or nirS) and nitrous oxide (nosZ) reductases to different physico-chemical parameters during agricultural waste composting.

    PubMed

    Zhang, Lihua; Zeng, Guangming; Zhang, Jiachao; Chen, Yaoning; Yu, Man; Lu, Lunhui; Li, Hui; Zhu, Yuan; Yuan, Yujie; Huang, Aizhi; He, Ling

    2015-05-01

    The present research was performed to clarify the changes of denitrifying genes (nirK, nirS, and nosZ) abundances under different physico-chemical parameters through evaluating the relationships between the genes abundances and parameters during agricultural waste composting. The genes abundances were determined by real-time quantitative PCR (qPCR). The correlations between physico-chemical parameters and denitrifying genes abundances were analysed by regression analysis. qPCR results showed that the nosZ gene abundance was higher than that of nirK and nirS genes. The nirK gene abundance was higher than nirS gene indicating that nitrite reducers with Cu-containing enzyme encoded by nirK gene were more of importance than those with cytochrome cd1 nitrite reductase encoded by nirS gene in the nitrite reduction step. Regression analysis suggested that (1) nirK gene abundance was correlated with pile temperature following quadratic model; (2) nirS gene abundance was linearly correlated with pile temperature and concentration of NH4 (+), while correlated with concentration of NO3 (-) and pH following inverse and quadratic model respectively; (3) nosZ gene abundance was quadratically correlated with pH and linearly correlated with water soluble carbon (WSC). PMID:25877886

  6. Inorganic nitrite attenuates NADPH oxidase-derived superoxide generation in activated macrophages via a nitric oxide-dependent mechanism.

    PubMed

    Yang, Ting; Peleli, Maria; Zollbrecht, Christa; Giulietti, Alessia; Terrando, Niccolo; Lundberg, Jon O; Weitzberg, Eddie; Carlström, Mattias

    2015-06-01

    Oxidative stress contributes to the pathogenesis of many disorders, including diabetes and cardiovascular disease. Immune cells are major sources of superoxide (O2(∙-)) as part of the innate host defense system, but exaggerated and sustained O2(∙-) generation may lead to progressive inflammation and organ injuries. Previous studies have proven organ-protective effects of inorganic nitrite, a precursor of nitric oxide (NO), in conditions manifested by oxidative stress and inflammation. However, the mechanisms are still not clear. This study aimed at investigating the potential role of nitrite in modulating NADPH oxidase (NOX) activity in immune cells. Mice peritoneal macrophages or human monocytes were activated by lipopolysaccharide (LPS), with or without coincubation with nitrite. O2(∙-) and peroxynitrite (ONOO(-)) formation were detected by lucigenin-based chemiluminescence and fluorescence techniques, respectively. The intracellular NO production was measured by DAF-FM DA fluorescence. NOX isoforms and inducible NO synthase (iNOS) expression were detected by qPCR. LPS increased both O2(∙-) and ONOO(-) production in macrophages, which was significantly reduced by nitrite (10µmol/L). Mechanistically, the effects of nitrite are (1) linked to increased NO generation, (2) similar to that observed with the NO donor DETA-NONOate, and (3) can be abolished by the NO scavenger carboxy-PTIO or by the xanthine oxidase (XO) inhibitor febuxostat. Nox2 expression was increased in activated macrophages, but was not influenced by nitrite. However, nitrite attenuated LPS-induced upregulation of iNOS expression. Similar to that observed in mice macrophages, nitrite also reduced O2(∙-) generation in LPS-activated human monocytes. In conclusion, XO-mediated reduction of nitrite attenuates NOX activity in activated macrophages, which may modulate the inflammatory response. PMID:25724690

  7. Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4’-sulfonyl derivative of L-methionine (dabsyl Met), the ...

  8. Evaluation of 5α-reductase inhibitory activity of certain herbs useful as antiandrogens.

    PubMed

    Nahata, A; Dixit, V K

    2014-08-01

    This study demonstrates 5α-reductase inhibitory activity of certain herbs useful in the management of androgenic disorders. Ganoderma lucidum (Curtis) P. Karst (GL), Urtica dioica Linn. (UD), Caesalpinia bonducella Fleming. (CB), Tribulus terrestris Linn. (TT), Pedalium murex Linn. (PM), Sphaeranthus indicus Linn. (SI), Cuscuta reflexa Roxb. (CR), Citrullus colocynthis Schrad. (CC), Benincasa hispida Cogn. (BH), Phyllanthus niruri Linn. (PN) and Echinops echinatus Linn. (EE) were included in the study. Petroleum ether, ethanol and aqueous extracts of these herbs were tested for their 5α-reductase inhibitory activity against the standard 5α-reductase inhibitor, finasteride. A biochemical method to determine the activity of 5α-reductase was used to evaluate the inhibition of different extracts to the enzyme. The optical density (OD) value of each sample was measured continuously with ultraviolet spectrophotometer for the reason that the substrate NADPH has a specific absorbance at 340 nm. As the enzyme 5α-reductase uses NADPH as a substrate, so in the presence of 5α-reductase inhibitor, the NADPH concentration will increase with the function of time. This method thus implicates the activity of 5α-reductase. The method proved to be extremely useful to screen the herbs for their 5α-reductase inhibitory potential. GL, UD, BH, SI and CR came out to be promising candidates for further exploring their antiandrogenic properties. PMID:23710567

  9. Reductive activation of E. coli respiratory nitrate reductase.

    PubMed

    Ceccaldi, Pierre; Rendon, Julia; Léger, Christophe; Toci, René; Guigliarelli, Bruno; Magalon, Axel; Grimaldi, Stéphane; Fourmond, Vincent

    2015-10-01

    Over the past decades, a number of authors have reported the presence of inactive species in as-prepared samples of members of the Mo/W-bisPGD enzyme family. This greatly complicated the spectroscopic studies of these enzymes, since it is impossible to discriminate between active and inactive species on the basis of the spectroscopic signatures alone. Escherichia coli nitrate reductase A (NarGHI) is a member of the Mo/W-bisPGD family that allows anaerobic respiration using nitrate as terminal electron acceptor. Here, using protein film voltammetry on NarGH films, we show that the enzyme is purified in a functionally heterogeneous form that contains between 20 and 40% of inactive species that activate the first time they are reduced. This activation proceeds in two steps: a non-redox reversible reaction followed by an irreversible reduction. By carefully correlating electrochemical and EPR spectroscopic data, we show that neither the two major Mo(V) signals nor those of the two FeS clusters that are the closest to the Mo center are associated with the two inactive species. We also conclusively exclude the possibility that the major "low-pH" and "high-pH" Mo(V) EPR signatures correspond to species in acid-base equilibrium. PMID:26073890

  10. Nitrite activates protein kinase A in normoxia to mediate mitochondrial fusion and tolerance to ischaemia/reperfusion

    PubMed Central

    Pride, Christelle Kamga; Mo, Li; Quesnelle, Kelly; Dagda, Ruben K.; Murillo, Daniel; Geary, Lisa; Corey, Catherine; Portella, Rafael; Zharikov, Sergey; St Croix, Claudette; Maniar, Salony; Chu, Charleen T.; K. H. Khoo, Nicholas; Shiva, Sruti

    2014-01-01

    Aims Nitrite (NO2–), a dietary constituent and nitric oxide (NO) oxidation product, mediates cardioprotection after ischaemia/reperfusion (I/R) in a number of animal models when administered during ischaemia or as a pre-conditioning agent hours to days prior to the ischaemic episode. When present during ischaemia, the reduction of nitrite to bioactive NO by deoxygenated haem proteins accounts for its protective effects. However, the mechanism of nitrite-induced pre-conditioning, a normoxic response which does not appear to require reduction of nitrite to NO, remains unexplored. Methods and results Using a model of hypoxia/reoxygenation (H/R) in cultured rat H9c2 cardiomyocytes, we demonstrate that a transient (30 min) normoxic nitrite treatment significantly attenuates cell death after a hypoxic episode initiated 1 h later. Mechanistically, this protection depends on the activation of protein kinase A, which phosphorylates and inhibits dynamin-related protein 1, the predominant regulator of mitochondrial fission. This results morphologically, in the promotion of mitochondrial fusion and functionally in the augmentation of mitochondrial membrane potential and superoxide production. We identify AMP kinase (AMPK) as a downstream target of the mitochondrial reactive oxygen species (ROS) generated and show that its oxidation and subsequent phosphorylation are essential for cytoprotection, as scavenging of ROS prevents AMPK activation and inhibits nitrite-mediated protection after H/R. The protein kinase A-dependent protection mediated by nitrite is reproduced in an intact isolated rat heart model of I/R. Conclusions These data are the first to demonstrate nitrite-dependent normoxic modulation of both mitochondrial morphology and function and reveal a novel signalling pathway responsible for nitrite-mediated cardioprotection. PMID:24081164

  11. Antitumor effect of synergistic contribution of nitrite and hydrogen peroxide in the plasma activated medium

    NASA Astrophysics Data System (ADS)

    Kurake, Naoyuki; Tanaka, Hiromasa; Ishikawa, Kenji; Nakamura, Kae; Kajiyama, Hiroaki; Kikkawa, Fumiaki; Kondo, Takashi; Mizuno, Masaaki; Takeda, Keigo; Kondo, Hiroki; Sekine, Makoto; Hori, Masaru

    2015-09-01

    Non-equilibrium atmospheric pressure plasmas (NEAPP) have been attracted attention in the noble application of cancer therapy. Although good effects of the Plasma-Activated-Medium (PAM) such as the selective antitumor effect and killing effect for the anticancer agent resistant cells were reported, a mechanism of this effect has not been still clarified yet. In this study, we have investigated a contribution of the reactive nitrogen and oxygen species (RNOS) generated in PAM such as hydrogen peroxide and nitrite. Those species generated in the PAM quantitatively measured by light absorbance of commercial regent. Moreover, viable cell count after cell culture with those RNOS intentionally added medium or PAM were also measured by MTS assay. Our NEAPP source generated hydrogen peroxide and nitrite with the generation ratio of 0.35 μM/s and 9.8 μM/s. In those RNOS, hydrogen peroxide has respective antitumor effect. On the other hands, nitrite has no antitumor effect singly. But, synergistically enhance the antitumor effect of hydrogen peroxide. Moreover, this effect of those RNOS also contribute for the selectively cancer killing effect of PAM.

  12. Effect of changing the nanoscale environment on activity and stability of nitrate reductase.

    PubMed

    Sachdeva, Veena; Hooda, Vinita

    2016-07-01

    Nitrate reductase (NR) is employed for fabrication of nitrate sensing devices in which the enzyme in immobilized form is used to catalyze the conversion of nitrate to nitrite in the presence of a suitable cofactor. So far, instability of immobilized NR due to the use of inappropriate immobilization matrices has limited the practical applications of these devices. Present study is an attempt to improve the kinetic properties and stability of NR using nanoscale iron oxide (nFe3O4) and zinc oxide (nZnO) particles. The desired nanoparticles were synthesized, surface functionalized, characterized and affixed onto the epoxy resin to yield two nanocomposite supports (epoxy/nFe3O4 and epoxy/nZnO) for immobilizing NR. Epoxy/nFe3O4 and epoxy/nZnO support could load as much as 35.8±0.01 and 33.20±0.01μg/cm(2) of NR with retention of about 93.72±0.50 and 84.81±0.80% of its initial activity respectively. Changes in surface morphology and chemical bonding structure of both the nanocomposite supports after addition of NR were confirmed by scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR). Optimum working conditions of pH, temperature and substrate concentration were ascertained for free as well as immobilized NR preparations. Further, storage stability at 4°C and thermal stability between 25-50°C were determined for all the NR preparations. Analytical applications of immobilized NR for determination of soil and water nitrates along with reusability data has been included to make sure the usefulness of the procedure. PMID:27233127

  13. Diversity in Overall Activity Regulation of Ribonucleotide Reductase*

    PubMed Central

    Jonna, Venkateswara Rao; Crona, Mikael; Rofougaran, Reza; Lundin, Daniel; Johansson, Samuel; Brännström, Kristoffer; Sjöberg, Britt-Marie; Hofer, Anders

    2015-01-01

    Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides, which are used as building blocks for DNA replication and repair. This process is tightly regulated via two allosteric sites, the specificity site (s-site) and the overall activity site (a-site). The a-site resides in an N-terminal ATP cone domain that binds dATP or ATP and functions as an on/off switch, whereas the composite s-site binds ATP, dATP, dTTP, or dGTP and determines which substrate to reduce. There are three classes of RNRs, and class I RNRs consist of different combinations of α and β subunits. In eukaryotic and Escherichia coli class I RNRs, dATP inhibits enzyme activity through the formation of inactive α6 and α4β4 complexes, respectively. Here we show that the Pseudomonas aeruginosa class I RNR has a duplicated ATP cone domain and represents a third mechanism of overall activity regulation. Each α polypeptide binds three dATP molecules, and the N-terminal ATP cone is critical for binding two of the dATPs because a truncated protein lacking this cone could only bind dATP to its s-site. ATP activates the enzyme solely by preventing dATP from binding. The dATP-induced inactive form is an α4 complex, which can interact with β2 to form a non-productive α4β2 complex. Other allosteric effectors induce a mixture of α2 and α4 forms, with the former being able to interact with β2 to form active α2β2 complexes. The unique features of the P. aeruginosa RNR are interesting both from evolutionary and drug discovery perspectives. PMID:25971975

  14. The nasB operon and nasA gene are required for nitrate/nitrite assimilation in Bacillus subtilis.

    PubMed Central

    Ogawa, K; Akagawa, E; Yamane, K; Sun, Z W; LaCelle, M; Zuber, P; Nakano, M M

    1995-01-01

    Bacillus subtilis can use either nitrate or nitrite as a sole source of nitrogen. The isolation of the nasABCDEF genes of B. subtilis, which are required for nitrate/nitrite assimilation, is reported. The probable gene products include subunits of nitrate/nitrite reductases and an enzyme involved in the synthesis of siroheme, a cofactor for nitrite reductase. PMID:7868621

  15. Chromate reductase activity in Streptomyces sp. MC1.

    PubMed

    Polti, Marta A; Amoroso, María J; Abate, Carlos M

    2010-02-01

    Biological transformation of Cr(VI) to Cr(III) by enzymatic reduction may provide a less costly and more environmentally friendly approach to remediation. In a previous report a Cr(VI) resistant actinomycete strain, Streptomyces sp. MC1, was able to reduce Cr(VI) present in a synthetic medium, soil extract and soil samples. This is the first time optimal conditions such as pH, temperature, growth phase and electron donor have been elucidated in vitro for Cr(VI) reduction by a streptomycete. Chromate reductase of Streptomyces sp. MC1 is a constitutive enzyme which was mainly associated with biomass and required NAD(P)H as an electron donor. It was active over a broad temperature (19-39 degrees C) and pH (5-8) range, and optimum conditions were 30 degrees C and pH 7. The enzyme was present in supernatant, pellet and cell free extract. Bioremediation with the enzyme was observed in non-compatible cell reproduction systems, conditions frequently found in contaminated environments. PMID:20339215

  16. A Periplasmic Complex of the Nitrite Reductase NirS, the Chaperone DnaK, and the Flagellum Protein FliC Is Essential for Flagellum Assembly and Motility in Pseudomonas aeruginosa

    PubMed Central

    Borrero-de Acuña, José Manuel; Molinari, Gabriella; Rohde, Manfred; Dammeyer, Thorben; Wissing, Josef; Jänsch, Lothar; Arias, Sagrario; Jahn, Martina; Schobert, Max; Timmis, Kenneth N.

    2015-01-01

    ABSTRACT Pseudomonas aeruginosa is a ubiquitously occurring environmental bacterium and opportunistic pathogen responsible for various acute and chronic infections. Obviously, anaerobic energy generation via denitrification contributes to its ecological success. To investigate the structural basis for the interconnection of the denitrification machinery to other essential cellular processes, we have sought to identify the protein interaction partners of the denitrification enzyme nitrite reductase NirS in the periplasm. We employed NirS as an affinity-purifiable bait to identify interacting proteins in vivo. Results obtained revealed that both the flagellar structural protein FliC and the protein chaperone DnaK form a complex with NirS in the periplasm. The interacting domains of NirS and FliC were tentatively identified. The NirS-interacting stretch of amino acids lies within its cytochrome c domain. Motility assays and ultrastructure analyses revealed that a nirS mutant was defective in the formation of flagella and correspondingly in swimming motility. In contrast, the fliC mutant revealed an intact denitrification pathway. However, deletion of the nirF gene, coding for a heme d1 biosynthetic enzyme, which leads to catalytically inactive NirS, did not abolish swimming ability. This pointed to a structural function for the NirS protein. FliC and NirS were found colocalized with DnaK at the cell surface of P. aeruginosa. A function of the detected periplasmic NirS-DnaK-FliC complex in flagellum formation and motility was concluded and discussed. IMPORTANCE Physiological functions in Gram-negative bacteria are connected with the cellular compartment of the periplasm and its membranes. Central enzymatic steps of anaerobic energy generation and the motility mediated by flagellar activity use these cellular structures in addition to multiple other processes. Almost nothing is known about the protein network functionally connecting these processes in the periplasm

  17. 5α-reductase inhibitors, antiviral and anti-tumor activities of some steroidal cyanopyridinone derivatives.

    PubMed

    Al-Mohizea, Abdullah M; Al-Omar, Mohamed A; Abdalla, Mohamed M; Amr, Abdel-Galil E

    2012-01-01

    We herein report the 5α-reductase inhibitors, antiviral and anti-tumor activities of some synthesized heterocyclic cyanopyridone and cyanothiopyridone derivatives fused with steroidal structure. Initially the acute toxicity of the compounds was assayed via the determination of their LD(50). All the compounds, except 3b, were interestingly less toxic than the reference drug (Prednisolone(®)). Seventeen heterocyclic derivatives containing a cyanopyridone or cyanothiopyridone rings fused to a steroidal moiety were synthesized and screened for their 5α-reductase inhibitors, antiviral and anti-tumor activities comparable to that of Anastrozole, Bicalutamide, Efavirenz, Capravirine, Ribavirin, Oseltamivir and Amantadine as the reference drugs. Some of the compounds exhibited better 5α-reductase inhibitors, antiviral and anti-tumor activities than the reference drugs. The detailed 5α-reductase inhibitors, antiviral and anti-tumor activities of the synthesized compounds were reported. PMID:22057085

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

    PubMed Central

    Zhou, Hui; Lin-Wang, Kui; Liao, Liao; Gu, Chao; Lu, Ziqi; Allan, Andrew C.; Han, Yuepeng

    2015-01-01

    Proanthocyanidins (PAs) are a group of natural phenolic compounds that have a great effect on both flavor and nutritious value of fruit. It has been shown that PA synthesis is regulated by R2R3-MYB transcription factors (TFs) via activation of PA-specific pathway genes encoding leucoanthocyanidin reductase and anthocyanidin reductase. Here, we report the isolation and characterization of a MYB gene designated PpMYB7 in peach. The peach PpMYB7 represents a new group of R2R3-MYB genes regulating PA synthesis in plants. It is able to activate transcription of PpLAR1 but not PpANR, and has a broader selection of potential bHLH partners compared with PpMYBPA1. Transcription of PpMYB7 can be activated by the peach basic leucine-zipper 5 TF (PpbZIP5) via response to ABA. Our study suggests a transcriptional network regulating PA synthesis in peach, with the results aiding the understanding of the functional divergence between R2R3-MYB TFs in plants. PMID:26579158

  19. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

    PubMed Central

    Steinkellner, Georg; Gruber, Christian C.; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Łyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-01-01

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts. PMID:24954722

  20. Increased 5. cap alpha. -reductase activity in idiopathic hirsutism

    SciTech Connect

    Serafini, P.; Lobo, R.A.

    1985-01-01

    In vitro, genital skin 5..cap alpha..-reductase activity (5..cap alpha..-RA) was measured in ten hirsute women with normal androgen levels (idiopathic hirsutism (IH)) and in ten hirsute women with elevated androgen levels (polycystic ovary syndrome (PCO)) in order to determine the influence of secreted androgens on 5..cap alpha..-RA. In vitro 5..cap alpha..-RA was assessed by incubations of skin with /sup 14/C-testosterone (T) for 2 hours, after which steroids were separated and the radioactivity of dihydrotestosterone (DHT) and 5..cap alpha..-androstane 3..cap alpha..-17..beta..-estradiol (3..cap alpha..-diol) in specific eluates were determined. All androgens were normal in IH with the exception of higher levels of 3..cap alpha..-diol glucuronide which were similar to the levels of PCO. The conversion ratio (CR) of T to DHT in IH and PCO were similar, yet significantly greater than the CR of control subjects. The CR of T to 3..cap alpha..-diol in IH and PCO were similar, yet higher than in control subjects. Serum androgens showed no correlation with 5..cap alpha..-RA, while the CR of T to DHT showed a significant positive correlation with the Ferriman and Gallwey score. The increased 5..cap alpha..-RA in IH appears to be independent of serum androgen levels and is, therefore, an inherent abnormality. The term idiopathic is a misnomer, because hirsutism in these patients may be explained on the basis of increased skin 5..cap alpha..-RA.

  1. Role of the denitrifying Haloarchaea in the treatment of nitrite-brines.

    PubMed

    Nájera-Fernández, Cindy; Zafrilla, Basilio; Bonete, María José; Martínez-Espinosa, Rosa María

    2012-09-01

    Haloferax mediterranei is a denitrifying halophilic archaeon able to reduce nitrate and nitrite under oxic and anoxic conditions. In the presence of oxygen, nitrate and nitrite are used as nitrogen sources for growth. Under oxygen scarcity, this haloarchaeon uses both ions as electron acceptors via a denitrification pathway. In the present work, the maximal nitrite concentration tolerated by this organism was determined by studying the growth of H. mediterranei in minimal medium containing 30, 40 and 50 mM nitrite as sole nitrogen source and under initial oxic conditions at 42 degrees C. The results showed the ability of H. mediterranei to withstand nitrite concentrations up to 50 mM. At the beginning of the incubation, nitrate was detected in the medium, probably due to the spontaneous oxidation of nitrite under the initial oxic conditions. The complete removal of nitrite and nitrate was accomplished in most of the tested conditions, except in culture medium containing 50 mM nitrite, suggesting that this concentration compromised the denitrification capacity of the cells. Nitrite and nitrate reductases activities were analyzed at different growth stages of H. mediterranei. In all cases, the activities of the respiratory enzymes were higher than their assimilative counterparts; this was especially the case for NirK. The denitrifying and possibly detoxifying role of this enzyme might explain the high nitrite tolerance of H. mediterranei. This archaeon was also able to remove 60% of the nitrate and 75% of the nitrite initially present in brine samples collected from a wastewater treatment facility. These results suggest that H. mediterranei, and probably other halophilic denitrifying Archaea, are suitable candidates for the bioremediation of brines with high nitrite and nitrate concentrations. PMID:23847815

  2. Endothelial human dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling

    PubMed Central

    Whitsett, Jennifer; Filho, Artur Rangel; Sethumadhavan, Savitha; Celinska, Joanna; Widlansky, Michael; Vásquez-Vivar, Jeannette

    2013-01-01

    Tetrahydrobiopterin (BH4) is required for NO synthesis and inhibition of superoxide release from eNOS. Clinical trials using BH4 to treat endothelial dysfunction have produced mixed results. Poor outcomes may be explained by the rapid systemic and cellular oxidation of BH4. One of the oxidation products of BH4, 7,8-dihydrobiopterin (7,8-BH2), is recycled back to BH4 by dihydrofolate reductase (DHFR). This enzyme is ubiquitously distributed and shows a wide range of activity depending on species-specific factors and cell type. Information about the kinetics and efficiency of BH4 recycling in human endothelial cells receiving BH4 treatment is lacking. To characterize this reaction, we applied a novel multi-electrode coulometric HPLC method that enabled the direct quantification of 7,8-BH2 and BH4 which is not possible with fluorescent-based methodologies. We found that basal untreated BH4 and 7,8-BH2 concentrations in human ECs is lower than bovine and murine endothelioma cells. Treatment of human ECs with BH4 transiently increased intracellular BH4 while accumulating the more stable 7,8-BH2. This was different from bovine or murine ECs that resulted in preferential BH4 increase. Using BH4 diastereomers, 6S-BH4 and 6R-BH4, the narrow contribution of enzymatic DHFR recycling to total intracellular BH4 was demonstrated. Reduction of 7,8-BH2 to BH4 occurs at very slow rates in cells and needs supra-physiological levels of 7,8-BH2, indicating this reaction is kinetically limited. Activity assays verified that hDHFR has very low affinity for 7,8-BH2 (DHF7,8-BH2) and folic acid inhibits 7,8-BH2 recycling. We conclude that low activity of endothelial DHFR is an important factor limiting the benefits of BH4 therapies which may be further aggravated by folate supplements. PMID:23707606

  3. Modulation of nitrate-nitrite conversion in the oral cavity.

    PubMed

    van Maanen, J M; van Geel, A A; Kleinjans, J C

    1996-01-01

    The formation of nitrite from ingested nitrate can give rise to the induction of methemoglobinemia and endogenous nitrosation resulting in the formation of carcinogenic N-nitroso compounds. We investigated the possibility of modulation of the conversion of nitrate into nitrite in the oral cavity in order to seek ways of reducing the formation of the deleterious nitrite. We investigated the effectiveness of several mouthwash solutions with antibacterial constituents on the reduction of nitrate into nitrite in the oral cavity. In 15 studied subjects, the mean percentage of salivary nitrate reduced to nitrite after ingestion of 235 mg (3.8 mmol) nitrate was found to be 16.1 +/- 6.2%. The use of an antiseptic mouthwash with active antibacterial constituent chlorhexidine resulted in an almost complete decrease of the mean percentage of reduced nitrate, to 0.9 +/- 0.8%. Mouthwash solutions with antibacterial component triclosan or antimicrobial enzymes amyloglucosidase and glucose oxidase did not affect the reduction of nitrate into nitrite. A toothpaste with active components triclosan and zinc citrate with synergistic antiplaque activity was also without effect. Use of a pH-regulating chewing gum resulted in a rise in the pH in the oral cavity from 6.8 to 7.3. At 30 min after nitrate ingestion, this rise was accompanied by a significant increase in the salivary nitrite concentration, which might be explained by the pH being close to the optimal pH for nitrate reductase of 8. In conclusion, a limited number of possibilities of modulation of the conversion of nitrate into nitrite in the oral cavity are available. PMID:8939344

  4. Exploring the Mechanisms of the Reductase Activity of Neuroglobin by Site-Directed Mutagenesis of the Heme Distal Pocket

    PubMed Central

    2016-01-01

    Neuroglobin (Ngb) is a six-coordinate globin that can catalyze the reduction of nitrite to nitric oxide. Although this reaction is common to heme proteins, the molecular interactions in the heme pocket that regulate this reaction are largely unknown. We have shown that the H64L Ngb mutation increases the rate of nitrite reduction by 2000-fold compared to that of wild-type Ngb [Tiso, M., et al. (2011) J. Biol. Chem. 286, 18277–18289]. Here we explore the effect of distal heme pocket mutations on nitrite reduction. For this purpose, we have generated mutations of Ngb residues Phe28(B10), His64(E7), and Val68(E11). Our results indicate a dichotomy in the reactivity of deoxy five- and six-coordinate globins toward nitrite. In hemoglobin and myoglobin, there is a correlation between faster rates and more negative potentials. However, in Ngb, reaction rates are apparently related to the distal pocket volume, and redox potential shows a poor relationship with the rate constants. This suggests a relationship between the nitrite reduction rate and heme accessibility in Ngb, particularly marked for His64(E7) mutants. In five-coordinate globins, His(E7) facilitates nitrite reduction, likely through proton donation. Conversely, in Ngb, the reduction mechanism does not rely on the delivery of a proton from the histidine side chain, as His64 mutants show the fastest reduction rates. In fact, the rate observed for H64A Ngb (1120 M–1 s–1) is to the best of our knowledge the fastest reported for a heme nitrite reductase. These differences may be related to a differential stabilization of the iron–nitrite complexes in five- and six-coordinate globins. PMID:25554946

  5. Regulation of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Human Fibroblasts by Lipoproteins

    PubMed Central

    Brown, Michael S.; Dana, Suzanna E.; Goldstein, Joseph L.

    1973-01-01

    The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), the rate-limiting enzyme of hepatic cholesterol biosynthesis, is suppressed in human fibroblasts cultured in the presence of serum. This enzyme activity increases by more than 10-fold after the removal of serum from the medium. The rise in enzyme activity requires de novo protein synthesis and is not accompanied by changes in the activities of several other cellular enzymes. The factor responsible for the suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured fibroblasts is present in the sera of at least four mammalian species, and in human serum it is found in the low-density lipoproteins. Human high-density lipoproteins, very low-density lipoproteins from chicken egg yolk, and the fraction of human serum containing no lipoproteins do not suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. PMID:4352976

  6. Phylogenomics of Mycobacterium Nitrate Reductase Operon.

    PubMed

    Huang, Qinqin; Abdalla, Abualgasim Elgaili; Xie, Jianping

    2015-07-01

    NarGHJI operon encodes a nitrate reductase that can reduce nitrate to nitrite. This process enhances bacterial survival by nitrate respiration under anaerobic conditions. NarGHJI operon exists in many bacteria, especially saprophytic bacteria living in soil which play a key role in the nitrogen cycle. Most actinomycetes, including Mycobacterium tuberculosis, possess NarGHJI operons. M. tuberculosis is a facultative intracellular pathogen that expands in macrophages and has the ability to persist in a non-replicative form in granuloma lifelong. Nitrogen and nitrogen compounds play crucial roles in the struggle between M. tuberculosis and host. M. tuberculosis can use nitrate as a final electron acceptor under anaerobic conditions to enhance its survival. In this article, we reviewed the mechanisms regulating nitrate reductase expression and affecting its activity. Potential genes involved in regulating the nitrate reductase expression in M. tuberculosis were identified. The conserved NarG might be an alternative mycobacterium taxonomic marker. PMID:25980349

  7. Color formation in nitrite-free dried hams as related to Zn-protoporphyrin IX and Zn-chelatase activity.

    PubMed

    Parolari, Giovanni; Benedini, Riccardo; Toscani, Tania

    2009-08-01

    The development of red pigment Zn-protoporphyrin IX (ZPP) in nitrite-free Parma hams was investigated in 5 leg muscles at several stages of processing and the activity of muscle Zn-chelatase was concurrently assayed for its potential role in ZPP formation. A steady increase of the pigment was observed throughout the manufacturing stages at mild temperatures while no development was observed during the prior cold resting phase. The enzyme was partly inactivated according to a muscle-dependent pattern, resulting in similar ZPP contents, hence color, in finished hams. It is concluded that enzyme-dependent synthesis of ZPP in nitrite-free dried hams contributes to color development, enabling muscles in dried hams to become more similar in redness than in green thighs. Therefore, checking raw meat for the enzyme content may be a means to control color formation in nitrite-free dry-cured meat derivatives. PMID:19723176

  8. Testosterone selectively affects aromatase and 5α-reductase activities in the green anole lizard brain

    PubMed Central

    Cohen, Rachel E.; Wade, Juli

    2011-01-01

    Testosterone (T) and its metabolites are important in the regulation of reproductive behavior in males of a variety of vertebrate species. Aromatase converts T to estradiol and 5α-reductase converts T to 5α-dihydrotestosterone (DHT). Male green anole reproduction depends on androgens, yet 5α-reductase in the brain is not sexually dimorphic and does not vary with season. In contrast, aromatase activity in the male brain is increased during the breeding compared to non-breeding season, and males have higher levels than females during the breeding season. Aromatase is important for female, but not male, sexual behaviors. The present experiment was conducted to determine whether 5α-reductase and aromatase are regulated by T. Enzyme activity was quantified in whole brain homogenates in both the breeding and non-breeding seasons in males and females that had been treated with either a T or blank implant. In males only, T increased 5α-reductase activity regardless of season and up-regulated aromatase during the breeding season specifically. Thus, regulation of both enzymes occurs in males, whereas females do not show parallel sensitivity to T. When considered with previous results, the data suggest that aromatase might influence a male function associated with the breeding season other than sexual behavior. 5α-Reductase can be mediated by T availability, but this regulation may not serve a sex- or season-specific purpose. PMID:19917285

  9. Gastric S-nitrosothiol formation drives the antihypertensive effects of oral sodium nitrite and nitrate in a rat model of renovascular hypertension.

    PubMed

    Pinheiro, Lucas C; Amaral, Jefferson H; Ferreira, Graziele C; Portella, Rafael L; Ceron, Carla S; Montenegro, Marcelo F; Toledo, Jose Carlos; Tanus-Santos, Jose E

    2015-10-01

    Many effects of nitrite and nitrate are attributed to increased circulating concentrations of nitrite, ultimately converted into nitric oxide (NO(•)) in the circulation or in tissues by mechanisms associated with nitrite reductase activity. However, nitrite generates NO(•) , nitrous anhydride, and other nitrosating species at low pH, and these reactions promote S-nitrosothiol formation when nitrites are in the stomach. We hypothesized that the antihypertensive effects of orally administered nitrite or nitrate involve the formation of S-nitrosothiols, and that those effects depend on gastric pH. The chronic effects of oral nitrite or nitrate were studied in two-kidney, one-clip (2K1C) hypertensive rats treated with omeprazole (or vehicle). Oral nitrite lowered blood pressure and increased plasma S-nitrosothiol concentrations independently of circulating nitrite levels. Increasing gastric pH with omeprazole did not affect the increases in plasma nitrite and nitrate levels found after treatment with nitrite. However, treatment with omeprazole severely attenuated the increases in plasma S-nitrosothiol concentrations and completely blunted the antihypertensive effects of nitrite. Confirming these findings, very similar results were found with oral nitrate. To further confirm the role of gastric S-nitrosothiol formation, we studied the effects of oral nitrite in hypertensive rats treated with the glutathione synthase inhibitor buthionine sulfoximine (BSO) to induce partial thiol depletion. BSO treatment attenuated the increases in S-nitrosothiol concentrations and antihypertensive effects of oral nitrite. These data show that gastric S-nitrosothiol formation drives the antihypertensive effects of oral nitrite or nitrate and has major implications, particularly to patients taking proton pump inhibitors. PMID:26159506

  10. Hydrogenase activity in Azospirillum brasilense is inhibited by nitrite, nitric oxide, carbon monoxide, and acetylene

    SciTech Connect

    Tibelius, K.H.; Knowles, R.

    1984-10-01

    Nitrite, NO, CO, and C/sub 2/H/sub 2/ inhibited O/sub 2/-dependent H/sub 2/ uptake (H/sup 3/H oxidation) in denitrifying Azospirillum brasilense Sp7 grown anaerobically on N/sub 2/O or NO/sub 3//sup -/. The apparent K/sub i/ values for inhibition of O/sub 2/-dependent H/sub 2/ uptake were 20 ..mu..M for NO/sub 2//sup -/, 0.4 ..mu..M for NO, 28 ..mu..M for CO, and 88 ..mu..M for C/sub 2/H/sub 2/. These inhibitors also affected methylene blue-dependent H/sub 2/ uptake, presumably by acting directly on the hydrogenase. Nitrite and NO inhibited H/sub 2/ uptake irreversibly, whereas inhibition due to CO was easily reversed by repeatedly evacuating and backfilling with N/sub 2/. The C/sub 2/H/sub 2/ inhibition was not readily reversed, partly due to difficulty in removing the last traces of this gas from solution. The NO/sub 2//sup -/ inhibition of malate-dependent respiration was readily reversed by repeatedly washing the cells, in contrast to the effect of NO/sub 2//sup -/ on H/sub 2/-dependent respiration. These results suggest that the low hydrogenase activities observed in NO/sub 3//sup -/-grown cultures of A. brasilense may be due to the irreversible inhibition of hydrogenase by NO/sub 2//sup -/ and NO produced by NO/sub 3//sup -/ reduction.

  11. Aggregate Size and Architecture Determine Microbial Activity Balance for One-Stage Partial Nitritation and Anammox ▿

    PubMed Central

    Vlaeminck, Siegfried E.; Terada, Akihiko; Smets, Barth F.; De Clippeleir, Haydée; Schaubroeck, Thomas; Bolca, Selin; Demeestere, Lien; Mast, Jan; Boon, Nico; Carballa, Marta; Verstraete, Willy

    2010-01-01

    Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing. PMID:19948857

  12. Mechanisms of Nitrite Bioactivation

    PubMed Central

    Kim-Shapiro, Daniel B.; Gladwin, Mark T.

    2014-01-01

    It is now accepted that the anion nitrite, once considered an inert oxidation product of nitric oxide (NO), contributes to hypoxic vasodilation, physiological blood pressure control, and redox signaling. As such, its application in therapeutics is being actively testing in pre-clinical models and in human phase I–II clinical trials. Major pathways for nitrite bioactivation involve its reduction to NO by members of the hemoglobin or molybdopterin family of proteins, or catalyzed dysproportionation. These conversions occur preferentially under hypoxic and acidic conditions. A number of enzymatic systems reduce nitrite to NO and their activity and importance are defined by oxygen tension, specific organ system and allosteric and redox effectors. In this work, we review different proposed mechanisms of nitrite bioactivation, focusing on analysis of kinetics and experimental evidence for the relevance of each mechanism under different conditions. PMID:24315961

  13. Molecular Characterization of the Nitrite-Reducing System of Staphylococcus carnosus

    PubMed Central

    Neubauer, H.; Pantel, I.; Götz, F.

    1999-01-01

    Characterization of a nitrite reductase-negative Staphylococcus carnosus Tn917 mutant led to the identification of the nir operon, which encodes NirBD, the dissimilatory NADH-dependent nitrite reductase; SirA, the putative oxidase and chelatase, and SirB, the uroporphyrinogen III methylase, both of which are necessary for biosynthesis of the siroheme prosthetic group; and NirR, which revealed no convincing similarity to proteins with known functions. We suggest that NirR is essential for nir promoter activity. In the absence of NirR, a weak promoter upstream of sirA seems to drive transcription of sirA, nirB, nirD, and sirB in the stationary-growth phase. In primer extension experiments one predominant and several weaker transcription start sites were identified in the nir promoter region. Northern blot analyses indicated that anaerobiosis and nitrite are induction factors of the nir operon: cells grown aerobically with nitrite revealed small amounts of full-length transcript whereas cells grown anaerobically with or without nitrite showed large amounts of full-length transcript. Although a transcript is detectable, no nitrite reduction occurs in cells grown aerobically with nitrite, indicating an additional oxygen-controlled step at the level of translation, enzyme folding, assembly, or insertion of prosthetic groups. The nitrite-reducing activity expressed during anaerobiosis is switched off reversibly when the oxygen tension increases, most likely due to competition for electrons with the aerobic respiratory chain. Another gene, nirC, is located upstream of the nir operon. nirC encodes a putative integral membrane-spanning protein of unknown function. A nirC mutant showed no distinct phenotype. PMID:10049379

  14. MEK2 regulates ribonucleotide reductase activity through functional interaction with ribonucleotide reductase small subunit p53R2.

    PubMed

    Piao, Chunmei; Youn, Cha-Kyung; Jin, Min; Yoon, Sang Pil; Chang, In-Youb; Lee, Jung Hee; You, Ho Jin

    2012-09-01

    The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRNA, dramatically decreased the serum-stimulated RNR activity. Moreover, p53R2 siRNA, but not R2 siRNA, significantly inhibits serum-stimulated RNR activity, indicating that p53R2 is specifically regulated by a MEK2-dependent pathway. Co-immunoprecipitation analyses revealed that the MEK2 segment comprising amino acids 65-171 is critical for p53R2-MEK2 interaction, and the binding domain of MEK2 is required for MEK2-mediated increased RNR activity. Phosphorylation of MEK1/2 was greatly augmented by ionizing radiation, and RNR activity was concurrently increased. Ionizing radiation-induced RNR activity was markedly attenuated by transfection of MEK2 or p53R2 siRNA, but not R2 siRNA. These data show that MEK2 is an endogenous regulator of p53R2 and suggest that MEK2 may associate with p53R2 and upregulate its activity. PMID:22895183

  15. The dihydrolipoamide dehydrogenase of Aeromonas caviae ST exhibits NADH-dependent tellurite reductase activity.

    PubMed

    Castro, Miguel E; Molina, Roberto; Díaz, Waldo; Pichuantes, Sergio E; Vásquez, Claudio C

    2008-10-10

    Potassium tellurite (K(2)TeO(3)) is extremely toxic for most forms of life and only a limited number of organisms are naturally resistant to the toxic effects of this compound. Crude extracts prepared from the environmental isolate Aeromonas caviae ST catalize the in vitro reduction of TeO32- in a NADH-dependent reaction. Upon fractionation by ionic exchange column chromatography three major polypeptides identified as the E1, E2, and E3 components of the pyruvate dehydrogenase (PDH) complex were identified in fractions exhibiting tellurite-reducing activity. Tellurite reductase and pyruvate dehydrogenase activities co-eluted from a Sephadex gel filtration column. To determine which component(s) of the PDH complex has tellurite reductase activity, the A. caviae ST structural genes encoding for E1 (aceE), E2 (aceF), and E3 (lpdA) were independently cloned and expressed in Escherichia coli and their gene products purified. Results indicated that tellurite reductase activity lies almost exclusively in the E3 component, dihydrolipoamide dehydrogenase. The E3 component of the PDH complex from E. coli, Zymomonas mobilis, Streptococcus pneumoniae, and Geobacillus stearothermophilus also showed NADH-dependent tellurite reductase in vitro suggesting that this enzymatic activity is widely distributed among microorganisms. PMID:18675788

  16. Importance of P450 reductase activity in determining sensitivity of breast tumour cells to the bioreductive drug, tirapazamine (SR 4233).

    PubMed Central

    Patterson, A. V.; Barham, H. M.; Chinje, E. C.; Adams, G. E.; Harris, A. L.; Stratford, I. J.

    1995-01-01

    P450 reductase (NADPH:cytochrome P450 reductase, EC 1.6.2.4) is known to be important in the reductive activation of the benzotriazene-di-N-oxide tirapazamine (SR 4233). Using a panel of six human breast adenocarcinoma cell lines we have examined the relationship between P450 reductase activity and sensitivity to tirapazamine. The toxicity of tirapazamine was found to correlate strongly with P450 reductase activity following an acute (3 h) exposure under hypoxic conditions, the drug being most toxic in the cell lines with the highest P450 reductase activity. A similar correlation was also observed following a chronic (96 h) exposure to the drug in air but not following acute (3 h) exposure in air. We have also determined the ability of lysates prepared from the cell lines to metabolise tirapazamine to its two-electron reduced product, SR 4317, under hypoxic conditions using NADPH as an electron donor. The rate of SR 4317 formation was found to correlate both with P450 reductase activity and with sensitivity to tirapazamine, the highest rates of SR 4317 formation being associated with the highest levels of P450 reductase activity and the greatest sensitivity to the drug. These findings indicate a major role for P450 reductase in determining the hypoxic toxicity of tirapazamine in breast tumour cell lines. Images Figure 4 PMID:7577460

  17. Ferric reductase activity and PsFRO1 sequence variation in pisum sps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Physiological studies in pea (Pisum sativum) suggest that the reduction of iron (Fe) is the rate-limiting physiological process in Fe acquisition by dicotyledonous plants. Previous molecular work suggests that ferric reductase activity is regulated at both the transcriptional and post-translational ...

  18. Glyphosate Effect on Shikimate, Nitrate Reductase Activity, Yield, and Seed Composition in Corn

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 2-yr field study investigated the effects of glyphosate drift rate on plant injury, shikimate accumulation, nitrate reductase activity, leaf nitrogen, yield, and seed composition in non-glyphosate-resistant (non-GR) corn (Zea mays L.) and the effects of glyphosate at label rates on nitrate reducta...

  19. Differential antioxidant and quinone reductase inducing activity of American, Asian, and Siberian ginseng

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antioxidant and quinone reductase (QR) inducing activities of American, Asian, and Siberian ginseng have been reported using various plant materials, solvents, and assays. To directly establish their comparative bioactivity, the effects of extracts obtained from acidified methanol (MeOH), a gas...

  20. EFFECT OF LINDANE ON INTESTINAL NITROREDUCTASE, AZO REDUCTASE, B-GLUCURONIDASE, DECHLORINASE AND DEHYDROCHLORINASE ACTIVITY

    EPA Science Inventory

    The effect of daily p.o. injections of 20 mg/kg lindane on nitroreductase, azo reductase, B-glucuronidase, dechlorinase and dehydrochlorinase enzyme activity in the rat intestinal tract vas investigated after 2 weeks and 5 weeks of treatment. Antibiotics were administered to half...

  1. Resolution of two native monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina and the sequence of two napA genes

    SciTech Connect

    Simpson, Philippa J.L.; McKinzie, Audra A.; Codd, Rachel; School of Medical Sciences and Bosch Institute, University of Sydney, NSW 2006

    2010-07-16

    Research highlights: {yields} Two monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina. {yields} Sequence of napA from napEDABC-type operon and napA from NapDAGHB-type operon. {yields} Isolation of NAP as NapA or NapAB correlated with NapA P47E amino acid substitution. -- Abstract: The reduction of nitrate to nitrite in the bacterial periplasm occurs in the 90 kDa NapA subunit of the periplasmic nitrate reductase (NAP) system. Most Shewanella genomes contain two nap operons: napEDABC and napDAGHB, which is an unusual feature of this genus. Two native, monomeric, 90 kDa nitrate reductase active proteins were resolved by hydrophobic interaction chromatography from aerobic cultures of Shewanella gelidimarina replete with reduced nitrogen compounds. The 90 kDa protein obtained in higher yield was characterized as NapA by electronic absorption and electron paramagnetic resonance spectroscopies and was identified by LC/MS/MS and MALDI-TOF/TOF MS as NapA from the napEDABC-type operon. The other 90 kDa protein, which was unstable and produced in low yields, was posited as NapA from the napDAGHB-type operon. Two napA genes have been sequenced from the napEDABC-type and napDAGHB-type operons of S. gelidimarina. Native NAP from S. putrefaciens was resolved as one NapA monomer and one NapAB heterodimer. Two amino acid substitutions in NapA correlated with the isolation of NAP as a NapA monomer or a NapAB heterodimer. The resolution of native, redox-active NapA isoforms in Shewanella provides new insight into the respiratory versatility of this genus, which has implications in bioremediation and the assembly of microbial fuel cells.

  2. Elevated CO2 levels affect the activity of nitrate reductase and carbonic anhydrase in the calcifying rhodophyte Corallina officinalis

    PubMed Central

    Hofmann, Laurie C.

    2013-01-01

    The concentration of CO2 in global surface ocean waters is increasing due to rising atmospheric CO2 emissions, resulting in lower pH and a lower saturation state of carbonate ions. Such changes in seawater chemistry are expected to impact calcification in calcifying marine organisms. However, other physiological processes related to calcification might also be affected, including enzyme activity. In a mesocosm experiment, macroalgal communities were exposed to three CO2 concentrations (380, 665, and 1486 µatm) to determine how the activity of two enzymes related to inorganic carbon uptake and nutrient assimilation in Corallina officinalis, an abundant calcifying rhodophyte, will be affected by elevated CO2 concentrations. The activity of external carbonic anhydrase, an important enzyme functioning in macroalgal carbon-concentrating mechanisms, was inversely related to CO2 concentration after long-term exposure (12 weeks). Nitrate reductase, the enzyme responsible for reduction of nitrate to nitrite, was stimulated by CO2 and was highest in algae grown at 665 µatm CO2. Nitrate and phosphate uptake rates were inversely related to CO2, while ammonium uptake was unaffected, and the percentage of inorganic carbon in the algal skeleton decreased with increasing CO2. The results indicate that the processes of inorganic carbon and nutrient uptake and assimilation are affected by elevated CO2 due to changes in enzyme activity, which change the energy balance and physiological status of C. officinalis, therefore affecting its competitive interactions with other macroalgae. The ecological implications of the physiological changes in C. officinalis in response to elevated CO2 are discussed. PMID:23314813

  3. Overexpression of Nitrate Reductase in Tobacco Delays Drought-Induced Decreases in Nitrate Reductase Activity and mRNA1

    PubMed Central

    Ferrario-Méry, Sylvie; Valadier, Marie-Hélène; Foyer, Christine H.

    1998-01-01

    Transformed (cauliflower mosaic virus 35S promoter [35S]) tobacco (Nicotiana plumbaginifolia L.) plants constitutively expressing nitrate reductase (NR) and untransformed controls were subjected to drought for 5 d. Drought-induced changes in biomass accumulation and photosynthesis were comparable in both lines of plants. After 4 d of water deprivation, a large increase in the ratio of shoot dry weight to fresh weight was observed, together with a decrease in the rate of photosynthetic CO2 assimilation. Foliar sucrose increased in both lines during water stress, but hexoses increased only in leaves from untransformed controls. Foliar NO3− decreased rapidly in both lines and was halved within 2 d of the onset of water deprivation. Total foliar amino acids decreased in leaves of both lines following water deprivation. After 4 d of water deprivation no NR activity could be detected in leaves of untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much more stable than NR activity. NR mRNA abundance increased in the leaves of the 35S-NR plants and remained constant in controls for the first 3 d of drought. On the 4th d, however, NR mRNA suddenly decreased in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls. The phosphorylation state of the protein was unchanged by long-term drought. There was a strong correlation between maximal extractable NR activity and ambient photosynthesis in both lines. We conclude that drought first causes increased NR protein turnover and then accelerates NR mRNA turnover. Constitutive NR expression temporarily delayed drought-induced losses in NR activity. 35S-NR expression may therefore allow more rapid recovery of N assimilation following short-term water deficit. PMID:9576799

  4. Isolation, modification, and aldose reductase inhibitory activity of rosmarinic acid derivatives from the roots of Salvia grandifolia.

    PubMed

    Kang, Jie; Tang, Yanbo; Liu, Quan; Guo, Nan; Zhang, Jian; Xiao, Zhiyan; Chen, Ruoyun; Shen, Zhufang

    2016-07-01

    To find aldose reductase inhibitors, two previously unreported compounds, grandifolias H and I, and five known compounds, including rosmarinic acid and rosmarinic acid derivatives, were isolated from the roots of Salvia grandifolia. A series of rosmarinic acid derivatives was obtained from rosmarinic acid using simple synthetic methods. The aldose reductase inhibitory activity of the isolated and synthesized compounds was assessed. Seven of the tested compounds showed moderate aldose reductase inhibition (IC50=0.06-0.30μM). The structure-activity relationship of aldose reductase inhibitory activity of rosmarinic acid derivatives was discussed for the first time. This study provided useful information that will facilitate the development of aldose reductase inhibitors. PMID:27233987

  5. Biochemical and antitumor activity of trimidox, a new inhibitor of ribonucleotide reductase.

    PubMed

    Szekeres, T; Gharehbaghi, K; Fritzer, M; Woody, M; Srivastava, A; van't Riet, B; Jayaram, H N; Elford, H L

    1994-01-01

    Trimidox (3,4,5-trihydroxybenzamidoxime), a newly synthesized analog of didox (N,3,4-trihydroxybenzamide) reduced the activity of ribonucleotide reductase (EC 1.17.4.1) in extracts of L1210 cells by 50% (50% growth-inhibitory concentration, IC50) at 5 microM, whereas hydroxyurea, the only ribonucleotide reductase inhibitor in clinical use, exhibited an IC50 of 500 microM. Ribonucleotide reductase activity was also measured in situ by incubating L1210 cells for 24 h with trimidox at 7.5 microM, a concentration that inhibits cell proliferation by 50% (IC50) or at 100 microM for 2 h; these concentrations resulted in a decrease in enzyme activity to 22% and 50% of the control value, respectively. Trimidox and hydroxyurea were cytotoxic to L1210 cells with IC50 values of 7.5 and 50 microM, respectively. Versus ribonucleotide reductase, trimidox and hydroxyurea yielded IC50 values of 12 and 87 microM, respectively. A dose-dependent increase in life span was observed in mice bearing intraperitoneally transplanted L1210 tumors. Trimidox treatment (200 mg/kg; q1dx9) significantly increased the life span of mice bearing L1210 leukemia (by 82% in male mice and 112% in female mice). The anti-tumor activity appeared more pronounced in female mice than in male mice. Viewed in concert, these findings suggest that trimidox is a new and potent inhibitor of ribonucleotide reductase and that it is a promising candidate for the chemotherapy of cancer in humans. PMID:8174204

  6. Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse peritoneal macrophages.

    PubMed Central

    Angelin, B

    1988-01-01

    The lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl-(HMG-) CoA reductase in cultured mouse peritoneal macrophages has been investigated. In contrast to what has been reported for other cells, HMG-CoA reductase activity is not suppressed by normal serum or by normal low density lipoproteins (LDL) from humans or dogs. Suppression of reductase activity occurred when cells were cultured in the presence of beta-migrating very low density lipoproteins (beta-VLDL) or LDL from hypercholesterolaemic dogs, or LDL modified by acetoacetylation. Human beta-VLDL from an atypical type III hyperlipoproteinaemic patient was also effective, as was apolipoprotein (apo) E-containing high density lipoproteins (HDL) from cholesterol-fed dogs (apo-E HDLc). The results indicate that cholesterol biosynthesis in mouse peritoneal macrophages is regulated by lipoprotein cholesterol entering via receptor-mediated endocytosis. Normal LDL were not effective because of the poor binding and uptake of these lipoproteins by the apo-B, E (LDL) receptor. Only beta-VLDL, apo-E HDLc, and hypercholesterolaemic LDL were avidly taken up by this receptor and were able to suppress HMG-CoA reductase. Acetoacetylated LDL were internalized via the acetyl-LDL (scavenger) receptor. Thus, mouse macrophages differ from human fibroblasts and smooth muscle cells in their physiological regulation of cholesterogenesis. PMID:3202831

  7. Anion selective optodes: development of a fluorescent fiber optic sensor for the determination of nitrite activity

    NASA Astrophysics Data System (ADS)

    Barker, Susan L. R.; Shortreed, Michael R.; Kopelman, Raoul

    1996-12-01

    The response of state of the art anion optodes often cannot be described in a thermodynamically exact manner because the ionic strength within the membrane phase of such optodes changes during the course of a titration. Incorporating lipophilic charge sites in the anion optode membranes provides a constant ionic strength in the membrane phase, the ability to measure anion activities, and a more thermodynamically describable system. This configuration has been used to create a micrometer-sized nitrite-selective optode. Recent elucidation of the many biological roles of nitric oxide (NO) has spurred interest in sensitive and selective detection of this molecule. In biological systems NO is converted to NO2- within 30 sec and the biological concentration of NO2- is normally on the micromolar level. The optode we have prepared contains a selective vitamin B12 derivative ionophore, a fluorescent chromoionophore (ETH 2439 or ETH 5350), and lipophilic charge sites. These components are entrapped in a highly plasticized PVC matrix which is placed on the distal end of the fiber. Sensor characteristics such as limit of detection and reversibility are presented.

  8. Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase.

    PubMed Central

    Grunwald, S K; Lies, D P; Roberts, G P; Ludden, P W

    1995-01-01

    Rhodospirillum rubrum strains that overexpress the enzymes involved in posttranslational nitrogenase regulation, dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG), were constructed, and the effect of this overexpression on in vivo DRAT and DRAG regulation was investigated. Broad-host-range plasmid constructs containing a fusion of the R. rubrum nifH promoter and translation initiation sequences to the second codon of draT, the first gene of the dra operon, were constructed. Overexpression plasmid constructs which overexpressed (i) only functional DRAT, (ii) only functional DRAG and presumably the putative downstream open reading frame (ORF)-encoded protein, or (iii) all three proteins were generated and introduced into wild-type R. rubrum. Overexpression of DRAT still allowed proper regulation of nitrogenase activity, with ADP-ribosylation of dinitrogenase reductase by DRAT occurring only upon dark or ammonium stimuli, suggesting that DRAT is still regulated upon overexpression. However, overexpression of DRAG and the downstream ORF altered nitrogenase regulation such that dinitrogenase reductase did not accumulate in the ADP-ribosylated form under inactivation conditions, suggesting that DRAG was constitutively active and that therefore DRAG regulation is altered upon overexpression. Proper DRAG regulation was observed in a strain overexpressing DRAT, DRAG, and the downstream ORF, suggesting that a proper balance of DRAT and DRAG levels is required for proper DRAG regulation. PMID:7836296

  9. Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF

    PubMed Central

    Nicke, Tristan; Schnitzer, Tobias; Münch, Karin; Adamczack, Julia; Haufschildt, Kristin; Buchmeier, Sabine; Kucklick, Martin; Felgenträger, Undine; Jänsch, Lothar; Riedel, Katharina; Layer, Gunhild

    2013-01-01

    The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS–NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV–visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS. PMID:23683062

  10. Analytical properties of some commercially available nitrate reductase enzymes evaluated as replacements for cadmium in automated, semiautomated, and manual colorimetric methods for determination of nitrate plus nitrite in water

    USGS Publications Warehouse

    Patton, Charles J.; Kryskalla, Jennifer R.

    2013-01-01

    A multiyear research effort at the U.S. Geological Survey (USGS) National Water Quality Laboratory (NWQL) evaluated several commercially available nitrate reductase (NaR) enzymes as replacements for toxic cadmium in longstanding automated colorimetric air-segmented continuous-flow analyzer (CFA) methods for determining nitrate plus nitrite (NOx) in water. This research culminated in USGS approved standard- and low-level enzymatic reduction, colorimetric automated discrete analyzer NOx methods that have been in routine operation at the NWQL since October 2011. The enzyme used in these methods (AtNaR2) is a product of recombinant expression of NaR from Arabidopsis thaliana (L.) Heynh. (mouseear cress) in the yeast Pichia pastoris. Because the scope of the validation report for these new automated discrete analyzer methods, published as U.S. Geological Survey Techniques and Methods 5–B8, was limited to performance benchmarks and operational details, extensive foundational research with different enzymes—primarily YNaR1, a product of recombinant expression of NaR from Pichia angusta in the yeast Pichia pastoris—remained unpublished until now. This report documents research and development at the NWQL that was foundational to development and validation of the discrete analyzer methods. It includes: (1) details of instrumentation used to acquire kinetics data for several NaR enzymes in the presence and absence of known or suspected inhibitors in relation to reaction temperature and reaction pH; and (2) validation results—method detection limits, precision and bias estimates, spike recoveries, and interference studies—for standard- and low-level automated colorimetric CFA-YNaR1 reduction NOx methods in relation to corresponding USGS approved CFA cadmium-reduction (CdR) NOx methods. The cornerstone of this validation is paired sample statistical and graphical analysis of NOx concentrations from more than 3,800 geographically and seasonally diverse surface

  11. MYC Phosphorylation, Activation, and Tumorigenic Potential in Hepatocellular Carcinoma are Regulated by HMG-CoA Reductase

    PubMed Central

    Cao, Zhongwei; Fan-Minogue, Hua; Bellovin, David I.; Yevtodiyenko, Aleksey; Arzeno, Julia; Yang, Qiwei; Gambhir, Sanjiv Sam; Felsher, Dean W.

    2011-01-01

    MYC is a potential target for many cancers but is not amenable to existing pharmacological approaches. Inhibition of HMG-CoA reductase by statins has shown potential efficacy against a number of cancers. Here, we demonstrate that inhibition of HMG-CoA reductase by atorvastatin blocks both MYC phosphorylation and activation, suppressing tumor initiation and growth in vivo in a transgenic model of MYC-induced HCC as well as in human HCC-derived cell lines. To confirm specificity, we show that the anti-tumor effects of atorvastatin are blocked by co-treatment with the HMG-CoA reductase product, Mevalonate. Moreover, by using a novel molecular imaging sensor, we confirm that inhibition of HMG-CoA reductase blocks MYC phosphorylation in vivo. Importantly, the introduction of phosphorylation mutants of MYC at Ser62 or Thr58 into tumors blocks their sensitivity to inhibition of HMG-CoA reductase. Finally, we demonstrate that inhibition of HMG-CoA reductase suppresses MYC phosphorylation through Rac GTPase. Therefore, HMG-CoA reductase is a critical regulator of MYC phosphorylation, activation, and tumorigenic properties. The inhibition of HMG-CoA reductase may be a useful target for the treatment of MYC-associated HCC as well as other tumors. PMID:21262914

  12. Post-translational Regulation of Nitrate Reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite, which is the first step in the nitrate assimilation pathway, but can also reduce nitrite to nitric oxide (NO), an important signaling molecule that is thought to mediate a wide array of of developmental and physiological processes...

  13. Immobilization of mercuric reductase from a pseudomonas putida strain on different activated carriers

    SciTech Connect

    Anspach, F.B.; Hueckel, M.; Brunke, M.

    1994-02-01

    Mercuric reductase was isolated from Pseudomonas putida KT2442::mer-73 and immobilized on chromatographic carriers activated by various methods. The immobilization methods for covalent coupling were compared with regard to preservation of enzymatic activity and coupling yields. Highest yields were obtained with carriers bearing the most reactive functional groups. Best results were achieved with tresyl chloride-activated carriers. The optimum binding conditions were found at pH 8. Application of the immobilized mercuric reductase for continuous treatment of Hg(II)-containing water was examined in a fixed bed reactor. Space-time yields up to 510 nmol/min{center_dot}mL were attained. The kinetics of immobilized enzyme systems were not diffusion-controlled. 22 refs., 7 figs., 2 tabs.

  14. Ferric reductase activity in Azotobacter vinelandii and its inhibition by Zn2+.

    PubMed

    Huyer, M; Page, W J

    1989-07-01

    Ferric reductase activity was examined in Azotobacter vinelandii and was found to be located in the cytoplasm. The specific activities of soluble cell extracts were not affected by the iron concentration of the growth medium; however, activity was inhibited by the presence of Zn2+ during cell growth and also by the addition of Zn2+ to the enzyme assays. Intracellular Fe2+ levels were lower and siderophore production was increased in Zn2+-grown cells. The ferric reductase was active under aerobic conditions, had an optimal pH of approximately 7.5, and required flavin mononucleotide and Mg2+ for maximum activity. The enzyme utilized NADH to reduce iron supplied as a variety of iron chelates, including the ferrisiderophores of A. vinelandii. The enzyme was purified by conventional protein purification techniques, and the final preparation consisted of two major proteins with molecular weights of 44,600 and 69,000. The apparent Km values of the ferric reductase for Fe3+ (supplied as ferric citrate) and NADH were 10 and 15.8 microM, respectively, and the data for the enzyme reaction were consistent with Ping Pong Bi Bi kinetics. The approximate Ki values resulting from inhibition of the enzyme by Zn2+, which was a hyperbolic (partial) mixed-type inhibitor, were 25 microM with respect to iron and 1.7 microM with respect to NADH. These results suggested that ferric reductase activity may have a regulatory role in the processes of iron assimilation in A. vinelandii. PMID:2525550

  15. Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn (Macrobrachium rosenbergii): effects on the oxidative stress, antioxidant enzymatic activities and apoptosis in haemocytes.

    PubMed

    Zhang, Yufan; Ye, Chaoxia; Wang, Anli; Zhu, Xuan; Chen, Changhong; Xian, Jianan; Sun, Zhenzhu

    2015-10-01

    The residual contaminators such as ammonia and nitrite are widely considered as relevant sources of aquatic environmental pollutants, posing a great threat to shrimp survival. To study the toxicological effects of ammonia and nitrite exposure on the innate immune response in invertebrates, we investigated the oxidative stress and apoptosis in haemocytes of freshwater prawn (Macrobrachium rosenbergii) under isolated and combined exposure to ammonia and nitrite in order to provide useful information about adult prawn immune responses. M. rosenbergii (13.44 ± 2.75 g) were exposed to 0, 5, and 25 mg/L total ammonia-N (TAN) and 0, 5, and 20 mg/L nitrite-N for 24 h. All ammonia concentrations were combined with all nitrite concentrations, making a total of nine treatments studied. Following the exposure treatment, antioxidant enzyme activity, reactive oxygen species (ROS) generation, nitric oxide (NO) generation, and apoptotic cell ratio of haemocytes were measured using flow cytometry. Results indicated that ROS generation was sensitive to the combined effect of ammonia and nitrite, which subsequently affected the Cu-Zn SOD activity. In addition, CAT showed the highest activity at 5 mg/L TAN while GPx decreased at 5 mg/L TAN and returned towards baseline at 25 mg/L. NO generation synchronized with the apoptotic cell ratio in haemocytes, indicating that NO production was closely associated with programmed cell death. Both NO production and apoptotic ratios significantly decreased following 25 mg/L TAN, which may be due to the antagonistic regulation of NO and GPx. We hypothesized that the toxicological effect of nitrite exhibited less change in physiological changes compared to that of ammonia, because of the high tolerance to nitrite exposure in mature M. rosenbergii and/or the competitive effects of chloride ions. Taken together, these results showed that ammonia and nitrite caused a series of combined oxidative stress and apoptosis in M. rosenbergi, but further

  16. Sodium nitrite exerts an antihypertensive effect and improves endothelial function through activation of eNOS in the SHR.

    PubMed

    Ling, Wei Chih; Murugan, Dharmani Devi; Lau, Yeh Siang; Vanhoutte, Paul M; Mustafa, Mohd Rais

    2016-01-01

    Sodium nitrite (NaNO2) induces relaxation in isolated arteries partly through an endothelium-dependent mechanism involving NO-eNOS-sGC-cGMP pathway. The present study was designed to investigate the effect of chronic NaNO2 administration on arterial systolic blood pressure (SBP) and vascular function in hypertensive rats. NaNO2 (150 mg L-1) was given in drinking water for four weeks to spontaneously (SHR) and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) treated hypertensive SD rats. Arterial SBP and vascular function in isolated aortae were studied. Total plasma nitrate/nitrite and vascular cyclic guanosine monophosphate (cGMP) levels were measured using commercially available assay kits. Vascular nitric oxide (NO) levels were evaluated by DAF-FM fluorescence while the proteins involved in endothelial nitric oxide synthase (eNOS) activation was determined by Western blotting. NaNO2 treatment reduced SBP, improved the impaired endothelium-dependent relaxation, increased plasma total nitrate/nitrite level and vascular tissue NO and cGMP levels in SHR. Furthermore, increased presence of phosphorylated eNOS and Hsp-90 was observed in NaNO2-treated SHR. The beneficial effect of nitrite treatment was not observed in L-NAME treated hypertensive SD rats. The present study provides evidence that chronic treatment of genetically hypertensive rats with NaNO2 improves endothelium-dependent relaxation in addition to its antihypertensive effect, partly through mechanisms involving activation of eNOS. PMID:27616322

  17. The Desulfuromonas acetoxidans Triheme Cytochrome c7 Produced in Desulfovibrio desulfuricans Retains Its Metal Reductase Activity

    PubMed Central

    Aubert, Corinne; Lojou, Elisabeth; Bianco, Pierre; Rousset, Marc; Durand, Marie-Claire; Bruschi, Mireille; Dolla, Alain

    1998-01-01

    Multiheme cytochrome c proteins that belong to class III have been recently shown to exhibit a metal reductase activity, which could be of great environmental interest, especially in metal bioremediation. To get a better understanding of these activities, the gene encoding cytochrome c7 from the sulfur-reducing bacterium Desulfuromonas acetoxidans was cloned from genomic DNA by PCR and expressed in Desulfovibrio desulfuricans G201. The expression system was based on the cyc transcription unit from Desulfovibrio vulgaris Hildenborough and led to the synthesis of holocytochrome c7 when transferred by electrotransformation into the sulfate reducer Desulfovibrio desulfuricans G201. The produced cytochrome was indistinguishable from the protein purified from Desulfuromonas acetoxidans cells with respect to several biochemical and biophysical criteria and exhibited the same metal reductase activities as determined from electrochemical experiments. This suggests that the molecule was correctly folded in the host organism. Desulfovibrio desulfuricans produces functional multiheme c-type cytochromes from bacteria belonging to a different genus and may be considered a suitable host for the heterologous biogenesis of multiheme c-type cytochromes for either structural or engineering studies. This report, which presents the first example of the transformation of a Desulfovibrio desulfuricans strain by electrotransformation, describes work that is the first necessary step of a protein engineering program that aims to specify the structural features that are responsible for the metal reductase activities of multiheme cytochrome c7. PMID:9546165

  18. Mineral supplementation increases erythrose reductase activity in erythritol biosynthesis from glycerol by Yarrowia lipolytica.

    PubMed

    Tomaszewska, Ludwika; Rymowicz, Waldemar; Rywińska, Anita

    2014-03-01

    The aim of this study was to examine the impact of divalent copper, iron, manganese, and zinc ions on the production of erythritol from glycerol by Yarrowia lipolytica and their effect on the activity of erythrose reductase. No inhibitory effect of the examined minerals on yeast growth was observed in the study. Supplementation with MnSO4 · 7H2O (25 mg l(-1)) increased erythritol production by Y. lipolytica by 14.5%. In the bioreactor culture with manganese ion addition, 47.1 g l(-1) of erythritol was produced from 100.0 g l(-1) of glycerol, which corresponded to volumetric productivity of 0.87 g l(-1) h(-1). The addition of Mn(2+) enhanced the intracellular activity of erythrose reductase up to 24.9 U g(-1) of dry weight of biomass (DW), hence, about 1.3 times more than in the control. PMID:24488778

  19. 5-alpha reductase inhibitors in patients on active surveillance: do the benefits outweigh the risk?

    PubMed

    Al Edwan, Ghazi; Fleshner, Neil

    2013-06-01

    Prostate cancer (PCa) is a slow, progressive disease. Prostate specific antigen testing, screening, and aggressive case identification has made PCa the most frequently diagnosed cancer. Concerns regarding overdiagnosis and overtreatment flourish on a large scale. In order to avoid overtreatment for those in whom therapeutic intervention is not required, active surveillance for eligible patients with the use of 5-alpha reductase can be considered a safe and a promising approach to delay the progression of the disease with minimal side effects. PMID:23579402

  20. Molecular cloning, expression and catalytic activity of a human AKR7 member of the aldo-keto reductase superfamily: evidence that the major 2-carboxybenzaldehyde reductase from human liver is a homologue of rat aflatoxin B1-aldehyde reductase.

    PubMed Central

    Ireland, L S; Harrison, D J; Neal, G E; Hayes, J D

    1998-01-01

    The masking of charged amino or carboxy groups by N-phthalidylation and O-phthalidylation has been used to improve the absorption of many drugs, including ampicillin and 5-fluorouracil. Following absorption of such prodrugs, the phthalidyl group is hydrolysed to release 2-carboxybenzaldehyde (2-CBA) and the pharmaceutically active compound; in humans, 2-CBA is further metabolized to 2-hydroxymethylbenzoic acid by reduction of the aldehyde group. In the present work, the enzyme responsible for the reduction of 2-CBA in humans is identified as a homologue of rat aflatoxin B1-aldehyde reductase (rAFAR). This novel human aldo-keto reductase (AKR) has been cloned from a liver cDNA library, and together with the rat protein, establishes the AKR7 family of the AKR superfamily. Unlike its rat homologue, human AFAR (hAFAR) appears to be constitutively expressed in human liver, and is widely expressed in extrahepatic tissues. The deduced human and rat protein sequences share 78% identity and 87% similarity. Although the two AKR7 proteins are predicted to possess distinct secondary structural features which distinguish them from the prototypic AKR1 family of AKRs, the catalytic- and NADPH-binding residues appear to be conserved in both families. Certain of the predicted structural features of the AKR7 family members are shared with the AKR6 beta-subunits of voltage-gated K+-channels. In addition to reducing the dialdehydic form of aflatoxin B1-8,9-dihydrodiol, hAFAR shows high affinity for the gamma-aminobutyric acid metabolite succinic semialdehyde (SSA) which is structurally related to 2-CBA, suggesting that hAFAR could function as both a SSA reductase and a 2-CBA reductase in vivo. This hypothesis is supported in part by the finding that the major peak of 2-CBA reductase activity in human liver co-purifies with hAFAR protein. PMID:9576847

  1. Studies on the nitrate reductase activities of the fruit and the source leaf in pepper

    SciTech Connect

    Achhireddy, N.R.; Beevers, L.; Fletcher, J.S.

    1983-12-01

    Nitrate reductase (NR) activity (NO/sub 2//sup -/ produced in the dark and under anaerobic conditions) of 30-day-old fruit of Capsicum annuum L. was 2.2% that in tissues of a single leaf adjacent to each fruit (33 vs. 1500 nmoles/hr-g fresh weight). The optimal NR activity in one source leaf could only account for about 17% of the fruit's total nitrogen accumulation, while the fruit's own NR activity was almost negligible. Covered and uncovered fruits did not differ significantly in NR activities. 19 references, 1 figure, 1 table.

  2. Distinguishing two groups of flavin reductases by analyzing the protonation state of an active site carboxylic acid.

    PubMed

    Dumit, Verónica I; Cortez, Néstor; Matthias Ullmann, G

    2011-07-01

    Flavin-containing reductases are involved in a wide variety of physiological reactions such as photosynthesis, nitric oxide synthesis, and detoxification of foreign compounds, including therapeutic drugs. Ferredoxin-NADP(H)-reductase (FNR) is the prototypical enzyme of this family. The fold of this protein is highly conserved and occurs as one domain of several multidomain enzymes such as the members of the diflavin reductase family. The enzymes of this family have emerged as fusion of a FNR and a flavodoxin. Although the active sites of these enzymes are very similar, different enzymes function in opposite directions, that is, some reduce oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) and some oxidize reduced nicotinamide adenine dinucleotide phosphate (NADPH). In this work, we analyze the protonation behavior of titratable residues of these enzymes through electrostatic calculations. We find that a highly conserved carboxylic acid in the active site shows a different titration behavior in different flavin reductases. This residue is deprotonated in flavin reductases present in plastids, but protonated in bacterial counterparts and in diflavin reductases. The protonation state of the carboxylic acid may also influence substrate binding. The physiological substrate for plastidic enzymes is NADP(+), but it is NADPH for the other mentioned reductases. In this article, we discuss the relevance of the environment of this residue for its protonation and its importance in catalysis. Our results allow to reinterpret and explain experimental data. PMID:21538544

  3. Process-driven bacterial community dynamics are key to cured meat colour formation by coagulase-negative staphylococci via nitrate reductase or nitric oxide synthase activities.

    PubMed

    Sánchez Mainar, María; Leroy, Frédéric

    2015-11-01

    The cured colour of European raw fermented meats is usually achieved by nitrate-into-nitrite reduction by coagulase-negative staphylococci (CNS), subsequently generating nitric oxide to form the relatively stable nitrosomyoglobin pigment. The present study aimed at comparing this classical curing procedure, based on nitrate reductase activity, with a potential alternative colour formation mechanism, based on nitric oxide synthase (NOS) activity, under different acidification profiles. To this end, meat models with and without added nitrate were fermented with cultures of an acidifying strain (Lactobacillus sakei CTC 494) and either a nitrate-reducing Staphylococcus carnosus strain or a rare NOS-positive CNS strain (Staphylococcus haemolyticus G110), or by relying on the background microbiota. Satisfactory colour was obtained in the models prepared with added nitrate and S. carnosus. In the presence of nitrate but absence of added CNS, however, cured colour was only obtained when L. sakei CTC 494 was also omitted. This was ascribed to the pH dependency of the emerging CNS background microbiota, selecting for nitrate-reducing Staphylococcus equorum strains at mild acidification conditions but for Staphylococcus saprophyticus strains with poor colour formation capability when the pH decrease was more rapid. This reliance of colour formation on the composition of the background microbiota was further explored by a side experiment, demonstrating the heterogeneity in nitrate reduction of a set of 88 CNS strains from different species. Finally, in all batches prepared with S. haemolyticus G110, colour generation failed as the strain was systematically outcompeted by the background microbiota, even when imposing milder acidification profiles. Thus, when aiming at colour formation through CNS metabolism, technological processing can severely interfere with the composition and functionality of the meat-associated CNS communities, for both nitrate reductase and NOS activities

  4. Major peptides from amaranth (Amaranthus cruentus) protein inhibit HMG-CoA reductase activity.

    PubMed

    Soares, Rosana Aparecida Manólio; Mendonça, Simone; de Castro, Luíla Ívini Andrade; Menezes, Amanda Caroline Cardoso Corrêa Carlos; Arêas, José Alfredo Gomes

    2015-01-01

    The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect. PMID:25690031

  5. Major Peptides from Amaranth (Amaranthus cruentus) Protein Inhibit HMG-CoA Reductase Activity

    PubMed Central

    Soares, Rosana Aparecida Manólio; Mendonça, Simone; de Castro, Luíla Ívini Andrade; Menezes, Amanda Caroline Cardoso Corrêa Carlos; Arêas, José Alfredo Gomes

    2015-01-01

    The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect. PMID:25690031

  6. Novel prenylated bichalcone and chalcone from Humulus lupulus and their quinone reductase induction activities.

    PubMed

    Yu, Liyan; Zhang, Fuxian; Hu, Zhijuan; Ding, Hui; Tang, Huifang; Ma, Zhongjun; Zhao, Xiaofeng

    2014-03-01

    A new prenylated chalcone xanthohumol M (1), a novel prenylated bichalcone humulusol (2) and six known chalcones (3-8) were found from Humulus lupulus. Their structures were determined by spectroscopic methods. All the chalcones' electrophilic abilities were assessed by GSH (glutathione) rapid screening, and their QR (quinone reductase) induction activities were evaluated using hepa 1c1c7 cells. The results of electrophilic assay and QR induction activity assay were quite well. New compounds 1 and 2, along with some known prenylated chalcones, displayed certain QR induction activity. PMID:24397993

  7. Molecular Underpinnings of Nitrite Effect on CymA-Dependent Respiration in Shewanella oneidensis

    PubMed Central

    Jin, Miao; Fu, Huihui; Yin, Jianhua; Yuan, Jie; Gao, Haichun

    2016-01-01

    Shewanella exhibit a remarkable versatility of respiration, with a diverse array of electron acceptors (EAs). In environments where these bacteria thrive, multiple EAs are usually present. However, we know little about strategies by which these EAs and their interaction affect ecophysiology of Shewanella. In this study, we demonstrate in the model strain, Shewanella oneidensis MR-1, that nitrite, not through nitric oxide to which it may convert, inhibits respiration of fumarate, and probably many other EAs whose reduction depends on quinol dehydrogenase CymA. This is achieved via the repression of cyclic adenosine monophosphate (cAMP) production, a second messenger required for activation of cAMP-receptor protein (Crp) which plays a primary role in regulation of respiration. If nitrite is not promptly removed, intracellular cAMP levels drop, and this impairs Crp activity. As a result, the production of nitrite reductase NrfA, CymA, and fumarate reductase FccA is substantially reduced. In contrast, nitrite can be simultaneously respired with trimethylamine N-oxide, resulting in enhanced biomass. PMID:27493647

  8. Aldose Reductase Inhibitory Activity of Compounds from  Zea mays L.

    PubMed Central

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

    2013-01-01

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

  9. Azo-reductase activated budesodine prodrugs for colon targeting.

    PubMed

    Marquez Ruiz, Juan F; Kedziora, Kinga; O'Reilly, Mary; Maguire, Jacqueline; Keogh, Brian; Windle, Henry; Kelleher, Dermot P; Gilmer, John F

    2012-12-15

    Budesodine is a synthetic glurocorticoid that undergoes substantial first pass metabolism, limiting systemic exposure. Its use in treatment of inflammatory bowel disease would benefit from a targeting strategy that could lead to a local topical effect, improving safety and increasing anti-inflammatory efficacy. A two-step prodrug strategy involving azoreduction/cyclization that we developed previously for prednisolone is here applied with some variations to budesonide. The budesodine prodrugs were tested using an in vitro azoreductase assay simulating human colonic microflora. The kinetics of amino steroid ester cyclization and its pH dependence was also evaluated. The stability of the prodrugs systems in simulated human duodenal and gastric fluid was evaluated to determine the likelihood of intact intestinal transit. The propionic acid derived prodrug 3 undergoes rapid activation by Clostridium perfingens and its putative reduction product cyclizes with acceptable rapidity when synthesized independently. These properties of 3 suggest that it has potential in management of ulcerative colitis. PMID:23122819

  10. Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

    USGS Publications Warehouse

    Stolz, J.F.; Gugliuzza, T.; Switzer, Blum J.; Oremland, R.; Martinez, Murillo F.

    1997-01-01

    The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b554 (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b556 (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth.

  11. Key Residues Regulating the Reductase Activity of the Human Mitochondrial Apoptosis Inducing Factor.

    PubMed

    Villanueva, Raquel; Ferreira, Patricia; Marcuello, Carlos; Usón, Alejandro; Miramar, M Dolores; Peleato, M Luisa; Lostao, Anabel; Susin, Santos A; Medina, Milagros

    2015-08-25

    The human Apoptosis Inducing Factor (hAIF) is a bifunctional NAD(P)H-dependent flavoreductase involved in both mitochondrial energy metabolism and caspase-independent cell death. Even though several studies indicate that both functions are redox controlled by NADH binding, the exact role of hAIF as a reductase in healthy mitochondria remains unknown. Upon reduction by NADH, hAIF dimerizes and produces very stable flavin/nicotinamide charge transfer complexes (CTC), by stacking of the oxidized nicotinamide moiety of the NAD(+) coenzyme against the re-face of the reduced flavin ring of its FAD cofactor. Such complexes are critical to restrict the hAIF efficiency as a reductase. The molecular basis of the hAIF reductase activity is here investigated by analyzing the role played by residues contributing to the interaction of the FAD isoalloxazine ring and of the nicotinamide moiety of NADH at the active site. Mutations at K177 and E314 produced drastic effects on the hAIF ability to retain the FAD cofactor, indicating that these residues are important to set up the holo-enzyme active site conformation. Characterization of P173G hAIF indicates that the stacking of P173 against the isoalloxazine ring is relevant to determine the flavin environment and to modulate the enzyme affinity for NADH. Finally, the properties of the F310G and H454S hAIF mutants indicate that these two positions contribute to form a compact active site essential for NADH binding, CTC stabilization, and NAD(+) affinity for the reduced state of hAIF. These features are key determinants of the particular behavior of hAIF as a NADH-dependent oxidoreductase. PMID:26237213

  12. Osmolarity and glucose differentially regulate aldose reductase activity in cultured mouse podocytes.

    PubMed

    Lewko, Barbara; Latawiec, Elżbieta; Maryn, Anna; Barczyńska, Anna; Pikuła, Michał; Zieliński, Maciej; Rybczyńska, Apolonia

    2011-01-01

    Podocyte injury is associated with progression of many renal diseases, including diabetic nephropathy. In this study we examined whether aldose reductase (AR), the enzyme implicated in diabetic complications in different tissues, is modulated by high glucose and osmolarity in podocyte cells. AR mRNA, protein expression, and activity were measured in mouse podocytes cultured in both normal and high glucose and osmolarity for 6 hours to 5 days. Hyperosmolarity acutely stimulated AR expression and activity, with subsequent increase of AR expression but decrease of activity. High glucose also elevated AR protein level; however, this was not accompanied by respective enzyme activation. Furthermore, high glucose appeared to counteract the osmolarity-dependent activation of AR. In conclusion, in podocytes AR is modulated by high glucose and increased osmolarity in a different manner. Posttranslational events may affect AR activity independent of enzyme protein amount. Activation of AR in podocytes may be implicated in diabetic podocytopathy. PMID:22253613

  13. Quinone Reductase 2 Is a Catechol Quinone Reductase

    SciTech Connect

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2008-09-05

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

  14. Conversion of NfsA, the Major Escherichia coli Nitroreductase, to a Flavin Reductase with an Activity Similar to That of Frp, a Flavin Reductase in Vibrio harveyi, by a Single Amino Acid Substitution

    PubMed Central

    Zenno, Shuhei; Kobori, Toshiro; Tanokura, Masaru; Saigo, Kaoru

    1998-01-01

    NfsA is the major oxygen-insensitive nitroreductase of Escherichia coli, similar in amino acid sequence to Frp, a flavin reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroreductase into a flavin reductase that is as active as the authentic Frp and a tartrazine reductase that is 30-fold more active than wild-type NfsA. PMID:9440535

  15. Adventitious Arsenate Reductase Activity of the Catalytic Domain of the Human Cdc25B and Cdc25C Phosphatases†

    PubMed Central

    Bhattacharjee, Hiranmoy; Sheng, Ju; Ajees, A. Abdul; Mukhopadhyay, Rita; Rosen, Barry P.

    2013-01-01

    A number of eukaryotic enzymes that function as arsenate reductases are homologues of the catalytic domain of the human Cdc25 phosphatase. For example, the Leishmania major enzyme LmACR2 is both a phosphatase and an arsenate reductase, and its structure bears similarity to the structure of the catalytic domain of human Cdc25 phosphatase. These reductases contain an active site C-X5-R signature motif, where C is the catalytic cysteine, the five X residues form a phosphate binding loop, and R is a highly conserved arginine, which is also present in human Cdc25 phosphatases. We therefore investigated the possibility that the three human Cdc25 isoforms might have adventitious arsenate reductase activity. The sequences for the catalytic domains of Cdc25A, -B, and -C were cloned individually into a prokaryotic expression vector, and their gene products were purified from a bacterial host using nickel affinity chromatography. While each of the three Cdc25 catalytic domains exhibited phosphatase activity, arsenate reductase activity was observed only with Cdc25B and -C. These two enzymes reduced inorganic arsenate but not methylated pentavalent arsenicals. Alteration of either the cysteine and arginine residues of the Cys-X5-Arg motif led to the loss of both reductase and phosphatase activities. Our observations suggest that Cdc25B and -C may adventitiously reduce arsenate to the more toxic arsenite and may also provide a framework for identifying other human protein tyrosine phosphatases containing the active site Cys-X5-Arg loop that might moonlight as arsenate reductases. PMID:20025242

  16. Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action.

    PubMed

    Lauer, T; Preik, M; Rassaf, T; Strauer, B E; Deussen, A; Feelisch, M; Kelm, M

    2001-10-23

    The plasma level of NO(x), i.e., the sum of NO(2)- and NO(3)-, is frequently used to assess NO bioavailability in vivo. However, little is known about the kinetics of NO conversion to these metabolites under physiological conditions. Moreover, plasma nitrite recently has been proposed to represent a delivery source for intravascular NO. We therefore sought to investigate in humans whether changes in NO(x) concentration are a reliable marker for endothelial NO production and whether physiological concentrations of nitrite are vasoactive. NO(2)- and NO(3)- concentrations were measured in blood sampled from the antecubital vein and brachial artery of 24 healthy volunteers. No significant arterial-venous gradient was observed for either NO(2)- or NO(3)-. Endothelial NO synthase (eNOS) stimulation with acetylcholine (1-10 microg/min) dose-dependently augmented venous NO(2)- levels by maximally 71%. This effect was paralleled by an almost 4-fold increase in forearm blood flow (FBF), whereas an equieffective dose of papaverine produced no change in venous NO(2)-. Intraarterial infusion of NO(2)- had no effect on FBF. NOS inhibition (N(G)-monomethyl-l-arginine; 4-12 micromol/min) dose-dependently reduced basal NO(2)- and FBF and blunted acetylcholine-induced vasodilation and NO release by more than 80% and 90%, respectively. In contrast, venous NO(3)- and total NO(x) remained unchanged as did systemic arterial NO(2)- and NO(3)- levels during all these interventions. FBF and NO release showed a positive association (r = 0.85; P < 0.001). These results contradict the current paradigm that plasma NO(3)- and/or total NO(x) are generally useful markers of endogenous NO production and demonstrate that only NO(2)- reflects acute changes in regional eNOS activity. Our results further demonstrate that physiological levels of nitrite are vasodilator-inactive. PMID:11606734

  17. Differentiation in the microbial ecology and activity of suspended and attached bacteria in a nitritation-anammox process.

    PubMed

    Park, Hongkeun; Sundar, Suneethi; Ma, Yiwei; Chandran, Kartik

    2015-02-01

    A directed differentiation between the biofilm and suspension was observed in the molecular microbial ecology and gene expression of different bacteria in a biofilm nitritation-anammox process operated at varying hydraulic residence times (HRT) and nitrogen loading rates (NLR). The highest degree of enrichment observed in the biofilm was of anaerobic ammonia-oxidizing bacteria (AMX) followed by that of Nitrospira spp. related nitrite-oxidizing bacteria (NOB). For AMX, a major shift from Candidatus "Brocadia fulgida" to Candidatus "Kuenenia stuttgartiensis" in both suspension and biofilm was observed with progressively shorter HRT, using discriminatory biomarkers targeting the hydrazine synthase (hzsA) gene. In parallel, expression of the hydrazine oxidoreductase gene (hzo), a functional biomarker for AMX energy metabolism, became progressively prominent in the biofilm. A marginal but statistically significant enrichment in the biofilm was observed for Nitrosomonas europaea related ammonia-oxidizing bacteria (AOB). In direct contrast to AMX, the gene expression of ammonia monooxygenase subunit A (amoA), a functional biomarker for AOB energy metabolism, progressively increased in suspension. Using gene expression and biomass concentration measures in conjunction, it was determined that signatures of AOB metabolism were primarily present in the biofilm throughout the study. On the other hand, AMX metabolism gradually shifted from being uniformly distributed in both the biofilm and suspension to primarily the biofilm at shorter HRTs and higher NLRs. These results therefore highlight the complexity and key differences in the microbial ecology, gene expression and activity between the biofilm and suspension of a nitritation-anammox process and the biokinetic and metabolic drivers for such niche segregation. PMID:25115980

  18. Glyphosate effect on shikimate, nitrate reductase activity, yield, and seed composition in corn.

    PubMed

    Reddy, Krishna N; Bellaloui, Nacer; Zablotowicz, Robert M

    2010-03-24

    When glyphosate is applied to glyphosate-resistant (GR) crops, drift to nonglyphosate-resistant (non-GR) crops may cause significant injury and reduce yields. Tools are needed to quantify injury and predict crop losses. In this study, glyphosate drift was simulated by direct application at 12.5% of the recommended label rate to non-GR corn (Zea mays L.) at 3 or 6 weeks after planting (WAP) during two field seasons in the Mississippi delta region of the southeastern USA. Visual plant injury, shikimate accumulation, nitrate reductase activity, leaf nitrogen, yield, and seed composition were evaluated. Effects were also evaluated in GR corn and GR corn with stacked glufosinate-resistant gene at the recommended label rate at 3 and 6 WAP. Glyphosate at 105 g ae/ha was applied once at 3 or 6 weeks after planting to non-GR corn. Glyphosate at 840 (lower label limit) or 1260 (upper label limit) g ae/ha was applied twice at 3 and 6 WAP to transgenic corn. Glyphosate caused injury (45-55%) and increased shikimate levels (24-86%) in non-GR compared to nontreated corn. In non-GR corn, glyphosate drift did not affect starch content but increased seed protein 8-21% while reducing leaf nitrogen reductase activity 46-64%, leaf nitrogen 7-16%, grain yield 49-54%, and seed oil 18-23%. In GR and GR stacked with glufosinate-resistant corn, glyphosate applied at label rates did not affect corn yield, leaf and seed nitrogen, or seed composition (protein, oil, and starch content). Yet, nitrate reductase activity was reduced 5-19% with glyphosate at 840 + 840 g/ha rate and 8-42% with glyphosate at 1260 + 1260 g/ha rate in both GR and GR stacked corn. These results demonstrate the potential for severe yield loss in non-GR corn exposed to glyphosate drift. PMID:20180575

  19. Human castration resistant prostate cancer rather prefer to decreased 5α-reductase activity

    PubMed Central

    Kosaka, Takeo; Miyajima, Akira; Nagata, Hirohiko; Maeda, Takahiro; Kikuchi, Eiji; Oya, Mototsugu

    2013-01-01

    Physiologically relevant steroid 5α-reductase (SRD5A) activity that is essential for dihydrotestosterone (DHT) biosynthesis in human castration-resistant prostate cancer (CRPC) has not been fully characterized yet. In this study to ascertain the potential SRD5A activity, we cultured two human CRPC cell lines, C4-2 and C4-2AT6, with the steroid precursor: 13C-[2,3,4]-androstenedione (13C-Adione), and analyzed the sequential biosynthesis of 13C-[2,3,4]-testosterone (13C-T) and 13C-[2,3,4]-DHT (13C-DHT) by liquid chromatography/mass spectrometry (LC/MS/MS). The 13C-DHT/13C-T concentration ratio detected by LC/MS/MS in C4-2AT6 cells appeared to reflect the SRD5A activity. The ratio in C4-2AT6 was significantly lower than that in C4-2. An increased concentration of DHT did not have a positive effect on cell proliferation, rather it exhibited inhibitory effects. 5α-reductase inhibitors did not have any inhibitory effect at clinically achievable concentrations. These results indicate that CRPC cells may have an unknown regulation system to protect themselves from an androgenic suppressive effect mediated by SRD5A activity. PMID:23429215

  20. The inhibitory activity of aldose reductase in vitro by constituents of Garcinia mangostana Linn.

    PubMed

    Fatmawati, Sri; Ersam, Taslim; Shimizu, Kuniyoshi

    2015-01-15

    We investigated aldose reductase inhibition of Garcinia mangostana Linn. from Indonesia. Dichloromethane extract of the root bark of this tree was found to demonstrate an IC50 value of 11.98 µg/ml for human aldose reductase in vitro. From the dichloromethane fraction, prenylated xanthones were isolated as potent human aldose reductase inhibitors. We discovered 3-isomangostin to be most potent against aldose reductase, with an IC50 of 3.48 µM. PMID:25636870

  1. Mutational analysis of the nor gene cluster which encodes nitric-oxide reductase from Paracoccus denitrificans.

    PubMed

    de Boer, A P; van der Oost, J; Reijnders, W N; Westerhoff, H V; Stouthamer, A H; van Spanning, R J

    1996-12-15

    The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower

  2. Activation of accumulated nitrite reduction by immobilized Pseudomonas stutzeri T13 during aerobic denitrification.

    PubMed

    Ma, Fang; Sun, Yilu; Li, Ang; Zhang, Xuening; Yang, Jixian

    2015-01-01

    The excellent removal efficiency of nitrate by the aerobic denitrifier, Pseudomonas stutzeri T13, was achieved in free cells system. However, poor nitrite reduction prevents efficient aerobic denitrification because of the nitrite accumulation. This problem could be conquered by immobilizing the cells on supports. In this study, strain T13 was immobilized by mycelial pellets (MPs), polyurethane foam cubes (PFCs) and sodium alginate beads (SABs). Higher removal percentages of TN in MP (43.78%), PFC (42.31%) and SAB (57.25%) systems were achieved compared with the free cell system (29.7%). Furthermore, the optimal condition for immobilized cell systems was as follows: 30°C, 100rpm shaking speed and pH 7. The shock-resistance of SAB system was relatively poor, which could collapse under either alkaline (pH=9) or high rotating (200rpm) conditions. The recycling experiments demonstrated that the high steady TN removal rate could be maintained for seven cycles in both MP and PFC systems. PMID:25827250

  3. Physicochemical nature of interfaces controlling ferredoxin NADP(+) reductase activity through its interprotein interactions with ferredoxin.

    PubMed

    Kinoshita, Misaki; Kim, Ju Yaen; Kume, Satoshi; Sakakibara, Yukiko; Sugiki, Toshihiko; Kojima, Chojiro; Kurisu, Genji; Ikegami, Takahisa; Hase, Toshiharu; Kimata-Ariga, Yoko; Lee, Young-Ho

    2015-10-01

    Although acidic residues of ferredoxin (Fd) are known to be essential for activities of various Fd-dependent enzymes, including ferredoxin NADP(+) reductase (FNR) and sulfite reductase (SiR), through electrostatic interactions with basic residues of partner enzymes, non-electrostatic contributions such as hydrophobic forces remain largely unknown. We herein demonstrated that intermolecular hydrophobic and charge-charge interactions between Fd and enzymes were both critical for enzymatic activity. Systematic site-directed mutagenesis, which altered physicochemical properties of residues on the interfaces of Fd for FNR /SiR, revealed various changes in activities of both enzymes. The replacement of serine 43 of Fd to a hydrophobic residue (S43W) and charged residue (S43D) increased and decreased FNR activity, respectively, while S43W showed significantly lower SiR activity without affecting SiR activity by S43D, suggesting that hydrophobic and electrostatic interprotein forces affected FNR activity. Enzyme kinetics revealed that changes in FNR activity by mutating Fd correlated with Km, but not with kcat or activation energy, indicating that interprotein interactions determined FNR activity. Calorimetry-based binding thermodynamics between Fd and FNR showed different binding modes of FNR to wild-type, S43W, or S43D, which were controlled by enthalpy and entropy, as shown by the driving force plot. Residue-based NMR spectroscopy of (15)N FNR with Fds also revealed distinct binding modes of each complex based on different directions of NMR peak shifts with similar overall chemical shift differences. We proposed that subtle adjustments in both hydrophobic and electrostatic forces were critical for enzymatic activity, and these results may be applicable to protein-based electron transfer systems. PMID:26087388

  4. Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide reductase.

    PubMed

    Kapetanaki, Sofia M; Field, Sarah J; Hughes, Ross J L; Watmough, Nicholas J; Liebl, Ursula; Vos, Marten H

    2008-01-01

    The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in approximately 13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site. PMID:18420024

  5. Arabidopsis nitrate reductase activity is stimulated by the E3 SUMO ligase AtSIZ1

    PubMed Central

    Park, Bong Soo; Song, Jong Tae; Seo, Hak Soo

    2011-01-01

    Small ubiquitin-related modifier (SUMO) is a small polypeptide that modulates protein activity and regulates hormone signalling, abiotic and biotic responses in plants. Here we show that AtSIZ regulates nitrogen assimilation in Arabidopsis through its E3 SUMO ligase function. Dwarf plants of siz1-2 flower early, show abnormal seed development and have high salicylic acid content and enhanced resistance to bacterial pathogens. These mutant phenotypes are reverted to wild-type phenotypes by exogenous ammonium but not by nitrate, phosphate or potassium. Decreased nitrate reductase activity in siz1-2 plants resulted in low nitrogen concentrations, low nitric oxide production and high nitrate content in comparison with wild-type plants. The nitrate reductases, NIA1 and NIA2, are sumoylated by AtSIZ1, which dramatically increases their activity. Both sumoylated and non-sumoylated NIA1 and NIA2 can form dimers. Our results indicate that AtSIZ1 positively controls nitrogen assimilation by promoting sumoylation of NRs in Arabidopsis. PMID:21772271

  6. Inducible chromate reductase exhibiting extracellular activity in Bacillus methylotrophicus for chromium bioremediation.

    PubMed

    Sandana Mala, John Geraldine; Sujatha, Dhanasingh; Rose, Chellan

    2015-01-01

    Chromium toxicity is one of the major causes of environmental pollution due to its heavy discharge in industrial wastewaters. Chromate reduction is a viable method to detoxify hexavalent chromium to nontoxic trivalent species mediated by enzymes and metabolites. A new Bacillus methylotrophicus strain was isolated from tannery sludge and was an efficient candidate for chromate reduction. An initial chromate reductase activity of 212.84 U/mg protein was obtained at 48 h in a low-cost defined medium formulation with 0.25 mM chromate. The extracellular enzyme was inducible at 12h substrate addition with 312.99 U/mg at 48 h. Reduced glutathione was required for enhanced specific activity of 356.48 U/mg. Enzyme activity was optimum at pH 7.0 and at 30 °C, and was stable in the presence of EDTA, DTT and metal ions. The enzyme exhibited a Vmax of 59.89 μM/min/mg protein and a Km of 86.5 μM, suggesting feasibility of the reaction with K₂Cr₂O₇ as substrate. Application of the crude reductase in tannery effluent resulted in 91.3% chromate reduction at 48 h. An enzyme-mediated chromate reduction process has therefore been developed for bioremediation of toxic chromium sp. in industrial effluents. PMID:24985094

  7. Arabidopsis nitrate reductase activity is stimulated by the E3 SUMO ligase AtSIZ1.

    PubMed

    Park, Bong Soo; Song, Jong Tae; Seo, Hak Soo

    2011-01-01

    Small ubiquitin-related modifier (SUMO) is a small polypeptide that modulates protein activity and regulates hormone signalling, abiotic and biotic responses in plants. Here we show that AtSIZ regulates nitrogen assimilation in Arabidopsis through its E3 SUMO ligase function. Dwarf plants of siz1-2 flower early, show abnormal seed development and have high salicylic acid content and enhanced resistance to bacterial pathogens. These mutant phenotypes are reverted to wild-type phenotypes by exogenous ammonium but not by nitrate, phosphate or potassium. Decreased nitrate reductase activity in siz1-2 plants resulted in low nitrogen concentrations, low nitric oxide production and high nitrate content in comparison with wild-type plants. The nitrate reductases, NIA1 and NIA2, are sumoylated by AtSIZ1, which dramatically increases their activity. Both sumoylated and non-sumoylated NIA1 and NIA2 can form dimers. Our results indicate that AtSIZ1 positively controls nitrogen assimilation by promoting sumoylation of NRs in Arabidopsis. PMID:21772271

  8. Nitrite reduction in paracoccus halodenitrificans: Evidence for the role of a cd-type cytochrome in ammonia formation

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Cronin, S. E.

    1984-01-01

    Cell-free extracts prepared from Paracoccus halodenitrificans catalyzed the reduction of nitrate to ammonia in the presence of dithionite and methyl viologen. Enzyme activity was located in the soluble fraction and was associated with a cytochrome whose spectral properties resembled those of a cd-type cytochrome. Unlike the sissimilatory cd-cytochrome nitrate reductase associated with the membrane fraction of P. halodenitrificans, this soluble cd-cytochrome did not reduce nitrite to nitrous oxide.

  9. Response to Arsenate Treatment in Schizosaccharomyces pombe and the Role of Its Arsenate Reductase Activity

    PubMed Central

    Matia-González, Ana M.; Sotelo, Jael; Zarco-Fernández, Sonia; Muñoz-Olivas, Riansares; Cámara, Carmen; Rodríguez-Gabriel, Miguel A.

    2012-01-01

    Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast. PMID:22912829

  10. Modulation of HMG-CoA reductase activity by pantetheine/pantethine.

    PubMed

    Cighetti, G; Del Puppo, M; Paroni, R; Galli Kienle, M

    1988-11-25

    The ability of pantetheine/pantethine to modulate the activity of HMG-CoA reductase (EC 1.1.1.34) was determined in vitro with rat liver microsomes. The decay of the activity was obtained with pantethine in the 10(-5)-10(-4) M range, whereas stimulation by pantetheine occurred at 10(-3)-10(-2) M, as previously reported for GSSG and GSH, respectively. Inhibition of HMG-CoA by pantethine in isolated liver cells was also investigated by measuring the enzyme activity in microsomes isolated from hepatocytes incubated without or with 1 mM pantethine under conditions previously shown by us to induce inhibition of cholesterol synthesis from acetate. The enzyme amount was not modified by pantethine, but in cells treated with the disulphide, the relative amounts of the thiolic active forms of the enzyme, both phosphorylated and dephosphorylated, were decreased to about half compared to controls. PMID:3196742

  11. Enhanced Xylitol Production by Mutant Kluyveromyces marxianus 36907-FMEL1 Due to Improved Xylose Reductase Activity.

    PubMed

    Kim, Jin-Seong; Park, Jae-Bum; Jang, Seung-Won; Ha, Suk-Jin

    2015-08-01

    A directed evolution and random mutagenesis were carried out with thermotolerant yeast Kluyveromyces marxianus ATCC 36907 for efficient xylitol production. The final selected strain, K. marxianus 36907-FMEL1, exhibited 120 and 39 % improvements of xylitol concentration and xylitol yield, respectively, as compared to the parental strain, K. marxianus ATCC 36907. According to enzymatic assays for xylose reductase (XR) activities, XR activity from K. marxianus 36907-FMEL1 was around twofold higher than that from the parental strain. Interestingly, the ratios of NADH-linked and NADPH-linked XR activities were highly changed from 1.92 to 1.30 when K. marxianus ATCC 36907 and K. marxianus 36907-FMEL1 were compared. As results of KmXYL1 genes sequencing, it was found that cysteine was substituted to tyrosine at position 36 after strain development which might cause enhanced XR activity from K. marxianus 36907-FMEL1. PMID:26043853

  12. Synthesis of benzothiadiazine derivatives exhibiting dual activity as aldose reductase inhibitors and antioxidant agents.

    PubMed

    Zhu, Shaojuan; Hao, Xin; Zhang, Shuzhen; Qin, Xiangyu; Chen, Xin; Zhu, Changjin

    2016-06-15

    Several multifunctional benzothiadiazine derivatives were synthesized and examined for their inhibition to the enzyme aldose reductase and in vitro antioxidant activity to identify novel drugs for diabetes and its complications. Most of them exhibited good inhibitory activity. Importantly, a number of compounds demonstrated strong antioxidant activity and one compound in particular was extremely active in the DPPH radical scavenging and MDA inhibition analysis. The DPPH radical scavenging rate with this compound was 98.0%, 92.3% and 42.1% at concentrations of 100μM, 10μM, and 1μM, respectively, and the initial reaction rate was faster than Trolox at a concentration of 10μM. PMID:27156769

  13. Methionine sulfoxide reductase: chemistry, substrate binding, recycling process and oxidase activity.

    PubMed

    Boschi-Muller, Sandrine; Branlant, Guy

    2014-12-01

    Three classes of methionine sulfoxide reductases are known: MsrA and MsrB which are implicated stereo-selectively in the repair of protein oxidized on their methionine residues; and fRMsr, discovered more recently, which binds and reduces selectively free L-Met-R-O. It is now well established that the chemical mechanism of the reductase step passes through formation of a sulfenic acid intermediate. The oxidized catalytic cysteine can then be recycled by either Trx when a recycling cysteine is operative or a reductant like glutathione in the absence of recycling cysteine which is the case for 30% of the MsrBs. Recently, it was shown that a subclass of MsrAs with two recycling cysteines displays an oxidase activity. This reverse activity needs the accumulation of the sulfenic acid intermediate. The present review focuses on recent insights into the catalytic mechanism of action of the Msrs based on kinetic studies, theoretical chemistry investigations and new structural data. Major attention is placed on how the sulfenic acid intermediate can be formed and the oxidized catalytic cysteine returns back to its reduced form. PMID:25108804

  14. Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis.

    PubMed

    Uthus, Eric O

    2010-06-01

    A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4'-sulfonyl derivative of l-methionine (dabsyl Met), the product of the enzymatic reactions when either dabsyl l-methionine S-sulfoxide or dabsyl l-methionine R-sulfoxide is used as a substrate. The method provides baseline resolution of the substrates and, therefore, can be used to easily determine the purity of the substrates. The method is rapid ( approximately 20min sample to sample), requires no column regeneration, and uses very small amounts of buffers. Separation was performed by using a 75-mum internal diameter polyimide-coated fused silica capillary (no inside coating) with 60cm total length (50cm to the detector window). Samples were separated at 22.5kV, and the separation buffer was 25mM KH(2)PO(4) (pH 8.0) containing 0.9ml of N-lauroylsarcosine (sodium salt, 30% [w/v] solution) per 100ml of buffer. Prior to use, the capillary was conditioned with the same buffer that also contained 25mM sodium dodecyl sulfate. The CE method is compared with high-performance liquid chromatography (HPLC) as determined by comparing results from measurements of hepatic enzyme activities in mice fed either deficient or adequate selenium. PMID:20167203

  15. Consequence of absence of nitrate reductase activity on photosynthesis in Nicotiana plumbaginifolia plants

    SciTech Connect

    Saux, C.; Lemoine, Y.; Marion-Poll, A.; Valadier, M.H.; Deng, M.; Morot-Gaudry, J.F.

    1987-05-01

    Chlorate-resistant Nicotiana plumbaginifolia (cv Viviani) mutants were found to be deficient in the nitrate reductase apoprotein (NR/sup -/ nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild-type Nicotiana tabacum plants. The grafts of mutant plants were chlorotic compared to the grafts of wild type. Mutant leaves did not accumulate nitrogen but contained less malate and more glutamine than wild leaves. They exhibited a slight increase of the proportion of the light-harvesting chlorophyll a/b protein complexes and a lowering of the efficiency of energy transfer between these complexes and the active centers. After a 3 second /sup 14/CO/sub 2/ pulse, the total /sup 14/C incorporation of the mutant leaves was approximately 20/sup 5/ of that of the control. The /sup 14/C was essentially recovered in ribulose bisphosphate in these plants. It was consistent with a decline of ribulose bisphosphate carboxylase activity observed in the mutant. After a 3 second /sup 14/CO/sub 2/ pulse followed by a 60 second chase with normal CO/sub 2/, /sup 14/C was mainly accumulated in starch which was labeled more in the mutant than in the wild type. These results confirm the observation that in the nitrate reductase deficient leaves, chloroplasts were loaded with large starch inclusions preceding disorganization of the photosynthetic apparatus.

  16. Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities.

    PubMed

    Kilgore, Matthew B; Holland, Cynthia K; Jez, Joseph M; Kutchan, Toni M

    2016-08-01

    Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities. Several groups of Amaryllidaceae alkaloids including the hemanthamine- and crinine-type alkaloids show promise as anticancer agents. Two reduction reactions are required for the production of these compounds: the reduction of norcraugsodine to norbelladine and the reduction of noroxomaritidine to normaritidine, with the enantiomer of noroxomaritidine dictating whether the derivatives will be the crinine-type or hemanthamine-type. It is also possible for the carbon-carbon double bond of noroxomaritidine to be reduced, forming the precursor for maritinamine or elwesine depending on the enantiomer reduced to an oxomaritinamine product. In this study, a short chain alcohol dehydrogenase/reductase that co-expresses with the previously discovered norbelladine 4'-O-methyltransferase from Narcissus sp. and Galanthus spp. was cloned and expressed in Escherichia coli Biochemical analyses and x-ray crystallography indicates that this protein functions as a noroxomaritidine reductase that forms oxomaritinamine from noroxomaritidine through a carbon-carbon double bond reduction. The enzyme also reduces norcraugsodine to norbelladine with a 400-fold lower specific activity. These studies identify a missing step in the biosynthesis of this pharmacologically important class of plant natural products. PMID:27252378

  17. Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter.

    PubMed Central

    Snape, J R; Walkley, N A; Morby, A P; Nicklin, S; White, G F

    1997-01-01

    Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively. PMID:9401040

  18. Crystal Structures of Manganese- and Cobalt-substituted Myoglobin in Complex with NO and Nitrite Reveal Unusual Ligand Conformations

    PubMed Central

    Zahran, Zaki N.; Chooback, Lilian; Copeland, Daniel M.; West, Ann H.; Richter-Addo, George B.

    2009-01-01

    Nitrite is now recognized as a storage pool of bioactive nitric oxide (NO). Hemoglobin (Hb) and myoglobin (Mb) convert, under certain conditions, nitrite to NO. This newly discovered nitrite reductase activity of Hb and Mb provides an attractive alternative to mammalian NO synthesis from the NO synthase pathway that requires dioxygen. We recently reported the X-ray crystal structure of the nitrite adduct of ferric horse heart Mb, and showed that the nitrite ligand binds in an unprecedented O-binding (nitrito) mode to the d5 ferric center in MbIII(ONO) (D. M. Copeland, A. Soares, A. H. West, G. B. Richter-Addo, J. Inorg. Biochem. 100 (2006) 1413-1425). We also showed that the distal pocket in Mb allows for different conformations of the NO ligand (120° and 144°) in MbIINO depending on the mode of preparation of the compound. In this article, we report the crystal structures of the nitrite and NO adducts of manganese-substituted hh Mb (a d4 system) and of the nitrite adduct of cobalt-substituted hh Mb (a d6 system). We show that the distal His64 residue directs the nitrite ligand towards the rare nitrito O-binding mode in MnIIIMb and CoIIIMb. We also report that the distal pocket residues allow a stabilization of an unprecendented bent MnNO moiety in MnIIMbNO. These crystal structural data, when combined with the data for the aquo, methanol, and azide MnMb derivatives, provide information on the role of distal pocket residues in the observed binding modes of nitrite and NO ligands to wild-type and metal-substituted Mb. PMID:17905436

  19. Membrane composition influences the activity of in vitro refolded human vitamin K epoxide reductase.

    PubMed

    Jaenecke, Frank; Friedrich-Epler, Beatrice; Parthier, Christoph; Stubbs, Milton T

    2015-10-27

    Human vitamin K epoxide reductase (hVKOR) is an integral membrane protein responsible for the maintenance of reduced vitamin K pools, a prerequisite for the action of γ-glutamyl carboxylase and hence for hemostasis. Here we describe the recombinant expression of hVKOR as an insoluble fusion protein in Escherichia coli, followed by purification and chemical cleavage under denaturing conditions. In vitro renaturation and reconstitution of purified solubilized hVKOR in phospholipids could be established to yield active protein. Crucially, the renatured enzyme is inhibited by the powerful coumarin anticoagulant warfarin, and we demonstrate that enzyme activity depends on lipid composition. The completely synthetic system for protein production allows a rational investigation of the multiple variables in membrane protein folding and paves the way for the provision of pure, active membrane protein for structural studies. PMID:26435421

  20. Oxidase, superoxide dismutase, and hydrogen peroxide reductase activities of methanobactin from types I and II methanotrophs.

    PubMed

    Choi, Dong W; Semrau, Jeremy D; Antholine, William E; Hartsel, Scott C; Anderson, Ryan C; Carey, Jeffrey N; Dreis, Ashley M; Kenseth, Erik M; Renstrom, Joel M; Scardino, Lori L; Van Gorden, Garrett S; Volkert, Anna A; Wingad, Aaron D; Yanzer, Paul J; McEllistrem, Marcus T; de la Mora, Arlene M; DiSpirito, Alan A

    2008-08-01

    Methanobactin (mb) is a copper-binding chromopeptide that appears to be involved in oxidation of methane by the membrane-associated or particulate methane monooxygenase (pMMO). To examine this potential physiological role, the redox and catalytic properties of mb from three different methanotrophs were examined in the absence and presence of O(2). Metal free mb from the type II methanotroph Methylosinus trichosporium OB3b, but not from the type I methanotrophs Methylococcus capsulatus Bath or Methylomicrobium album BG8, were reduced by a variety of reductants, including NADH and duroquinol, and catalyzed the reduction of O(2) to O(2)(-). Copper-containing mb (Cu-mb) from all three methanotrophs showed several interesting properties, including reductase dependent oxidase activity, dismutation of O(2)(-) to H(2)O(2), and the reductant dependent reduction of H(2)O(2) to H(2)O. The superoxide dismutase-like and hydrogen peroxide reductase activities of Cu-mb were 4 and 1 order(s) of magnitude higher, respectively, than the observed oxidase activity. The results demonstrate that Cu-mb from all three methanotrophs are redox-active molecules and oxygen radical scavengers, with the capacity to detoxify both superoxide and hydrogen peroxide without the formation of the hydroxyl radicals associated with Fenton reactions. As previously observed with Cu-mb from Ms. trichosporium OB3b, Cu-mb from both type I methanotrophs stimulated pMMO activity. However, in contrast to previous studies using mb from Ms. trichosporium OB3b, pMMO activity was not inhibited by mb from the two type I methanotrophs at low copper to mb ratios. PMID:18372044

  1. Metal complexes with 2-acetylpyridine-N(4)-orthochlorophenylthiosemicarbazone: cytotoxicity and effect on the enzymatic activity of thioredoxin reductase and glutathione reductase.

    PubMed

    Parrilha, Gabrieli L; Ferraz, Karina S O; Lessa, Josane A; de Oliveira, Kely Navakoski; Rodrigues, Bernardo L; Ramos, Jonas P; Souza-Fagundes, Elaine M; Ott, Ingo; Beraldo, Heloisa

    2014-09-12

    Metal complexes with 2-acetylpyridine-N(4)-orthochlorophenylthiosemicarbazone (H2Ac4oClPh) were assayed for their cytotoxicity against MCF-7 breast adenocarcinoma and HT-29 colon carcinoma cells. The thiosemicarbazone and most of the complexes were highly cytotoxic. H2Ac4oClPh and its gallium(III) and tin(IV) complexes did not show any inhibitory activity against thioredoxin reductase (TrxR) and glutathione reductase (GR). The palladium(II), platinum(II) and bismuth(III) complexes inhibited TrxR at micromolar concentrations but not GR. The antimony(III) and gold(III) complexes strongly inhibited TrxR at submicromolar doses with GR inhibition at higher concentrations. The selectivity of these complexes for TrxR suggests metal binding to a selenol residue in the active site of the enzyme. TrxR inhibition is likely a contributing factor to the mode of action of the gold and antimony derivatives. PMID:25058344

  2. Determination of oenothein B as the active 5-alpha-reductase-inhibiting principle of the folk medicine Epilobium parviflorum.

    PubMed

    Lesuisse, D; Berjonneau, J; Ciot, C; Devaux, P; Doucet, B; Gourvest, J F; Khemis, B; Lang, C; Legrand, R; Lowinski, M; Maquin, P; Parent, A; Schoot, B; Teutsch, G

    1996-05-01

    Several extracts from Epilobium parviflorum, a plant used in Central Europe for the treatment of prostate disorders, were evaluated in a biochemical assay with 5-alpha-reductase. The aqueous extract displaying inhibition of the enzyme was analyzed, the fraction responsible for this activity was purified, and the active compound identified as a macrocyclic tannin, oenothein B (1). PMID:8778238

  3. Xanthones with quinone reductase-inducing activity from the fruits of Garcinia mangostana (Mangosteen).

    PubMed

    Chin, Young-Won; Jung, Hyun-Ah; Chai, Heebyung; Keller, William J; Kinghorn, A Douglas

    2008-02-01

    Bioactivity-guided fractionation of a dichloromethane-soluble extract of Garcinia mangostana fruits has led to the isolation and identification of five compounds, including two xanthones, 1,2-dihydro-1,8,10-trihydroxy-2-(2-hydroxypropan-2-yl)-9-(3-methylbut-2-enyl)furo[3,2-a]xanthen-11-one (1) and 6-deoxy-7-demethylmangostanin (2), along with three known compounds, 1,3,7-trihydroxy-2,8-di-(3-methylbut-2-enyl)xanthone (3), mangostanin (4), and alpha-mangostin (5). The structures of compounds 1 and 2 were determined from analysis of their spectroscopic data. All isolated compounds in the present study together with eleven other compounds previously isolated from the pericarp of mangosteen, were tested in an in vitro quinone reductase-induction assay using murine hepatoma cells (Hepa 1c1c7) and an in vitro hydroxyl radical antioxidant assay. Of these, compounds 1-4 induced quinone reductase (concentration to double enzyme induction, 0.68-2.2microg/mL) in Hepa 1c1c7 cells and gamma-mangostin (6) exhibited hydroxyl radical-scavenging activity (IC50, 0.20microg/mL). PMID:17991497

  4. Esculetin, a Coumarin Derivative, Inhibits Aldose Reductase Activity in vitro and Cataractogenesis in Galactose-Fed Rats

    PubMed Central

    Kim, Chan-Sik; Kim, Junghyun; Lee, Yun Mi; Sohn, Eunjin; Kim, Jin Sook

    2016-01-01

    Naturally occurring coumarin compounds have received substantial attention due to their pharmaceutical effects. Esculetin is a coumarin derivative and a polyphenol compound that is used in a variety of therapeutic and pharmacological strategies. However, its effect on aldose reductase activity remains poorly understood. In this study, the potential beneficial effects of esculetin on lenticular aldose reductase were investigated in galactose-fed (GAL) rats, an animal model of sugar cataracts. Cataracts were induced in Sprague-Dawley (SD) rats via a 50% galactose diet for 2 weeks, and groups of GAL rats were orally treated with esculetin (10 or 50 mg/kg body weight). In vehicle-treated GAL rats, lens opacification was observed, and swelling and membrane rupture of the lens fiber cells were increased. Additionally, aldose reductase was highly expressed in the lens epithelium and superficial cortical fibers during cataract development in the GAL rats. Esculetin reduced rat lens aldose reductase (RLAR) activity in vitro, and esculetin treatment significantly inhibited lens opacity, as well as morphological alterations, such as swelling, vacuolation and liquefaction of lens fibers, via the inhibition of aldose reductase in the GAL rats. These results indicate that esculetin is a useful treatment for galactose-induced cataracts. PMID:26902086

  5. Esculetin, a Coumarin Derivative, Inhibits Aldose Reductase Activity in vitro and Cataractogenesis in Galactose-Fed Rats.

    PubMed

    Kim, Chan-Sik; Kim, Junghyun; Lee, Yun Mi; Sohn, Eunjin; Kim, Jin Sook

    2016-03-01

    Naturally occurring coumarin compounds have received substantial attention due to their pharmaceutical effects. Esculetin is a coumarin derivative and a polyphenol compound that is used in a variety of therapeutic and pharmacological strategies. However, its effect on aldose reductase activity remains poorly understood. In this study, the potential beneficialeffects of esculetin on lenticular aldose reductase were investigated in galactose-fed (GAL) rats, an animal model of sugar cataracts. Cataracts were induced in Sprague-Dawley (SD) rats via a 50% galactose diet for 2 weeks, and groups of GAL rats were orally treated with esculetin (10 or 50 mg/kg body weight). In vehicle-treated GAL rats, lens opacificationwas observed, and swelling and membrane rupture of the lens fibercells were increased. Additionally, aldose reductase was highly expressed in the lens epithelium and superficialcortical fibersduring cataract development in the GAL rats. Esculetin reduced rat lens aldose reductase (RLAR) activity in vitro, and esculetin treatment significanty inhibited lens opacity, as well as morphological alterations, such as swelling, vacuolation and liquefaction of lens fibers,via the inhibition of aldose reductase in the GAL rats. These results indicate that esculetin is a useful treatment for galactose-induced cataracts. PMID:26902086

  6. Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS–NO–nitrite signaling

    PubMed Central

    Yazji, Ibrahim; Sodhi, Chhinder P.; Lee, Elizabeth K.; Good, Misty; Egan, Charlotte E.; Afrazi, Amin; Neal, Matthew D.; Jia, Hongpeng; Lin, Joyce; Branca, Maria F.; Prindle, Thomas; Richardson, Ward M.; Ozolek, John; Billiar, Timothy R.; Binion, David G.; Gladwin, Mark T.; Hackam, David J.

    2013-01-01

    Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS−/− mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate—a precursor for enteral generation of nitrite and nitric oxide—and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate–nitrite–NO signaling. PMID:23650378

  7. Selective nitrite reduction at heterobimetallic CoMg complexes.

    PubMed

    Uyeda, Christopher; Peters, Jonas C

    2013-08-14

    Heme-containing nitrite reductases bind and activate nitrite by a mechanism that is proposed to involve interactions with Brønsted acidic residues in the secondary coordination sphere. To model this functionality using synthetic platforms that incorporate a Lewis acidic site, heterobimetallic CoMg complexes supported by diimine-dioxime ligands are described. The neutral (μ-NO2)CoMg species 3 is synthesized from the [(μ-OAc)(Br)CoMg](+) complex 1 by a sequence of one-electron reduction and ligand substitution reactions. Data are presented for a redox series of nitrite adducts, featuring a conserved μ-(η(1)-N:η(1)-O)-NO2 motif, derived from this synthon. Conditions are identified for the proton-induced N-O bond heterolysis of bound NO2(-) in the most reduced member of this series, affording the [(NO)(Cl)CoMg(H2O)](+) complex 6. Reduction of this complex followed by protonation leads to the evolution of free N2O. On the basis of these stoichiometric reactivity studies, the competence of complex 1 as a NO2(-) reduction catalyst is evaluated using electrochemical methods. In bulk electrolysis experiments, conducted at -1.2 V vs SCE using Et3NHCl as a proton source, N2O is produced selectively without the competing formation of NH3, NH2OH, or H2. PMID:23865638

  8. Thioredoxin reductase.

    PubMed

    Mustacich, D; Powis, G

    2000-02-15

    The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases. PMID:10657232

  9. Hexavalent Chromate Reductase Activity in Cell Free Extracts of Penicillium sp.

    PubMed Central

    Arévalo-Rangel, Damaris L.; Cárdenas-González, Juan F.; Martínez-Juárez, Víctor M.; Acosta-Rodríguez, Ismael

    2013-01-01

    A chromium-resistant fungus isolated from contaminated air with industrial vapors can be used for reducing toxic Cr(VI) to Cr(III). This study analyzes in vitro reduction of hexavalent chromium using cell free extract(s) of the fungus that was characterized based on optimal temperature, pH, use of electron donors, metal ions and initial Cr(VI) concentration in the reaction mixture. This showed the highest activity at 37°C and pH 7.0; there is an increase in Cr(VI) reductase activity with addition of NADH as an electron donor, and it was highly inhibited by Hg2+, Ca2+ and Mg2+, and azide, EDTA, and KCN. PMID:24027493

  10. Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones.

    PubMed

    Johansson, Renzo; Jonna, Venkateswara Rao; Kumar, Rohit; Nayeri, Niloofar; Lundin, Daniel; Sjöberg, Britt-Marie; Hofer, Anders; Logan, Derek T

    2016-06-01

    Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone. PMID:27133024

  11. Nitrite in feed: From Animal health to human health

    SciTech Connect

    Cockburn, Andrew; Brambilla, Gianfranco; Fernández, Maria-Luisa; Arcella, Davide; Peteghem, Carlos van; Dorne, Jean-Lou

    2013-08-01

    Nitrite is widely consumed from the diet by animals and humans. However the largest contribution to exposure results from the in vivo conversion of exogenously derived nitrate to nitrite. Because of its potential to cause to methaemoglobin (MetHb) formation at excessive levels of intake, nitrite is regulated in feed and water as an undesirable substance. Forages and contaminated water have been shown to contain high levels of nitrate and represent the largest contributor to nitrite exposure for food-producing animals. Interspecies differences in sensitivity to nitrite intoxication principally result from physiological and anatomical differences in nitrite handling. In the case of livestock both pigs and cattle are relatively susceptible. With pigs this is due to a combination of low levels of bacterial nitrite reductase and hence potential to reduce nitrite to ammonia as well as reduced capacity to detoxify MetHb back to haemoglobin (Hb) due to intrinsically low levels of MetHb reductase. In cattle the sensitivity is due to the potential for high dietary intake and high levels of rumen conversion of nitrate to nitrite, and an adaptable gut flora which at normal loadings shunts nitrite to ammonia for biosynthesis. However when this escape mechanism gets overloaded, nitrite builds up and can enter the blood stream resulting in methemoglobinemia. Looking at livestock case histories reported in the literature no-observed-effect levels of 3.3 mg/kg body weight (b.w.) per day for nitrite in pigs and cattle were estimated and related to the total daily nitrite intake that would result from complete feed at the EU maximum permissible level. This resulted in margins of safety of 9-fold and 5-fold for pigs and cattle, respectively. Recognising that the bulkiness of animal feed limits their consumption, these margins in conjunction with good agricultural practise were considered satisfactory for the protection of livestock health. A human health risk assessment was also

  12. Nitrite in feed: from animal health to human health.

    PubMed

    Cockburn, Andrew; Brambilla, Gianfranco; Fernández, Maria-Luisa; Arcella, Davide; Bordajandi, Luisa R; Cottrill, Bruce; van Peteghem, Carlos; Dorne, Jean-Lou

    2013-08-01

    Nitrite is widely consumed from the diet by animals and humans. However the largest contribution to exposure results from the in vivo conversion of exogenously derived nitrate to nitrite. Because of its potential to cause to methaemoglobin (MetHb) formation at excessive levels of intake, nitrite is regulated in feed and water as an undesirable substance. Forages and contaminated water have been shown to contain high levels of nitrate and represent the largest contributor to nitrite exposure for food-producing animals. Interspecies differences in sensitivity to nitrite intoxication principally result from physiological and anatomical differences in nitrite handling. In the case of livestock both pigs and cattle are relatively susceptible. With pigs this is due to a combination of low levels of bacterial nitrite reductase and hence potential to reduce nitrite to ammonia as well as reduced capacity to detoxify MetHb back to haemoglobin (Hb) due to intrinsically low levels of MetHb reductase. In cattle the sensitivity is due to the potential for high dietary intake and high levels of rumen conversion of nitrate to nitrite, and an adaptable gut flora which at normal loadings shunts nitrite to ammonia for biosynthesis. However when this escape mechanism gets overloaded, nitrite builds up and can enter the blood stream resulting in methemoglobinemia. Looking at livestock case histories reported in the literature no-observed-effect levels of 3.3mg/kg body weight (b.w.) per day for nitrite in pigs and cattle were estimated and related to the total daily nitrite intake that would result from complete feed at the EU maximum permissible level. This resulted in margins of safety of 9-fold and 5-fold for pigs and cattle, respectively. Recognising that the bulkiness of animal feed limits their consumption, these margins in conjunction with good agricultural practise were considered satisfactory for the protection of livestock health. A human health risk assessment was also

  13. Isolation, characterization, and biological activity of ferredoxin-NAD+ reductase from the methane oxidizer Methylosinus trichosporium OB3b.

    PubMed Central

    Chen, Y P; Yoch, D C

    1989-01-01

    A ferredoxin-NAD+ oxidoreductase (EC 1.18.1.3) has been isolated from extracts of the obligate methanotroph Methylosinus trichosporium OB3b. This enzyme was shown to couple electron flow from formate dehydrogenase (NAD+ requiring) to ferredoxin. Ferredoxin-NAD+ reductase was purified to homogeneity by conventional chromatography techniques and was shown to be a flavoprotein with a molecular weight of 36,000 +/- 1,000. This ferredoxin reductase was specific for NADH (Km, 125 microM) and coupled electron flow to the native ferredoxin and to ferredoxins from spinach, Clostridium pasteurianum, and Rhodospirillum rubrum (ferredoxin II). M. trichosporium ferredoxin saturated the ferredoxin-NAD+ reductase at a concentration 2 orders of magnitude lower (3 nM) than did spinach ferredoxin (0.4 microM). Ferredoxin-NAD+ reductase also had transhydrogenase activity which transferred electrons and protons from NADH to thionicotinamide adenine dinucleotide phosphate (Km, 9 microM) and from NADPH to 3-acetylpyridine adenine dinucleotide (Km, 16 microM). Reconstitution of a soluble electron transport pathway that coupled formate oxidation to ferredoxin reduction required formate dehydrogenase, NAD+, and ferredoxin-NAD+ reductase. Images PMID:2768195

  14. The Capacity of Red Blood Cells to Reduce Nitrite Determines Nitric Oxide Generation under Hypoxic Conditions

    PubMed Central

    Fens, Marcel H.; Larkin, Sandra K.; Oronsky, Bryan; Scicinski, Jan; Morris, Claudia R.; Kuypers, Frans A.

    2014-01-01

    Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release. PMID:25007272

  15. Regulation of assimilatory nitrate reductase activity in soil by microbial assimilation of ammonium.

    PubMed Central

    McCarty, G W; Bremner, J M

    1992-01-01

    It is well established that assimilatory nitrate reductase (ANR) activity in soil is inhibited by ammonium (NH4+). To elucidate the mechanism of this inhibition, we studied the effect of L-methionine sulfoximine (MSX), an inhibitor of NH4+ assimilation by microorganisms, on assimilatory reduction of nitrate (NO3-) in aerated soil slurries treated with NH4+. We found that NH4+ strongly inhibited ANR activity in these slurries and that MSX eliminated this inhibition. We also found that MSX induced dissimilatory reduction of NO3- to NH4+ in soil and that the NH4+ thus formed had no effect on the rate of NO-3 reduction. We concluded from these observations that the inhibition of ANR activity by NH4+ is due not to NH4+ per se but to products formed by microbial assimilation of NH4+. This conclusion was supported by a study of the effects of early products of NH4+ assimilation (L amino acids) on ANR activity in soil, because this study showed that the biologically active, L isomers of glutamine and asparagine strongly inhibited ANR activity, whereas the D isomers of these amino acids had little effect on ANR activity. Evidence that ANR activity is regulated by the glutamine formed by NH4+ assimilation was provided by studies showing that inhibitors of glutamine metabolism (azaserine, albizziin, and aminooxyacetate) inhibited ANR activity in soil treated with NO3- but did not do so in the presence of MSX. PMID:11607250

  16. Evolutionary origins of retinoid active short-chain dehydrogenases/reductases of SDR16C family.

    PubMed

    Belyaeva, Olga V; Chang, Chenbei; Berlett, Michael C; Kedishvili, Natalia Y

    2015-06-01

    Vertebrate enzymes that belong to the 16C family of short-chain dehydrogenases/reductases (SDR16C) were shown to play an essential role in the control of retinoic acid (RA) levels during development. To trace the evolution of enzymatic function of SDR16C family, and to examine the origins of the pathway for RA biosynthesis from vitamin A, we identified putative SDR16C enzymes through the extensive search of available genome sequencing data in a subset of species representing major metazoan phyla. The phylogenetic analysis revealed that enzymes from protostome, non-chordate deuterostome and invertebrate chordate species are found in three clades of SDR16C family containing retinoid active enzymes, which are retinol dehydrogenase 10 (RDH10), retinol dehydrogenases E2 (RDHE2) and RDHE2-similar, and dehydrogenase reductase (SDR family) member 3 (DHRS3). For the initial functional analysis, we cloned RDH10- and RDHE2-related enzymes from the early developmental stages of a non-chordate deuterostome, green sea urchin Lytechinus variegatus, and an invertebrate chordate, sea squirt Ciona intestinalis. In situ hybridization revealed that these proteins are expressed in a pattern relevant to development, while assays performed on proteins expressed in mammalian cell culture showed that they possess retinol-oxidizing activity as their vertebrate homologs. The existence of invertebrate homologs of DHRS3 was inferred from the analysis of phylogeny and cofactor-binding residues characteristic of preference for NADP(H). The presence of invertebrate homologs in the DHRS3 group of SDR16C is interesting in light of the complex mutually activating interaction, which we have recently described for human RDH10 and DHRS3 enzymes. Further functional analysis of these homologs will establish whether this interaction evolved to control retinoid homeostasis only in vertebrates, or is also conserved in pre-vertebrates. PMID:25451586

  17. Effect of Pharmaceutical Potential Endocrine Disruptor Compounds on Protein Disulfide Isomerase Reductase Activity Using Di-Eosin-Oxidized-Glutathion

    PubMed Central

    Klett, Danièle; Cahoreau, Claire; Villeret, Mélanie; Combarnous, Yves

    2010-01-01

    Background Protein Disulfide Isomerase (PDI) in the endoplasmic reticulum of all cells catalyzes the rearrangement of disulfide bridges during folding of membrane and secreted proteins. As PDI is also known to bind various molecules including hormones such as estradiol and thyroxin, we considered the hypothesis that adverse effects of endocrine-disrupter compounds (EDC) could be mediated through their interaction with PDI leading to defects in membrane or secreted proteins. Methodology/Principal Findings Taking advantage of the recent description of the fluorescence self quenched substrate di-eosin-oxidized-glutathion (DiE-GSSG), we determined kinetically the effects of various potential pharmaceutical EDCs on the in-vitro reductase activity of bovine liver PDI by measuring the fluorescence of the reaction product (E-GSH). Our data show that estrogens (ethynylestradiol and bisphenol-A) as well as indomethacin exert an inhibition whereas medroxyprogesteroneacetate and nortestosterone exert a potentiation of bovine PDI reductase activity. Conclusions The present data indicate that the tested EDCs could not only affect endocrine target cells through nuclear receptors as previously shown, but could also affect these and all other cells by positively or negatively affecting PDI activity. The substrate DiE-GSSG has been demonstrated to be a convenient substrate to measure PDI reductase activity in the presence of various potential EDCs. It will certainely be usefull for the screening of potential effect of all kinds of chemicals on PDI reductase activity. PMID:20209080

  18. From Alcohol Dehydrogenase to a “One-way” Carbonyl Reductase by Active-site Redesign

    PubMed Central

    Klimacek, Mario; Nidetzky, Bernd

    2010-01-01

    Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol (Kint) was altered dramatically (104- to 105-fold) from being balanced in the wild-type enzyme (Kint ≈ 3) to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. The change in Kint reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn → Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD+-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn→Asp substitutions behaved as a slow “fructose reductase” at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8–10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively “one-way” reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis. PMID:20639204

  19. Methionine sulfoxide reductase regulates brain catechol-O-methyl transferase activity.

    PubMed

    Moskovitz, Jackob; Walss-Bass, Consuelo; Cruz, Dianne A; Thompson, Peter M; Bortolato, Marco

    2014-10-01

    Catechol-O-methyl transferase (COMT) plays a key role in the degradation of brain dopamine (DA). Specifically, low COMT activity results in higher DA levels in the prefrontal cortex (PFC), thereby reducing the vulnerability for attentional and cognitive deficits in both psychotic and healthy individuals. COMT activity is markedly reduced by a non-synonymous single-nucleotide polymorphism (SNP) that generates a valine-to-methionine substitution on the residue 108/158, by means of as-yet incompletely understood post-translational mechanisms. One post-translational modification is methionine sulfoxide, which can be reduced by the methionine sulfoxide reductase (Msr) A and B enzymes. We used recombinant COMT proteins (Val/Met108) and mice (wild-type (WT) and MsrA knockout) to determine the effect of methionine oxidation on COMT activity and COMT interaction with Msr, through a combination of enzymatic activity and Western blot assays. Recombinant COMT activity is positively regulated by MsrA, especially under oxidative conditions, whereas brains of MsrA knockout mice exhibited lower COMT activity (as compared with their WT counterparts). These results suggest that COMT activity may be reduced by methionine oxidation, and point to Msr as a key molecular determinant for the modulation of COMT activity in the brain. The role of Msr in modulating cognitive functions in healthy individuals and schizophrenia patients is yet to be determined. PMID:24735585

  20. Characterization of nitrite uptake in Arabidopsis thaliana: evidence for a nitrite-specific transporter.

    PubMed

    Kotur, Zorica; Siddiqi, Yaeesh M; Glass, Anthony D M

    2013-10-01

    Nitrite-specific plasma membrane transporters have been described in bacteria, algae and fungi, but there is no evidence of a nitrite-specific plasma membrane transporter in higher plants. We have used 13NO2(-) to characterize nitrite influx into roots of Arabidopsis thaliana. Hydroponically grown Arabidopsis mutants, defective in high-affinity nitrate transport, were used to distinguish between nitrate and nitrite uptake by means of the short-lived tracers 13NO2(-) and 13NO3(-). This approach allowed us to characterize a nitrite-specific transporter. The Atnar2.1-2 mutant, lacking a functional high-affinity nitrate transport system, is capable of nitrite influx that is constitutive and thermodynamically active. The corresponding fluxes conform to a rectangular hyperbola, exhibiting saturation at concentrations above 200 μM (Km = 185 μM and Vmax = 1.89 μmol g(-1) FW h(-1)). Nitrite influx via the putative nitrite transporter is not subject to competitive inhibition by nitrate but is downregulated after 6 h exposure to ammonium. These results signify the existence of a nitrite-specific transporter in Arabidopsis. This transporter enables Atnar2.1-2 mutants, which are incapable of sustained growth on low nitrate, to maintain significant growth on low nitrite. In wild-type plants, this nitrite flux may increase nitrogen acquisition and also participate in the induction of genes specifically induced by nitrite. PMID:23763619

  1. Inhibitory activity of reuterin, nisin, lysozyme and nitrite against vegetative cells and spores of dairy-related Clostridium species.

    PubMed

    Avila, Marta; Gómez-Torres, Natalia; Hernández, Marta; Garde, Sonia

    2014-02-17

    The butyric acid fermentation, responsible for late blowing of cheese, is caused by the outgrowth in cheese of some species of Clostridium, resulting in texture and flavor defects and economical losses. The aim of this study was to evaluate the effectiveness of different antimicrobial compounds against vegetative cells and spores of C. tyrobutyricum, C. butyricum, C. beijerinckii and C. sporogenes strains isolated from cheeses with late blowing defect. Minimal inhibitory concentration (MIC) for reuterin, nisin, lysozyme and sodium nitrite were determined against Clostridium strains in milk and modified RCM (mRCM) after 7d exposure. Although the sensitivity of Clostridium to the tested antimicrobials was strain-dependent, C. sporogenes and C. beijerinckii generally had higher MIC values than the rest of Clostridium species. The majority of Clostridium strains were more resistant to antimicrobials in milk than in mRCM, and vegetative cells exhibited higher sensitivity than spores. Reuterin (MIC values 0.51-32.5 mM) and nisin (MIC values 0.05-12.5 μg/ml) were able to inhibit the growth of vegetative cells and spores of all assayed Clostridium strains in milk and mRCM. Strains of C. tyrobutyricum exhibited the highest sensitivity to lysozyme (MIC values<0.20-400 μg/ml) and sodium nitrite (MIC values 18.75-150 μg/ml). These results suggest that reuterin and nisin, with a broad inhibitory activity spectrum against Clostridium spp. spores and vegetative cells, may be the best options to control Clostridium growth in dairy products and to prevent associated spoilage, such as late blowing defect of cheese. However, further studies in cheese would be necessary to validate this hypothesis. PMID:24361835

  2. Disappearance of chloramines in the presence of bromide and nitrite. [Ammoniacal monochloramine, diethylchloramine, and chloramines produced by chlorinating a real and synthetic secondary (activated sludge) municipal waste effluent

    SciTech Connect

    Valentine, R.L.

    1982-01-01

    Batch experiments were used to study the reduction of chloramines in the presence of bromide and nitrite. Chloramines studies were ammoniacal monochloramine, diethylchloramine (DECA), and those produced by chlorinating a real and synthetic secondary (activated sludge) municipal waste effluent. Oxidant concentrations were measured using the DPD-FAS (N,N-diethyl-p-phenylenediamine, Ferrous Ammonium Sulfate) titrimetric procedure and/or spectrophotometrically. The degradation of NH/sub 2/Cl in the presence of bromide was found to occur via a mechanism consistent with a rate limiting step involving monochlorammonium ion (NH/sub 3/Cl/sup +/) and bromide ion. Experimental evidence suggests that the mixed haloamine, NHBrCl, was produced as an unstable intermediate. The oxidation of bromide by DECA did not occur by a mechanism similar to that describing the oxidation of bromide by NH/sub 2/Cl. The rate was not affected by added ammonia and was slower than that observed for comparable NH/sub 2/Cl-Br/sup -/ reactions. Chloramine loss in organic rich effluents was greatly accelerated by bromide addition. The reaction is not dependent on excess ammonia and is slower than that observed for a pure NH/sub 2/Cl-Br/sup -/ solution. Monochloramine can rapidly disappear in the presence of nitrite. The rates are too fast to be due solely to the hydrolysis of monochloramine. The presence of relatively small concentrations of nitrite can greatly accelerate the loss of NH/sub 2/Cl in the presence of bromide. Nitrite is not significantly consumed. Nitrite appears to increase the rate of bromide oxidation in a parallel acid catalyzed reaction mechanism which involves a rate limiting step described by a first order dependence on nitrite but no dependence on bromide. Empirical rate expressions and rate constants were determined for each reaction. 54 figures, 17 tables.

  3. Utilization of iron-catecholamine complexes involving ferric reductase activity in Listeria monocytogenes.

    PubMed Central

    Coulanges, V; Andre, P; Ziegler, O; Buchheit, L; Vidon, D J

    1997-01-01

    Listeria monocytogenes is a ubiquitous potentially pathogenic organism requiring iron for growth and virulence. Although it does not produce siderophores, L. monocytogenes is able to obtain iron by using either exogenous siderophores produced by various microorganisms or natural catechol compounds widespread in the environment. In the presence of tropolone, an iron-chelating agent, growth of L. monocytogenes is completely inhibited. However, the growth inhibition can be relieved by the addition of dopamine or norepinephrine under their different isomeric forms, while the catecholamine derivatives 4-hydroxy-3-methoxyphenylglycol and normetanephrine did not relieve the inhibitory effect of tropolone. Preincubation of L. monocytogenes with chlorpromazine and yohimbine did not antagonize the growth-promoting effect of catecholamines in iron-complexed medium. In addition, norepinephrine stimulated the growth-promoting effect induced by human transferrin in iron-limited medium. Furthermore, dopamine and norepinephrine allowed 55Fe uptake by iron-deprived bacterial cells. The uptake of iron was energy dependent, as indicated by inhibition of 55Fe uptake at 0 degrees C as well as by preincubating the bacteria with KCN. Inhibition of 55Fe uptake by L. monocytogenes was also observed in the presence of Pt(II). Moreover, when assessed by a whole-cell ferric reductase assay, reductase activity of L. monocytogenes was inhibited by Pt(II). These data demonstrate that dopamine and norepinephrine can function as siderophore-like compounds in L. monocytogenes owing to their ortho-diphenol function and that catecholamine-mediated iron acquisition does not involve specific catecholamine receptors but acts through a cell-bound ferrireductase activity. PMID:9199450

  4. Growth, photosynthesis, nitrogen partitioning and responses to CO2 enrichment in barley mutants lacking NADH-dependent nitrate reductase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We examined plant growth, photosynthesis and leaf constituents of both the wild type (WT) and two mutant lines of barley (Hordeum vulgare L. cv. Steptoe) with defects in NADH-dependent nitrate reductase (NADH-NAR) activity. The first mutant, nar1, had a lesion within the NAR structural gene and the...

  5. The Thiol Reductase Activity of YUCCA6 Mediates Delayed Leaf Senescence by Regulating Genes Involved in Auxin Redistribution.

    PubMed

    Cha, Joon-Yung; Kim, Mi R; Jung, In J; Kang, Sun B; Park, Hee J; Kim, Min G; Yun, Dae-Jin; Kim, Woe-Yeon

    2016-01-01

    Auxin, a phytohormone that affects almost every aspect of plant growth and development, is biosynthesized from tryptophan via the tryptamine, indole-3-acetamide, indole-3-pyruvic acid, and indole-3-acetaldoxime pathways. YUCCAs (YUCs), flavin monooxygenase enzymes, catalyze the conversion of indole-3-pyruvic acid (IPA) to the auxin (indole acetic acid). Arabidopsis thaliana YUC6 also exhibits thiol-reductase and chaperone activity in vitro; these activities require the highly conserved Cys-85 and are essential for scavenging of toxic reactive oxygen species (ROS) in the drought tolerance response. Here, we examined whether the YUC6 thiol reductase activity also participates in the delay in senescence observed in YUC6-overexpressing (YUC6-OX) plants. YUC6 overexpression delays leaf senescence in natural and dark-induced senescence conditions by reducing the expression of SENESCENCE-ASSOCIATED GENE 12 (SAG12). ROS accumulation normally occurs during senescence, but was not observed in the leaves of YUC6-OX plants; however, ROS accumulation was observed in YUC6-OX(C85S) plants, which overexpress a mutant YUC6 that lacks thiol reductase activity. We also found that YUC6-OX plants, but not YUC6-OX(C85S) plants, show upregulation of three genes encoding NADPH-dependent thioredoxin reductases (NTRA, NTRB, and NTRC), and GAMMA-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), encoding an enzyme involved in redox signaling. We further determined that excess ROS accumulation caused by methyl viologen treatment or decreased glutathione levels caused by buthionine sulfoximine treatment can decrease the levels of auxin efflux proteins such as PIN2-4. The expression of PINs is also reduced in YUC6-OX plants. These findings suggest that the thiol reductase activity of YUC6 may play an essential role in delaying senescence via the activation of genes involved in redox signaling and auxin availability. PMID:27242830

  6. The Thiol Reductase Activity of YUCCA6 Mediates Delayed Leaf Senescence by Regulating Genes Involved in Auxin Redistribution

    PubMed Central

    Cha, Joon-Yung; Kim, Mi R.; Jung, In J.; Kang, Sun B.; Park, Hee J.; Kim, Min G.; Yun, Dae-Jin; Kim, Woe-Yeon

    2016-01-01

    Auxin, a phytohormone that affects almost every aspect of plant growth and development, is biosynthesized from tryptophan via the tryptamine, indole-3-acetamide, indole-3-pyruvic acid, and indole-3-acetaldoxime pathways. YUCCAs (YUCs), flavin monooxygenase enzymes, catalyze the conversion of indole-3-pyruvic acid (IPA) to the auxin (indole acetic acid). Arabidopsis thaliana YUC6 also exhibits thiol-reductase and chaperone activity in vitro; these activities require the highly conserved Cys-85 and are essential for scavenging of toxic reactive oxygen species (ROS) in the drought tolerance response. Here, we examined whether the YUC6 thiol reductase activity also participates in the delay in senescence observed in YUC6-overexpressing (YUC6-OX) plants. YUC6 overexpression delays leaf senescence in natural and dark-induced senescence conditions by reducing the expression of SENESCENCE-ASSOCIATED GENE 12 (SAG12). ROS accumulation normally occurs during senescence, but was not observed in the leaves of YUC6-OX plants; however, ROS accumulation was observed in YUC6-OXC85S plants, which overexpress a mutant YUC6 that lacks thiol reductase activity. We also found that YUC6-OX plants, but not YUC6-OXC85S plants, show upregulation of three genes encoding NADPH-dependent thioredoxin reductases (NTRA, NTRB, and NTRC), and GAMMA-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), encoding an enzyme involved in redox signaling. We further determined that excess ROS accumulation caused by methyl viologen treatment or decreased glutathione levels caused by buthionine sulfoximine treatment can decrease the levels of auxin efflux proteins such as PIN2-4. The expression of PINs is also reduced in YUC6-OX plants. These findings suggest that the thiol reductase activity of YUC6 may play an essential role in delaying senescence via the activation of genes involved in redox signaling and auxin availability. PMID:27242830

  7. Activities of nitrate reductase and glutamine synthetase in rice seedlings during cyanide metabolism.

    PubMed

    Yu, Xiao-Zhang; Zhang, Fu-Zhong

    2012-07-30

    A study was conducted to investigate activities of nitrate reductase (NR) and glutamine synthetase (GS) in plants during cyanide metabolism. Young rice seedlings (Oryza sativa L. cv. XZX 45) were grown in the nutrient solutions containing KNO(3) or NH(4)Cl and treated with free cyanide (KCN). Cyanide in solutions and in plant materials was analyzed to estimate the phyto-assimilation potential. Activities of NR and GS in different parts of rice seedlings were assayed in vivo. Seedlings grown on NH(4)(+) showed significantly higher relative growth rate than those on NO(3)(-) (p<0.05) in the presence of exogenous cyanide. The metabolic rates of cyanide by seedlings were all positively correlated to the concentrations supplied. A negligible difference was observed between the two treatments with nitrate and ammonium (p>0.05). Enzymatic assays showed that cyanide (≥0.97mg CN L(-1)) impaired NR activity significantly in both roots and shoots (p<0.05). The effect of cyanide on GS activity in roots was more evident at 1.93mg CN L(-1), suggesting that NR activity was more susceptible to change from cyanide application than GS activity. The results observed here suggest that the exogenous cyanide, which to a certain level has a beneficial role in plant nutrition. PMID:22633925

  8. Human biliverdin reductase, a previously unknown activator of protein kinase C betaII.

    PubMed

    Maines, Mahin D; Miralem, Tihomir; Lerner-Marmarosh, Nicole; Shen, Jenny; Gibbs, Peter E M

    2007-03-16

    Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) betaII, activated by insulin and free radicals (Miralem, T., Hu, Z., Torno, M. D., Lelli, K. M., and Maines, M. D. (2005) J. Biol. Chem. 280, 17084-17092; Lerner-Marmarosh, N., Shen, J., Torno, M. D., Kravets, A., Hu, Z., and Maines, M. D. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7109-7114). Here, by using 293A cells co-transfected with pcDNA3-hBVR and PKC betaII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC betaII, but not the reductase and PKC zeta, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC betaII K371R mutant protein ("kinase-dead") was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC betaII for myelin basic protein; activation was independent of phospholipids and extended to the phosphorylation of S2, a PKC-specific substrate. The increase in substrate phosphorylation was blocked by specific inhibitors of conventional PKCs and attenuated by sihBVR. The effect of the latter could be rescued by subsequent overexpression of hBVR. To a large extent, the activation was a function of the hBVR N-terminal chain of valines and intact ATP-binding site and the cysteine-rich C-terminal segment. The cobalt protoporphyrin-activated hBVR phosphorylated a threonine in a peptide corresponding to the Thr500 in the human PKC betaII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC betaII nor PKC zeta activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR.PKC betaII immunocomplex. The potential biological relevance of the hBVR activation of PKC betaII was suggested by the finding that in cells transfected with the PKC betaII, h

  9. The relationship between hypertension and plasma allantoin, uric acid, xanthine oxidase activity and nitrite, and their predictive capacity in severe preeclampsia.

    PubMed

    Elmas, Oguz; Elmas, Onur; Aliciguzel, Yakup; Simsek, Tayyup

    2016-01-01

    It is controversial that uric acid (UA) levels are related to the severity of hypertension in preeclampsia (PE). Our aim in this study was to determine whether UA, xanthine oxidase activity (XOA), allantoin and nitrite levels are related to arterial blood pressure (BP) in PE. We formed a control group (n = 20) and a PE group (n = 20) for the study. Their BPs and plasma UA, XOA, allantoin and nitrite levels were measured. The values from the control and PE pregnant women were assessed via a Wilcoxon matched-pairs test. A Pearson correlation test was also performed. In addition, the diagnostic value of these tests was evaluated via receiver operating characteristic (ROC) analysis. The BP, UA, XOA and allantoin levels in the PE patients were found to be higher when compared with those of the pregnant controls. The UA, XOA and allantoin levels showed high correlations with BP in cases of PE. However, there was no superiority among the correlations. No differences were observed between the groups in terms of nitrite levels and the relationship between nitrite and BP. UA, XOA and allantoin levels may be high due to placental cell death because of abnormal trophoblastic activity observed in PE. Moreover, the reactive oxygen products that are created during the genetic material degradation may explain how UA, XOA and allantoin levels are related to BP. According to ROC analysis, UA, XOA and allantoin assays are reliable predictors for the determination of PE. PMID:26366935

  10. Mechanisms for nitrite loss from the stomach.

    PubMed

    Licht, W R; Schultz, D S; Fox, J G; Tannenbaum, S R; Deen, W M

    1986-10-01

    Nitrite loss from the stomach was studied using dogs equipped with Thomas cannulas for direct access to the stomach lumen. Solutions containing sodium nitrite and non-absorbable volume marker (polyethylene glycol, PEG) were infused into the stomach, and samples were taken over 60 min to determine the concentration of 'total nitrite' (including NO2-, HNO2 and other species in equilibrium with NO2-) and rate of dilution of the stomach contents as a function of time. Changes in stomach volume were also measured. Nitrite loss was found to be very rapid, with total nitrite concentrations declining to less than half the initial levels in 10 min. The decay in total nitrite concentrations was due predominantly to gastric absorption, with small additional contributions from dilution of the stomach contents (inferred from PEG concentrations) and chemical reactions (from in vitro kinetic data). Results for initial nitrite concentrations varying over a range of 0.15-4.5 mM showed absorption to be first order in total nitrite. The permeability-area product for nitrite absorption (PA) was about 0.6 l/h, and was unaffected by the addition of 1 mM SCN- or Cl-. All of these results are consistent with nitrite absorption in the form of NO2- or HNO2. Buffering the infusate with HCO3- to increase luminal pH from approximately 2 to 7 caused a three-fold reduction in the apparent value of PA. When pentagastrin was used to stimulate acid secretion, nitrite absorption was only half as fast as when acid secretion was inhibited with cimetidine, or when no drug was given. This effect could not be explained by variations in luminal pH, and suggests that acid secretion either decreases PA or is accompanied by active secretion of nitrite. Based on these data, a mathematical model was developed to stimulate the physical and chemical factors governing nitrite concentrations in the stomach. PMID:3757171

  11. An ene reductase from Clavispora lusitaniae for asymmetric reduction of activated alkenes.

    PubMed

    Ni, Yan; Yu, Hui-Lei; Lin, Guo-Qiang; Xu, Jian-He

    2014-03-01

    A putative ene reductase gene from Clavispora lusitaniae was heterologously overexpressed in Escherichia coli, and the encoded protein (ClER) was purified and characterized for its biocatalytic properties. This NADPH-dependent flavoprotein was identified with reduction activities toward a diverse range of activated alkenes including conjugated enones, enals, maleimide derivative and α,β-unsaturated carboxylic esters. The purified ClER exhibited a relatively high activity of 7.3 U mg(prot)⁻¹ for ketoisophorone while a remarkable catalytic efficiency (k(cat)/K(m)=810 s⁻¹ mM⁻¹) was obtained for 2-methyl-cinnamaldehyde due to the high affinity. A series of prochiral activated alkenes were stereoselectively reduced by ClER furnishing the corresponding saturated products in up to 99% ee. The practical applicability of ClER was further evaluated for the production of (R)-levodione, a valuable chiral compound, from ketoisophorone. Using the crude enzyme of ClER and glucose dehydrogenase (GDH), 500 mM of ketoisophorone was efficiently converted to (R)-levodione with excellent stereoselectivity (98% ee) within 1h. All these positive features demonstrate a high synthetic potential of ClER in the asymmetric reduction of activated alkenes. PMID:24564901

  12. Effect of dietary supplementation of vitamin C on growth, reactive oxygen species, and antioxidant enzyme activity of Apostichopus japonicus (Selenka) juveniles exposed to nitrite

    NASA Astrophysics Data System (ADS)

    Luo, Zuoyong; Wang, Baojie; Liu, Mei; Jiang, Keyong; Liu, Mingxing; Wang, Lei

    2014-07-01

    Different amounts of vitamin C were added to diets fed to juveniles (2.5 ± 0.15 g) of sea cucumber Apostichopus japonic u s (Selenka) in an attempt to reduce the stress response of specimens exposed to nitrite stress. A commercial feed was used as the control diet and three experimental diets were made by supplementing 1 000, 1 500, or 2 000 mg vitamin C/kg diet to control diet separately in a 45-day experiment. Sea cucumbers were exposed to three different levels (0.5, 1.0, and 1.5 mg/L) of nitrite stress for 4, 8, and 12 h at four time intervals (0, 15, 30, and 45 d). Growth of the animals was recorded during the experiment. Reactive oxygen species (ROS) (i.e. hydroxyl free radical (-OH), malondialdehyde (MDA) and total antioxidant capacity (T-AOC)) and antioxidant enzyme activities (i.e., superoxide dismutase (SOD) and catalase (CAT)) were measured. Response surface methodology (RSM) was used to analyze the effect of multiple factors on ROS indices and enzyme activities. Weight gain (WG) and special growth rate (SGR) of vitamin C supplementation groups were significantly higher than those of control group ( P < 0.05). The levels of -OH and MDA increased under exposure time extending and nitrite concentration increasing, whereas T-AOC level decreased. SOD and CAT activities increased at 4 h and 8 h and decreased at 12 h. During the days in which the animal consumed experimental diets, the levels of -OH and MDA decreased and that of T-AOC increased. This result suggests that diets containing vitamin C could reduce the nitrite stress response in the animals and increase their antioxidant capacity. The multifactor regression equation of growth performance, ROS indices, and duration of feeding results suggest that vitamin C supplementation of 1 400-2 000 mg/kg diet for 29-35 days could reduce effectively the effects of nitrite exposure.

  13. Influence of the enzyme dissimilatory sulfite reductase on stable isotope fractionation during sulfate reduction

    NASA Astrophysics Data System (ADS)

    Mangalo, Muna; Einsiedl, Florian; Meckenstock, Rainer U.; Stichler, Willibald

    2008-03-01

    The stable isotopes of sulfate are often used as a tool to assess bacterial sulfate reduction on the macro scale. However, the mechanisms of stable isotope fractionation of sulfur and oxygen at the enzymatic level are not yet fully understood. In batch experiments with water enriched in 18O we investigated the effect of different nitrite concentrations on sulfur isotope fractionation by Desulfovibrio desulfuricans. With increasing nitrite concentrations, we found sulfur isotope enrichment factors ranging from -11.2 ± 1.8‰ to -22.5 ± 3.2‰. Furthermore, the δ18O values in the remaining sulfate increased from approximately 50-120‰ when 18O-enriched water was supplied. Since 18O-exchange with ambient water does not take place in sulfate, but rather in intermediates of the sulfate reduction pathway (e.g. SO32-), we suggest that nitrite affects the steady-state concentration and the extent of reoxidation of the metabolic intermediate sulfite to sulfate during sulfate reduction. Given that nitrite is known to inhibit the production of the enzyme dissimilatory sulfite reductase, our results suggest that the activity of the dissimilatory sulfite reductase regulates the kinetic isotope fractionation of sulfur and oxygen during bacterial sulfate reduction. Our novel results also imply that isotope fractionation during bacterial sulfate reduction strongly depends on the cell internal enzymatic regulation rather than on the physico-chemical features of the individual enzymes.

  14. Induction of quinone reductase activity by stilbene analogs in mouse Hepa 1c1c7 cells.

    PubMed

    Heo, Y H; Kim, S; Park, J E; Jeong, L S; Lee, S K

    2001-12-01

    Based on the potential cancer chemopreventive activity of resveratrol, a trihydroxystilbene with the induction of quinone reductase activity, this study was designed to determine if stilbene-related compounds were inducers of phase II detoxifying metabolic enzyme quinone reductase (QR) in the mouse hepatoma Hepa 1c1c7 cells. Among the thirteen compounds tested, several compounds including 3,4,5,3',5'-pentamethoxy-trans-stilbene were found to potentially induce QR activity in this cell line. In addition, substitution with 3-thiofurane ring instead of phenyl ring in the stilbene skeleton also exhibited potential induction of QR activity. This result will give primary information to design the potential inducers of QR activity in the stilbene analogs. PMID:11794542

  15. Inhibitory effects of Zingiber officinale Roscoe derived components on aldose reductase activity in vitro and in vivo.

    PubMed

    Kato, Atsushi; Higuchi, Yasuko; Goto, Hirozo; Kizu, Haruhisa; Okamoto, Tadashi; Asano, Naoki; Hollinshead, Jackie; Nash, Robert J; Adachi, Isao

    2006-09-01

    Ginger (Zingiber officinale Roscoe) continues to be used as an important cooking spice and herbal medicine around the world. Scientific research has gradually verified the antidiabetic effects of ginger. Especially gingerols, which are the major components of ginger, are known to improve diabetes including the effect of enhancement against insulin-sensitivity. Aldose reductase inhibitors have considerable potential for the treatment of diabetes, without increased risk of hypoglycemia. The assay for aldose reductase inhibitors in ginger led to the isolation of five active compounds including 2-(4-hydroxy-3-methoxyphenyl)ethanol (2) and 2-(4-hydroxy-3-methoxyphenyl)ethanoic acid (3). Compounds 2 and 3 were good inhibitors of recombinant human aldose reductase, with IC50 values of 19.2 +/- 1.9 and 18.5 +/- 1.1 microM, respectively. Furthermore, these compounds significantly suppressed not only sorbitol accumulation in human erythrocytes but also lens galactitol accumulation in 30% of galactose-fed cataract rat model. A structure-activity relationship study revealed that the applicable side alkyl chain length and the presence of a C3 OCH3 group in the aromatic ring are essential features for enzyme recognition and binding. These results suggested that it would contribute to the protection against or improvement of diabetic complications for a dietary supplement of ginger or its extract containing aldose reductase inhibitors. PMID:16939321

  16. Activity landscape analysis of novel 5[Formula: see text]-reductase inhibitors.

    PubMed

    Naveja, J Jesús; Cortés-Benítez, Francisco; Bratoeff, Eugene; Medina-Franco, José L

    2016-08-01

    Inhibitors of the enzyme 5[Formula: see text]-reductase (5aR) are promising therapeutic agents for the treatment of benign prostatic hyperplasia (BPH) and prostate cancer. The lack of structural data of the enzyme 5aR prompts the application of ligand-based approaches to systematically explore the activity landscape of 5aR inhibitors. As part of an effort to develop inhibitors of this enzyme for the treatment of BPH, herein we discuss a chemoinformatic-based analysis of the activity landscape of a novel set of 53 novel pregnane and androstene compounds. It was found that, in general, for each pair of compounds in the set, as the structure similarity of the compounds increases the corresponding potency difference decreases. These results are in agreement with an overall smooth activity landscape. However, two potent activity cliff generators were identified pointing to specific small structural changes that have a large impact on the inhibition of 5aR. PMID:26829939

  17. 21 CFR 181.34 - Sodium nitrite and potassium nitrite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium nitrite and potassium nitrite. 181.34... nitrite and potassium nitrite. Sodium nitrite and potassium nitrite are subject to prior sanctions issued... without sodium or potassium nitrate, in the curing of red meat and poultry products....

  18. Annual pattern of nitrate reductase activity in needles of high-elevation red spruce trees

    SciTech Connect

    Tjoelker, M.G.; Norby, R.J. ); DiCosty, R.J. ); Weerasuriya, Y. )

    1989-04-01

    To assess the ability of foliar nitrate reductase (NR) as a biochemical marker for the impact of nitrogen oxide pollutants on high-elevation forests, we measured needle NR activity in red spruce (Picea rubens Sarg.) saplings at two stands in the Great Smoky Mountains National Park (1,935 m, 1,720 m). Seven times between September 1987 and 1988, branches were cut from selected saplings, and NR activity was assayed on current-year needles, using an in vivo method. NR activity increased to maximum values of 60 nmol g{sup {minus}1} h{sup {minus}1} in late summer of both years and then declined by 85 percent in October 1987 and 65 percent in September 1988. Although NR activity was 30 percent great in red spruce at the high site relative to the low site in September and October 1987, NR activity dropped to 10 nmol g{sup {minus}1} h{sup {minus}1} at both sites in November 1987. No difference between the sites were evident in 1988. The seasonal pattern of needle NR activity at these sites may be due to ontogenetic changes in needle N metabolism and/or extrinsic variation in temperature or nitrogen oxide deposition. Characterization of nitrogen oxide pollutant levels and exposure episodes at high-elevation sites may aid in assessing seasonal and site variation in NR activity and the likelihood of needle NR induction by uptake of nitrogen oxides. These measurements of NR activity indicate that red spruce are capable of reducing nitrate in foliage under field conditions and that the nitrate assimilation capacity varies seasonally.

  19. Differential thioredoxin reductase activity from human normal hepatic and hepatoma cell lines.

    PubMed

    Jung, Haeng-Im; Lim, Hye-Won; Kim, Byung-Chul; Park, Eun-Hee; Lim, Chang-Jin

    2004-04-30

    Thioredoxin reductase (TrxR), a component of the thioredoxin system, including thioredoxin (Trx) and NADPH, catalyzes the transfer of electrons from NADPH to Trx, acts as a reductant of disulfide-containing proteins and participates in the defense system against oxidative stresses. In this study, the regulation pattern of TrxR in the presence of various stressful reagents was compared between Chang (human normal hepatic cell) and HepG2 (human hepatoma cell) cell lines. Aluminum chloride (0.5 mM) and zinc chloride (0.5 mM) enhanced the TrxR activity in the Chang cell line to a higher degree than in the HepG2 cell line, but cupric chloride (0.2 mM) and cadmium chloride (0.1 mM) enhanced the TrxR activity in the HepG2 cell line to a greater degree. The TrxR activities in both Chang and HepG2 cell lines were similarly induced by treatment with sodium selenite (0.02 mM) and menadione (0.5 and 1.0 mM). Lipopolysaccharide (2 micro g/m1) increased the TrxR activity upto 4.02- and 2.2-fold in the Chang and HepG2 cell lines, respectively, in time-dependent manners. Hydrogen peroxide (5 mM) markedly enhanced the TrxR activity in the HepG2 cell line, but not in the Chang cell line. NO-generating sodium nitroprusside (3.0 and 6.0 mM) induced TrxR activities in both human liver cell lines. The TrxR activity was also induced in human liver cells under limited growth conditions by serum deprivation. These results imply that the TrxR activities in normal hepatic and hepatoma cell lines are subject to different regulatory responses to various stresses. PMID:15118998

  20. Bioactive fraction of Saraca indica prevents diabetes induced cataractogenesis: An aldose reductase inhibitory activity

    PubMed Central

    Somani, Gauresh; Sathaye, Sadhana

    2015-01-01

    Background: The present study was designed to investigate the effect of Saraca indica (SI) flowers extract and different bioactive fraction on in vitro aldose reductase (AR) inhibitory activity, high glucose-induced cataract in goat lens and in vivo streptozotocin (STZ; 45 mg/kg, i.p) induced cataract in rats. Methods: Extract of flowers of SI tested for inhibition against rat lens AR. Furthermore, bioactive fraction was investigated against high glucose-induced opacification of the lens in vitro lens culture and STZ induced diabetic cataract in rats. Identification of the bioactive component was attempted through high-performance thin-layer chromatography, high-performance liquid chromatography and liquid chromatography-mass spectrometry analysis. Results: Ethyl acetate fraction of S. indica (EASI) produced maximum inhibition that may be due to high phenolic content. Goat lenses in media containing glucose developed a distinctly opaque ring in 72 h and treatment with EASI fraction lowered lens opacity in 72 h. Prolonged treatment with EASI to STZ-induced diabetic rats inhibited the AR activity and delayed cataract progression in a dose dependent manner. Conclusion: Ethyl acetate fraction of S. indica fraction has potential to inhibit rat lens AR enzyme and prevent cataractogenesis not only in goat lens model (in vitro), but also in STZ induced diabetic rats (in vivo). This study is suggestive of the anticataract activity of EASI fraction that could be attributed to the phytoconstituents present in the same. PMID:25709218

  1. Free energy simulations of active-site mutants of dihydrofolate reductase.

    PubMed

    Doron, Dvir; Stojković, Vanja; Gakhar, Lokesh; Vardi-Kilshtain, Alexandra; Kohen, Amnon; Major, Dan Thomas

    2015-01-22

    This study employs hybrid quantum mechanics-molecular mechanics (QM/MM) simulations to investigate the effect of mutations of the active-site residue I14 of E. coli dihydrofolate reductase (DHFR) on the hydride transfer. Recent kinetic measurements of the I14X mutants (X = V, A, and G) indicated slower hydride transfer rates and increasingly temperature-dependent kinetic isotope effects (KIEs) with systematic reduction of the I14 side chain. The QM/MM simulations show that when the original isoleucine residue is substituted in silico by valine, alanine, or glycine (I14V, I14A, and I14G DHFR, respectively), the free energy barrier height of the hydride transfer reaction increases relative to the wild-type enzyme. These trends are in line with the single-turnover rate measurements reported for these systems. In addition, extended dynamics simulations of the reactive Michaelis complex reveal enhanced flexibility in the mutants, and in particular for the I14G mutant, including considerable fluctuations of the donor-acceptor distance (DAD) and the active-site hydrogen bonding network compared with those detected in the native enzyme. These observations suggest that the perturbations induced by the mutations partly impair the active-site environment in the reactant state. On the other hand, the average DADs at the transition state of all DHFR variants are similar. Crystal structures of I14 mutants (V, A, and G) confirmed the trend of increased flexibility of the M20 and other loops. PMID:25382260

  2. Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli

    PubMed Central

    Rodriguez, Gabriel M.; Atsumi, Shota

    2015-01-01

    Advances in synthetic biology and metabolic engineering have enabled the construction of novel biological routes to valuable chemicals using suitable microbial hosts. Aldehydes serve as chemical feedstocks in the synthesis of rubbers, plastics, and other larger molecules. Microbial production of alkanes is dependent on the formation of a fatty aldehyde intermediate which is converted to an alkane by an aldehyde deformylating oxygenase (ADO). However, microbial hosts such as Escherichia coli are plagued by many highly active endogenous aldehyde reductases (ALRs) that convert aldehydes to alcohols, which greatly complicates strain engineering for aldehyde and alkane production. It has been shown that the endogenous ALR activity outcompetes the ADO enzyme for fatty aldehyde substrate. The large degree of ALR redundancy coupled with an incomplete database of ALRs represents a significant obstacle in engineering E. coli for either aldehyde or alkane production. In this study, we identified 44 ALR candidates encoded in the E. coli genome using bioinformatics tools, and undertook a comprehensive screening by measuring the ability of these enzymes to produce isobutanol. From the pool of 44 candidates, we found five new ALRs using this screening method (YahK, DkgA, GldA, YbbO, and YghA). Combined deletions of all 13 known ALRs resulted in a 90–99% reduction in endogenous ALR activity for a wide range of aldehyde substrates (C2–C12). Elucidation of the ALRs found in E. coli could guide one in reducing competing alcohol formation during alkane or aldehyde production. PMID:25108218

  3. Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Lang, F.

    1991-01-01

    A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography. The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide. Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present. Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition. The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000). Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt.

  4. HMG-CoA reductase inhibitory activity and phytocomponent investigation of Basella alba leaf extract as a treatment for hypercholesterolemia.

    PubMed

    Baskaran, Gunasekaran; Salvamani, Shamala; Ahmad, Siti Aqlima; Shaharuddin, Noor Azmi; Pattiram, Parveen Devi; Shukor, Mohd Yunus

    2015-01-01

    The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase is the key enzyme of the mevalonate pathway that produces cholesterol. Inhibition of HMG-CoA reductase reduces cholesterol biosynthesis in the liver. Synthetic drugs, statins, are commonly used for the treatment of hypercholesterolemia. Due to the side effects of statins, natural HMG-CoA reductase inhibitors of plant origin are needed. In this study, 25 medicinal plant methanol extracts were screened for anti-HMG-CoA reductase activity. Basella alba leaf extract showed the highest inhibitory effect at about 74%. Thus, B. alba was examined in order to investigate its phytochemical components. Gas chromatography with tandem mass spectrometry and reversed phase high-performance liquid chromatography analysis revealed the presence of phenol 2,6-bis(1,1-dimethylethyl), 1-heptatriacotanol, oleic acid, eicosyl ester, naringin, apigenin, luteolin, ascorbic acid, and α-tocopherol, which have been reported to possess antihypercholesterolemic effects. Further investigation of in vivo models should be performed in order to confirm its potential as an alternative treatment for hypercholesterolemia and related cardiovascular diseases. PMID:25609924

  5. HMG-CoA reductase inhibitory activity and phytocomponent investigation of Basella alba leaf extract as a treatment for hypercholesterolemia

    PubMed Central

    Baskaran, Gunasekaran; Salvamani, Shamala; Ahmad, Siti Aqlima; Shaharuddin, Noor Azmi; Pattiram, Parveen Devi; Shukor, Mohd Yunus

    2015-01-01

    The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase is the key enzyme of the mevalonate pathway that produces cholesterol. Inhibition of HMG-CoA reductase reduces cholesterol biosynthesis in the liver. Synthetic drugs, statins, are commonly used for the treatment of hypercholesterolemia. Due to the side effects of statins, natural HMG-CoA reductase inhibitors of plant origin are needed. In this study, 25 medicinal plant methanol extracts were screened for anti-HMG-CoA reductase activity. Basella alba leaf extract showed the highest inhibitory effect at about 74%. Thus, B. alba was examined in order to investigate its phytochemical components. Gas chromatography with tandem mass spectrometry and reversed phase high-performance liquid chromatography analysis revealed the presence of phenol 2,6-bis(1,1-dimethylethyl), 1-heptatriacotanol, oleic acid, eicosyl ester, naringin, apigenin, luteolin, ascorbic acid, and α-tocopherol, which have been reported to possess antihypercholesterolemic effects. Further investigation of in vivo models should be performed in order to confirm its potential as an alternative treatment for hypercholesterolemia and related cardiovascular diseases. PMID:25609924

  6. Potentiation of the reductase activity of protein disulphide isomerase (PDI) by 19-nortestosterone, bacitracin, fluoxetine, and ammonium sulphate.

    PubMed

    Hassan, Maya Haj; Alvarez, Eva; Cahoreau, Claire; Klett, Danièle; Lecompte, François; Combarnous, Yves

    2011-10-01

    Protein disulphide isomerase (PDI) in the endoplasmic reticulum catalyzes the rearrangement of disulphide bridges during folding of secreted proteins. It binds various molecules that inhibit its activity. But here, we looked for molecules that would potentiate its activity. PDI reductase activity was measured in vitro using di-eosin-oxidized glutathione as substrate. Its classical inhibitor bacitracin was found to exert a biphasic effect: stimulatory at low concentrations (∼10(-6) M) and inhibitory only at higher concentrations (∼10(-4)-10(-3) M). The weak oestrogenic molecule bisphenol A was found to exert a weak inhibitory effect on PDI reductase activity relative to the strong oestrogens, ethynylestradiol, and diethylstilbestrol. Like 19-nortestosterone, fluoxetine was found to exert a potentiating effect on PDI reductase activity and their potentiating effects could be reversed by increasing concentrations of oestrogens. In conclusion, this paper provides the first identification of potentiators of PDI activity that are potential pharmaceuticals against pathologies affecting protein folding such as Alzheimer's disease. PMID:21250820

  7. Targeted Mutations of Bacillus anthracis Dihydrofolate Reductase Condense Complex Structure-Activity Relationships

    SciTech Connect

    J Beierlein; N Karri; A Anderson

    2011-12-31

    Several antifolates, including trimethoprim (TMP) and a series of propargyl-linked analogues, bind dihydrofolate reductase from Bacillus anthracis (BaDHFR) with lower affinity than is typical in other bacterial species. To guide lead optimization for BaDHFR, we explored a new approach to determine structure-activity relationships whereby the enzyme is altered and the analogues remain constant, essentially reversing the standard experimental design. Active site mutants of the enzyme, Ba(F96I)DHFR and Ba(Y102F)DHFR, were created and evaluated with enzyme inhibition assays and crystal structures. The affinities of the antifolates increase up to 60-fold with the Y102F mutant, suggesting that interactions with Tyr 102 are critical for affinity. Crystal structures of the enzymes bound to TMP and propargyl-linked inhibitors reveal the basis of TMP resistance and illuminate the influence of Tyr 102 on the lipophilic linker between the pyrimidine and aryl rings. Two new inhibitors test and validate these conclusions and show the value of the technique for providing new directions during lead optimization.

  8. Role of active site loop in coenzyme binding and flavin reduction in cytochrome P450 reductase.

    PubMed

    Mothersole, Robert G; Meints, Carla E; Louder, Alex; Wolthers, Kirsten R

    2016-09-15

    Cytochrome P450 reductase (CPR) contains a loop within the active site (comprising Asp(634), Ala(635), Arg(636) and Asn(637); human CPR numbering) that relocates upon NADPH binding. Repositioning of the loop triggers the reorientation of an FAD-shielding tryptophan (Trp(679)) to a partially stacked conformer, reducing the energy barrier for displacement of the residue by the NADPH nicotinamide ring: an essential step for hydride transfer. We used site-directed mutagenesis and kinetic analysis to investigate if the amino acid composition of the loop influences the catalytic properties of CPR. The D634A and D634N variants elicited a modest increase in coenzyme binding affinity coupled with a 36- and 10-fold reduction in cytochrome c(3+) turnover and a 17- and 3-fold decrease in the pre-steady state rate of flavin reduction. These results, in combination with a reduction in the kinetic isotope effect for hydride transfer, suggest that diminished activity is due to destabilization of the partially stacked conformer of Trp(677) and slower release of NADP(+). In contrast, R636A, R636S and an A635G/R636S double mutant led to a modest increase in cytochrome c(3+) reduction, which is linked to weaker coenzyme binding and faster interflavin electron transfer. A potential mechanism by which Arg(636) influences catalysis is discussed. PMID:27461959

  9. Glutathione-dependent extracellular ferric reductase activities in dimorphic zoopathogenic fungi

    PubMed Central

    Zarnowski, Robert; Woods, Jon P.

    2009-01-01

    In this study, extracellular glutathione-dependent ferric reductase (GSH-FeR) activities in different dimorphic zoopathogenic fungal species were characterized. Supernatants from Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis and Sporothrix schenckii strains grown in their yeast form were able to reduce iron enzymically with glutathione as a cofactor. Some variations in the level of reduction were noted amongst the strains. This activity was stable in acidic, neutral and slightly alkaline environments and was inhibited when trivalent aluminium and gallium ions were present. Using zymography, single bands of GSH-FeRs with apparent molecular masses varying from 430 to 460 kDa were identified in all strains. The same molecular mass range was determined by size exclusion chromatography. These data demonstrate that dimorphic zoopathogenic fungi produce and secrete a family of similar GSH-FeRs that may be involved in the acquisition and utilization of iron. Siderophore production by these and other fungi has sometimes been considered to provide a full explanation of iron acquisition in these organisms. Our work reveals an additional common mechanism that may be biologically and pathogenically important. Furthermore, while some characteristics of these enzymes such as extracellular location, cofactor utilization and large size are not individually unique, when considered together and shared across a range of fungi, they represent an important novel physiological feature. PMID:16000713

  10. Glucose and collagen regulate human platelet activity through aldose reductase induction of thromboxane

    PubMed Central

    Tang, Wai Ho; Stitham, Jeremiah; Gleim, Scott; Di Febbo, Concetta; Porreca, Ettore; Fava, Cristiano; Tacconelli, Stefania; Capone, Marta; Evangelista, Virgilio; Levantesi, Giacomo; Wen, Li; Martin, Kathleen; Minuz, Pietro; Rade, Jeffrey; Patrignani, Paola; Hwa, John

    2011-01-01

    Diabetes mellitus is associated with platelet hyperactivity, which leads to increased morbidity and mortality from cardiovascular disease. This is coupled with enhanced levels of thromboxane (TX), an eicosanoid that facilitates platelet aggregation. Although intensely studied, the mechanism underlying the relationship among hyperglycemia, TX generation, and platelet hyperactivity remains unclear. We sought to identify key signaling components that connect high levels of glucose to TX generation and to examine their clinical relevance. In human platelets, aldose reductase synergistically modulated platelet response to both hyperglycemia and collagen exposure through a pathway involving ROS/PLCγ2/PKC/p38α MAPK. In clinical patients with platelet activation (deep vein thrombosis; saphenous vein graft occlusion after coronary bypass surgery), and particularly those with diabetes, urinary levels of a major enzymatic metabolite of TX (11-dehydro-TXB2 [TX-M]) were substantially increased. Elevated TX-M persisted in diabetic patients taking low-dose aspirin (acetylsalicylic acid, ASA), suggesting that such patients may have underlying endothelial damage, collagen exposure, and thrombovascular disease. Thus, our study has identified multiple potential signaling targets for designing combination chemotherapies that could inhibit the synergistic activation of platelets by hyperglycemia and collagen exposure. PMID:22005299

  11. Glucose and collagen regulate human platelet activity through aldose reductase induction of thromboxane.

    PubMed

    Tang, Wai Ho; Stitham, Jeremiah; Gleim, Scott; Di Febbo, Concetta; Porreca, Ettore; Fava, Cristiano; Tacconelli, Stefania; Capone, Marta; Evangelista, Virgilio; Levantesi, Giacomo; Wen, Li; Martin, Kathleen; Minuz, Pietro; Rade, Jeffrey; Patrignani, Paola; Hwa, John

    2011-11-01

    Diabetes mellitus is associated with platelet hyperactivity, which leads to increased morbidity and mortality from cardiovascular disease. This is coupled with enhanced levels of thromboxane (TX), an eicosanoid that facilitates platelet aggregation. Although intensely studied, the mechanism underlying the relationship among hyperglycemia, TX generation, and platelet hyperactivity remains unclear. We sought to identify key signaling components that connect high levels of glucose to TX generation and to examine their clinical relevance. In human platelets, aldose reductase synergistically modulated platelet response to both hyperglycemia and collagen exposure through a pathway involving ROS/PLCγ2/PKC/p38α MAPK. In clinical patients with platelet activation (deep vein thrombosis; saphenous vein graft occlusion after coronary bypass surgery), and particularly those with diabetes, urinary levels of a major enzymatic metabolite of TX (11-dehydro-TXB2 [TX-M]) were substantially increased. Elevated TX-M persisted in diabetic patients taking low-dose aspirin (acetylsalicylic acid, ASA), suggesting that such patients may have underlying endothelial damage, collagen exposure, and thrombovascular disease. Thus, our study has identified multiple potential signaling targets for designing combination chemotherapies that could inhibit the synergistic activation of platelets by hyperglycemia and collagen exposure. PMID:22005299

  12. CIPK23 is involved in iron acquisition of Arabidopsis by affecting ferric chelate reductase activity.

    PubMed

    Tian, Qiuying; Zhang, Xinxin; Yang, An; Wang, Tianzuo; Zhang, Wen-Hao

    2016-05-01

    Iron deficiency is one of the major limiting factors affecting quality and production of crops in calcareous soils. Numerous signaling molecules and transcription factors have been demonstrated to play a regulatory role in adaptation of plants to iron deficiency. However, the mechanisms underlying the iron deficiency-induced physiological processes remain to be fully dissected. Here, we demonstrated that the protein kinase CIPK23 was involved in iron acquisition. Lesion of CIPK23 rendered Arabidopsis mutants hypersensitive to iron deficiency, as evidenced by stronger chlorosis in young leaves and lower iron concentration than wild-type plants under iron-deficient conditions by down-regulating ferric chelate reductase activity. We found that iron deficiency evoked an increase in cytosolic Ca(2+) concentration and the elevated Ca(2+) would bind to CBL1/CBL9, leading to activation of CIPK23. These novel findings highlight the involvement of calcium-dependent CBL-CIPK23 complexes in the regulation of iron acquisition. Moreover, mutation of CIPK23 led to changes in contents of mineral elements, suggesting that CBL-CIPK23 complexes could be as "nutritional sensors" to sense and regulate the mineral homeostasis in Arabisopsis. PMID:26993237

  13. Activity improvement of a Kluyveromyces lactis aldo-keto reductase KlAKR via rational design.

    PubMed

    Luo, Xi; Wang, Ya-Jun; Shen, Wei; Zheng, Yu-Guo

    2016-04-20

    Optically pure t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate ((R)-1b) is the key chiral precursor for atorvastatin calcium, the most widely used cholesterol-lowering drug. Wild-type aldo-keto reductase KlAKR from Kluyveromyces lactis has ideal diastereoselectivity toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a, dep>99.5%) but poor activity. A rational engineering was used to improve the KlAKR activity. Based on homology modeling and molecular docking, two amino acid residues (295 and 296) were selected as mutation sites, and two rounds of site-saturation mutagenesis were performed. Among the mutants, KlAKR-Y295W/W296L exhibited the highest catalytic efficiency (kcat/Km) toward 1a up to 12.37s(-1)mM(-1), which was 11.25-fold higher than that of wild-type KlAKR. Moreover, the majority of mutations have no negative impact on stereoselectivity. Using KlAKR-Y295W/W296L coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for cofactor regeneration, (R)-1b was accumulated up to 162.7mM with dep value above 99.5%. KlAKR-Y295W/W296L represents a robust tool for (R)-1b synthesis. PMID:26959479

  14. Latent nitrate reductase activity is associated with the plasma membrane of corn roots

    NASA Technical Reports Server (NTRS)

    Ward, M. R.; Grimes, H. D.; Huffaker, R. C.

    1989-01-01

    Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.

  15. S-Nitrosation of Conserved Cysteines Modulates Activity and Stability of S-Nitrosoglutathione Reductase (GSNOR).

    PubMed

    Guerra, Damian; Ballard, Keith; Truebridge, Ian; Vierling, Elizabeth

    2016-05-01

    The free radical nitric oxide (NO(•)) regulates diverse physiological processes from vasodilation in humans to gas exchange in plants. S-Nitrosoglutathione (GSNO) is considered a principal nitroso reservoir due to its chemical stability. GSNO accumulation is attenuated by GSNO reductase (GSNOR), a cysteine-rich cytosolic enzyme. Regulation of protein nitrosation is not well understood since NO(•)-dependent events proceed without discernible changes in GSNOR expression. Because GSNORs contain evolutionarily conserved cysteines that could serve as nitrosation sites, we examined the effects of treating plant (Arabidopsis thaliana), mammalian (human), and yeast (Saccharomyces cerevisiae) GSNORs with nitrosating agents in vitro. Enzyme activity was sensitive to nitroso donors, whereas the reducing agent dithiothreitol (DTT) restored activity, suggesting that catalytic impairment was due to S-nitrosation. Protein nitrosation was confirmed by mass spectrometry, by which mono-, di-, and trinitrosation were observed, and these signals were sensitive to DTT. GSNOR mutants in specific non-zinc-coordinating cysteines were less sensitive to catalytic inhibition by nitroso donors and exhibited reduced nitrosation signals by mass spectrometry. Nitrosation also coincided with decreased tryptophan fluorescence, increased thermal aggregation propensity, and increased polydispersity-properties reflected by differential solvent accessibility of amino acids important for dimerization and the shape of the substrate and coenzyme binding pockets as assessed by hydrogen-deuterium exchange mass spectrometry. Collectively, these data suggest a mechanism for NO(•) signal transduction in which GSNOR nitrosation and inhibition transiently permit GSNO accumulation. PMID:27064847

  16. A Continuous Spectrophotometric Assay for APS Reductase Activity with Sulfite-Selective Probes

    PubMed Central

    Paritala, Hanumantharao; Carroll, Kate S.

    2013-01-01

    Mycobacterium tuberculosis (Mtb) adenosine 5′-phosphosulfate (APS) reductase (EC number 1.8.4.10), (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of essential reduced sulfur-containing biomolecules, such as cysteine, and is essential for survival in the latent phase of TB infection. Despite the importance of APR to Mtb, and other bacterial pathogens, current assay methods depend on use of [35S]-labeled APS or shunt AMP to a coupled-enzyme system. Both methods are cumbersome and require the use of expensive reagents. Here we report the development of a continuous spectrophotometric method for measuring APR activity by using novel sulfite-selective colorimetric or “off-on” fluorescent levulinate-based probes. The APR activity can thus be followed by monitoring the increase in absorbance or fluorescence of the resulting phenolate product. Using this assay, we determined Michelis-Menten kinetic constants (Km, kcat, kcat/Km) and apparent inhibition constant (Ki) for adenosine 5′-diphosphate (ADP), which compared favorably to values obtained in the gold-standard radioactive assay. The newly developed assay is robust and easy to perform with a simple spectrophotometer. PMID:23711725

  17. S-nitrosation of conserved cysteines modulates activity and stability of S-nitrosoglutathione reductase (GSNOR)

    PubMed Central

    Guerra, Damian; Ballard, Keith; Truebridge, Ian; Vierling, Elizabeth

    2016-01-01

    The free radical nitric oxide (NO•) regulates diverse physiological processes from vasodilation in humans to gas exchange in plants. S-nitrosoglutathione (GSNO) is considered a principal nitroso reservoir due to its chemical stability. GSNO accumulation is attenuated by GSNO reductase (GSNOR), a cysteine-rich cytosolic enzyme. Regulation of protein nitrosation is not well understood since NO•-dependent events proceed without discernible changes in GSNOR expression. Because GSNORs contain evolutionarily-conserved cysteines that could serve as nitrosation sites, we examined the effects of treating plant (Arabidopsis thaliana), mammalian (human), and yeast (Saccharomyces cerevisiae) GSNORs with nitrosating agents in vitro. Enzyme activity was sensitive to nitroso donors, while the reducing agent dithiothreitol (DTT) restored activity, suggesting catalytic impairment was due to S-nitrosation. Protein nitrosation was confirmed by mass spectrometry, by which mono-, di-, and tri-nitrosation were observed, and these signals were sensitive to DTT. GSNOR mutants in specific non-zinc coordinating cysteines were less sensitive to catalytic inhibition by nitroso donors and exhibited reduced nitrosation signals by mass spectrometry. Nitrosation also coincided with decreased tryptophan fluorescence, increased thermal aggregation propensity, and increased polydispersity—properties reflected by differential solvent accessibility of amino acids important for dimerization and the shape of the substrate and coenzyme binding pockets as assessed by hydrogen-deuterium exchange mass spectrometry. Collectively, these data suggest a mechanism for NO• signal transduction in which GSNOR nitrosation and inhibition transiently permit GSNO accumulation. PMID:27064847

  18. Activity of glutathione peroxidase, glutathione reductase, and lipid peroxidation in erythrocytes in workers exposed to lead.

    PubMed

    Kasperczyk, Slawomir; Kasperczyk, Aleksandra; Ostalowska, Alina; Dziwisz, Maria; Birkner, Ewa

    2004-01-01

    The aim of this study was to estimate the activity of glutathione peroxidase (GPx), glutathione reductase (GR), and malondialdehyde (MDA) in erythrocytes in healthy male employees of zinc and lead steelworks who were occupationally exposed to lead over a long period of time (about 15 yr). Workers were divided into two subgroups: the first included employees with low exposure to lead (LL) (n=75) with blood lead level PbB=25-40 microg/dL and the second with high exposure to lead (HL) (n=62) with PbB over 40 microg/dL. Administration workers (n=35) with normal levels of PbB and zinc protoporphyrin in blood (ZPP) in blood were the control group. The activity of GPx significantly increased in LL when compared to the control group (p<0.001) and decreased when compared to the HL group (p=0.036). There were no significant changes in activity of GR in the study population. MDA erythrocyte concentration significantly increased in the HL group compared to the control (p=0.014) and to the LL group (p=0.024). For the people with low exposure to lead (PbB=25-40 microg/dL), the increase of activity of GPx by about 79% in erythrocytes prevented lipid peroxidation and it appears to be the adaptive mechanism against the toxic effect of lead. People with high exposure to lead (with PbB over 40 microg/dL) have shown an increase in MDA concentration in erythrocytes by about 91%, which seems to have resulted from reduced activity of GPx and the lack of increase in activity of GR in blood red cells. PMID:15621928

  19. Effects of Elevated Cytosolic Glutathione Reductase Activity on the Cellular Glutathione Pool and Photosynthesis in Leaves under Normal and Stress Conditions.

    PubMed

    Foyer, C; Lelandais, M; Galap, C; Kunert, K J

    1991-11-01

    Tobacco (Nicotiana tabacum var Samsun) was transformed using the bacterial gor gene coding for the enzyme glutathione reductase. Transgenic plants were selected by their kanamycin resistence and expression of the bacterial gor gene. After separation by isoelectric focusing techniques, leaf extracts from transgenic plants having both native and bacterial glutathione reductase activity gave, in addition to the six bands of the native enzyme, two further closely running isoenzymes. These additional bands originating from the expression of the bacterial gor gene were nonchloroplastic. Leaves from transgenic plants had two- to 10-fold higher glutathione reductase activity than non-transgenic controls. The amount of extractable glutathione reductase activity obtained in transgenic plants was dependent on leaf age and the conditions to which leaves were exposed. Both light and exposure to methylviologen increased leaf glutathione reductase activity. Elevated levels of cytosolic glutathione reductase activity in transgenic plants had no effect on the amount or reduction state of the reduced glutathione/oxidized glutathione pool under optimal conditions or oxidative conditions induced by methylviologen. The glutathione pool was unaltered despite the oxidation-dependent loss of CO(2) assimilation and oxidation of enzymes involved in photosynthesis. However, the reduction state of the ascorbate pool was greater in transgenic plants relative to nontransgenic controls following illumination of methylviologen-treated leaf discs. Therefore, we conclude that in the natural state glutathione reductase is present in tobacco at levels above those required for maximal operation of the ascorbate-glutathione pathway. PMID:16668524

  20. Platelet Inhibition by Nitrite Is Dependent on Erythrocytes and Deoxygenation

    PubMed Central

    Srihirun, Sirada; Sriwantana, Thanaporn; Unchern, Supeenun; Kittikool, Dusadee; Noulsri, Egarit; Pattanapanyasat, Kovit; Fucharoen, Suthat; Piknova, Barbora; Schechter, Alan N.; Sibmooh, Nathawut

    2012-01-01

    Background Nitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated. Methodology/Finding Platelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 µM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes. Conclusion Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia. PMID:22276188

  1. Microbial activity balance in size fractionated suspended growth biomass from full-scale sidestream combined nitritation-anammox reactors.

    PubMed

    Shi, Yijing; Wells, George; Morgenroth, Eberhard

    2016-10-01

    The purpose of this study was to determine the abundance, distribution and activity of aerobic ammonia-oxidizing bacteria (AOB) and anammox in size fractionated aggregates from full-scale suspended growth combined nitritation-anammox sidestream reactors. Plants with or without a cyclone device were also studied to assess a purported enrichment of anammox granules. Specific aerobic ammonium oxidation rates (p=0.01) and specific oxygen uptake rates (p=0.02) were significantly greater in flocs than in granules. AOB abundance measured using quantitative FISH was significantly higher in flocs than in granules (p=0.01). Conversely, anammox abundance was significantly greater in granules (p=0.03). The average ratio of anammox/AOB in systems employing hydrocyclone separation devices was 2.4, significantly higher (p=0.02) than the average ratio (0.5) in a system without a hydrocyclone. Our results demonstrate substantial functional and population-level segregation between floccular and granular fractions, and provide a key corroboration that cyclone separation devices can increase anammox levels in such systems. PMID:27347796

  2. Effect of temperature on AOB activity of a partial nitritation SBR treating landfill leachate with extremely high nitrogen concentration.

    PubMed

    Gabarró, J; Ganigué, R; Gich, F; Ruscalleda, M; Balaguer, M D; Colprim, J

    2012-12-01

    This study investigates the effects of temperature on ammonia oxidizing bacteria activity in a partial nitritation (PN) sequencing batch reactor. Stable PN was achieved in a 250 L SBR with a minimum operating volume of 111L treating mature landfill leachate containing an ammonium concentration of around 6000 mg N-NH(4)(+)L(-1) at both 25 and 35 °C. A suitable influent to feed an anammox reactor was achieved in both cases. A kinetic model was applied to study the influence of free ammonia (FA), the free nitrous acid (FNA) inhibition, and the inorganic carbon (IC) limitation. NH(4)(+) and NO(2)(-) concentrations were similar at 25 and 35 °C experiments (about 2500 mg N-NH(4)(+)L(-1) and 3500 mg N-NO(2)(-)L(-1)), FA and FNA concentrations differed due to the strong temperature dependence. FNA was the main source of inhibition at 25 °C, while at 35 °C combined FA and FNA inhibition occurred. DGGE results demonstrated that PN-SBR sludge was enriched on the same AOB phylotypes in both experiments. PMID:23079415

  3. Molecular characterization of thioredoxin-1 and thioredoxin reductase activity in mud crab Scylla paramamosain.

    PubMed

    Hu, J H; Zhang, F Y; Jiang, K J; Fang, Y B; Wang, J; Zhao, M; Qiao, Z G; Ma, L B

    2014-01-01

    The thioredoxin (Trx) system consists of thioredoxin reductase (TrxR), Trx, and nicotinamide adenine dinucleotide phosphate (NADPH). TrxR is an NADPH-dependent oxidoreductase. Trx is a ubiquitous small protein with a redox-active disulfide bridge that plays important regulatory roles in some vital metabolic reactions. In this study, a cDNA sequence (SpTrx1) showing high identity to the first Trx gene was isolated from a hepatopancreas cDNA library of the mud crab Scylla paramamosain. The full-length cDNA of SpTrx1 consisted of 672 bp and contained a complete open reading frame of 318 bp encoding a polypeptide of 105 amino acids. Quantitative real-time polymerase chain reaction analysis revealed that SpTrx1 expression was ubiquitous in various organs of S. paramamosain, including the gill, muscle, heart, hemolymph, testis, and hepatopancreas. SpTrx1 expression was upregulated significantly after Vibrio parahaemolyticus challenge: it obviously rose at 48 h and reached the highest level at 72 h. Furthermore, TrxR activity was detected in the gill, heart, muscle, hemolymph, and hepatopancreas. The relative TrxR activity in different tissues after V. parahaemolyticus injection had the same tendency in each tissue (P < 0.01) as SpTrx1 expression. The TrxR activity increased 2 h after injection, peaked at 8 h, slowly decreased from 12 to 24 h, and returned to normal levels at 48 h. The consistency of the expression between the Trx transcript and TrxR activity demonstrated that Trx was closely related to TrxR in the Trx system in S. paramamosain, suggesting that it may participate in the immune system of mud crabs. PMID:25501236

  4. Human Aldo-Keto Reductases and the Metabolic Activation of Polycyclic Aromatic Hydrocarbons

    PubMed Central

    2015-01-01

    Aldo-keto reductases (AKRs) are promiscuous NAD(P)(H) dependent oxidoreductases implicated in the metabolic activation of polycyclic aromatic hydrocarbons (PAH). These enzymes catalyze the oxidation of non-K-region trans-dihydrodiols to the corresponding o-quinones with the concomitant production of reactive oxygen species (ROS). The PAH o-quinones are Michael acceptors and can form adducts but are also redox-active and enter into futile redox cycles to amplify ROS formation. Evidence exists to support this metabolic pathway in humans. The human recombinant AKR1A1 and AKR1C1–AKR1C4 enzymes all catalyze the oxidation of PAH trans-dihydrodiols to PAH o-quinones. Many human AKRs also catalyze the NADPH-dependent reduction of the o-quinone products to air-sensitive catechols, exacerbating ROS formation. Moreover, this pathway of PAH activation occurs in a panel of human lung cell lines, resulting in the production of ROS and oxidative DNA damage in the form of 8-oxo-2′-deoxyguanosine. Using stable-isotope dilution liquid chromatography tandem mass spectrometry, this pathway of benzo[a]pyrene (B[a]P) metabolism was found to contribute equally with the diol-epoxide pathway to the activation of this human carcinogen in human lung cells. Evaluation of the mutagenicity of anti-B[a]P-diol epoxide with B[a]P-7,8-dione on p53 showed that the o-quinone produced by AKRs was the more potent mutagen, provided that it was permitted to redox cycle, and that the mutations observed were G to T transversions, reminiscent of those observed in human lung cancer. It is concluded that there is sufficient evidence to support the role of human AKRs in the metabolic activation of PAH in human lung cell lines and that they may contribute to the causation of human lung cancer. PMID:25279998

  5. In vitro activity of almond skin polyphenols for scavenging free radicals and inducing quinone reductase.

    PubMed

    Chen, C-Y Oliver; Blumberg, Jeffrey B

    2008-06-25

    Observational studies and clinical trials suggest nut intake, including almonds, is associated with an enhancement in antioxidant defense and a reduction in the risk of cancer and cardiovascular disease. Almond skins are rich in polyphenols (ASP) that may contribute to these putative benefits. To assess their potential mechanisms of action, we tested the in vitro effect of ASP extracted with methanol (M) or a gastrointestinal juice mimic (GI) alone or in combination with vitamins C (VC) or E (VE) (1-10 micromol/L) on scavenging free radicals and inducing quinone reductase (QR). Flavonoid profiles from ASP-M and -GI extracts were different from one another. ASP-GI was more potent in scavenging HOCl and ONOO (-) radicals than ASP-M. In contrast, ASP-M increased and ASP-GI decreased QR activity in Hepa1c1c7 cells. Adding VC or VE to ASP produced a combination- and dose-dependent action on radical scavenging and QR induction. In comparison to their independent actions, ASP-M plus VC were less potent in scavenging DPPH, HOCl, ONOO (-), and O 2 (-) (*). However, the interaction between ASP-GI plus VC promoted their radical scavenging activity. Combining ASP-M plus VC resulted in a synergistic interaction, inducing QR activity, but ASP-GI plus VC had an antagonistic effect. On the basis of their total phenolic content, the measures of total antioxidant activity of ASP-M and -GI were comparable. Thus, in vitro, ASP act as antioxidants and induce QR activity, but these actions are dependent upon their dose, method of extraction, and interaction with antioxidant vitamins. PMID:18512942

  6. Human aldo-keto reductases and the metabolic activation of polycyclic aromatic hydrocarbons.

    PubMed

    Penning, Trevor M

    2014-11-17

    Aldo-keto reductases (AKRs) are promiscuous NAD(P)(H) dependent oxidoreductases implicated in the metabolic activation of polycyclic aromatic hydrocarbons (PAH). These enzymes catalyze the oxidation of non-K-region trans-dihydrodiols to the corresponding o-quinones with the concomitant production of reactive oxygen species (ROS). The PAH o-quinones are Michael acceptors and can form adducts but are also redox-active and enter into futile redox cycles to amplify ROS formation. Evidence exists to support this metabolic pathway in humans. The human recombinant AKR1A1 and AKR1C1-AKR1C4 enzymes all catalyze the oxidation of PAH trans-dihydrodiols to PAH o-quinones. Many human AKRs also catalyze the NADPH-dependent reduction of the o-quinone products to air-sensitive catechols, exacerbating ROS formation. Moreover, this pathway of PAH activation occurs in a panel of human lung cell lines, resulting in the production of ROS and oxidative DNA damage in the form of 8-oxo-2'-deoxyguanosine. Using stable-isotope dilution liquid chromatography tandem mass spectrometry, this pathway of benzo[a]pyrene (B[a]P) metabolism was found to contribute equally with the diol-epoxide pathway to the activation of this human carcinogen in human lung cells. Evaluation of the mutagenicity of anti-B[a]P-diol epoxide with B[a]P-7,8-dione on p53 showed that the o-quinone produced by AKRs was the more potent mutagen, provided that it was permitted to redox cycle, and that the mutations observed were G to T transversions, reminiscent of those observed in human lung cancer. It is concluded that there is sufficient evidence to support the role of human AKRs in the metabolic activation of PAH in human lung cell lines and that they may contribute to the causation of human lung cancer. PMID:25279998

  7. Evidence for Increased 5α-Reductase Activity During Early Childhood in Daughters of Women With Polycystic Ovary Syndrome

    PubMed Central

    Torchen, Laura C.; Idkowiak, Jan; Fogel, Naomi R.; O'Neil, Donna M.; Shackleton, Cedric H. L.; Arlt, Wiebke

    2016-01-01

    Context: Polycystic ovary syndrome (PCOS) is a heritable, complex genetic disease. Animal models suggest that androgen exposure at critical developmental stages contributes to disease pathogenesis. We hypothesized that genetic variation resulting in increased androgen production produces the phenotypic features of PCOS by programming during critical developmental periods. Although we have not found evidence for increased in utero androgen levels in cord blood in the daughters of women with PCOS (PCOS-d), target tissue androgen production may be amplified by increased 5α-reductase activity analogous to findings in adult affected women. It is possible to noninvasively test this hypothesis by examining urinary steroid metabolites. Objective: We performed this study to investigate whether PCOS-d have altered androgen metabolism during early childhood. Design, Setting, and Participants: Twenty-one PCOS-d, 1–3 years old, and 36 control girls of comparable age were studied at an academic medical center. Main Outcome Measures: Urinary steroid metabolites were measured by gas chromatography/mass spectrometry. Twenty-four hour steroid excretion rates and precursor to product ratios suggestive of 5α-reductase and 11β-hydroxysteroid dehydrogenase activities were calculated. Results: Age did not differ but weight for length Z-scores were higher in PCOS-d compared to control girls (P = .02). PCOS-d had increased 5α-tetrahydrocortisol:tetrahydrocortisol ratios (P = .04), suggesting increased global 5α-reductase activity. There was no evidence for differences in 11β-hydroxysteroid dehydrogenase activity. Steroid metabolite excretion was not correlated with weight. Conclusions: Our findings suggest that differences in androgen metabolism are present in early childhood in PCOS-d. Increased 5α-reductase activity could contribute to the development of PCOS by amplifying target tissue androgen action. PMID:26990942

  8. Protective effect of salivary nitrate and microbial nitrate reductase activity against caries.

    PubMed

    Doel, J J; Hector, M P; Amirtham, C V; Al-Anzan, L A; Benjamin, N; Allaker, R P

    2004-10-01

    To test the hypothesis that a combination of high salivary nitrate and high nitrate-reducing capacity are protective against dental caries, 209 children attending the Dental Institute, Barts and The London NHS Trust were examined. Salivary nitrate and nitrite levels, counts of Streptococcus mutans and Lactobacillus spp., and caries experience were recorded. Compared with control subjects, a significant reduction in caries experience was found in patients with high salivary nitrate and high nitrate-reducing ability. Production of nitrite from salivary nitrate by commensal nitrate-reducing bacteria may limit the growth of cariogenic bacteria as a result of the production of antimicrobial oxides of nitrogen, including nitric oxide. PMID:15458501

  9. [Effect of anticancer agents on rat prostate. Evaluation of organ weight, histological finding and 5 alpha-reductase activities].

    PubMed

    Takeda, M; Hosaka, M; Kitajima, N; Noguchi, K; Fujii, H; Oshima, H; Harada, M

    1985-01-01

    To evaluate the effect of anticancer chemotherapeutic antigens on rat prostate, ten kinds of anticancer agents corresponding to the dose generally used for humans were intraperitoneally injected to 63-day-old Wistar rats. The anticancer agents were administered as follows: Cyclophosphamide (CPM) was used at the dose of 8 mg/kg for 7 days. Methotrexate (MTX), actinomycin-D (ACD) and cis-platinum (CDDP), 163 micrograms/kg, 8 micrograms/kg and 833 micrograms/kg for 5 days, respectively. Nitrogen mustard (NM), bleomycin (BLM), peplomycin (PLM), adriamycin (ADM), vincristine (VCR), and vinblastine (VBL), 500 micrograms/kg, 250 micrograms/kg, 170 micrograms/kg, 2.5 mg/kg, 33 micrograms/kg and 83 micrograms/kg, twice in a week, respectively. The rats were killed on the fifth day after completion of the schedule. Then, the weight of the body, the prostate, the epididymis and the adrenal gland were measured. In addition, 5 alpha-reductase activities and histological findings in the prostate were examined. For determination of 5 alpha-reductase activities, cell-free homogenate obtained from the rat ventral prostate was incubated with C14-testosterone at 37 degrees C for 30 minutes in an atmosphere of 95% of O2 and 5% of CO2. Subsequently, the metabolites from testosterone were separated and purified with thin layer chromatography using the solvent system with benzene acetone, 4:1 (v/v). 5 alpha-Reductase activity was determined with the sum of dihydrotestosterone (DHT) and androstanediol converted from testosterone and indicated as pmol product/mg protein. The 5 alpha-reductase activity was employed as a biological marker for the degree of androgenic dependency in the prostate. The results were summarized as follows. CDDP significantly reduced the weight of the body (p less than 0.001, n = 7), but not the activity of 5 alpha-reductase. NM and VBL had a specific action to reduce the weight of the prostate (p less than 0.01, n = 8) without causing loss of body weight. NM and

  10. Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars

    PubMed Central

    Bicho, Paul A.; Runnals, P. Lynn; Cunningham, J. Douglas; Lee, Hung

    1988-01-01

    The induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars was investigated in the yeasts Pachysolen tannophilus and Pichia stipitis. Enzyme activities induced on d-xylose served as the controls. In both yeasts, d-glucose, d-mannose, and 2-deoxyglucose inhibited enzyme induction by d-xylose to various degrees. Cellobiose, l-arabinose, and d-galactose were not inhibitory. In liquid batch culture, P. tannophilus utilized d-glucose and d-mannose rapidly and preferentially over d-xylose, while d-galactose consumption was poor and lagged behind that of the pentose sugar. In P. stipitis, all three hexoses were used preferentially over d-xylose. The results showed that the repressibility of xylose reductase and xylitol dehydrogenase may limit the potential of yeast fermentation of pentose sugars in hydrolysates of lignocellulosic substrates. PMID:16347538

  11. Tobacco Nectarin III is a bifunctional enzyme with monodehydroascorbate reductase and carbonic anhydrase activities.

    PubMed

    Carter, Clay J; Thornburg, Robert W

    2004-02-01

    Tobacco plants secrete a limited array of proteins (nectarins) into their floral nectar. N-terminal sequencing of the Nectarin II ( NEC2; 35kD) and the Nectarin III ( NEC3; 40kD) proteins revealed that they both share identity with dioscorin, the major soluble protein of yam tubers. These sequences also revealed that NEC2 is a breakdown product of NEC3. Using these N-terminal peptide sequences, degenerate oligonucleotides were designed that permitted the isolation of a partial NEC3 cDNA. This cDNA was then used to probe a nectary specific cDNA library and a full-length NEC3 cDNA clone was isolated. Complete sequence analysis confirmed the identity of NEC3 as a dioscorin-like protein. MALDI-TOF mass spectrometric fingerprinting of tryptic peptides derived from the purified NEC3 confirmed that this protein was encoded by the isolated cDNA. NEC3 was shown to possess both carbonic anhydrase and monodehydroascorbate reductase activities. RT-PCR based expression analyses demonstrated that NEC3 transcript is expressed throughout nectary development as well as in other floral organs. A proposed function in the maintenance of pH and oxidative balance in nectar is discussed. PMID:15284496

  12. Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site

    PubMed Central

    Barrack, Keri L.; Tulloch, Lindsay B.; Burke, Lynsey-Ann; Fyfe, Paul K.; Hunter, William N.

    2011-01-01

    Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species, protozoa that are responsible for a range of serious diseases found in tropical and subtropical parts of the world. As part of a structure-based approach to inhibitor development, specifically targeting Leishmania species, well ordered crystals of L. donovani PTR1 were sought to support the characterization of complexes formed with inhibitors. An efficient system for recombinant protein production was prepared and the enzyme was purified and crystallized in an orthorhombic form with ammonium sulfate as the precipitant. Diffraction data were measured to 2.5 Å resolution and the structure was solved by molecular replacement. However, a sulfate occupies a phosphate-binding site used by NADPH and occludes cofactor binding. The nicotinamide moiety is a critical component of the active site and without it this part of the structure is disordered. The crystal form obtained under these conditions is therefore unsuitable for the characterization of inhibitor complexes. PMID:21206018

  13. A role for 5alpha-reductase activity in the development of male homosexuality?

    PubMed

    Alias, A G

    2004-12-01

    Higher body hair with lower mesmorphism ratings were observed in Caucasian homosexual men compared with the general male population, reflecting elevated 5alpha-reductase (5alphaR) activity, and higher dihydrotestosterone-to-testosterone (DHT-to-T) ratio, in sharp contrast to 46,XY 5alphaR 2 deficiency subjects, who are often born with ambiguous, or female genitalia, but tend to grow up to be muscular, heterosexual men with very little body hair, or beard. One study also showed them scoring around dull normal IQs. A greater prevalence of liberal body hair growth in men with higher IQs and/or educational levels was also observed in several samples. The exceptions to this statistical trend are too unsettling, however. Nevertheless, the results of a number of published studies, including one showing higher DHT-to-T ratio in homosexual men, done with different objectives over a span of 80 years, together strongly support these findings. Furthermore, in an animal model, "cognitive-enhancing effects" of "5alpha-reduced androgen [metabolites]" were recently demonstrated. PMID:15677419

  14. Non-native ligands define the active site of Pennisetum glaucum (L.) R. Br dehydroascorbate reductase.

    PubMed

    Krishna Das, Bhaba; Kumar, Amit; Maindola, Priyank; Mahanty, Srikrishna; Jain, S K; Reddy, Mallireddy K; Arockiasamy, Arulandu

    2016-05-13

    Dehydroascorbate reductase (DHAR), a member of the glutathione-S-transferase (GST) family, reduces dehydroascorbate (DHA) to ascorbate (AsA; Vitamin-C) in a glutathione (GSH)-dependent manner and in doing so, replenishes the critical AsA pool of the cell. To understand the enzyme mechanism in detail, we determined the crystal structure of a plant DHAR from Pennisetum glaucum (PgDHAR) using Iodide-Single Anomalous Dispersion (SAD) and Molecular replacement methods, in two different space groups. Here, we show PgDHAR in complex with two non-native ligands, viz. an acetate bound at the G-site, which resembles the γ-carboxyl moiety of GSH, and a glycerol at the H-site, which shares the backbone of AsA. We also show that, in the absence of bound native substrates, these non-native ligands help define the critical 'hook points' in the DHAR enzyme active site. Further, our data suggest that these non-native ligands can act as the logical bootstrapping points for iterative design of inhibitors/analogs for DHARs. PMID:27067046

  15. Response of AMP-activated protein kinase and energy metabolism to acute nitrite exposure in the Nile tilapia Oreochromis niloticus.

    PubMed

    Xu, Zhixin; Li, Erchao; Xu, Chang; Gan, Lei; Qin, Jian G; Chen, Liqiao

    2016-08-01

    Adenosine monophosphate-activated protein kinase (AMPK) is a prevalent mammalian energy metabolism sensor, but little is known about its role as an energy sensor in fish experiencing stress. We aimed to study AMPK in Oreochromis niloticus on both the molecular and the physical level. We found that the cDNAs encoding the AMPKα1 and AMPKα2 variants of the O. niloticus catalytic α subunit were 1753bp and 2563 bp long and encoded 571 and 557 amino acids, respectively. Both the AMPKα1 and the AMPKα2 isoform possess structural features similar to mammalian AMPKα, including a phosphorylation site at Thr172 in the N-terminus, and exhibit high homology with other fish and vertebrate AMPKα sequences (81.3%-98.1%). mRNA encoding the AMPKα isoforms was widely expressed in various tissues with distinctive patterns. AMPKα1 and AMPKα2 were primarily expressed in the intestines and brain, respectively. Under acute nitrite challenge, the mRNA encoding the AMPKα isoforms, as well as AMPK activity, changed over time. Its recovery period in freshwater, combined with the fact that it is highly conserved, suggests that fish AMPK, like its mammalian orthologues, acts as an energy metabolism sensor. Furthermore, subsequent decreases in AMPK mRNA levels and activity suggested that its action was transient but efficient. Physically, glucose, lactic acid and TGs in plasma, as well as energy materials in the hepatopancreas and muscle, were significantly altered over time, indicating changes in energy metabolism during the experimental period. These data have enabled us to characterize energy utilization in O. niloticus and further illustrate the role of fish AMPK as an energy sensor. This study provides new insight into energy metabolism and sensing by AMPK in teleost and necessitates further study of the multiple physiologic roles of AMPK in fish. PMID:27262938

  16. A Cu4S Model for the Nitrous Oxide Reductase Active Sites Supported Only by Nitrogen Ligands†

    PubMed Central

    Johnson, Brittany J.; Antholine, William E.; Lindeman, Sergey V.; Mankad, Neal P.

    2016-01-01

    To model the (His)7Cu4Sn (n = 1 or 2) active sites of nitrous oxide reductase, the first Cu4(μ4-S) cluster supported only by nitrogen donors has been prepared using amidinate supporting ligands. Structural, magnetic, spectroscopic, and computational characterization is reported. Electrochemical data indicates that the 2-hole model complex can be reduced reversibly to the 1-hole state and irreversibly to the fully reduced state. PMID:26111160

  17. A Cu4S model for the nitrous oxide reductase active sites supported only by nitrogen ligands.

    PubMed

    Johnson, Brittany J; Antholine, William E; Lindeman, Sergey V; Mankad, Neal P

    2015-07-28

    To model the (His)7Cu4Sn (n = 1 or 2) active sites of nitrous oxide reductase, the first Cu4(μ4-S) cluster supported only by nitrogen donors has been prepared using amidinate supporting ligands. Structural, magnetic, spectroscopic, and computational characterization is reported. Electrochemical data indicates that the 2-hole model complex can be reduced reversibly to the 1-hole state and irreversibly to the fully reduced state. PMID:26111160

  18. Kinetics of Hydrogen Atom Abstraction from Substrate by an Active Site Thiyl Radical in Ribonucleotide Reductase

    PubMed Central

    2015-01-01

    Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Active E. coli class Ia RNR is an α2β2 complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 → [β-W48] → β-Y356 → α-Y731 → α-Y730 → α-C439) that spans ∼35 Å. To unmask PCET kinetics from rate-limiting conformational changes, we prepared a photochemical RNR containing a [ReI] photooxidant site-specifically incorporated at position 355 ([Re]-β2), adjacent to PCET pathway residue Y356 in β. [Re]-β2 was further modified by replacing Y356 with 2,3,5-trifluorotyrosine to enable photochemical generation and spectroscopic observation of chemically competent tyrosyl radical(s). Using transient absorption spectroscopy, we compare the kinetics of Y· decay in the presence of substrate and wt-α2, Y731F-α2 ,or C439S-α2, as well as with 3′-[2H]-substrate and wt-α2. We find that only in the presence of wt-α2 and the unlabeled substrate do we observe an enhanced rate of radical decay indicative of forward radical propagation. This observation reveals that cleavage of the 3′-C–H bond of substrate by the transiently formed C439· thiyl radical is rate-limiting in forward PCET through α and has allowed calculation of a lower bound for the rate constant associated with this step of (1.4 ± 0.4) × 104 s–1. Prompting radical propagation with light has enabled observation of PCET events heretofore inaccessible, revealing active site chemistry at the heart of RNR catalysis. PMID:25353063

  19. Probing the Electrostatics of Active Site Microenvironments along the Catalytic Cycle for Escherichia coli Dihydrofolate Reductase

    PubMed Central

    2015-01-01

    Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and 13C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor–acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for

  20. Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity

    SciTech Connect

    Y Lin; N Yeung; Y Gao; K Miner; L Lei; H Robinson; Y Lu

    2011-12-31

    A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called Fe{sub B}Mb(-His)). A high resolution (1.65 {angstrom}) crystal structure of Cu(II)-CN{sup -}-Fe{sub B}Mb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe{sub B}Mb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-Fe{sub B}Mb(-His) and Fe(II)-Fe{sub B}Mb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N{sub 2}O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-Fe{sub B}Mb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-Fe{sub B}Mb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.

  1. Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity

    SciTech Connect

    Lin, Y.W.; Robinson, H.; Yeung, N.; Gao, Y.-G.; Miner, K. D.; Lei, L.; Lu, Y.

    2010-07-28

    A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called Fe{sub B}Mb(-His)). A high resolution (1.65 {angstrom}) crystal structure of Cu(II)-CN?-Fe{sub B}Mb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe{sub B}Mb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-Fe{sub B}Mb(-His) and Fe(II)-Fe{sub B}Mb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N{sub 2}O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-Fe{sub B}Mb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-Fe{sub B}Mb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.

  2. Exogenous Methyl Jasmonate Treatment Increases Glucosinolate Biosynthesis and Quinone Reductase Activity in Kale Leaf Tissue

    PubMed Central

    Ku, Kang-Mo; Jeffery, Elizabeth H.; Juvik, John A.

    2014-01-01

    Methyl jasmonate (MeJA) spray treatments were applied to the kale varieties ‘Dwarf Blue Curled Vates’ and ‘Red Winter’ in replicated field plantings in 2010 and 2011 to investigate alteration of glucosinolate (GS) composition in harvested leaf tissue. Aqueous solutions of 250 µM MeJA were sprayed to saturation on aerial plant tissues four days prior to harvest at commercial maturity. The MeJA treatment significantly increased gluconasturtiin (56%), glucobrassicin (98%), and neoglucobrassicin (150%) concentrations in the apical leaf tissue of these genotypes over two seasons. Induction of quinone reductase (QR) activity, a biomarker for anti-carcinogenesis, was significantly increased by the extracts from the leaf tissue of these two cultivars. Extracts of apical leaf tissues had greater MeJA mediated increases in phenolics, glucosinolate concentrations, GS hydrolysis products, and QR activity than extracts from basal leaf tissue samples. The concentration of the hydrolysis product of glucoraphanin, sulforphane was significantly increased in apical leaf tissue of the cultivar ‘Red Winter’ in both 2010 and 2011. There was interaction between exogenous MeJA treatment and environmental conditions to induce endogenous JA. Correlation analysis revealed that indole-3-carbanol (I3C) generated from the hydrolysis of glucobrassicin significantly correlated with QR activity (r = 0.800, P<0.001). Concentrations required to double the specific QR activity (CD values) of I3C was calculated at 230 µM, which is considerably weaker at induction than other isothiocyanates like sulforphane. To confirm relationships between GS hydrolysis products and QR activity, a range of concentrations of MeJA sprays were applied to kale leaf tissues of both cultivars in 2011. Correlation analysis of these results indicated that sulforaphane, NI3C, neoascorbigen, I3C, and diindolylmethane were all significantly correlated with QR activity. Thus, increased QR activity may be due to

  3. Interaction with extracellular matrix proteins influences Lsh/Ity/Bcg (candidate Nramp) gene regulation of macrophage priming/activation for tumour necrosis factor-alpha and nitrite release.

    PubMed

    Formica, S; Roach, T I; Blackwell, J M

    1994-05-01

    The murine resistance gene Lsh/Ity/Bcg regulates activation of macrophages for tumour necrosis factor-alpha (TNF-alpha)-dependent production of nitric oxide mediating antimicrobial activity against Leishmania, Salmonella and Mycobacterium. As Lsh is differentially expressed in macrophages from different tissue sites, experiments were performed to determine whether interaction with extracellular matrix (ECM) proteins would influence the macrophage TNF-alpha response. Plating of bone marrow-derived macrophages onto purified fibrinogen or fibronectin-rich L929 cell-derived matrices, but not onto mannan, was itself sufficient to stimulate TNF-alpha release, with significantly higher levels released from congenic B10.L-Lshr compared to C57BL/10ScSn (Lshs) macrophages. Only macrophages plated onto fibrinogen also released measurable levels of nitrites, again higher in Lshr compared to Lshs macrophages. Addition of interferon-gamma (IFN-gamma), but not bacterial lipopolysaccharide or mycobacterial lipoarabinomannan, as a second signal enhanced the TNF-alpha and nitrite responses of macrophages plated onto fibrinogen, particularly in the Lshr macrophages. Interaction with fibrinogen and fibronectin also primed macrophages for an enhanced TNF-alpha response to leishmanial parasites, but this was only translated into enhanced nitrite responses in the presence of IFN-gamma. In these experiments, Lshr macrophages remained superior in their TNF-alpha responses throughout, but to a degree which reflected the magnitude of the difference observed on ECM alone. Hence, the specificity for the enhanced TNF-alpha responses of Lshr macrophages lay in their interaction with fibrinogen and fibronectin ECM, while a differential nitrite response was only observed with fibrinogen and/or IFN-gamma. The results are discussed in relation to the possible function of the recently cloned candidate gene Nramp, which has structural identity to eukaryote transporters and an N-terminal cytoplasmic

  4. 21 CFR 181.34 - Sodium nitrite and potassium nitrite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium nitrite and potassium nitrite. 181.34...-Sanctioned Food Ingredients § 181.34 Sodium nitrite and potassium nitrite. Sodium nitrite and potassium... fixatives and preservative agents, with or without sodium or potassium nitrate, in the curing of red...

  5. 21 CFR 181.34 - Sodium nitrite and potassium nitrite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium nitrite and potassium nitrite. 181.34...-Sanctioned Food Ingredients § 181.34 Sodium nitrite and potassium nitrite. Sodium nitrite and potassium... fixatives and preservative agents, with or without sodium or potassium nitrate, in the curing of red...

  6. 21 CFR 181.34 - Sodium nitrite and potassium nitrite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium nitrite and potassium nitrite. 181.34...-Sanctioned Food Ingredients § 181.34 Sodium nitrite and potassium nitrite. Sodium nitrite and potassium... fixatives and preservative agents, with or without sodium or potassium nitrate, in the curing of red...

  7. 21 CFR 181.34 - Sodium nitrite and potassium nitrite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium nitrite and potassium nitrite. 181.34...-Sanctioned Food Ingredients § 181.34 Sodium nitrite and potassium nitrite. Sodium nitrite and potassium... fixatives and preservative agents, with or without sodium or potassium nitrate, in the curing of red...

  8. Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity.

    PubMed

    Chi, Jen-Chih; Roeper, Juliane; Schwarz, Guenter; Fischer-Schrader, Katrin

    2015-03-01

    14-3-3 proteins represent a family of ubiquitous eukaryotic proteins involved in numerous signal transduction processes and metabolic pathways. One important 14-3-3 target in higher plants is nitrate reductase (NR), whose activity is regulated by different physiological conditions. Intra-molecular electron transfer in NR is inhibited following 14-3-3 binding to a conserved phospho-serine motif located in hinge 1, a surface exposed loop between the catalytic molybdenum and central heme domain. Here we describe a novel 14-3-3 binding site within the NR N-terminus, an acidic motif conserved in NRs of higher plants, which significantly contributes to 14-3-3-mediated inhibition of NR. Deletion or mutation of the N-terminal acidic motif resulted in a significant loss of 14-3-3 mediated inhibition of Ser534 phosphorylated NR-Mo-heme (residues 1-625), a previously established model of NR regulation. Co-sedimentation and crosslinking studies with NR peptides comprising each of the two binding motifs demonstrated direct binding of either peptide to 14-3-3. Surface plasmon resonance spectroscopy disclosed high-affinity binding of 14-3-3ω to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3ω variant retained interaction to the acidic motif, but lost binding to the phospho-hinge motif. To our knowledge, NR is the first enzyme that harbors two independent 14-3-3 binding sites with different affinities, which both need to be occupied by 14-3-3ω to confer full inhibition of NR activity under physiological conditions. PMID:25578809

  9. Melatonin Reduces Cataract Formation and Aldose Reductase Activity in Lenses of Streptozotocin-induced Diabetic Rat

    PubMed Central

    Khorsand, Marjan; Akmali, Masoumeh; Sharzad, Sahab; Beheshtitabar, Mojtaba

    2016-01-01

    Background: The relationship between the high activity of aldose reductase (AR) and diabetic cataract formation has been previously investigated. The purpose of the present study was to determine the preventing effect of melatonin on streptozotocin (STZ)-induced diabetic cataract in rats. Methods: 34 adult healthy male Sprague-Dawely rats were divided into four groups. Diabetic control and diabetic+melatonin received a single dose of STZ (50 mg/kg, intraperitoneally), whereas the normal control and normal+melatonin received vehicle. The melatonin groups were gavaged with melatonin (5 mg/kg) daily for a period of 8 weeks, whereas the rats in the normal control and diabetic control groups received only the vehicle. The rats’ eyes were examined every week and cataract formation scores (0-4) were determined by slit-lamp microscope. At the end of the eighth week, the rats were sacrificed and markers of the polyol pathway and antioxidative (Glutathione, GSH) in their lens were determined. The levels of blood glucose, HbA1c and plasma malondialdhyde (MDA), as a marker of lipid peroxidation, were also measured. Results: Melatonin prevented STZ-induced hyperglycemia by decreased blood glucose and HbA1c levels. Slit lamp examination indicated that melatonin delayed cataract progression in diabetic rats. The results revealed that melatonin feeding increased the GSH levels, decreased the activities of AR and sorbitol dehydrogenase (SDH) and sorbitol formation in catractous lenses as well as plasma MDA content. Conclusion: In summary, for the first time we demonstrated that melatonin delayed the formation and progression of cataract in diabetic rat lenses. PMID:27365552

  10. Dissection of malonyl-coenzyme A reductase of Chloroflexus aurantiacus results in enzyme activity improvement.

    PubMed

    Liu, Changshui; Wang, Qi; Xian, Mo; Ding, Yamei; Zhao, Guang

    2013-01-01

    The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2)G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K cat/K m value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems. PMID:24073271

  11. Mercury Resistance and Mercuric Reductase Activities and Expression among Chemotrophic Thermophilic Aquificae

    PubMed Central

    Freedman, Zachary; Zhu, Chengsheng

    2012-01-01

    Mercury (Hg) resistance (mer) by the reduction of mercuric to elemental Hg is broadly distributed among the Bacteria and Archaea and plays an important role in Hg detoxification and biogeochemical cycling. MerA is the protein subunit of the homodimeric mercuric reductase (MR) enzyme, the central function of the mer system. MerA sequences in the phylum Aquificae form the deepest-branching lineage in Bayesian phylogenetic reconstructions of all known MerA homologs. We therefore hypothesized that the merA homologs in two thermophilic Aquificae, Hydrogenobaculum sp. strain Y04AAS1 (AAS1) and Hydrogenivirga sp. strain 128-5-R1-1 (R1-1), specified Hg resistance. Results supported this hypothesis, because strains AAS1 and R1-1 (i) were resistant to >10 μM Hg(II), (ii) transformed Hg(II) to Hg(0) during cellular growth, and (iii) possessed Hg-dependent NAD(P)H oxidation activities in crude cell extracts that were optimal at temperatures corresponding with the strains' optimal growth temperatures, 55°C for AAS1 and 70°C for R1-1. While these characteristics all conformed with the mer system paradigm, expression of the Aquificae mer operons was not induced by exposure to Hg(II) as indicated by unity ratios of merA transcripts, normalized to gyrA transcripts for hydrogen-grown AAS1 cultures, and by similar MR specific activities in thiosulfate-grown cultures with and without Hg(II). The Hg(II)-independent expression of mer in the deepest-branching lineage of MerA from bacteria whose natural habitats are Hg-rich geothermal environments suggests that regulated expression of mer was a later innovation likely in environments where microorganisms were intermittently exposed to toxic concentrations of Hg. PMID:22773655

  12. Interaction of Product Analogues With the Active Site of Rhodobacter Sphaeroides Dimethyl Sulfoxide Reductase

    SciTech Connect

    George, G.N.; Nelson, K.J.; Harris, H.H.; Doonan, C.J.; Rajagopalan, K.V.; /Saskatchewan U. /Duke U. /Sydney U.

    2007-07-09

    We report a structural characterization using X-ray absorption spectroscopy of Rhodobacter sphaeroides dimethylsulfoxide (DMSO) reductase reduced with trimethylarsine, and show that this is structurally analogous to the physiologically relevant dimethylsulfide-reduced DMSO reductase. Our data unambiguously indicate that these species should be regarded as formal MoIV species, and indicate a classical coordination complex of trimethylarsine oxide, with no special structural distortions. The similarity of the trimethylarsine and dimethylsulfide complexes suggests in turn that the dimethylsulfide reduced enzyme possesses a classical coordination of DMSO with no special elongation of the S-O bond, as previously suggested.

  13. Imaging the activity of nitrate reductase by means of a scanning electrochemical microscope.

    PubMed

    Zaumseil, J; Wittstock, G; Bahrs, S; Steinrücke, P

    2000-06-01

    Scanning electrochemical microscopy (SECM) was used to characterize immobilized nitrate reductase (NaR) from Pseudonomonas stutzeri (E.C. 1.7.99.4). Nitrate reductase with membrane fragment was embedded in a polyurethane hydrogel in a capillary and solubilized NaR without membrane fragment was covalently coupled to a diaminoethyl-cellulose-carbamitate film on glass. After systematic studies of possible mediators, SECM feedback imaging of both forms of immobilized NaR was accomplished with methylviologen as redox mediator. PMID:11225859

  14. Interaction of Product Analogs with the Active Site of Rhodobacter sphaeroides Dimethylsulfoxide Reductase

    PubMed Central

    George, Graham N.; Nelson, Kimberly Johnson; Harris, Hugh H.; Doonan, Christian J.; Rajagopalan, K.V.

    2007-01-01

    We report a structural characterization using X-ray absorption spectroscopy of Rhodobacter sphaeroides dimethylsulfoxide (DMSO) reductase reduced with trimethylarsine, and show that this is structurally analogous to the physiologically relevant dimethylsulfide-reduced DMSO reductase. Our data unambiguously indicate that these species should be regarded as formal MoIV species, and indicate a classical coordination complex of trimethylarsine oxide, with no special structural distortions. The similarity of the trimethylarsine and dimethylsulfide complexes suggests in turn that the dimethylsulfide reduced enzyme possesses a classical coordination of DMSO with no special elongation of the S—O bond, as previously suggested. PMID:17361996

  15. Nitrite impacts the survival of Mycobacterium tuberculosis in response to isoniazid and hydrogen peroxide

    PubMed Central

    Cunningham-Bussel, Amy; Bange, Franz C; Nathan, Carl F

    2013-01-01

    When access to molecular oxygen is restricted, Mycobacterium tuberculosis (Mtb) can respire an alternative electron acceptor, nitrate. We found that Mtb within infected primary human macrophages in vitro at physiologic tissue oxygen tensions respired nitrate, generating copious nitrite. A strain of Mtb lacking a functioning nitrate reductase was more susceptible than wild-type Mtb to treatment with isoniazid during infection of macrophages. Likewise, nitrate reductase-deficient Mtb was more susceptible to isoniazid than wild-type Mtb in axenic culture, and more resistant to hydrogen peroxide. These phenotypes were reversed by the addition of exogenous nitrite. Further investigation suggested that nitrite might inhibit the bacterial catalase. To the extent that Mtb itself is the most relevant source of nitrite acting within Mtb, these findings suggest that inhibitors of Mtb's nitrate transporter or nitrate reductase could enhance the efficacy of isoniazid. PMID:24019302

  16. Biosynthesis of catalytically active rat testosterone 5. alpha. -reductase in microinjected Xenopus oocytes: Evidence for tissue-specific differences in translatable mRNA

    SciTech Connect

    Farkash, Y.; Soreq, H.; Orly, J. )

    1988-08-01

    The enzyme 4-ene-3-ketosteroid-5{alpha}-oxidoreductase plays a key role in androgen-dependent target tissues, where it catalyzes the conversion of testosterone to the biologically active dihydrotestosterone. The regulation of 5{alpha}-reductase expression has not been studied at the molecular level as the enzyme is a membrane protein that is labile in cell-free homogenates. The authors developed a sensitive bioassay of the enzyme activity expressed in Xenopus oocytes microinjected with rat liver and prostate mRNA. After microinjection, incubation of intact oocytes in the presence of ({sup 3}H)testosterone revealed the in ovo appearance of active 5{alpha}-reductase. Polyandenylylated RNA was fractionated by sucrose gradient centrifugation, and the enzymatic activity was shown to be encoded by a 1,600- to 2,000-base-pair fraction of hepatic poly(A){sup +} RNA. 5{alpha}-Reductase mRNA was most efficiently translated when up to 80 ng of RNA was injected per oocyte. In the injected oocytes, 5{alpha}-reductase mRNA was found to be a short-lived molecule whereas its in ovo translatable 5{alpha}-reductase protein exhibited stable enzymatic activity for over 40 hr. Moreover, the levels of translatable tissue-specific 5{alpha}-reductase mRNAs as monitored in the Xenopus oocytes correlated with the variable 5{alpha}-reductase activities in female rat liver, male rat liver, and prostate homogenates. Altogether, these results provide supporting evidence in favor of the transcriptional control of 5{alpha}-reductase expression in rat tissues.

  17. Nitrite in organ protection

    PubMed Central

    Rassaf, Tienush; Ferdinandy, Peter; Schulz, Rainer

    2014-01-01

    In the last decade, the nitrate-nitrite-nitric oxide pathway has emerged to therapeutical importance. Modulation of endogenous nitrate and nitrite levels with the subsequent S-nitros(yl)ation of the downstream signalling cascade open the way for novel cytoprotective strategies. In the following, we summarize the actual literature and give a short overview on the potential of nitrite in organ protection. PMID:23826831

  18. Functional complementation of a nitrate reductase defective mutant of a green alga Dunaliella viridis by introducing the nitrate reductase gene.

    PubMed

    Sun, Yu; Gao, Xiaoshu; Li, Qiyun; Zhang, Qingqi; Xu, Zhengkai

    2006-08-01

    Nitrate reductase (NR) catalyzes NAD (P) H dependent reduction of nitrate to nitrite. Transformation systems have been established in several species of green algae by nitrate reductase gene functional complementation. In this report, an endogenous NR cDNA (3.4 kb) and a genomic fragment (14.6 kb) containing the NR gene (DvNIA1) were isolated from the D. viridis cDNA and genomic libraries respectively. Southern blot and Northern blot analyses showed that this gene exists as a single copy in D. viridis and is induced by nitrate. To obtain a NR defective mutant as a recipient strain, D. viridis cells were treated with a chemical mutagen and then cultured on a chlorate-containing plate to enrich chlorate tolerant mutants. Southern analysis showed that one isolate, B14, had a deletion in the DvNIA1 gene region. Using electroporation conditions determined in this laboratory, plasmid pDVNR containing the intact DvNIA1 gene has been electroporated into the defective mutant B14. Strains retaining a nitrate assimilation phenotype were obtained from nitrate plates after spreading the electroporated cells. In some individual strains, transcription of the introduced gene was detected. NR activity in these strains was slightly higher than that in the defective B14 cell, but excretion of nitrite into culture media was almost as high as that of the wild-type cell. Possible episomal presence of the introduced DNA in D. viridis is discussed. PMID:16797881

  19. Polymorphism in methylenetetrahydrofolate reductase, plasminogen activator inhibitor-1, and apolipoprotein E in hemodialysis patients.

    PubMed

    Al-Muhanna, Fahad; Al-Mueilo, Samir; Al-Ali, Amein; Larbi, Emmanuel; Rubaish, Abdullah; Abdulmohsen, Mohammed Fakhry; Al-Zahrani, Alhussain; Al-Ateeq, Suad

    2008-11-01

    The methylenetetrahydrofolate reductase (MTHFR) gene polymorphism, apolipoprotein E (apo epsilon4) gene polymorphism and polymorphism of plasminogen activator inhibitor-1 (PAI-1) have been shown to be associated with end-stage renal disease (ESRD). To determine the prevalence of these mutations in Saudi patients with ESRD on hemodialysis, we studied the allelic frequency and genotype distribution in patients receiving hemodialysis and in a control group, all residing in the Eastern Province of Saudi Arabia. The genotypes were determined using allele specific hybridization procedures and were confirmed by restriction fragment length polymorphism. The T allele frequency and homozygous genotype of MTHFR in ESRD patients were 14% and 2.4%, respectively compared to 13.4% and 0%, respectively in the control group. The allele frequency and homozygous genotype of 4G/4G PAI-1 gene polymorphism were 46.4% and 4.8% respectively in ESRD patients compared to 57.1% and 32% respectively in the control group. The apo s4 allele frequency and homozygous genotype distribution in hemodialysis patients were 7% and 2.4%, respectively compared to 13% and 2% in the control group. Although allele frequency of C677T of MTHFR was statistically similar in the hemodialysis patients and in the control group, the homozygotes T allele genotype was over represented in the hemodialysis group compared to normal. The prevalence of PAI-1 4G/4G polymorphism in ESRD patients was lower when compared to the control group. The prevalence of apo s4 allele did not differ significantly between the two groups. The present results demonstrate that all three studied polymorphic mutations are present in our population and that they may contribute to the etiology of the disease in our area. PMID:18974580

  20. Simultaneous binding of coenzyme and two ligand molecules into the active site of fungal trihydroxynaphthalene reductase.

    PubMed

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

    2009-03-16

    We present here a kinetic characterization of the oxidation of the artificial substrate 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one in the presence of NADP(+) by trihydroxynaphthalene reductase from the filamentous fungus Curvularia lunata. Although the experimental data were gathered by conventional equipment and were only available for the reaction in one direction, the analysis confirms the bi-bi reaction mechanism and yields estimates of kinetic parameters of the intermediates. It is based on an independent estimation of coenzyme binding constants and on a sequential analysis of three portions of the progress curves, from the beginning of the reaction until equilibrium is reached. First, the plateaus are used to determine the overall equilibrium constant of the non-catalyzed reaction. Then, the dissociation constants of the oxidized and reduced cofactor are estimated by titration. Subsequently, the initial parts of the progress curves are analyzed using the rate equation that is derived under combined assumptions of equilibrium and steady state. The macroscopic relations obtained are then fixed in the final progress curve analysis where the information for only two remaining rate constants is extracted from their curved portions by fitting numerically solved model-specific differential equations to the data. At pH 8, the overall equilibrium largely favours the oxidized substrate and reduced cofactor, and the activity of the holoenzyme is inhibited by high substrate concentrations. Substrate inhibition can be discriminated from true cooperativity through the effects of apigenin, a flavonoid inhibitor that is structurally similar, but larger, than the substrate used in the study. PMID:19071099

  1. Iridoid Synthase Activity Is Common among the Plant Progesterone 5β-Reductase Family.

    PubMed

    Munkert, Jennifer; Pollier, Jacob; Miettinen, Karel; Van Moerkercke, Alex; Payne, Richard; Müller-Uri, Frieder; Burlat, Vincent; O'Connor, Sarah E; Memelink, Johan; Kreis, Wolfgang; Goossens, Alain

    2014-09-19

    Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, among which the anti-cancer drugs vinblastine and vincristine. The monoterpenoid branch of the alkaloid pathway leads to the secoiridoid secologanin and involves the enzyme iridoid synthase (IS), a member of the progesterone 5β-reductase (P5βR) family. IS reduces 8-oxogeranial to iridodial. Through transcriptome mining, we show that IS belongs to a family of six C. roseus P5βR genes. Characterisation of recombinant CrP5βR proteins demonstrates that all but CrP5βR3 can reduce progesterone, and thus can be classified as P5βRs. Three of them, namely CrP5βR1, CrP5βR2 and CrP5βR4, could also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5βR5) in secoiridoid synthesis. In depth functional analysis by subcellular protein localisation, gene expression analysis, in situ hybridisation and virus-induced gene silencing, indicates that besides IS, CrP5βR4 may also participate in secoiridoid biosynthesis. Finally, we cloned a set of P5βR genes from angiosperm plant species not known to produce iridoids and demonstrate that the corresponding recombinant proteins are also capable of using 8-oxogeranial as a substrate. This suggests that 'IS activity' is intrinsic to angiosperm P5βR proteins and has evolved early during evolution. PMID:25239067

  2. Iridoid synthase activity is common among the plant progesterone 5β-reductase family.

    PubMed

    Munkert, Jennifer; Pollier, Jacob; Miettinen, Karel; Van Moerkercke, Alex; Payne, Richard; Müller-Uri, Frieder; Burlat, Vincent; O'Connor, Sarah E; Memelink, Johan; Kreis, Wolfgang; Goossens, Alain

    2015-01-01

    Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, including the anti-cancer drugs vinblastine and vincristine. The monoterpenoid branch of the alkaloid pathway leads to the secoiridoid secologanin and involves the enzyme iridoid synthase (IS), a member of the progesterone 5β-reductase (P5βR) family. IS reduces 8-oxogeranial to iridodial. Through transcriptome mining, we show that IS belongs to a family of six C. roseus P5βR genes. Characterization of recombinant CrP5βR proteins demonstrates that all but CrP5βR3 can reduce progesterone and thus can be classified as P5βRs. Three of them, namely CrP5βR1, CrP5βR2, and CrP5βR4, can also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5βR5) in secoiridoid synthesis. In-depth functional analysis by subcellular protein localization, gene expression analysis, in situ hybridization, and virus-induced gene silencing indicate that besides IS, CrP5βR4 may also participate in secoiridoid biosynthesis. We cloned a set of P5βR genes from angiosperm plant species not known to produce iridoids and demonstrate that the corresponding recombinant proteins are also capable of using 8-oxogeranial as a substrate. This suggests that IS activity is intrinsic to angiosperm P5βR proteins and has evolved early during evolution. PMID:25578278

  3. Pivotal role of dihydrofolate reductase knockdown in the anticancer activity of 2-hydroxyoleic acid

    PubMed Central

    Lladó, Victoria; Terés, Silvia; Higuera, Mónica; Álvarez, Rafael; Noguera-Salva, Maria Antònia; Halver, John E.; Escribá, Pablo V.; Busquets, Xavier

    2009-01-01

    α-Hydroxy-9-cis-octadecenoic acid, a synthetic fatty acid that modifies the composition and structure of lipid membranes. 2-Hydroxyoleic acid (HOA) generated interest due to its potent, yet nontoxic, anticancer activity. It induces cell cycle arrest in human lung cancer (A549) cells and apoptosis in human leukemia (Jurkat) cells. These two pathways may explain how HOA induces regression of a variety of cancers. We showed that HOA repressed the expression of dihydrofolate reductase (DHFR), the enzyme responsible for tetrahydrofolate (THF) synthesis. Folinic acid, which readily produces THF without the participation of DHFR, reverses the antitumor effects of HOA in A549 and Jurkat cells, as well as the inhibitory influence on cyclin D and cdk2 in A549 cells, and on DNA and PARP degradation in Jurkat cells. This effect was very specific, because either elaidic acid (an analog of HOA) or other lipids, failed to alter A549 or Jurkat cell growth. THF is a cofactor necessary for DNA synthesis. Thus, impairment of DNA synthesis appears to be a common mechanism involved in the different responses elicited by cancer cells following treatment with HOA, namely cell cycle arrest or apoptosis. Compared with other antifolates, such as methotrexate, HOA did not directly inhibit DHFR but rather, it repressed its expression, a mode of action that offers certain therapeutic advantages. These results not only demonstrate the effect of a fatty acid on the expression of DHFR, but also emphasize the potential of HOA to be used as a wide-spectrum drug against cancer. PMID:19666584

  4. Biliverdin reductase, a novel regulator for induction of activating transcription factor-2 and heme oxygenase-1.

    PubMed

    Kravets, Anatoliy; Hu, Zhenbo; Miralem, Tihomir; Torno, Michael D; Maines, Mahin D

    2004-05-01

    Biliverdin IXalpha reductase (BVR) catalyzes reduction of the HO activity product, biliverdin, to bilirubin. hBVR is a serine/threonine kinase that contains a bZip domain. Presently, regulation of gene expression by hBVR was examined. 293A cells were infected with adenovirus-doxycycline (Ad-Dox)-inducible hBVR cDNA. High level expression of hBVR was determined at mRNA, protein, and activity levels 8 h after induction. Cell signal transduction microarray analysis of cells infected with expression or with the control Ad-inverted (INV)-hBVR vector identified ATF-2 among several up-regulated genes. ATF-2 is a bZip transcription factor for activation of cAMP response element (CRE) and a dimeric partner to c-jun in MAPK pathway that regulates the stress protein, HO-1, expression. Northern and Western blot analyses showed increases of approximately 10-fold in ATF-2 mRNA and protein at 16 and 24 h after Dox addition. Ad-INV-hBVR did not effect ATF-2 expression. In hBVR-infected cells, levels of HO-1 mRNA and protein were increased. In vitro translated hBVR and nuclear extract containing hBVR in gel mobility-shift assay bound to AP-1 sites in the ATF-2 promoter region and to an oligonucleotide containing the CRE site. Both bindings could be competed out by excess unlabeled probe; in the presence of hBVR antibody, they displayed shifted bands. Co-transfection of hBVR with ATF-2 or c-jun promoters caused a severalfold increase in luciferase activity. hBVR modulation of ATF-2 and HO-1 expression suggests it has a potential role in regulation of AP-1 and cAMP-regulated genes and a role in cell signaling. We propose that increased expression of the protein can be used to alter the gene expression profile in the cell. PMID:14988408

  5. Magnesium Deficiency and High Light Intensity Enhance Activities of Superoxide Dismutase, Ascorbate Peroxidase, and Glutathione Reductase in Bean Leaves 1

    PubMed Central

    Cakmak, Ismail; Marschner, Horst

    1992-01-01

    The influence of varied Mg supply (10-1000 micromolar) and light intensity (100-580 microeinsteins per square meter per second) on the concentrations of ascorbate (AsA) and nonprotein SH-compounds and the activities of superoxide dismutase (SOD; EC 1.15.11) and the H2O2 scavenging enzymes, AsA peroxidase (EC 1.11.1.7), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were studied in bean (Phaseolus vulgaris L.) leaves over a 13-day period. The concentrations of AsA and SH-compounds and the activities of SOD and H2O2 scavenging enzymes increased with light intensity, in particular in Mg-deficient leaves. Over the 12-day period of growth for a given light intensity, the concentrations of AsA and SH-compounds and the activities of these enzymes remained more or less constant in Mg-sufficient leaves. In contrast, in Mg-deficient leaves, a progressive increase was recorded, particularly in concentrations of AsA and activities of AsA peroxidase and glutathione reductase, whereas the activities of guaiacol peroxidase and catalase were only slightly enhanced. Partial shading of Mg-deficient leaf blades for 4 days prevented chlorosis, and the activities of the O2.− and H2O2 scavenging enzymes remained at a low level. The results demonstrate the role of both light intensity and Mg nutritional status on the regulation of O2.− and H2O2 scavenging enzymes in chloroplasts. PMID:16668779

  6. Human biliverdin reductase: a member of the insulin receptor substrate family with serine/threonine/tyrosine kinase activity.

    PubMed

    Lerner-Marmarosh, Nicole; Shen, Jenny; Torno, Michael D; Kravets, Anatoliy; Hu, Zhenbo; Maines, Mahin D

    2005-05-17

    We describe here the tyrosine kinase activity of human biliverdin reductase (BVR) and its potential role in the insulin-signaling pathway. BVR is both a substrate for insulin receptor (IR) tyrosine kinase (IRK) activity and a kinase for serine phosphorylation of IR substrate 1 (IRS-1). Our previous studies have revealed serine/threonine kinase activity of BVR. Y198, in the YMKM motif found in the C-terminal domain of BVR, is shown to be a substrate for insulin-activated IRK. This motif in IRS proteins provides a docking site for proteins that contain a Src homology 2 domain. Additionally, Y228 in the YLSF sequence and Y291 are IRK substrates; the former sequence provides optimum recognition motif in the tyrosine phosphatase, SHP-1, and for SHC (Src homology 2 domain containing transfroming protein 1). BVR autophosphorylates N-terminal tyrosines Y72 and Y83. Serine residues in IRS-1 are targets for BVR phosphorylation, and point mutation of serine residues in the kinase domain of the reductase inhibits phosphotransferase activity. Because tyrosine phosphorylation of IRS-1 activates the insulin signaling pathway and serine phosphorylation of IRS-1 blocks insulin action, our findings that insulin increases BVR tyrosine phosphorylation and that there is an increase in glucose uptake in response to insulin when expression of BVR is "knocked down" by small interfering RNA suggest a potential role for BVR in the insulin signaling pathway. PMID:15870194

  7. Diurnal variation in the fraction of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the active form in the mammary gland of the lactating rat.

    PubMed Central

    Smith, R A; Middleton, B; West, D W

    1986-01-01

    'Expressed' and 'total' activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in freeze-clamped samples of mammary glands from lactating rats at intervals throughout the 24 h light/dark cycle. 'Expressed' activities were measured in microsomal fractions isolated and assayed in the presence of 100 mM-KF. 'Total' activities were determined in microsomal preparations from the same homogenates but washed free of KF and incubated with exogenously added sheep liver phosphoprotein phosphatase before assay. Both 'expressed' and 'total' activities of HMG-CoA reductase underwent a diurnal cycle, which had a major peak 6 h into the light phase and a nadir 15 h later, i.e. 9 h into the dark period. Both activities showed a secondary peak of activity (around 68% of the maximum activity) at the time of changeover from dark to light, with a trough in the value of the 'expressed' activity that was close to the nadir value. 'Expressed' activity was lower than 'total' at all time points, indicating the presence of enzyme molecules inactivated by covalent phosphorylation. Nevertheless the 'expressed'/'total' activity ratio was comparatively constant and varied only between 43% and 75%. Immunotitration of enzyme activity, with antiserum raised in sheep against purified rat liver HMG-CoA reductase, confirmed the presence of both active and inactive forms of the enzyme and indicated that at the peak and nadir the variation in 'expressed' HMG-CoA reductase activity resulted from changes in the total number of enzyme molecules rather than from covalent modification. The sample obtained after 3 h of the light phase exhibited an anomalously low 'total' HMG-CoA reductase activity, which could be increased when Cl- replaced F- in the homogenization medium. The result suggests that at that time the activity of the enzyme could be regulated by mechanisms other than covalent phosphorylation or degradation. PMID:3814075

  8. Identification of one-electron reductases that activate both the hypoxia prodrug SN30000 and diagnostic probe EF5.

    PubMed

    Wang, Jingli; Guise, Chris P; Dachs, Gabi U; Phung, Yen; Hsu, Annie Huai-Ling; Lambie, Neil K; Patterson, Adam V; Wilson, William R

    2014-10-15

    SN30000 is a second-generation benzotriazine-N-oxide hypoxia-activated prodrug scheduled for clinical trial. Previously we showed that covalent binding of the hypoxia probe EF5 predicts metabolic activation of SN30000 in a panel of cancer cell lines under anoxia, suggesting that they are activated by the same reductases. However the identity of these reductases is unknown. Here, we test whether forced expression of nine oxidoreductases with known or suspected roles in bioreductive prodrug metabolism (AKR1C3, CYB5R3, FDXR, MTRR, NDOR1, NOS2A, NQO1, NQO2 and POR) enhances oxic or anoxic reduction of SN30000 and EF5 by HCT116 cells. Covalent binding of (14)C-EF5 and reduction of SN30000 to its 1-oxide and nor-oxide metabolites was highly selective for anoxia in all lines, with significantly elevated anoxic metabolism of both compounds in lines over-expressing POR, MTRR, NOS2A or NDOR1. There was a strong correlation between EF5 binding and SN30000 metabolism under anoxia across the cell lines (R(2)=0.84, p=0.0001). Antiproliferative potency of SN30000 under anoxia was increased most strongly by overexpression of MTRR and POR. Transcript abundance in human tumours, evaluated using public domain mRNA expression data, was highest for MTRR, followed by POR, NOS2A and NDOR1, with little variation between tumour types. Immunostaining of tissue microarrays demonstrated variable MTRR protein expression across 517 human cancers with most displaying low expression. In conclusion, we have identified four diflavin reductases (POR, MTRR, NOS2A and NDOR1) capable of reducing both SN30000 and EF5, further supporting use of 2-nitroimidazole probes to predict the ability of hypoxic cells to activate SN30000. PMID:25130546

  9. Genome sequence of the chemolithoautotrophic nitrite-oxidizing bacterium Nitrobacter winogradskyi Nb-255

    SciTech Connect

    Hauser, Loren John; Land, Miriam L; Larimer, Frank W; Arp, D J; Hickey, W J

    2006-03-01

    The alphaproteobacterium Nitrobacter winogradskyi (ATCC 25391) is a gram-negative facultative chemolithoautotroph capable of extracting energy from the oxidation of nitrite to nitrate. Sequencing and analysis of its genome revealed a single circular chromosome of 3,402,093 bp encoding 3,143 predicted proteins. There were extensive similarities to genes in two alphaproteobacteria, Bradyrhizobium japonicum USDA110 (1,300 genes) and Rhodopseudomonas palustris CGA009 CG (815 genes). Genes encoding pathways for known modes of chemolithotrophic and chemoorganotrophic growth were identified. Genes encoding multiple enzymes involved in anapleurotic reactions centered on C2 to C4 metabolism, including a glyoxylate bypass, were annotated. The inability of N. winogradskyi to grow on C6 molecules is consistent with the genome sequence, which lacks genes for complete Embden-Meyerhof and Entner-Doudoroff pathways, and active uptake of sugars. Two gene copies of the nitrite oxidoreductase, type I ribulose-1,5-bisphosphate carboxylase/oxygenase, cytochrome c oxidase, and gene homologs encoding an aerobic-type carbon monoxide dehydrogenase were present. Similarity of nitrite oxidoreductases to respiratory nitrate reductases was confirmed. Approximately 10% of the N. winogradskyi genome codes for genes involved in transport and secretion, including the presence of transporters for various organic-nitrogen molecules. The N. winogradskyi genome provides new insight into the phylogenetic identity and physiological capabilities of nitrite-oxidizing bacteria. The genome will serve as a model to study the cellular and molecular processes that control nitrite oxidation and its interaction with other nitrogen-cycling processes.

  10. Hydrogen Bonding Networks Tune Proton-Coupled Redox Steps during the Enzymatic Six-Electron Conversion of Nitrite to Ammonia

    PubMed Central

    2015-01-01

    Multielectron multiproton reactions play an important role in both biological systems and chemical reactions involved in energy storage and manipulation. A key strategy employed by nature in achieving such complex chemistry is the use of proton-coupled redox steps. Cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron seven-proton reduction of nitrite to ammonia. While a catalytic mechanism for ccNiR has been proposed on the basis of studies combining computation and crystallography, there have been few studies directly addressing the nature of the proton-coupled events that are predicted to occur along the nitrite reduction pathway. Here we use protein film voltammetry to directly interrogate the proton-coupled steps that occur during nitrite reduction by ccNiR. We find that conversion of nitrite to ammonia by ccNiR adsorbed to graphite electrodes is defined by two distinct phases; one is proton-coupled, and the other is not. Mutation of key active site residues (H257, R103, and Y206) modulates these phases and specifically alters the properties of the detected proton-dependent step but does not inhibit the ability of ccNiR to conduct the full reduction of nitrite to ammonia. We conclude that the active site residues examined are responsible for tuning the protonation steps that occur during catalysis, likely through an extensive hydrogen bonding network, but are not necessarily required for the reaction to proceed. These results provide important insight into how enzymes can specifically tune proton- and electron transfer steps to achieve high turnover numbers in a physiological pH range. PMID:25137350

  11. Both Plant and Bacterial Nitrate Reductases Contribute to Nitric Oxide Production in Medicago truncatula Nitrogen-Fixing Nodules1[W][OA

    PubMed Central

    Horchani, Faouzi; Prévot, Marianne; Boscari, Alexandre; Evangelisti, Edouard; Meilhoc, Eliane; Bruand, Claude; Raymond, Philippe; Boncompagni, Eric; Aschi-Smiti, Samira; Puppo, Alain; Brouquisse, Renaud

    2011-01-01

    Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions. PMID:21139086

  12. The changes of nitrate reductase activity in soils under Robinia pseudoacacia shelterbelt and in adjoining cultivated field

    NASA Astrophysics Data System (ADS)

    Wojciech Szajdak, Lech; Gaca, Wioletta

    2010-05-01

    The investigations were carried out in Dezydery Chlapowski Agroecological Landscape Park in Turew (40 km South-West of Poznań, West Polish Lowlands, 16° 45 E and 52° 01 N). Intensively agriculture is observed in this region. Characteristic features of this landscape are shelterbelts created in the XIX century by the general Dezydery Chlapowski. Soil samples were taken from Robinia pseudoacacia shelterbelt and from adjoining cultivated field. This is 200 - years old shelterbelt consists mainly of Robinia pseudacacia and small admixture of Quercus robur and Quercus petraea. It is 2 kilometers length and 36 meters width. Shelterbelts and adjoining cultivated fields were introduced on Hapludalfs soils (according to FAO classification). The aim of this study was to evaluate the effect of moisture and nitrogen concentrations on the changes of nitrate reductase activity in soil under shelterbelt and in adjoining cultivated field. The experiments were carried out in two different moisture content. The first was field-moist and the second was 15% moisture content. In this study three different contents of nitrogen in the form of urea (organic form of nitrogen) were investigated: field concentration, after addition of 0.25% and 0.5% of nitrogen. Activity of nitrate reductase changes in different interval of time were measured. Rate constant of reactions was calculated for the changes of nitrate reductase activity. Our results have shown that this process runs according to the equation rate of first-order kinetic reaction model. The first-order reaction rate constants increases with the changes of moisture content from field-moist to 15% in soil under shelterbelt. In soil under adjoining cultivated field raise of the moisture content from field-moist to 15% causes an increase of the first-order reaction rate constants higher than in soil under shelterbelt. The processes of the changes of nitrate reductase activity 15% moisture content of the soil under shelterbelt and in

  13. Antihyperlipidemic Activity of Aloe succotrina in Rats: Possibly Mediated by Inhibition of HMG-CoA Reductase

    PubMed Central

    Lamba, Deepak; Kumar, Ramesh; Nath, Pashupati; Gauttam, Satyaprakash

    2014-01-01

    The present study was designed to investigate antihyperlipidemic activity of dried pulp of Aloe succotrina leaves in Wistar albino rats. Hyperlipidemia was induced in rats by feeding them high fat diet (HFD) or D-fructose (25% w/v) for 4 successive weeks. From 15th to 28th day, dried pulp (100 and 200 mg/kg, p.o) and atorvastatin (10 mg/kg, p.o.) per se were administered 2 h prior to feeding rats with HFD or fructose. Aloe succotrina did not significantly decrease the body weight of rats. The dried pulp and atorvastatin per se significantly decreased relative liver weight but did not significantly affect relative heart weight. HFD or fructose significantly increased serum total cholesterol, triglycerides, LDL-c, and VLDL, and decreased HDL-c; significantly increased liver MDA and decreased GSH levels. The dried pulp (200 mg/kg p.o.) significantly reversed high fat diet-induced and fructose-induced hyperlipidemia and atherogenic index. Aloe succotrina significantly decreased HMG Co-A reductase activity. Antihyperlipidemic effect of the dried pulp was comparable to atorvastatin. Thus, Aloe succotrina produced significant antihyperlipidemic activity in both HFD and fructose-induced hyperlipidemic rats, possibly through normalization of serum lipid profile, HMG-CoA reductase inhibitory activity, and amelioration of oxidative stress in liver. PMID:24693447

  14. Inhibitory Activities of Phenolic Compounds Isolated from Adina rubella Leaves Against 5α-Reductase Associated with Benign Prostatic Hypertrophy.

    PubMed

    Yin, Jun; Heo, Jun Hyeok; Hwang, Yoon Jeong; Le, Thi Tam; Lee, Min Won

    2016-01-01

    Adina rubella Hance (AR), a plant native to Korea, has been used as traditional medicine for dysentery, eczema, intoxication, and external hemorrhages. Previous phytochemical studies of AR have reported several components, including terpenoids, phenolics, and alkaloids. The current study evaluated the anti-oxidative and anti-inflammatory activities and 5α-reductase inhibition of isolated compounds of AR leaves to find a potential therapeutic agent for benign prostatic hypertrophy (BPH). Repeated chromatographic isolation of an 80% acetone extract of AR leaves yielded seven phenolic compounds: caffeic acid (1), chlorogenic acid (2), methyl chlorogenate (3), quercetin-3-rutinoside (4), kaempferol-3-O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside (5), hyperoside (6), and grandifloroside (7). Compound 7 is a novel compound in AR. Caffeoyl derivatives 1-3 and 7 showed good anti-oxidative activities. In particular, caffeic acid (1) and grandifloroside (7) showed potent anti-inflammatory activities, and 7 also exhibited potent inhibitory activity against TNF-α and 5α-reductase. Our results show that the extract and grandifloroside (7) from leaves of AR might be developed as a source of potent anti-oxidative and anti-inflammatory agents and therapeutic agent for BPH. PMID:27399661

  15. Effects of SRT and DO on N2O reductase activity in an anoxic-oxic activated sludge system.

    PubMed

    Noda, N; Kaneko, N; Mikami, M; Kimochi, Y; Tsuneda, S; Hirata, A; Mizuochi, M; Inamori, Y

    2003-01-01

    Nitrous oxide (N2O) is emitted from wastewater treatment processes, and is known to be a green house gas contributing to global warming. It is thus important to develop technology that can suppress N2O emission. The effects of sludge retention time (SRT) and dissolved oxygen (DO) on N2O emission in an anoxic-oxic activated sludge system were estimated. Moreover, the microbial community structure in the sludge, which plays an important role in N2O suppression, was clarified based on nitrous oxide reductase (nosZ) gene analysis by molecular biological techniques. The results showed that under low SRT conditions, nitrification efficiency was reduced and the N2O emission rate in the oxic reactors was increased. It was also observed that N2O emission was enhanced under low DO conditions, where the available oxygen is insufficient for nitrification. Moreover, molecular analysis revealed that the clones identified in this study were closely related to Ralstonia eutropha and Paracoccus denitrificans. The fact that the identified sequences are not closely related to known culturable denitrifier nosZ sequences indicates a substantial in situ diversity of denitrifiers contributing to N2O suppression, which are not reflected in the cultivatable fraction of the population. The further application of these new molecular techniques should serve to enhance our knowledge of the microbial community of denitrifying bacteria contributing to N2O suppression in wastewater treatment systems. PMID:14753557

  16. Evaluation of the impact on food safety of a Lactobacillus coryniformis strain from pickled vegetables with degradation activity against nitrite and other undesirable compounds.

    PubMed

    Fang, Fang; Feng, Tingting; Du, Guocheng; Chen, Jian

    2016-04-01

    Four strains of lactic acid bacteria showing antimicrobial activity against some food-spoilage microorganisms or pathogens, including both Gram-negative and -positive strains, were isolated from naturally fermented pickled vegetables and a traditional cheese product. Among these isolates, Lactobacillus coryniformis strain BBE-H3, characterised previously to be a non-biogenic amine producer, showed a high level of activity in degrading sodium nitrite and exhibited the ability to eliminate ethyl carbamate and one of its precursors, urea. The antimicrobial substance produced by L. coryniformis BBE-H3 was found to be active at an acidic pH range of 4.0-4.5. The antimicrobial activity of this strain decreased differentially after treatment with proteolytic enzymes (pepsin, papain, trypsin and proteinase K), implying this growth inhibitory compound is either a protein or a polypeptide. The results of this study show the suitability of L. coryniformis BBE-H3 as a starter in food manufacturing processes, and demonstrate its potential role in eliminating food origin carcinogens such as sodium nitrite and ethyl carbamate. PMID:26898528

  17. Mercuric reductase activity and evidence of broad-spectrum mercury resistance among clinical isolates of rapidly growing mycobacteria

    SciTech Connect

    Steingrube, V.A.; Wallace, R.J. Jr.; Steele, L.C.; Pang, Y.J. )

    1991-05-01

    Resistance to mercury was evaluated in 356 rapidly growing mycobacteria belonging to eight taxonomic groups. Resistance to inorganic Hg2+ ranged from 0% among the unnamed third biovariant complex of Mycobacterium fortuitum to 83% among M. chelonae-like organisms. With cell extracts and 203Hg(NO3)2 as the substrate, mercuric reductase (HgRe) activity was demonstrable in six of eight taxonomic groups. HgRe activity was inducible and required NADPH or NADH and a thiol donor for optimai activity. Species with HgRe activity were also resistant to organomercurial compounds, including phenylmercuric acetate. Attempts at intraspecies and intragenus transfer of HgRe activity by conjugation or transformation were unsuccessful. Mercury resistance is common in rapidly growing mycobacteria and appears to function via the same inducible enzyme systems already defined in other bacterial species. This system offers potential as a strain marker for epidemiologic investigations and for studying genetic systems in rapidly growing mycobacteria.

  18. Dihydropteridine reductase activity in dried blood spots: effects of aging and senile dementia of the Alzheimer type.

    PubMed Central

    Jeeps, C M; Silcox, A; Lloyd, B; Clayton, B E

    1986-01-01

    Dihydropteridine reductase (EC 1.6.99.7) (DHPR) activity was measured in blood spots from 50 neonates, 52 healthy adults aged 30-62 years, and 21 elderly controls aged 67-97 years, as well as 32 demented patients of whom 25 had senile dementia of the Alzheimer type. Enzyme activity was stable for seven days at 4 degrees C and for at least 14 days at -20 degrees C. No important difference was found between the DHPR activity of venous and capillary blood. DHPR activity was considerably lower in the healthy adult group compared with neonates and the elderly group, and there was no sex difference at any age. The erythrocyte DHPR activity of patients with senile dementia of the Alzheimer type was similar to that of elderly controls. This result differs from that previously reported for leucocytes. PMID:3950042

  19. Light effects and diel variations of nitrate reductase activity in phytoplankton from the northwest Africa upwelling region

    NASA Astrophysics Data System (ADS)

    Martinez, Rosa; Packard, Theodore T.; Blasco, Dolors

    1987-06-01

    Light kinetics and diel cycles of nitrate reductase (NR) activity were studied in the upwelling ecosystem off northwest Africa. The activity of the enzyme showed a strong response to light at low intensities but became saturated at light intensities above 15-30% of the incident light intensity. At higher irradiances, NR activity showed photoinhibition. At sea surface irradiances an average inhibition of 32% in the NR activity was observed. Diel cycles of NR activity exhibited the following characteristics: a low pre-dawn value (0.03-0.009 μmol NO 3-N (μg Chl α) -1), a rapid increase with the onset of daylight, a maximum before noon (tripling the dark value), a secondary maximum in the early afternoon, and an afternoon-evening decrease that coincided with the decrease in sunlight. To simulate these characteristics a continuous polynomial function of light and time was developed.

  20. Inhabitancy of active Nitrosopumilus-like ammonia-oxidizing archaea and Nitrospira nitrite-oxidizing bacteria in the sponge Theonella swinhoei.

    PubMed

    Feng, Guofang; Sun, Wei; Zhang, Fengli; Karthik, Loganathan; Li, Zhiyong

    2016-01-01

    Nitrification directly contributes to the ammonia removal in sponges, and it plays an indispensable role in sponge-mediated nitrogen cycle. Previous studies have demonstrated genomic evidences of nitrifying lineages in the sponge Theonella swinhoei. However, little is known about the transcriptional activity of nitrifying community in this sponge. In this study, combined DNA- and transcript-based analyses were performed to reveal the composition and transcriptional activity of the nitrifiers in T. swinhoei from the South China Sea. Transcriptional activity of ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in this sponge were confirmed by targeting their nitrifying genes,16S rRNA genes and their transcripts. Phylogenetic analysis coupled with RDP rRNA classification indicated that archaeal 16S rRNA genes, amoA (the subunit of ammonia monooxygenase) genes and their transcripts were closely related to Nitrosopumilus-like AOA; whereas nitrifying bacterial 16S rRNA genes, nxrB (the subunit of nitrite oxidoreductase) genes and their transcripts were closely related to Nitrospira NOB. Quantitative assessment demonstrated relative higher abundances of nitrifying genes and transcripts of Nitrosopumilus-like AOA than those of Nitrospira NOB in this sponge. This study illustrated the transcriptional potentials of Nitrosopumilus-like archaea and Nitrospira bacteria that would predominantly contribute to the nitrification functionality in the South China Sea T. swinhoei. PMID:27113140

  1. Inhabitancy of active Nitrosopumilus-like ammonia-oxidizing archaea and Nitrospira nitrite-oxidizing bacteria in the sponge Theonella swinhoei

    PubMed Central

    Feng, Guofang; Sun, Wei; Zhang, Fengli; Karthik, Loganathan; Li, Zhiyong

    2016-01-01

    Nitrification directly contributes to the ammonia removal in sponges, and it plays an indispensable role in sponge-mediated nitrogen cycle. Previous studies have demonstrated genomic evidences of nitrifying lineages in the sponge Theonella swinhoei. However, little is known about the transcriptional activity of nitrifying community in this sponge. In this study, combined DNA- and transcript-based analyses were performed to reveal the composition and transcriptional activity of the nitrifiers in T. swinhoei from the South China Sea. Transcriptional activity of ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in this sponge were confirmed by targeting their nitrifying genes,16S rRNA genes and their transcripts. Phylogenetic analysis coupled with RDP rRNA classification indicated that archaeal 16S rRNA genes, amoA (the subunit of ammonia monooxygenase) genes and their transcripts were closely related to Nitrosopumilus-like AOA; whereas nitrifying bacterial 16S rRNA genes, nxrB (the subunit of nitrite oxidoreductase) genes and their transcripts were closely related to Nitrospira NOB. Quantitative assessment demonstrated relative higher abundances of nitrifying genes and transcripts of Nitrosopumilus-like AOA than those of Nitrospira NOB in this sponge. This study illustrated the transcriptional potentials of Nitrosopumilus-like archaea and Nitrospira bacteria that would predominantly contribute to the nitrification functionality in the South China Sea T. swinhoei. PMID:27113140

  2. Alkylating activity in food products--especially sauerkraut and sour fermented dairy products--after incubation with nitrite under quasi-gastric conditions.

    PubMed

    Groenen, P J; Busink, E

    1988-03-01

    N-Nitroso compounds may well rank high among the genotoxic carcinogens present in our environment. Small amounts of such compounds may be formed in the human stomach after consumption of high-nitrate vegetables. Volatile nitrosamines can be conveniently determined but reliable methods of analysis for non-volatile N-nitroso compounds are still lacking. In this study we have used the 4-(4-nitrobenzyl)pyridine test to look for the formation of alkylating compounds such as N-nitroso-N-methylurea in a wide range of food products after incubation with nitrite under simulated gastric conditions. Our results indicate that many food products do not form alkylating compounds in appreciable amounts, even though the nitrite concentration used was five to ten times that found in saliva after a high-nitrate meal. Comparatively strong alkylating activity, however, was detected after incubation of samples of sauerkraut, certain dairy products (yoghurt, biogarde, quark, buttermilk and milk), wine and smoked mackerel. Samples of sauerkraut incubated with simulated gastric fluid, but without (added) nitrite, also displayed appreciable alkylating activity. The formation of alkylating substances in non-fat yoghurt was markedly inhibited by addition of ascorbic acid. The identity of the alkylating agents remains unknown. The isolation procedure was optimized for N-nitroso-N-methylurea, but will certainly result in the isolation of other compounds, such as C-nitroso-, C-nitro- or perhaps even C-nitroso-C'-nitro-compounds as well. Biogenic amines, glucosinolates, indole derivatives or other compounds may be involved as precursors. If alkylating agents are formed in vivo after ingestion of high-nitrate vegetables or drinking water, this is likely to occur only when the food products mentioned above are ingested simultaneously with or shortly after the nitrate load and not appreciably (except perhaps in the case of sauerkraut) when they are ingested alone, without a nitrate source. The

  3. Human brain aldehyde reductases: relationship to succinic semialdehyde reductase and aldose reductase.

    PubMed

    Hoffman, P L; Wermuth, B; von Wartburg, J P

    1980-08-01

    Human brain contains multiple forms of aldehyde-reducing enzymes. One major form (AR3), as previously shown, has properties that indicate its identity with NADPH-dependent aldehyde reductase isolated from brain and other organs of various species; i.e., low molecular weight, use of NADPH as the preferred cofactor, and sensitivity to inhibition by barbiturates. A second form of aldehyde reductase ("SSA reductase") specifically reduces succinic semialdehyde (SSA) to produce gamma-hydroxybutyrate. This enzyme form has a higher molecular weight than AR3, and uses NADH as well as NADPH as cofactor. SSA reductase was not inhibited by pyrazole, oxalate, or barbiturates, and the only effective inhibitor found was the flavonoid quercetine. Although AR3 can also reduce SSA, the relative specificity of SSA reductase may enhance its in vivo role. A third form of human brain aldehyde reductase, AR2, appears to be comparable to aldose reductases characterized in several species, on the basis of its activity pattern with various sugar aldehydes and its response to characteristic inhibitors and activators, as well as kinetic parameters. This enzyme is also the most active in reducing the aldehyde derivatives of biogenic amines. These studies suggest that the various forms of human brain aldehyde reductases may have specific physiological functions. PMID:6778961

  4. [Illumination's effect on the growth and nitrate reductase activity of typical red-tide algae in the East China Sea].

    PubMed

    Li, Hong-mei; Shi, Xiao-yong; Ding, Yan-yan; Tang, Hong-jie

    2013-09-01

    Two typical red-tide algae, Skeletonema costatum and Prorocentrum donghaiense were selected as studied objects. The nitrate reductase activity (NRA) and the growth of the two algae under different illuminations through incubation experiment were studied. The illumination condition was consistent with in situ. Results showed that P. donghaiense and S. costatum could grow normally in the solar radiation ranged from 30-60 W x m(-2), and the growth curve was "S" type. However, when solar radiation was below 9 W x m(-2), the two alga could hardly grow. In the range of 0-60 W x m(-2), three parameters (NRAmax, micro(max), Bf) increased with the increasing of light intensity, indicating that the light intensity can influence the grow of alga indirectly through influencing the nitrate reductase activity. The micro(max) and NRAmax in unite volume of Skeletonema costatum were higher than those of Prorocentrum donghaiense, indicating that Skeletonema costatum can better utilize the nitrate than Prorocentrum donghaiense. PMID:24288981

  5. Probing the Active Site of Candida Glabrata Dihydrofolate Reductase with High Resolution Crystal Structures and the Synthesis of New Inhibitors

    SciTech Connect

    Liu, J.; Bolstad, D; Smith, A; Priestley, N; Wright, D; Anderson, A

    2009-01-01

    Candida glabrata, a fungal strain resistant to many commonly administered antifungal agents, has become an emerging threat to human health. In previous work, we validated that the essential enzyme, dihydrofolate reductase, is a drug target in C. glabrata. Using a crystal structure of dihydrofolate reductase from C. glabrata bound to an initial lead compound, we designed a class of biphenyl antifolates that potently and selectively inhibit both the enzyme and the growth of the fungal culture. In this work, we explore the structure-activity relationships of this class of antifolates with four new high resolution crystal structures of enzyme:inhibitor complexes and the synthesis of four new inhibitors. The designed inhibitors are intended to probe key hydrophobic pockets visible in the crystal structure. The crystal structures and an evaluation of the new compounds reveal that methyl groups at the meta and para positions of the distal phenyl ring achieve the greatest number of interactions with the pathogenic enzyme and the greatest degree of selectivity over the human enzyme. Additionally, antifungal activity can be tuned with substitution patterns at the propargyl and para-phenyl positions.

  6. The nasFEDCBA operon for nitrate and nitrite assimilation in Klebsiella pneumoniae M5al.

    PubMed

    Lin, J T; Goldman, B S; Stewart, V

    1994-05-01

    Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources through the nitrate assimilation pathway. We previously identified structural genes for assimilatory nitrate and nitrite reductases, nasA and nasB, respectively. We report here our further identification of four genes, nasFEDC, upstream of the nasBA genes. The nasFEDCBA genes probably form an operon. Mutational and complementation analyses indicated that both the nasC and nasA genes are required for nitrate assimilation. The predicted NASC protein is homologous to a variety of NADH-dependent oxidoreductases. Thus, the NASC protein probably mediates electron transfer from NADH to the NASA protein, which contains the active site for nitrate reduction. The deduced NASF, NASE, and NASD proteins are homologous to the NRTA, NRTB, and NRTD proteins, respectively, that are involved in nitrate uptake in Synechococcus sp. (T. Omata, X. Andriesse, and A. Hirano, Mol. Gen. Genet. 236:193-202, 1993). Mutational and complementation studies indicated that the nasD gene is required for nitrate but not nitrite assimilation. By analogy with the Synechococcus nrt genes, we propose that the nasFED genes are involved in nitrate transport in K. pneumoniae. PMID:8169203

  7. The nasFEDCBA operon for nitrate and nitrite assimilation in Klebsiella pneumoniae M5al.

    PubMed Central

    Lin, J T; Goldman, B S; Stewart, V

    1994-01-01

    Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources through the nitrate assimilation pathway. We previously identified structural genes for assimilatory nitrate and nitrite reductases, nasA and nasB, respectively. We report here our further identification of four genes, nasFEDC, upstream of the nasBA genes. The nasFEDCBA genes probably form an operon. Mutational and complementation analyses indicated that both the nasC and nasA genes are required for nitrate assimilation. The predicted NASC protein is homologous to a variety of NADH-dependent oxidoreductases. Thus, the NASC protein probably mediates electron transfer from NADH to the NASA protein, which contains the active site for nitrate reduction. The deduced NASF, NASE, and NASD proteins are homologous to the NRTA, NRTB, and NRTD proteins, respectively, that are involved in nitrate uptake in Synechococcus sp. (T. Omata, X. Andriesse, and A. Hirano, Mol. Gen. Genet. 236:193-202, 1993). Mutational and complementation studies indicated that the nasD gene is required for nitrate but not nitrite assimilation. By analogy with the Synechococcus nrt genes, we propose that the nasFED genes are involved in nitrate transport in K. pneumoniae. PMID:8169203

  8. Substrate-dependent modulation of the enzymatic catalytic activity: reduction of nitrate, chlorate and perchlorate by respiratory nitrate reductase from Marinobacter hydrocarbonoclasticus 617.

    PubMed

    Marangon, Jacopo; Paes de Sousa, Patrícia M; Moura, Isabel; Brondino, Carlos D; Moura, José J G; González, Pablo J

    2012-07-01

    The respiratory nitrate reductase complex (NarGHI) from Marinobacter hydrocarbonoclasticus 617 (Mh, formerly Pseudomonas nautica 617) catalyzes the reduction of nitrate to nitrite. This reaction is the first step of the denitrification pathway and is coupled to the quinone pool oxidation and proton translocation to the periplasm, which generates the proton motive force needed for ATP synthesis. The Mh NarGH water-soluble heterodimer has been purified and the kinetic and redox properties have been studied through in-solution enzyme kinetics, protein film voltammetry and spectropotentiometric redox titration. The kinetic parameters of Mh NarGH toward substrates and inhibitors are consistent with those reported for other respiratory nitrate reductases. Protein film voltammetry showed that at least two catalytically distinct forms of the enzyme, which depend on the applied potential, are responsible for substrate reduction. These two forms are affected differentially by the oxidizing substrate, as well as by pH and inhibitors. A new model for the potential dependence of the catalytic efficiency of Nars is proposed. PMID:22561116

  9. Compensating for the absence of selenocysteine in high-molecular weight thioredoxin reductases: the electrophilic activation hypothesis.

    PubMed

    Lothrop, Adam P; Snider, Gregg W; Flemer, Stevenson; Ruggles, Erik L; Davidson, Ronald S; Lamb, Audrey L; Hondal, Robert J

    2014-02-01

    Mammalian thioredoxin reductase (TR) is a pyridine disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys). Selenium is a Janus-faced element because it is both highly nucleophilic and highly electrophilic. Cys orthologs of Sec-containing enzymes may compensate for the absence of a Sec residue by making the active site Cys residue more (i) nucleophilic, (ii) electrophilic, or (iii) reactive by increasing both S-nucleophilicity and S-electrophilicity. It has already been shown that the Cys ortholog TR from Drosophila melanogaster (DmTR) has increased S-nucleophilicity [Gromer, S., Johansson, L., Bauer, H., Arscott, L. D., Rauch, S., Ballou, D. P., Williams, C. H., Jr., Schrimer, R. H., and Arnér, E. S (2003) Active sites of thioredoxin reductases: Why selenoproteins? Proc. Natl. Acad. Sci. U.S.A. 100, 12618-12623]. Here we present evidence that DmTR also enhances the electrophilicity of Cys490 through the use of an "electrophilic activation" mechanism. This mechanism is proposed to work by polarizing the disulfide bond that occurs between Cys489 and Cys490 in the C-terminal redox center by the placement of a positive charge near Cys489. This polarization renders the sulfur atom of Cys490 electron deficient and enhances the rate of thiol/disulfide exchange that occurs between the N- and C-terminal redox centers. Our hypothesis was developed by using a strategy of homocysteine (hCys) for Cys substitution in the Cys-Cys redox dyad of DmTR to differentiate the function of each Cys residue. The results show that hCys could substitute for Cys490 with little loss of thioredoxin reductase activity, but that substitution of hCys for Cys489 resulted in a 238-fold reduction in activity. We hypothesize that replacement of Cys489 with hCys destroys an interaction between the sulfur atom of Cys489 and His464 crucial for the proposed electrophilic activation mechanism. This electrophilic activation

  10. Compensating for the Absence of Selenocysteine in High-Molecular Weight Thioredoxin Reductases: The Electrophilic Activation Hypothesis

    PubMed Central

    2015-01-01

    Mammalian thioredoxin reductase (TR) is a pyridine disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys). Selenium is a Janus-faced element because it is both highly nucleophilic and highly electrophilic. Cys orthologs of Sec-containing enzymes may compensate for the absence of a Sec residue by making the active site Cys residue more (i) nucleophilic, (ii) electrophilic, or (iii) reactive by increasing both S-nucleophilicity and S-electrophilicity. It has already been shown that the Cys ortholog TR from Drosophila melanogaster (DmTR) has increased S-nucleophilicity [Gromer, S., Johansson, L., Bauer, H., Arscott, L. D., Rauch, S., Ballou, D. P., Williams, C. H., Jr., Schrimer, R. H., and Arnér, E. S (2003) Active sites of thioredoxin reductases: Why selenoproteins? Proc. Natl. Acad. Sci. U.S.A. 100, 12618–12623]. Here we present evidence that DmTR also enhances the electrophilicity of Cys490 through the use of an “electrophilic activation” mechanism. This mechanism is proposed to work by polarizing the disulfide bond that occurs between Cys489 and Cys490 in the C-terminal redox center by the placement of a positive charge near Cys489. This polarization renders the sulfur atom of Cys490 electron deficient and enhances the rate of thiol/disulfide exchange that occurs between the N- and C-terminal redox centers. Our hypothesis was developed by using a strategy of homocysteine (hCys) for Cys substitution in the Cys-Cys redox dyad of DmTR to differentiate the function of each Cys residue. The results show that hCys could substitute for Cys490 with little loss of thioredoxin reductase activity, but that substitution of hCys for Cys489 resulted in a 238-fold reduction in activity. We hypothesize that replacement of Cys489 with hCys destroys an interaction between the sulfur atom of Cys489 and His464 crucial for the proposed electrophilic activation mechanism. This electrophilic

  11. The effect of nitrite inhibition on the anammox process.

    PubMed

    Lotti, T; van der Star, W R L; Kleerebezem, R; Lubello, C; van Loosdrecht, M C M

    2012-05-15

    The negative effect of nitrite on anammox activity has been reported widely during the past decade. Although the adverse effect is clear, conflicting reports exist on the level at which it occurs and its reversible/irreversible nature. An in depth study on nitrite inhibition therefore was performed in which the influence of environmental factors was evaluated. Anammox activity was measured in anammox granules by continuously monitored standardized manometric batch tests extending the interpretation by evaluation of lag times, maximum conversion rates during the tests and substrates/product conversion ratios. The granules where obtained from a one-stage anammox reactor, the dominant anammox organisms belonged to the Brocadia type. The observed 50% activity inhibition for nitrite (IC(50)) was 0.4 g N L(-1). The activity recovered fully after removal of the nitrite. Conversion in fresh medium after exposure to up to 6 g NO(2)(-)-N L(-1) for 24 h showed less then 60% loss of activity. Presence of ammonium during nitrite (2 g N L(-1)) exposure resulted in a stronger loss of activity after nitrite exposure (50% and 30% in presence and absence of ammonium respectively). Presence of oxygen during nitrite incubation led to a maximum activity reduction of 32%. The recovery after exposure indicates that the adverse effect of nitrite is reversible and thus inhibitory rather than toxic in nature. Similarities between exposure at three different pH-values indicate that nitrite rather than nitrous acid is the actual inhibiting compound. PMID:22424965

  12. Asymmetric Reduction of Activated Alkenes by Pentaerythritol Tetranitrate Reductase: Specificity and Control of Stereochemical Outcome by Reaction Optimisation

    PubMed Central

    Fryszkowska, Anna; Toogood, Helen; Sakuma, Michiyo; Gardiner, John M.; Stephens, Gill M.; Scrutton, Nigel S.

    2009-01-01

    We show that pentaerythritol tetranitrate reductase (PETNR), a member of the ‘ene’ reductase old yellow enzyme family, catalyses the asymmetric reduction of a variety of industrially relevant activated α,β-unsaturated alkenes including enones, enals, maleimides and nitroalkenes. We have rationalised the broad substrate specificity and stereochemical outcome of these reductions by reference to molecular models of enzyme-substrate complexes based on the crystal complex of the PETNR with 2-cyclohexenone 4a. The optical purity of products is variable (49–99% ee), depending on the substrate type and nature of substituents. Generally, high enantioselectivity was observed for reaction products with stereogenic centres at Cβ (>99% ee). However, for the substrates existing in two isomeric forms (e.g., citral 11a or nitroalkenes 18–19a), an enantiodivergent course of the reduction of E/Z-forms may lead to lower enantiopurities of the products. We also demonstrate that the poor optical purity obtained for products with stereogenic centres at Cα is due to non-enzymatic racemisation. In reactions with ketoisophorone 3a we show that product racemisation is prevented through reaction optimisation, specifically by shortening reaction time and through control of solution pH. We suggest this as a general strategy for improved recovery of optically pure products with other biocatalytic conversions where there is potential for product racemisation. PMID:20396613

  13. Anti-HMG-CoA Reductase, Antioxidant, and Anti-Inflammatory Activities of Amaranthus viridis Leaf Extract as a Potential Treatment for Hypercholesterolemia.

    PubMed

    Salvamani, Shamala; Gunasekaran, Baskaran; Shukor, Mohd Yunus; Shaharuddin, Noor Azmi; Sabullah, Mohd Khalizan; Ahmad, Siti Aqlima

    2016-01-01

    Inflammation and oxidative stress are believed to contribute to the pathology of several chronic diseases including hypercholesterolemia (elevated levels of cholesterol in blood) and atherosclerosis. HMG-CoA reductase inhibitors of plant origin are needed as synthetic drugs, such as statins, which are known to cause adverse effects on the liver and muscles. Amaranthus viridis (A. viridis) has been used from ancient times for its supposedly medically beneficial properties. In the current study, different parts of A. viridis (leaf, stem, and seed) were evaluated for potential anti-HMG-CoA reductase, antioxidant, and anti-inflammatory activities. The putative HMG-CoA reductase inhibitory activity of A. viridis extracts at different concentrations was determined spectrophotometrically by NADPH oxidation, using HMG-CoA as substrate. A. viridis leaf extract revealed the highest HMG-CoA reductase inhibitory effect at about 71%, with noncompetitive inhibition in Lineweaver-Burk plot analysis. The leaf extract showed good inhibition of hydroperoxides, 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and ferric ion radicals in various concentrations. A. viridis leaf extract was proven to be an effective inhibitor of hyaluronidase, lipoxygenase, and xanthine oxidase enzymes. The experimental data suggest that A. viridis leaf extract is a source of potent antioxidant and anti-inflammatory agent and may modulate cholesterol metabolism by inhibition of HMG-CoA reductase. PMID:27051453

  14. Anti-HMG-CoA Reductase, Antioxidant, and Anti-Inflammatory Activities of Amaranthus viridis Leaf Extract as a Potential Treatment for Hypercholesterolemia

    PubMed Central

    Salvamani, Shamala; Gunasekaran, Baskaran; Shukor, Mohd Yunus; Shaharuddin, Noor Azmi; Sabullah, Mohd Khalizan

    2016-01-01

    Inflammation and oxidative stress are believed to contribute to the pathology of several chronic diseases including hypercholesterolemia (elevated levels of cholesterol in blood) and atherosclerosis. HMG-CoA reductase inhibitors of plant origin are needed as synthetic drugs, such as statins, which are known to cause adverse effects on the liver and muscles. Amaranthus viridis (A. viridis) has been used from ancient times for its supposedly medically beneficial properties. In the current study, different parts of A. viridis (leaf, stem, and seed) were evaluated for potential anti-HMG-CoA reductase, antioxidant, and anti-inflammatory activities. The putative HMG-CoA reductase inhibitory activity of A. viridis extracts at different concentrations was determined spectrophotometrically by NADPH oxidation, using HMG-CoA as substrate. A. viridis leaf extract revealed the highest HMG-CoA reductase inhibitory effect at about 71%, with noncompetitive inhibition in Lineweaver-Burk plot analysis. The leaf extract showed good inhibition of hydroperoxides, 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and ferric ion radicals in various concentrations. A. viridis leaf extract was proven to be an effective inhibitor of hyaluronidase, lipoxygenase, and xanthine oxidase enzymes. The experimental data suggest that A. viridis leaf extract is a source of potent antioxidant and anti-inflammatory agent and may modulate cholesterol metabolism by inhibition of HMG-CoA reductase. PMID:27051453

  15. Purification and characterization of 3-hydroxymethylglutaryl-coenzyme A reductase of Schistosoma mansoni: regulation of parasite enzyme activity differs from mammalian host.

    PubMed

    Chen, G Z; Foster, L; Bennett, J L

    1991-07-01

    The enzyme 3-hydroxymethylglutaryl-CoA (HMG-CoA) reductase plays a critical role in regulating the production of cholesterol, dolichols, and ubiquinones in mammals. The inhibition of this enzyme in Schistosoma mansoni is accompanied by a cessation of egg production by the female parasite and a reduced ability of the parasite to properly glycoslyate their proteins. Furthermore, we recently demonstrated that mevinolin, if given continuously over a period of 10-14 days, is a potent antischistosomal drug. In this paper, we describe the properties of purified HMG-CoA reductase from S. mansoni. Using affinity chromatography, we were able to obtain a 417-fold purification of the enzyme which had Km values similar to the rat enzyme for HMG-CoA and NADPH. The Ki value for mevinolin, a potent and selective inhibitor of the rat reductase (Ki = 0.6 nM), was significantly higher (Ki = 46 nM) for the schistosome enzyme. SDS-PAGE and HPLC of the purified enzyme resulted in the appearance of a single protein, which had a molecular weight (66,000) in the range reported for the rat enzyme. Parasite reductase activity, unlike that of its host, did not display a circadian rhythm. Furthermore, agents which elevate (cholestyramine) or decrease (cholesterol) mammalian reductase activity had no effect on the parasite enzyme. Our results suggest that the mechanism which regulates production of the parasite's enzyme may differ from its mammalian host. PMID:1905241

  16. Sequence and properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2.

    PubMed Central

    French, C E; Nicklin, S; Bruce, N C

    1996-01-01

    Pentaerythritol tetranitrate reductase, which reductively liberates nitrite from nitrate esters, is related to old yellow enzyme. Pentaerythritol tetranitrate reductase follows a ping-pong mechanism with competitive substrate inhibition by NADPH, is strongly inhibited by steroids, and is capable of reducing the unsaturated bond of 2-cyclohexen-1-one. PMID:8932320

  17. Supercritical fluid extraction of grape seeds: extract chemical composition, antioxidant activity and inhibition of nitrite production in LPS-stimulated Raw 264.7 cells.

    PubMed

    Pérez, Concepción; Ruiz del Castillo, María Luisa; Gil, Carmen; Blanch, Gracia Patricia; Flores, Gema

    2015-08-01

    Grape by-products are a rich source of bioactive compounds having broad medicinal properties, but are usually wasted from juice/wine processing industries. The present study investigates the use of supercritical fluid extraction (SFE) for obtaining an extract rich in bioactive compounds. First, some variables involved in the extraction were applied. SFE conditions were selected based on the oil mass yield, fatty acid profile and total phenolic composition. As a result, 40 °C and 300 bar were selected as operational conditions. The phenolic composition of the grape seed oil was determined using LC-DAD. The antioxidant activity was determined by ABTS and DPPH assays. For the anti-inflammatory activity the inhibition of nitrite production was assessed. The grape seed oil extracted was rich in phenolic compounds and fatty acids with significant antioxidant and anti-inflammatory activities. From these results, added economic value to this agroindustrial residue is proposed using environmentally friendly techniques. PMID:26130020

  18. Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro.

    PubMed

    Rockel, Peter; Strube, Frank; Rockel, Andra; Wildt, Juergen; Kaiser, Werner M

    2002-01-01

    NO (nitric oxide) production from sunflower plants (Helianthus annuus L.), detached spinach leaves (Spinacia oleracea L.), desalted spinach leaf extracts or commercial maize (Zea mays L.) leaf nitrate reductase (NR, EC 1.6.6.1) was continuously followed as NO emission into the gas phase by chemiluminescence detection, and its response to post-translational NR modulation was examined in vitro and in vivo. NR (purified or in crude extracts) in vitro produced NO at saturating NADH and nitrite concentrations at about 1% of its nitrate reduction capacity. The K(m) for nitrite was relatively high (100 microM) compared to nitrite concentrations in illuminated leaves (10 microM). NO production was competitively inhibited by physiological nitrate concentrations (K(i)=50 microM). Importantly, inactivation of NR in crude extracts by protein phosphorylation with MgATP in the presence of a protein phosphatase inhibitor also inhibited NO production. Nitrate-fertilized plants or leaves emitted NO into purified air. The NO emission was lower in the dark than in the light, but was generally only a small fraction of the total NR activity in the tissue (about 0.01-0.1%). In order to check for a modulation of NO production in vivo, NR was artificially activated by treatments such as anoxia, feeding uncouplers or AICAR (a cell permeant 5'-AMP analogue). Under all these conditions, leaves were accumulating nitrite to concentrations exceeding those in normal illuminated leaves up to 100-fold, and NO production was drastically increased especially in the dark. NO production by leaf extracts or intact leaves was unaffected by nitric oxide synthase inhibitors. It is concluded that in non-elicited leaves NO is produced in variable quantities by NR depending on the total NR activity, the NR activation state and the cytosolic nitrite and nitrate concentration. PMID:11741046

  19. Tetrathionate reductase of Salmonella thyphimurium: a molybdenum containing enzyme

    SciTech Connect

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

    1986-04-29

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

  20. Effect of temperature on anoxic metabolism of nitrites to nitrous oxide by polyphosphate accumulating organisms.

    PubMed

    Miao, Zhijia; Zeng, Wei; Wang, Shuying; Peng, Yongzhen; Cao, Guihua; Weng, Dongchen; Xue, Guisong; Yang, Qing

    2014-02-01

    Temperature is an important physical factor, which strongly influences biomass and metabolic activity. In this study, the effects of temperature on the anoxic metabolism of nitrite (NO2(-)) to nitrous oxide (N2O) by polyphosphate accumulating organisms, and the process of the accumulation of N2O (during nitrite reduction), which acts as an electron acceptor, were investigated using 91% +/- 4% Candidatus Accumulibacter phosphatis sludge. The results showed that N2O is accumulated when Accumulibacter first utilize nitrite instead of oxygen as the sole electron acceptor during the denitrifying phosphorus removal process. Properties such as nitrite reduction rate, phosphorus uptake rate, N2O reduction rate, and polyhydroxyalkanoate degradation rate were all influenced by temperature variation (over the range from 10 to 30 degrees C reaching maximum values at 25 degrees C). The reduction rate of N2O by N2O reductase was more sensitive to temperature when N2O was utilized as the sole electron acceptor instead of N2O, and the N2O reduction rates, ranging from 0.48 to 3.53 N20-N/(hr x g VSS), increased to 1.45 to 8.60 mg N2O-N/(hr x g VSS). The kinetics processes for temperature variation of 10 to 30 degrees C were (theta1 = 1.140-1.216 and theta2 = 1.139-1.167). In the range of 10 degrees C to 30 degrees C, almost all of the anoxic stoichiometry was sensitive to temperature changes. In addition, a rise in N2O reduction activity leading to a decrease in N2O accumulation in long term operations at the optimal temperature (27 degrees C calculated by the Arrhenius model). PMID:25076517

  1. Construction of effective disposable biosensors for point of care testing of nitrite.

    PubMed

    Monteiro, Tiago; Rodrigues, Patrícia R; Gonçalves, Ana Luisa; Moura, José J G; Jubete, Elena; Añorga, Larraitz; Piknova, Barbora; Schechter, Alan N; Silveira, Célia M; Almeida, M Gabriela

    2015-09-01

    In this paper we aim to demonstrate, as a proof-of-concept, the feasibility of the mass production of effective point of care tests for nitrite quantification in environmental, food and clinical samples. Following our previous work on the development of third generation electrochemical biosensors based on the ammonia forming nitrite reductase (ccNiR), herein we reduced the size of the electrodes' system to a miniaturized format, solved the problem of oxygen interference and performed simple quantification assays in real samples. In particular, carbon paste screen printed electrodes (SPE) were coated with a ccNiR/carbon ink composite homogenized in organic solvents and cured at low temperatures. The biocompatibility of these chemical and thermal treatments was evaluated by cyclic voltammetry showing that the catalytic performance was higher with the combination acetone and a 40°C curing temperature. The successful incorporation of the protein in the carbon ink/solvent composite, while remaining catalytically competent, attests for ccNiR's robustness and suitability for application in screen printed based biosensors. Because the direct electrochemical reduction of molecular oxygen occurs when electroanalytical measurements are performed at the negative potentials required to activate ccNiR (ca.-0.4V vs Ag/AgCl), an oxygen scavenging system based on the coupling of glucose oxidase and catalase activities was successfully used. This enabled the quantification of nitrite in different samples (milk, water, plasma and urine) in a straightforward way and with small error (1-6%). The sensitivity of the biosensor towards nitrite reduction under optimized conditions was 0.55 A M(-1) cm(-2) with a linear response range 0.7-370 μM. PMID:26003719

  2. Metabolism of hydroxypyruvate in a mutant of barley lacking NADH-dependent hydroxypyruvate reductase, an important photorespiratory enzyme activity

    SciTech Connect

    Murray, A.J.S.; Blackwell, R.D.; Lea, P.J. )

    1989-09-01

    A mutant of barley (Hordeum vulgare L.), LaPr 88/29, deficient in NADH-dependent hydroxypyruvate reductase (HPR) activity has been isolated. The activities of both NADH (5%) and NADPH-dependent (19%) HPR were severely reduced in this mutant compared to the wild type. Although lacking an enzyme in the main carbon pathway of photorespiration, this mutant was capable of CO{sub 2} fixation rates equivalent to 75% of that of the wild type, in normal atmospheres and 50% O{sub 2}. There also appeared to be little disruption to the photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C)serine feeding were similar in both mutant and wild-type leaves. When leaves of LaPr 88/29 were fed either ({sup 14}C)serine or {sup 14}CO{sub 2}, the accumulation of radioactivity was in serine and not in hydroxypyruvate, although the mutant was still able to metabolize over 25% of the supplied ({sup 14}C)serine into sucrose. After 3 hours in air the soluble amino acid pool was almost totally dominated by serine and glycine. LaPr 88/29 has also been used to show that NADH-glyoxylate reductase and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-dependent HPR activity is due to the NADH-dependent enzyme. We also suggest that the alternative NADPH activity can metabolize a proportion, but not all, of the hydroxypyruvate produced during photorespiration and may thus form a useful backup to the NADH-dependent enzyme under conditions of maximal photorespiration.

  3. Relationship of changing delta 4-steroid 5 alpha-reductase activity to (125I)iododeoxyuridine uptake during regeneration of involuted rat prostates

    SciTech Connect

    Kitahara, S.; Higashi, Y.; Takeuchi, S.; Oshima, H. )

    1989-04-01

    To elucidate the phenotypic expression of proliferating prostatic cells, rats were castrated, and the regenerating process of involuted ventral prostates during testosterone propionate (TP) administration was investigated by examining morphology, (5-{sup 125}I)iododeoxyuridine ({sup 125}I-UdR) uptake, DNA content, weight, acid phosphatase, and delta 4-steroid 5 alpha-reductase (5 alpha-reductase) activities. Morphologically, TP treatment initially increased the number of epithelial cells lining glandular lobules and subsequently restored the shape of epithelial cells. {sup 125}I-UdR uptake peaked on Day 3 of TP treatment and stayed at higher levels than for uncastrated controls until Day 14 of treatment. Prostatic weight, protein content, acid phosphatase, and DNA content returned to uncastrated control levels by Day 14 of TP treatment. TP administration markedly stimulated prostatic 5 alpha-reductase activity, which peaked on the Day 5 of treatment and decreased to uncastrated control levels by Day 14 of treatment. It is concluded that TP administration to castrated rats initially induced active mitotic division of the remaining stem cells, followed by formation of differentiated functional epithelial cells. Prostatic 5 alpha-reductase was highly active at the initial phase of active mitotic cell division. The major portion of the increased enzyme activity can be regarded as a phenotypic expression of stem or transient cells of prostatic epithelium.

  4. FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR)

    PubMed Central

    Lawrence, Andrew D.; Taylor, Samantha L.; Scott, Alan; Rowe, Michelle L.; Johnson, Christopher M.; Rigby, Stephen E. J.; Geeves, Michael A.; Pickersgill, Richard W.; Howard, Mark J.; Warren, Martin J.

    2014-01-01

    Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I). PMID:24909839

  5. Periplasmic Nitrate Reductase (NapABC Enzyme) Supports Anaerobic Respiration by Escherichia coli K-12

    PubMed Central

    Stewart, Valley; Lu, Yiran; Darwin, Andrew J.

    2002-01-01

    Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especially Paracoccus pantotrophus. Whole-genome sequencing of Escherichia coli revealed the structural genes napFDAGHBC, which encode NapABC enzyme and associated electron transfer components. E. coli also expresses two membrane-bound proton-translocating nitrate reductases, encoded by the narGHJI and narZYWV operons. We measured reduced viologen-dependent nitrate reductase activity in a series of strains with combinations of nar and nap null alleles. The napF operon-encoded nitrate reductase activity was not sensitive to azide, as shown previously for the P. pantotrophus NapA enzyme. A strain carrying null alleles of narG and narZ grew exponentially on glycerol with nitrate as the respiratory oxidant (anaerobic respiration), whereas a strain also carrying a null allele of napA did not. By contrast, the presence of napA+ had no influence on the more rapid growth of narG+ strains. These results indicate that periplasmic nitrate reductase, like fumarate reductase, can function in anaerobic respiration but does not constitute a site for generating proton motive force. The time course of Φ(napF-lacZ) expression during growth in batch culture displayed a complex pattern in response to the dynamic nitrate/nitrite ratio. Our results are consistent with the observation that Φ(napF-lacZ) is expressed preferentially at relatively low nitrate concentrations in continuous cultures (H. Wang, C.-P. Tseng, and R. P. Gunsalus, J. Bacteriol. 181:5303-5308, 1999). This finding and other considerations support the hypothesis that NapABC enzyme may function in E. coli when low nitrate concentrations limit the bioenergetic efficiency of nitrate respiration via NarGHI enzyme. PMID:11844760

  6. Synergistic Inhibition of Microbial Sulfide Production by Combinations of the Metabolic Inhibitor Nitrite and Biocides▿

    PubMed Central

    Greene, E. Anne; Brunelle, Veronique; Jenneman, Gary E.; Voordouw, Gerrit

    2006-01-01

    MICs of six broad-spectrum biocides and two specific metabolic inhibitors and fractional inhibitory concentration indexes (FICIs) for controlling a sulfide-producing consortium were determined. Nitrite was synergistic (FICI < 1) with all but one biocide due to its specific inhibition of dissimilatory sulfite reductase. Hence, combining nitrite with biocides allows more efficient and cost-effective control of sulfate-reducing bacteria. PMID:16997976

  7. Modeling the Impact of Ingoing Sodium Nitrite, Sodium Ascorbate, and Residual Nitrite Concentrations on Growth Parameters of Listeria monocytogenes in Cooked, Cured Pork Sausage.

    PubMed

    King, Amanda M; Glass, Kathleen A; Milkowski, Andrew L; Seman, Dennis L; Sindelar, Jeffrey J

    2016-02-01

    Sodium nitrite has been identified as a key antimicrobial ingredient to control pathogens in ready-to-eat (RTE) meat and poultry products, including Listeria monocytogenes. This study was designed to more clearly elucidate the relationship between chemical factors (ingoing nitrite, ascorbate, and residual nitrite) and L. monocytogenes growth in RTE meats. Treatments of cooked, cured pork sausage (65% moisture, 1.8% salt, pH 6.6, and water activity 0.98) were based on response surface methodology with ingoing nitrite and ascorbate concentrations as the two main factors. Concentrations of nitrite and ascorbate, including star points, ranged from 0 to 352 and 0 to 643 ppm, respectively. At one of two time points after manufacturing (days 0 and 28), half of each treatment was surface inoculated to target 3 log CFU/g of a five-strain L. monocytogenes cocktail, vacuum packaged, and stored at 7°C for up to 4 weeks. Growth of L. monocytogenes was measured twice per week, and enumerations were used to estimate lag time and growth rates for each treatment. Residual nitrite concentrations were measured on days 0, 4, 7, 14, 21, and 28, and nitrite depletion rate was estimated by using first-order kinetics. The response surface methodology was used to model L. monocytogenes lag time and growth rate based on ingoing nitrite, ascorbate, and the residual nitrite remaining at the point of inoculation. Modeling results showed that lag time was impacted by residual nitrite concentration remaining at inoculation, as well as the squared term of ingoing nitrite, whereas growth rate was affected by ingoing nitrite concentration but not by the remaining residual nitrite at the point of inoculation. Residual nitrite depletion rate was dependent upon ingoing nitrite concentration and was only slightly affected by ascorbate concentration. This study confirmed that ingoing nitrite concentration influences L. monocytogenes growth in RTE products, yet residual nitrite concentration contributes

  8. The ferredoxin-binding site of ferredoxin: Nitrite oxidoreductase. Differential chemical modification of the free enzyme and its complex with ferredoxin.

    PubMed Central

    Dose, M M; Hirasawa, M; Kleis-SanFrancisco, S; Lew, E L; Knaff, D B

    1997-01-01

    Spinach (Spinacea oleracea) leaf ferredoxin (Fd)-dependent nitrite reductase was treated with either the arginine-modifying reagent phenyl-glyoxal or the lysine-modifying reagent pyridoxal-5'-phosphate under conditions where only the Fd-binding affinity of the enzyme was affected and where complex formation between Fd and the enzyme prevented the inhibition by either reagent. Modification with [14C]phenylglyoxal allowed the identification of two nitrite reductase arginines, R375 and R556, that are protected by Fd against labeling. Modification of nitrite reductase with pyridoxal-5'-phosphate, followed by reduction with NaBH4, allowed the identification of a lysine, K436, that is protected by Fd against labeling. Positive charges are present at these positions in all of the Fd-dependent nitrite reductase for which sequences are available, suggesting that these amino acids are directly involved in electrostatic binding of Fd to the enzyme. PMID:9232882

  9. Implications and problems in analysing cytotoxic activity of hydroxyurea in combination with a potential inhibitor of ribonucleotide reductase.

    PubMed

    Nocentini, G; Barzi, A; Franchetti, P

    1990-01-01

    The cytotoxicity of hydroxyurea in combination with 2.2'-bipyridyl-6-carbothioamide (a potential inhibitor of ribonucleotide reductase) on P388 murine leukemia is reported. Synergistic activity was studied using various interpretations of the isobologram method and the combination index method. We evaluated the pros and cons of these methods and their overall usefulness. In our opinion, to obtain all possible information from a compound association, it is important to choose a formally correct method that (a) can quantitatively evaluate synergism or antagonism, (b) may offer the possibility of averaging final results, (c) needs a minimal amount of experimental data, and (d) is rapid. Moreover, we emphasize both the utility of testing at least three molar ratios of compound association and the importance of carefully choosing the fractional inhibition used in calculating the combination effect. Such evaluation of drug combinations gives information essential to the preparation of new anticancer drug regimens and to the early assessment of biochemical interactions. PMID:2208576

  10. Induction of quinone reductase activity by psoralidin isolated from Psoralea corylifolia in mouse hepa 1c1c7 cells.

    PubMed

    Lee, Sung-Jin; Nam, Kung-Woo; Mar, Woongchon

    2009-07-01

    Quinone reductase (QR) is a protective phase II enzyme against mutagens and carcinogens which is inducible by a number of chemical compounds in plants. This study was carried out to investigate effects of the fractions from the seeds of Psoralea corylifolia on the induction of QR with Hepa 1c1c7 murine hepatoma cell line. The ethyl acetate-soluble fraction of the methanolic extract from the seeds was found to induce QR and the concentration of 1.5 fold QR induction (1.5 FIC) was 1.2 mug/mL. We obtained as an active compound, psoralidin, isolated from the ethyl acetate-soluble fraction after further sequential fractionation with column chromatography and 1.5 FIC of psoralidin was 0.5 mug/mL. The seeds of Psoralea corylifolia and psoralidin might be a candidate for developing QR inducers. PMID:19641888

  11. Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions

    PubMed Central

    Ebert, Maximilian C C J C; Morley, Krista L; Volpato, Jordan P; Schmitzer, Andreea R; Pelletier, Joelle N

    2015-01-01

    Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such “half-native” tetramers were shown to procure native-like catalytic activity, with similar KM values but kcat values 5- to 33-fold lower, illustrating a high tolerance for active-site substitutions. The selected variants showed a reduced thermal stability (Tm ∼12°C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active-site substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native-like catalytic rate and thus ensure cellular proliferation. PMID:25401264

  12. Osmotic Stress, not Aldose Reductase Activity, Directly induces Growth Factors and MAPK Signaling changes during Sugar Cataract Formation

    PubMed Central

    Zhang, Peng; Xing, Kuiyi; Randazzo, James; Blessing, Karen; Lou, Marjorie F.; Kador, Peter F.

    2012-01-01

    In sugar cataract formation in rats, aldose reductase (AR) actitvity is not only linked to lenticular sorbitol (diabetic) or galactitol (galactosemic) formation but also to signal transduction changes, cytotoxic signals and activation of apoptosis. Using both in vitro and in vivo techniques, the interrelationship between AR activity, polyol (sorbitol and galactitol) formation, osmotic stress, growth factor induction, and cell signaling changes have been investigated. For in vitro studies, lenses from Sprague Dawley rats were cultured for up to 48 hrs in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galctose with/without the aldose reductase inhibitors AL1576 or tolrestat, the sorbitol dehydrogenase inhibitor (SDI) CP-470,711, or 15 mM mannitol (osmotic-compensated media). For in vivo studies, lenses were obtained from streptozotocin-induced diabetic Sprague Dawley rats fed diet with/without the ARIs AL1576 or tolrestat for 10 weeks. As expected, lenses cultured in high glucose / galactose media or from untreated diabetic rats all showed a decrease in the GSH pool that was lessened by ARI treatment. Lenses either from diabetic rats or from glucose/galactose culture conditions showed increased expression of basic-FGF, TGF-β, and increased signaling through P-Akt, P-ERK1/2 and P-SAPK/JNK which were also normalized by ARIs to the expression levels observed in non-diabetic controls. Culturing rat lenses in osomotically compensated media containing 30 mM glucose or galactose did not lead to increased growth factor expression or altered signaling. These studies indicate that it is the biophysical response of the lens to osmotic stress that results in an increased intralenticular production of basic-FGF and TGF-β and the altered cytotoxic signaling that is observed during sugar cataract formation. PMID:22710095

  13. Adaptation of cytochrome-b5 reductase activity and methaemoglobinaemia in areas with a high nitrate concentration in drinking-water.

    PubMed Central

    Gupta, S. K.; Gupta, R. C.; Seth, A. K.; Gupta, A. B.; Bassin, J. K.; Gupta, A.

    1999-01-01

    An epidemiological investigation was undertaken in India to assess the prevalence of methaemoglobinaemia in areas with high nitrate concentration in drinking-water and the possible association with an adaptation of cytochrome-b5 reductase. Five areas were selected, with average nitrate ion concentrations in drinking-water of 26, 45, 95, 222 and 459 mg/l. These areas were visited and house schedules were prepared in accordance with a statistically designed protocol. A sample of 10% of the total population was selected in each of the areas, matched for age and weight, giving a total of 178 persons in five age groups. For each subject, a detailed history was documented, a medical examination was conducted and blood samples were taken to determine methaemoglobin level and cytochrome-b5 reductase activity. Collected data were subjected to statistical analysis to test for a possible relationship between nitrate concentration, cytochrome-b5 reductase activity and methaemoglobinaemia. High nitrate concentrations caused methaemoglobinaemia in infants and adults. The reserve of cytochrome-b5 reductase activity (i.e. the enzyme activity not currently being used, but which is available when needed; for example, under conditions of increased nitrate ingestion) and its adaptation with increasing water nitrate concentration to reduce methaemoglobin were more pronounced in children and adolescents. PMID:10534899

  14. YNL134C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity for detoxification of furfural derived from lignocellulosic biomass.

    PubMed

    Zhao, Xianxian; Tang, Juan; Wang, Xu; Yang, Ruoheng; Zhang, Xiaoping; Gu, Yunfu; Li, Xi; Ma, Menggen

    2015-05-01

    Furfural and 5-hydroxymethylfurfural (HMF) are the two main aldehyde compounds derived from pentoses and hexoses, respectively, during lignocellulosic biomass pretreatment. These two compounds inhibit microbial growth and interfere with subsequent alcohol fermentation. Saccharomyces cerevisiae has the in situ ability to detoxify furfural and HMF to the less toxic 2-furanmethanol (FM) and furan-2,5-dimethanol (FDM), respectively. Herein, we report that an uncharacterized gene, YNL134C, was highly up-regulated under furfural or HMF stress and Yap1p and Msn2/4p transcription factors likely controlled its up-regulated expression. Enzyme activity assays showed that YNL134C is an NADH-dependent aldehyde reductase, which plays a role in detoxification of furfural to FM. However, no NADH- or NADPH-dependent enzyme activity was observed for detoxification of HMF to FDM. This enzyme did not catalyse the reverse reaction of FM to furfural or FDM to HMF. Further studies showed that YNL134C is a broad-substrate aldehyde reductase, which can reduce multiple aldehydes to their corresponding alcohols. Although YNL134C is grouped into the quinone oxidoreductase family, no quinone reductase activity was observed using 1,2-naphthoquinone or 9,10-phenanthrenequinone as a substrate, and phylogenetic analysis indicates that it is genetically distant to quinone reductases. Proteins similar to YNL134C in sequence from S. cerevisiae and other microorganisms were phylogenetically analysed. PMID:25656244

  15. Characterizing and predicting carboxylic acid reductase activity for diversifying bioaldehyde production.

    PubMed

    Moura, Matthew; Pertusi, Dante; Lenzini, Stephen; Bhan, Namita; Broadbelt, Linda J; Tyo, Keith E J

    2016-05-01

    Chemicals with aldehyde moieties are useful in the synthesis of polymerization reagents, pharmaceuticals, pesticides, flavors, and fragrances because of their high reactivity. However, chemical synthesis of aldehydes from carboxylic acids has unfavorable thermodynamics and limited specificity. Enzymatically catalyzed reductive bioaldehyde synthesis is an attractive route that overcomes unfavorable thermodynamics by ATP hydrolysis in ambient, aqueous conditions. Carboxylic acid reductases (Cars) are particularly attractive, as only one enzyme is required. We sought to increase the knowledge base of permitted substrates for four Cars. Additionally, the Lys2 enzyme family was found to be mechanistically the same as Cars and two isozymes were also tested. Our results show that Cars prefer molecules where the carboxylic acid is the only polar/charged group. Using this data and other published data, we develop a support vector classifier (SVC) for predicting Car reactivity and make predictions on all carboxylic acid metabolites in iAF1260 and Model SEED. Biotechnol. Bioeng. 2016;113: 944-952. © 2015 Wiley Periodicals, Inc. PMID:26479709

  16. Profiles of Glucosinolates, Their Hydrolysis Products, and Quinone Reductase Inducing Activity from 39 Arugula (Eruca sativa Mill.) Accessions.

    PubMed

    Ku, Kang-Mo; Kim, Moo Jung; Jeffery, Elizabeth H; Kang, Young-Hwa; Juvik, John A

    2016-08-31

    Glucosinolates, their hydrolysis product concentrations, and the quinone reductase (QR) inducing activity of extracts of leaf tissue were assayed from 39 arugula (Eruca sativa Mill.) accessions. Arugula accessions from Mediterranean countries (n = 16; Egypt, Greece, Italy, Libya, Spain, and Turkey) and Northern Europe (n = 2; Poland and United Kingdom) were higher in glucosinolates and their hydrolysis products, especially glucoraphanin and sulforaphane, compared to those from Asia (n = 13; China, India, and Pakistan) and Middle East Asia (n = 8; Afghanistan, Iran, and Israel). The QR inducing activity was also the highest in Mediterranean and Northern European arugula accessions, possibly due to a significant positive correlation between sulforaphane and QR inducing activity (r = 0.54). No nitrile hydrolysis products were found, suggesting very low or no epithiospecifier protein activity from these arugula accessions. Broad sense heritability (H(2)) was estimated to be 0.91-0.98 for glucoinolates, 0.55-0.83 for their hydrolysis products, and 0.90 for QR inducing activity. PMID:27523193

  17. Differential activation of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes by wounding and pathogen challenge.

    PubMed Central

    Yang, Z; Park, H; Lacy, G H; Cramer, C L

    1991-01-01

    Potato genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were expressed in response to pathogen, elicitor, and wounding. HMGR catalyzes the rate-limiting step in isoprenoid biosynthesis leading to accumulation of phytoalexins and steroid glycoalkaloids. Wounding caused increases in HMGR mRNA levels. A rapid and transient peak occurred 30 minutes after wounding, followed by a slower peak at 14 hours; both were correlated with increased enzyme activity. Induction of HMGR mRNA by the soft rot pathogen Erwinia carotovora subsp carotovora or arachidonic acid began 8 hours after challenge and continued through 22 hours. Potato HMGR is encoded by a gene family. An HMGR gene-specific probe was used to demonstrate that one isogene of the HMGR family is pathogen activated and is distinct from isogene(s) that are wound activated. This provides evidence that defense-related increases in HMGR activity are due to mRNA level increases and that HMGR isogenes are activated differentially by wounding or pathogen challenge. PMID:1840919

  18. HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via geranylgeranylation and RhoA activation

    SciTech Connect

    Al-Haidari, Amr A.; Syk, Ingvar; Thorlacius, Henrik

    2014-03-28

    Highlights: • Simvastatin blocked CCL17-induced and CCR4-dependent RhoA activation in HT29 cells. • CCL17/CCR4-mediated migration of colon cancer cells was antagonised by simvastatin. • Cell migration recovered by adding Mevalonate and geranylgeranyl pyrophosphate. • Targeting HMG-CoA reductase might be useful to inhibit colon cancer metastasis. - Abstract: Background: Simvastatin is widely used to lower cholesterol levels in patients with cardiovascular diseases, although accumulating evidence suggests that statins, such as simvastatin, also exert numerous anti-tumoral effects. Aim: The aim of this study was to examine the effect of simvastatin on colon cancer cell migration. Methods: Migration assays were performed to evaluate CCL17-induced colon cancer cell (HT-29) chemotaxis. In vitro tumor growth and apoptosis were assessed using a proliferation assay and annexin V assay, respectively. Active RhoA protein levels in CCL17-stimulated colon cancer cells were quantified using a G-LISA assay. Results: We found that simvastatin dose-dependently decreased CCL17-induced colon cancer cell migration. Simvastatin had no effect on colon cancer cell proliferation or apoptosis. Inhibition of beta chemokine receptor 4, CCR4, reduced CCL17-evoked activation of RhoA in colon cancer cells. Moreover, administration of mevalonate reversed the inhibitory effect of simvastatin on CCL17-induced colon cancer cell migration. Interestingly, co-incubation with geranylgeranyl pyrophosphate (GGPP) antagonized the inhibitory impact of simvastatin on colon cancer cell migration triggered by CCL17. Moreover, we observed that simvastatin decreased CCL17-induced activation of RhoA in colon cancer cells. Administration of mevalonate and GGPP reversed the inhibitory effect of simvastatin on CCL17-provoked RhoA activation in colon cancer cells. Conclusions: Taken together, our findings show for the first time that HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via

  19. Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

    PubMed

    Vothknecht, U C; Kannangara, C G; von Wettstein, D

    1996-08-20

    delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(Glu) and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air. PMID:8799193

  20. Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase.

    PubMed Central

    Cooper, J; Conner, J; Clements, J B

    1995-01-01

    We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases. PMID:7609068

  1. Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512.

    PubMed

    Fujiwara, T; Fukumori, Y

    1996-04-01

    A highly active nitric oxide reductase was purified from Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha, which was anaerobically cultivated in the presence of nitrate. The enzyme was composed of two subunits with molecular masses of 34 and 15 kDa and contained two hemes b and one heme c per molecule. Copper was not found in the enzyme. The spectral properties suggested that one of the two hemes b and heme c were in six-coordinated low-spin states and another heme b was in a five-coordinated high-spin state and reacted with carbon monoxide. The enzyme showed high cytochrome c-nitric oxide oxidoreductase activity and formed nitrous oxide from nitric oxide with the expected stoichiometry when P. denitrificans ATCC 35512 ferrocytochrome c-550 was used as the electron donor. The V max and Km values for nitric oxide were 84 micromol of nitric oxide per min/mg of protein and 0.25 microM, respectively. Furthermore, the enzyme showed ferrocytochrome c-550-O2 oxidoreductase activity with a V max of 8.4 micromol of O2 per min/mg of protein and a Km value of 0.9 mM. Both activities were 50% inhibited by about 0.3 mM KCN. PMID:8606159

  2. In Silico Screening, Structure-Activity Relationship, and Biologic Evaluation of Selective Pteridine Reductase Inhibitors Targeting Visceral Leishmaniasis▿ †

    PubMed Central

    Kaur, Jaspreet; Kumar, Pranav; Tyagi, Sargam; Pathak, Richa; Batra, Sanjay; Singh, Prashant; Singh, Neeloo

    2011-01-01

    In this study we utilized the concept of rational drug design to identify novel compounds with optimal selectivity, efficacy and safety, which would bind to the target enzyme pteridine reductase 1 (PTR1) in Leishmania parasites. Twelve compounds afforded from Baylis-Hillman chemistry were docked by using the QUANTUM program into the active site of Leishmania donovani PTR1 homology model. The biological activity for these compounds was estimated in green fluorescent protein-transfected L. donovani promastigotes, and the most potential analogue was further investigated in intracellular amastigotes. Structure-activity relationship based on homology model drawn on our recombinant enzyme was substantiated by recombinant enzyme inhibition assay and growth of the cell culture. Flow cytometry results indicated that 7-(4-chlorobenzyl)-3-methyl-4-(4-trifluoromethyl-phenyl)-3,4,6,7,8,9-hexahydro-pyrimido[1,2-a]pyrimidin-2-one (compound 7) was 10 times more active on L. donovani amastigotes (50% inhibitory concentration [IC50] = 3 μM) than on promastigotes (IC50 = 29 μM). Compound 7 exhibited a Ki value of 0.72 μM in a recombinant enzyme inhibition assay. We discovered that novel pyrimido[1,2-a]pyrimidin-2-one systems generated from the allyl amines afforded from the Baylis-Hillman acetates could have potential as a valuable pharmacological tool against the neglected disease visceral leishmaniasis. PMID:21115787

  3. Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

    SciTech Connect

    Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

    1987-07-01

    The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

  4. New evidence of similarity between human and plant steroid metabolism: 5alpha-reductase activity in Solanum malacoxylon.

    PubMed

    Rosati, Fabiana; Danza, Giovanna; Guarna, Antonio; Cini, Nicoletta; Racchi, Milvia Luisa; Serio, Mario

    2003-01-01

    The physiological role of steroid hormones in humans is well known, and the metabolic pathway and mechanisms of action are almost completely elucidated. The role of plant steroid hormones, brassinosteroids, is less known, but an increasing amount of data on brassinosteroid biosynthesis is showing unexpected similarities between human and plant steroid metabolic pathways. Here we focus our attention on the enzyme 5alpha-reductase (5alphaR) for which a plant ortholog of the mammalian system, DET2, was recently described in Arabidopsis thaliana. We demonstrate that campestenone, the natural substrate of DET2, is reduced to 5alpha-campestanone by both human 5alphaR isozymes but with different affinities. Solanum malacoxylon, which is a calcinogenic plant very active in the biosynthesis of vitamin D-like molecules and sterols, was used to study 5alphaR activity. Leaves and calli were chosen as examples of differentiated and undifferentiated tissues, respectively. Two separate 5alphaR activities were found in calli and leaves of Solanum using campestenone as substrate. The use of progesterone allowed the detection of both activities in calli. Support for the existence of two 5alphaR isozymes in S. malacoxylon was provided by the differential actions of inhibitors of the human 5alphaR in calli and leaves. The evidence for the presence of two isozymes in different plant tissues extends the analogies between plant and mammalian steroid metabolic pathways. PMID:12488348

  5. Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512.

    PubMed Central

    Fujiwara, T; Fukumori, Y

    1996-01-01

    A highly active nitric oxide reductase was purified from Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha, which was anaerobically cultivated in the presence of nitrate. The enzyme was composed of two subunits with molecular masses of 34 and 15 kDa and contained two hemes b and one heme c per molecule. Copper was not found in the enzyme. The spectral properties suggested that one of the two hemes b and heme c were in six-coordinated low-spin states and another heme b was in a five-coordinated high-spin state and reacted with carbon monoxide. The enzyme showed high cytochrome c-nitric oxide oxidoreductase activity and formed nitrous oxide from nitric oxide with the expected stoichiometry when P. denitrificans ATCC 35512 ferrocytochrome c-550 was used as the electron donor. The V max and Km values for nitric oxide were 84 micromol of nitric oxide per min/mg of protein and 0.25 microM, respectively. Furthermore, the enzyme showed ferrocytochrome c-550-O2 oxidoreductase activity with a V max of 8.4 micromol of O2 per min/mg of protein and a Km value of 0.9 mM. Both activities were 50% inhibited by about 0.3 mM KCN. PMID:8606159

  6. E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

    SciTech Connect

    Fang, Zejun; Gong, Chaoju; Liu, Hong; Zhang, Xiaomin; Mei, Lingming; Song, Mintao; Qiu, Lanlan; Luo, Shuchai; Zhu, Zhihua; Zhang, Ronghui; Gu, Hongqian; Chen, Xiang

    2015-08-21

    As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration and

  7. Synthesis, in vitro antitumor activity, dihydrofolate reductase inhibition, DNA intercalation and structure-activity relationship studies of 1,3,5-triazine analogues.

    PubMed

    Singla, Prinka; Luxami, Vijay; Paul, Kamaldeep

    2016-01-15

    A series of triazine-benzimidazoles with 4-fluoroaniline substitution has been designed and synthesized. These compounds were further substituted with different primary and secondary amines. The structures of newly synthesized compounds were confirmed by (1)H, (13)C NMR, mass spectrometry and, in case of compound 18, by single crystal X-ray diffraction analysis. The newly synthesized compounds were evaluated against 60 human tumor cell lines at one dose and five dose concentration levels. Compounds 7, 8 and 22 have been found to be the most active antitumor agents with GI50 values of 1.77, 1.94 and 2.87μM, respectively. The synthesized compounds were then evaluated for their inhibitory activity to mammalian dihydrofolate reductase. Compound 22 was depicted as the most active compound for the inhibition of dihydrofolate reductase with IC50 value of 2.0nM. DNA binding studies were also revealed strong interacting properties of triazine derivatives towards calf thymus-DNA. PMID:26670841

  8. Spectrophotometric method for the assay of steroid 5α-reductase activity of rat liver and prostate microsomes.

    PubMed

    Iwai, Atsushi; Yoshimura, Teruki; Wada, Keiji; Watabe, Satoshi; Sakamoto, Yuki; Ito, Etsuro; Miura, Toshiaki

    2013-01-01

    A simple spectrophotometric method for the assay of steroid 5α-reductase (5α-SR) was developed in which 5α-dihydrotestosterone (5α-DHT) and 5α-androstane-3α,17β-diol (5α-diol), metabolites formed in the NADPH-dependent reduction of testosterone with enzyme sources of 5α-SR, were measured by enzymatic cycling using 3α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-NAD) and NADH. It was found that 5α-SR activity was proportional to the accumulated thio-NADH having an absorption maximum at 400 nm. Because of the high cycling rate (> 600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of 5α-DHT and 5α-diol at the picomole level without separation from excess testosterone. The present method was readily applicable to the assay of 5α-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of 5α-SR. PMID:23574674

  9. Thyroid-stimulating hormone decreases HMG-CoA reductase phosphorylation via AMP-activated protein kinase in the liver

    PubMed Central

    Zhang, Xiujuan; Song, Yongfeng; Feng, Mei; Zhou, Xinli; Lu, Yingli; Gao, Ling; Yu, Chunxiao; Jiang, Xiuyun; Zhao, Jiajun

    2015-01-01

    Cholesterol homeostasis is strictly regulated through the modulation of HMG-CoA reductase (HMGCR), the rate-limiting enzyme of cholesterol synthesis. Phosphorylation of HMGCR inactivates it and dephosphorylation activates it. AMP-activated protein kinase (AMPK) is the major kinase phosphorylating the enzyme. Our previous study found that thyroid-stimulating hormone (TSH) increased the hepatocytic HMGCR expression, but it was still unclear whether TSH affected hepatic HMGCR phosphorylation associated with AMPK. We used bovine TSH (bTSH) to treat the primary mouse hepatocytes and HepG2 cells with or without constitutively active (CA)-AMPK plasmid or protein kinase A inhibitor (H89), and set up the TSH receptor (Tshr)-KO mouse models. The p-HMGCR, p-AMPK, and related molecular expression were tested. The ratios of p-HMGCR/HMGCR and p-AMPK/AMPK decreased in the hepatocytes in a dose-dependent manner following bTSH stimulation. The changes above were inversed when the cells were treated with CA-AMPK plasmid or H89. In Tshr-KO mice, the ratios of liver p-HMGCR/HMGCR and p-AMPK/AMPK were increased relative to the littermate wild-type mice. Consistently, the phosphorylation of acetyl-CoA carboxylase, a downstream target molecule of AMPK, increased. All results suggested that TSH could regulate the phosphorylation of HMGCR via AMPK, which established a potential mechanism for hypercholesterolemia involved in a direct action of the TSH in the liver. PMID:25713102

  10. Influence of seasonal variation and methyl jasmonate mediated induction of glucosinolate biosynthesis on quinone reductase activity in broccoli florets.

    PubMed

    Ku, Kang Mo; Jeffery, Elizabeth H; Juvik, John A

    2013-10-01

    Methyl jasmonate spray treatments (250 μM) were utilized to alter glucosinolate composition in the florets of the commercial broccoli F1 hybrids 'Pirate', 'Expo', 'Green Magic', 'Imperial', and 'Gypsy' grown in replicated field plantings in 2009 and 2010. MeJA treatment significantly increased glucoraphanin (11%), gluconasturtiin (59%), and neoglucobrassicin (248%) concentrations and their hydrolysis products including sulforaphane (152%), phenethyl isothiocyanate (318%), N-methoxyindole-3-carbinol (313%), and neoascorbigen (232%) extracted from florets of these genotypes over two seasons. Increased quinone reductase (QR) activity was significantly correlated with increased levels of sulforaphane, N-methoxyindole-3-carbinol, and neoascorbigen. Partitioning experiment-wide trait variances indicated that the variability in concentrations of sulforaphane (29%), neoascorbigen (48%), and QR activity (72%) was influenced by year-associated weather variables, whereas variation in neoglucobrassicin (63%) and N-methoxyindole-3-carbinol (46%) concentrations was primarily attributed to methyl jasmonate treatment. These results suggest that methyl jasmonate treatment can enhance QR inducing activity by increased hydrolysis of glucoraphanin into sulforaphane and the hydrolysis products of neoglucobrassicin. PMID:24032372

  11. Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella tularensis: Residence Time and in Vivo Activity

    SciTech Connect

    Lu, H.; England, K; Ende, C; Truglio, J; Luckner, S; Reddy, B; Marlenee, N; Knudson, S; Knudson, D; et. al.

    2009-01-01

    Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 ?g mL-1. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.

  12. Thioredoxin reductase activity may be more important than GSH level in protecting human lens epithelial cells against UVA light.

    PubMed

    Padgaonkar, Vanita A; Leverenz, Victor R; Bhat, Aparna V; Pelliccia, Sara E; Giblin, Frank J

    2015-01-01

    This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L-buthionine-(S,R)-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O2 , 3% and 20%, were employed during a 1 h exposure of the cells to 25 J cm(-2) of UVA radiation (338-400 nm wavelength, peak at 365 nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O2 compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA-induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA-induced cell damage. PMID:25495870

  13. Thioredoxin Reductase Activity may be More Important than GSH Level in Protecting Human Lens Epithelial Cells Against UVA Light

    PubMed Central

    Padgaonkar, Vanita A.; Leverenz, Victor R.; Bhat, Aparna V.; Pelliccia, Sara E.; Giblin, Frank J.

    2014-01-01

    This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L-buthionine-(S,R)-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O2, 3% and 20%, were employed during a 1 hr exposure of the cells to 25 J/cm2 of UVA radiation (338-400nm wavelength, peak at 365nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O2 compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA-induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well-tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA-induced cell damage. PMID:25495870

  14. Rational design of broad spectrum antibacterial activity based on a clinically relevant enoyl-acyl carrier protein (ACP) reductase inhibitor.

    PubMed

    Schiebel, Johannes; Chang, Andrew; Shah, Sonam; Lu, Yang; Liu, Li; Pan, Pan; Hirschbeck, Maria W; Tareilus, Mona; Eltschkner, Sandra; Yu, Weixuan; Cummings, Jason E; Knudson, Susan E; Bommineni, Gopal R; Walker, Stephen G; Slayden, Richard A; Sotriffer, Christoph A; Tonge, Peter J; Kisker, Caroline

    2014-06-01

    Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms. PMID:24739388

  15. Influence of Volatile Fatty Acids on Nitrite Accumulation by a Pseudomonas stutzeri Strain Isolated from a Denitrifying Fluidized Bed Reactor

    PubMed Central

    van Rijn, J.; Tal, Y.; Barak, Y.

    1996-01-01

    Intermediate nitrite accumulation during denitrification by Pseudomonas stutzeri isolated from a denitrifying fluidized bed reactor was examined in the presence of different volatile fatty acids. Nitrite accumulated when acetate or propionate served as the carbon and electron source but did not accumulate in the presence of butyrate, valerate, or caproate. Nitrite accumulation in the presence of acetate was caused by differences in the rates of nitrate and nitrite reduction and, in addition, by competition between nitrate and nitrite reduction pathways for electrons. Incubation of the cells with butyrate resulted in a slower nitrate reduction rate and a faster nitrite reduction rate than incubation with acetate. Whereas nitrate inhibited the nitrite reduction rate in the presence of acetate, no such inhibition was found in butyrate-supplemented cells. Cytochromes b and c were found to mediate electron transport during nitrate reduction by the cells. Cytochrome c was reduced via a different pathway when nitrite-reducing cells were incubated with acetate than when they were incubated with butyrate. Furthermore, addition of antimycin A to nitrite-reducing cells resulted in partial inhibition of electron transport to cytochrome c in acetate-supplemented cells but not in butyrate-supplemented cells. On the basis of these findings, we propose that differences in intermediate nitrite accumulation are caused by differences in electron flow to nitrate and nitrite reductases during oxidation of either acetate or butyrate. PMID:16535368

  16. Chromate/Nitrite Interactions in Shewanella Oneidensis MR-1: Evidence for Multiple Cr(VI) Reduction Mechanisms Dependent on Physiological Growth Conditions

    SciTech Connect

    Apel, William Arnold; Viamajala, S.; Peyton, Brent Michael; Petersen, J. N.

    2002-06-01

    Inhibition of hexavalent chromium [Cr(VI)] reduction due to nitrate and nitrite was observed during tests with Shewanella oneidensis MR-1 (previously named Shewanella putrefaciens MR-1 and henceforth referred to as MR-1). Initial Cr(VI) reduction rates were measured at various nitrite concentrations, and a mixed inhibition kinetic model was used to determine the kinetic parameters-maximum Cr(VI) reduction rate and inhibition constant [V(max,Cr(VI)) and K(i,Cr(VI))]. Values of V(max,Cr(VI)) and K(i,Cr(VI)) obtained with MR-1 cultures grown under denitrifying conditions were observed to be significantly different from the values obtained when the cultures were grown with fumarate as the terminal electron acceptor. It was also observed that a single V(max,Cr(VI)) and K(i,Cr(VI)) did not adequately describe the inhibition kinetics of either nitrate-grown or fumarate-grown cultures. The inhibition patterns indicate that Cr(VI) reduction in MR-1 is likely not limited to a single pathway, but occurs via different mechanisms some of which are dependent on growth conditions. Inhibition of nitrite reduction due to the presence of Cr(VI) was also studied, and the kinetic parameters V(max,NO2) and K(i,NO2) were determined. It was observed that these coefficients also differed significantly between MR-1 grown under denitrifying conditions and fumarate reducing conditions. The inhibition studies suggest the involvement of nitrite reductase in Cr(VI) reduction. Because nitrite reduction is part of the anaerobic respiration process, inhibition due to Cr(VI) might be a result of interaction with the components of the anaerobic respiration pathway such as nitrite reductase. Also, differences in the degree of inhibition of nitrite reduction activity by chromate at different growth conditions suggest that the toxicity mechanism of Cr(VI) might also be dependent on the conditions of growth. Cr(VI) reduction has been shown to occur via different pathways, but to our knowledge, multiple

  17. Coordinated regulation of the Neisseria gonorrhoeae-truncated denitrification pathway by the nitric oxide-sensitive repressor, NsrR, and nitrite-insensitive NarQ-NarP.

    PubMed

    Overton, Tim W; Whitehead, Rebekah; Li, Ying; Snyder, Lori A S; Saunders, Nigel J; Smith, Harry; Cole, Jeff A

    2006-11-01

    Neisseria gonorrhoeae survives anaerobically by reducing nitrite to nitrous oxide catalyzed by the nitrite and nitric oxide reductases, AniA and NorB. P(aniA) is activated by FNR (regulator of fumarate and nitrate reduction), the two-component regulatory system NarQ-NarP, and induced by nitrite; P(norB) is induced by NO independently of FNR by an uncharacterized mechanism. We report the results of microarray analysis, bioinformatic analysis, and chromatin immunoprecipitation, which revealed that only five genes with readily identified NarP-binding sites are differentially expressed in narP(+) and narP strains. These include three genes implicated in the truncated gonococcal denitrification pathway: aniA, norB, and narQ. We also report that (i) nitrite induces aniA transcription in a narP mutant; (ii) nitrite induction involves indirect inactivation by nitric oxide of a gonococcal repressor, NsrR, identified from a multigenome bioinformatic study; (iii) in an nsrR mutant, aniA, norB, and dnrN (encoding a putative reactive nitrogen species response protein) were expressed constitutively in the absence of nitrite, suggesting that NsrR is the only NO-sensing transcription factor in N. gonorrhoeae; and (iv) NO rather than nitrite is the ligand to which NsrR responds. When expressed in Escherichia coli, gonococcal NarQ and chimaeras of E. coli and gonococcal NarQ are ligand-insensitive and constitutively active: a "locked-on" phenotype. We conclude that genes involved in the truncated denitrification pathway of N. gonorrhoeae are key components of the small NarQP regulon, that NarP indirectly regulates P(norB) by stimulating NO production by AniA, and that NsrR plays a critical role in enabling gonococci to evade NO generated as a host defense mechanism. PMID:16954205

  18. Simple and rapid method for detection of nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium canettii grown in the Bactec MGIT960 system.

    PubMed

    Goh, Khye Seng; Rastogi, Nalin

    2010-05-01

    Mycobacterium tuberculosis reduces nitrate very strongly as compared to Mycobacterium bovis and M. bovis BCG. Nitrate reductase, in conjunction with niacin accumulation, constitutes one of the major biochemical tests used in clinical microbiology laboratories to differentiate M. tuberculosis from other members of the M. tuberculosis complex, as well as nontuberculous Mycobacteria. Determination of nitrate reductase activity is currently performed using cultures grown on solid media with a slow detection time and the need for large quantities of bacilli, as otherwise the test is not reliable. Hereby, we propose a nitrate reduction test coupled to Bactec MGIT960 system as a simple, rapid and economic method with a total gain of time of about 3 to 4weeks over the conventional solid medium. In our study, almost all the M. tuberculosis and Mycobacterium canettii strains gave a strongly positive nitrate reductase result within 1day of positive detection by the MGIT960 system. In contrast, M. bovis, M. bovis BCG and M. africanum strains remained negative even after 14days of incubation. The possibility to detect nitrate reductase within 1 to 3days of a positive culture using MGIT960 opens new perspectives with the possibility of confirming M. tuberculosis - starting directly from pathological specimens. PMID:20298726

  19. Structural and mechanistic insights on nitrate reductases.

    PubMed

    Coelho, Catarina; Romão, Maria João

    2015-12-01

    Nitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data. PMID:26362109

  20. Radiation inactivation analysis of assimilatory NADH:nitrate reductase. Apparent functional sizes of partial activities associated with intact and proteolytically modified enzyme

    SciTech Connect

    Solomonson, L.P.; McCreery, M.J.; Kay, C.J.; Barber, M.J.

    1987-06-25

    Recently we demonstrated that target sizes for the partial activities of nitrate reductase were considerably smaller than the 100-kDa subunit which corresponded to the target size of the full (physiologic) activity NADH:nitrate reductase. These results suggested that the partial activities resided on functionally independent domains and that radiation inactivation may be due to localized rather than extensive damage to protein structure. The present study extends these observations and addresses several associated questions. Monophasic plots were observed over a wide range of radiation doses, suggesting a single activity component in each case. No apparent differences were observed over a 10-fold range of concentration for each substrate, suggesting that the observed slopes were not due to marked changes in Km values. Apparent target sizes estimated for partial activities associated with native enzyme and with limited proteolysis products of native enzyme suggested that the functional size obtained by radiation inactivation analysis is independent of the size of the polypeptide chain. The presence of free radical scavengers during irradiation reduced the apparent target size of both the physiologic and partial activities by an amount ranging from 24 to 43%, suggesting that a free radical mechanism is at least partially responsible for the inactivation. Immunoblot analysis of nitrate reductase irradiated in the presence of free radical scavengers revealed formation of distinct bands at 90, 75, and 40 kDa with increasing doses of irradiation rather than complete destruction of the polypeptide chain.

  1. Kinetic evidence that methionine sulfoxide reductase A can reveal its oxidase activity in the presence of thioredoxin.

    PubMed

    Kriznik, Alexandre; Boschi-Muller, Sandrine; Branlant, Guy

    2014-04-15

    The mouse methionine sulfoxide reductase A (MsrA) belongs to the subclass of MsrAs with one catalytic and two recycling Cys corresponding to Cys51, Cys198 and Cys206 in Escherichia coli MsrA, respectively. It was previously shown that in the absence of thioredoxin, the mouse and the E. coli MsrAs, which reduce two mol of methionine-O substrate per mol of enzyme, displays an in vitro S-stereospecific methionine oxidase activity. In the present study carried out with E. coli MsrA, kinetic evidence are presented which show that formation of the second mol of Ac-L-Met-NHMe is rate-limiting in the absence of thioredoxin. In the presence of thioredoxin, the overall rate-limiting step is associated with the thioredoxin-recycling process. Kinetic arguments are presented which support the accumulation of the E. coli MsrA under Cys51 sulfenic acid state in the presence of Trx. Thus, the methionine oxidase activity could be operative in vivo without the action of a regulatory protein in order to block the action of Trx as previously proposed. PMID:24632144

  2. Involvement of tristetraprolin in transcriptional activation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin

    SciTech Connect

    Ness, Gene C.; Edelman, Jeffrey L.; Brooks, Patricia A.

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer siRNAs to tristetraprolin blocks transcription of HMGR in vivo in rat liver. Black-Right-Pointing-Pointer siRNAs to tristetraprolin inhibits insulin activation of HMGR transcription. Black-Right-Pointing-Pointer Insulin acts to rapidly increase tristetraprolin in liver nuclear extracts. -- Abstract: Several AU-rich RNA binding element (ARE) proteins were investigated for their possible effects on transcription of hepatic 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR) in normal rats. Using in vivo electroporation, four different siRNAs to each ARE protein were introduced together with HMGR promoter (-325 to +20) luciferase construct and compared to saline controls. All four siRNAs to tristetraprolin (TTP) completely eliminated transcription from the HMGR promoter construct. Since insulin acts to rapidly increase hepatic HMGR transcription, the effect of TTP siRNA on induction by insulin was tested. The 3-fold stimulation by insulin was eliminated by this treatment. In comparison, siRNA to AU RNA binding protein/enoyl coenzyme A hydratase (AUH) had no effect. These findings indicate a role for TTP in the insulin-mediated activation of hepatic HMGR transcription.

  3. A novel thiol-reductase activity of Arabidopsis YUC6 confers drought tolerance independently of auxin biosynthesis

    PubMed Central

    Cha, Joon-Yung; Kim, Woe-Yeon; Kang, Sun Bin; Kim, Jeong Im; Baek, Dongwon; Jung, In Jung; Kim, Mi Ri; Li, Ning; Kim, Hyun-Jin; Nakajima, Masatoshi; Asami, Tadao; Sabir, Jamal S. M.; Park, Hyeong Cheol; Lee, Sang Yeol; Bohnert, Hans J.; Bressan, Ray A.; Pardo, Jose M.; Yun, Dae-Jin

    2015-01-01

    YUCCA (YUC) proteins constitute a family of flavin monooxygenases (FMOs), with an important role in auxin (IAA) biosynthesis. Here we report that Arabidopsis plants overexpressing YUC6 display enhanced IAA-related phenotypes and exhibit improved drought stress tolerance, low rate of water loss and controlled ROS accumulation under drought and oxidative stresses. Co-overexpression of an IAA-conjugating enzyme reduces IAA levels but drought stress tolerance is unaffected, indicating that the stress-related phenotype is not based on IAA overproduction. YUC6 contains a previously unrecognized FAD- and NADPH-dependent thiol-reductase activity (TR) that overlaps with the FMO domain involved in IAA biosynthesis. Mutation of a conserved cysteine residue (Cys-85) preserves FMO but suppresses TR activity and stress tolerance, whereas mutating the FAD- and NADPH-binding sites, that are common to TR and FMO domains, abolishes all outputs. We provide a paradigm for a single protein playing a dual role, regulating plant development and conveying stress defence responses. PMID:26314500

  4. A novel thiol-reductase activity of Arabidopsis YUC6 confers drought tolerance independently of auxin biosynthesis.

    PubMed

    Cha, Joon-Yung; Kim, Woe-Yeon; Kang, Sun Bin; Kim, Jeong Im; Baek, Dongwon; Jung, In Jung; Kim, Mi Ri; Li, Ning; Kim, Hyun-Jin; Nakajima, Masatoshi; Asami, Tadao; Sabir, Jamal S M; Park, Hyeong Cheol; Lee, Sang Yeol; Bohnert, Hans J; Bressan, Ray A; Pardo, Jose M; Yun, Dae-Jin

    2015-01-01

    YUCCA (YUC) proteins constitute a family of flavin monooxygenases (FMOs), with an important role in auxin (IAA) biosynthesis. Here we report that Arabidopsis plants overexpressing YUC6 display enhanced IAA-related phenotypes and exhibit improved drought stress tolerance, low rate of water loss and controlled ROS accumulation under drought and oxidative stresses. Co-overexpression of an IAA-conjugating enzyme reduces IAA levels but drought stress tolerance is unaffected, indicating that the stress-related phenotype is not based on IAA overproduction. YUC6 contains a previously unrecognized FAD- and NADPH-dependent thiol-reductase activity (TR) that overlaps with the FMO domain involved in IAA biosynthesis. Mutation of a conserved cysteine residue (Cys-85) preserves FMO but suppresses TR activity and stress tolerance, whereas mutating the FAD- and NADPH-binding sites, that are common to TR and FMO domains, abolishes all outputs. We provide a paradigm for a single protein playing a dual role, regulating plant development and conveying stress defence responses. PMID:26314500

  5. Solvent as a probe of active site motion and chemistry during the hydrogen tunnelling reaction in morphinone reductase.

    PubMed

    Hay, Sam; Pudney, Christopher R; Sutcliffe, Michael J; Scrutton, Nigel S

    2008-09-15

    The reductive half-reaction of morphinone reductase involves a hydride transfer from enzyme-bound beta-nicotinamide adenine dinucleotide (NADH) to a flavin mononucleotide (FMN). We have previously demonstrated that this step proceeds via a quantum mechanical tunnelling mechanism. Herein, we probe the effect of the solvent on the active site chemistry. The pK(a) of the reduced FMN N1 is 7.4+/-0.7, based on the pH-dependence of the FMN midpoint potential. We rule out that protonation of the reduced FMN N1 is coupled to the preceding H-transfer as both the rate and temperature-dependence of the reaction are insensitive to changes in solution pH above and below this pK(a). Further, the solvent kinetic isotope effect is approximately 1.0 and both the 1 degrees and 2 degrees KIEs are insensitive to solution pH. The effect of the solvent's dielectric constant is investigated and the rate of H-transfer is found to be unaffected by changes in the dielectric constant between approximately 60 and 80. We suggest that, while there is crystallographic evidence for some water in the active site, the putative promoting motion involved in the H-tunnelling reaction is insensitive to such changes. PMID:18668493

  6. A continuous spectrophotometric assay for NADPH-cytochrome P450 reductase activity using 1,1-diphenyl-2-picrylhydrazyl.

    PubMed

    Yim, Sung-Kun; Yun, Su-Jung; Yun, Chul-Ho

    2004-09-30

    NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450, and catalyzes the one-electron reduction of many drugs and foreign compounds. Various forms of spectrophotometric titration have been performed to investigate the electron-accepting properties of CPR, particularly, to examine its ability to reduce cytochrome c and ferricyanide. In this study, the reduction of 1,1-diphenyl-2-picrylhydrazyl (DPPH) by CPR was assessed as a means of monitoring CPR activity. The principle advantage of DPPH is that its reduction can be assayed directly in the reaction medium by a continuous spectrophotometry. Thus, electrons released from NADPH by CPR were transferred to DPPH, and DPPH reduction was then followed spectrophotometrically by measuring A(520) reduction. Optimal assay concentrations of DPPH, CPR, potassium phosphate buffer, and NADPH were first established. DPPH reduction activity was found to depend upon the strength of the buffer used, which was optimal at 100 mM potassium phosphate and pH 7.6. The extinction coefficient of DPPH was 4.09mM(-1) cm(-1). DPPH reduction followed classical Michaelis-Menten kinetics (K(m) = 28 microM, k(cat) = 1690 min(-1)). This method uses readily available materials, and has the additional advantages of being rapid and inexpensive. PMID:15479629

  7. Dissimilatory arsenate reductase activity and arsenate-respiring bacteria in bovine rumen fluid, hamster feces, and the termite hindgut

    USGS Publications Warehouse

    Herbel, M.J.; Switzer, Blum J.; Hoeft, S.E.; Cohen, S.M.; Arnold, L.L.; Lisak, J.; Stolz, J.F.; Oremland, R.S.

    2002-01-01

    Bovine rumen fluid and slurried hamster feces completely reduced millimolar levels of arsenate to arsenite upon incubation under anoxic conditions. This activity was strongly inhibited by autoclaving or aerobic conditions, and partially inhibited by tungstate or chloramphenicol. The rate of arsenate reduction was faster in feces from a population of arsenate-watered (100 ppm) hamsters compared to a control group watered without arsenate. Using radioisotope methods, arsenate reductase activity in hamster feces was also detected at very low concentrations of added arsenate (???10 ??M). Bacterial cultures were isolated from these materials, as well as from the termite hindgut, that grew using H2 as their electron donor, acetate as their carbon source, and arsenate as their respiratory electron acceptor. The three cultures aligned phylogenetically either with well-established enteric bacteria, or with an organism associated with feedlot fecal wastes. Because arsenite is transported across the gut epithelium more readily than arsenate, microbial dissimilatory reduction of arsenate in the gut may promote the body's absorption of arsenic and hence potentiate its toxicity. ?? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

  8. Plant Thioredoxin CDSP32 Regenerates 1-Cys Methionine Sulfoxide Reductase B Activity through the Direct Reduction of Sulfenic Acid*

    PubMed Central

    Tarrago, Lionel; Laugier, Edith; Zaffagnini, Mirko; Marchand, Christophe H.; Le Maréchal, Pierre; Lemaire, Stéphane D.; Rey, Pascal

    2010-01-01

    Thioredoxins (Trxs) are ubiquitous enzymes catalyzing the reduction of disulfide bonds, thanks to a CXXC active site. Among their substrates, 2-Cys methionine sulfoxide reductases B (2-Cys MSRBs) reduce the R diastereoisomer of methionine sulfoxide (MetSO) and possess two redox-active Cys as follows: a catalytic Cys reducing MetSO and a resolving one, involved in disulfide bridge formation. The other MSRB type, 1-Cys MSRBs, possesses only the catalytic Cys, and their regeneration mechanisms by Trxs remain unclear. The plant plastidial Trx CDSP32 is able to provide 1-Cys MSRB with electrons. CDSP32 includes two Trx modules with one potential active site 219CGPC222 and three extra Cys. Here, we investigated the redox properties of recombinant Arabidopsis CDSP32 and delineated the biochemical mechanisms of MSRB regeneration by CDSP32. Free thiol titration and 4-acetamido-4′-maleimidyldistilbene-2,2′-disulfonic acid alkylation assays indicated that the Trx possesses only two redox-active Cys, very likely the Cys219 and Cys222. Protein electrophoresis analyses coupled to mass spectrometry revealed that CDSP32 forms a heterodimeric complex with MSRB1 via reduction of the sulfenic acid formed on MSRB1 catalytic Cys after MetSO reduction. MSR activity assays using variable CDSP32 amounts revealed that MSRB1 reduction proceeds with a 1:1 stoichiometry, and redox titrations indicated that CDSP32 and MSRB1 possess midpoints potentials of −337 and −328 mV at pH 7.9, respectively, indicating that regeneration of MSRB1 activity by the Trx through sulfenic acid reduction is thermodynamically feasible in physiological conditions. PMID:20236937

  9. Loss of nitrous oxide reductase in Pseudomonas aeruginosa cultured under N sub 2 O as determined by rocket immunoelectrophoresis

    SciTech Connect

    SooHoo, C.K.; Hollocher, T.C. )

    1990-11-01

    Pseudomonas aeruginosa is unusual among denitrifiers in that it grows poorly on N{sub 2}O alone but can grow efficiently on the N{sub 2}O produced as a metabolic intermediate during the reduction of nitrate or nitrite to N{sub 2}. Previous studies in our laboratory have determined that the immediate cause for poor growth on N{sub 2}O is a progressive loss of nitrous oxide uptake and reductase activities in cells grown under N{sub 2}O. In this study, we determined by using rocket immunoelectrophoresis that the mass ratio of nitrous oxide reductase to total protein in the soluble protein fraction of Pseudomonas aeruginosa P2 was highest in cells grown on nitrate, decreased in cells grown on N{sub 2}O following the exhaustion of the initial charge of nitrate, and was nearly zero in cells exposed solely to N{sub 2}O.

  10. Control of 3-Hydroxy-3-Methylglutaryl-CoA Reductase Activity in Cultured Human Fibroblasts by Very Low Density Lipoproteins of Subjects with Hypertriglyceridemia

    PubMed Central

    Gianturco, Sandra H.; Gotto, Antonio M.; Jackson, Richard L.; Patsch, Josef R.; Sybers, Harley D.; Taunton, O. David; Yeshurun, Daniel L.; Smith, Louis C.

    1978-01-01

    Very low density lipoproteins (VLDL) and low density lipoproteins (LDL) from human normolipemic plasma, and the VLDL, the intermediate density lipoprotein (IDL), and LDL from patients with Type III hyperlipoproteinemic plasma were tested for their abilities to suppress the activity of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase in cultured human fibroblasts from normal subjects and a Type III patient. Regulation of cholesterol synthesis in the fibroblasts of a patient with Type III hyperlipoproteinemia appears to be normal. VLDL from normal subjects, isolated by angle head ultracentrifugation (d < 1.006) or by gel filtration on BioGel A-5m, were about 5 times less effective than LDL in suppressing HMG-CoA reductase activity, based on protein content, in agreement with previous reports with normal fibroblasts. Zonal centrifugation of normal VLDL isolated by both methods showed that the VLDL contained IDL. Normal VLDL from the angle head rotor, refractionated by the zonal method, had little, if any, ability to suppress the HMG-CoA reductase activity in either normal or Type III fibroblasts. VLDL, IDL, and LDL fractionated by zonal ultracentrifugation from Type III plasma gave half-maximum inhibition at 0.2-0.5 μg of protein/ml, indistinguishable from the suppression caused by normal LDL. Type III VLDL did not suppress HMG-CoA reductase in mutant LDL receptor-negative fibroblasts. Zonally isolated VLDL obtained from one Type IV and one Type V patient gave half-maximal suppression at 5 and 0.5 μg of protein/ml, respectively. Molecular diameters and apoprotein compositions of the zonally isolated normal and Type III VLDL were similar; the major difference in composition was that Type III VLDL contained more cholesteryl esters and less triglyceride than did normal VLDL. The compositions and diameters of the Type IV and Type V VLDL were similar to normal VLDL. These findings show that the basic defect in Type III hyperlipoproteinemia is qualitatively

  11. The Chinese Hamster Dihydrofolate Reductase Replication Origin Beta Is Active at Multiple Ectopic Chromosomal Locations and Requires Specific DNA Sequence Elements for Activity

    PubMed Central

    Altman, Amy L.; Fanning, Ellen

    2001-01-01

    To identify cis-acting genetic elements essential for mammalian chromosomal DNA replication, a 5.8-kb fragment from the Chinese hamster dihydrofolate reductase (DHFR) locus containing the origin beta (ori-β) initiation region was stably transfected into random ectopic chromosomal locations in a hamster cell line lacking the endogenous DHFR locus. Initiation at ectopic ori-β in uncloned pools of transfected cells was measured using a competitive PCR-based nascent strand abundance assay and shown to mimic that at the endogenous ori-β region in Chinese hamster ovary K1 cells. Initiation activity of three ectopic ori-β deletion mutants was reduced, while the activity of another deletion mutant was enhanced. The results suggest that a 5.8-kb fragment of the DHFR ori-β region is sufficient to direct initiation and that specific DNA sequences in the ori-β region are required for efficient initiation activity. PMID:11158297

  12. Post-translational control of nitrate reductase activity responding to light and photosynthesis evolved already in the early vascular plants.

    PubMed

    Nemie-Feyissa, Dugassa; Królicka, Adriana; Førland, Nina; Hansen, Margarita; Heidari, Behzad; Lillo, Cathrine

    2013-05-01

    Regulation of nitrate reductase (NR) by reversible phosphorylation at a conserved motif is well established in higher plants, and enables regulation of NR in response to rapid fluctuations in light intensity. This regulation is not conserved in algae NR, and we wished to test the evolutionary origin of the regulatory mechanism by physiological examination of ancient land plants. Especially a member of the lycophytes is of interest since their NR is candidate for regulation by reversible phosphorylation based on sequence analysis. We compared Selaginella kraussiana, a member of the lycophytes and earliest vascular plants, with the angiosperm Arabidopsis thaliana, and also tested the moss Physcomitrella patens. Interestingly, optimization of assay conditions revealed that S. kraussiana NR used NADH as an electron donor like A. thaliana, whereas P. patens NR activity depended on NADPH. Examination of light/darkness effects showed that S. kraussiana NR was rapidly regulated similar to A. thaliana NR when a differential (Mg(2+) contra EDTA) assay was used to reveal activity state of NR. This implies that already existing NR enzyme was post-translationally activated by light in both species. Light had a positive effect also on de novo synthesis of NR in S. kraussiana, which could be shown after the plants had been exposed to a prolonged dark period (7 days). Daily variations in NR activity were mainly caused by post-translational modifications. As for angiosperms, the post-translational light activation of NR in S. kraussiana was inhibited by 3-(3,4-dichlorophenyl)-1*1-dimethylurea (DCMU), an inhibitor of photosynthesis and stomata opening. Evolutionary, a post-translational control mechanism for NR have occurred before or in parallel with development of vascular tissue in land plants, and appears to be part of a complex mechanisms for coordination of CO2 and nitrogen metabolism in these plants. PMID:23395536

  13. Biological Nitrogen Removal through Nitritation Coupled with Thiosulfate-Driven Denitritation.

    PubMed

    Qian, Jin; Zhou, Junmei; Zhang, Zhen; Liu, Rulong; Wang, Qilin

    2016-01-01

    A novel biological nitrogen removal system based on nitritation coupled with thiosulfate-driven denitritation (Nitritation-TDD) was developed to achieve a high nitrogen removal rate and low sludge production. A nitritation sequential batch reactor (nitritation SBR) and an anoxic up-flow sludge bed (AnUSB) reactor were applied for effective nitritation and denitritation, respectively. Above 75% nitrite was accumulated in the nitritation SBR with an influent ammonia loading rate of 0.43 kg N/d/m(3). During Nitritation-TDD operation, particle sizes (d50) of the sludge decreased from 406 to 225 um in nitritation SBR and from 327-183 um in AnUSB reactor. Pyrosequencing tests revealed that ammonium-oxidizing bacteria (AOB) population was stabilized at approximately 7.0% (calculated as population of AOB-related genus divided by the total microbial population) in the nitritation SBR. In contrast, nitrite-oxidizing bacteria (NOB) population decreased from 6.5-0.6% over the same time, indicating the effective nitrite accumulation in the nitritation SBR. Thiobacillus, accounting for 34.2% in the AnUSB reactor, was mainly responsible for nitrogen removal via autotrophic denitritation, using an external source of thiosulfate as electron donor. Also, it was found that free nitrous acid could directly affect the denitritation activity. PMID:27272192

  14. Biological Nitrogen Removal through Nitritation Coupled with Thiosulfate-Driven Denitritation

    PubMed Central

    Qian, Jin; Zhou, Junmei; Zhang, Zhen; Liu, Rulong; Wang, Qilin

    2016-01-01

    A novel biological nitrogen removal system based on nitritation coupled with thiosulfate-driven denitritation (Nitritation-TDD) was developed to achieve a high nitrogen removal rate and low sludge production. A nitritation sequential batch reactor (nitritation SBR) and an anoxic up-flow sludge bed (AnUSB) reactor were applied for effective nitritation and denitritation, respectively. Above 75% nitrite was accumulated in the nitritation SBR with an influent ammonia loading rate of 0.43 kg N/d/m3. During Nitritation-TDD operation, particle sizes (d50) of the sludge decreased from 406 to 225 um in nitritation SBR and from 327–183 um in AnUSB reactor. Pyrosequencing tests revealed that ammonium-oxidizing bacteria (AOB) population was stabilized at approximately 7.0% (calculated as population of AOB-related genus divided by the total microbial population) in the nitritation SBR. In contrast, nitrite-oxidizing bacteria (NOB) population decreased from 6.5–0.6% over the same time, indicating the effective nitrite accumulation in the nitritation SBR. Thiobacillus, accounting for 34.2% in the AnUSB reactor, was mainly responsible for nitrogen removal via autotrophic denitritation, using an external source of thiosulfate as electron donor. Also, it was found that free nitrous acid could directly affect the denitritation activity. PMID:27272192

  15. Nitrogen removal performance and microbial distribution in pilot- and full-scale integrated fixed-biofilm activated sludge reactors based on nitritation-anammox process.

    PubMed

    Zhang, Liang; Zhang, Shujun; Peng, Yongzhen; Han, Xiaoyu; Gan, Yiping

    2015-11-01

    Nitritation-anammox process was successfully established in pilot- and full-scale integrated fixed-film activated sludge (IFAS) reactors. An average nitrogen removal efficiency of 80% was achieved under ammonium loading rate of 0.7-1.3kgN/(m(3)d) in the pilot-scale reactor (12m(3)). Moreover, molecular analysis showed that ammonium oxidizing bacteria (AOB) were more abundant in the activated sludge while anammox bacteria were primarily located in the biofilm. The segregation of AOB and anammox bacteria enhanced the nitrogen removal rate and operational stability. Furthermore, a full-scale IFAS reactor of 500m(3) was set-up to treat sludge dewatering liquors. An average nitrogen removal efficiency of 85% and a nitrogen removal rate of 0.48kgN/(m(3)d) were achieved after inoculation. It was noted that high influent suspended solids would seriously affect the performance of the IFAS system. Therefore, a pre-treatment was proposed to reduce suspended solid in the full-scale application. PMID:26278191

  16. Inhibitors of 7-Dehydrocholesterol Reductase: Screening of a Collection of Pharmacologically Active Compounds in Neuro2a Cells.

    PubMed

    Kim, Hye-Young H; Korade, Zeljka; Tallman, Keri A; Liu, Wei; Weaver, C David; Mirnics, Karoly; Porter, Ned A

    2016-05-16

    A small library of pharmacologically active compounds (the NIH Clinical Collection) was assayed in Neuro2a cells to determine their effect on the last step in the biosynthesis of cholesterol, the transformation of 7-dehydrocholesterol (7-DHC) to cholesterol promoted by 7-dehydrocholesterol reductase, DHCR7. Of some 727 compounds in the NIH Clinical Collection, over 30 compounds significantly increased 7-DHC in Neuro2a cells when assayed at 1 μM. Active compounds that increased 7-DHC with a Z-score of +3 or greater generally gave rise to modest decreases in desmosterol and increases in lanosterol levels. Among the most active compounds identified in the library were the antipsychotic, antidepressant, and anxiolytic compounds that included perospirone, nefazodone, haloperidol, aripiprazole, trazodone, and buspirone. Fluoxetine and risperidone were also active at 1 μM, and another 10 compounds in this class of pharmaceuticals were identified in the screen at concentrations of 10 μM. Increased levels of 7-DHC are associated with Smith-Lemli-Opitz syndrome (SLOS), a human condition that results from a mutation in the gene that encodes DHCR7. The SLOS phenotype includes neurological deficits and congenital malformations, and it is linked to a higher incidence of autism spectrum disorder. The significance of the current study is that it identifies common pharmacological compounds that may induce a biochemical presentation similar to SLOS. Little is known about the side effects of elevated 7-DHC postdevelopmentally, and the elevated 7-DHC that results from exposure to these compounds may also be a confounder in the diagnosis of SLOS. PMID:27097157

  17. In vivo protection of activated Tyr22-dihydrofolate reductase gene-modified canine T lymphocytes from methotrexate

    PubMed Central

    Gori, Jennifer L.; Beard, Brian C.; Williams, Nathaniel P.; Ironside, Christina; Swanson, Debra; McIvor, R. Scott; Kiem, HP

    2013-01-01

    Background Nonmyeloablative allogeneic hematopoietic stem cell (HSC) transplantation can cure malignant and nonmalignant diseases affecting the hematopoietic system, such as severe combined immunodeficiencies, aplastic anemia and hemoglobinopathies. Although nonmyeloablative is favored over myeloablative transplantation for many patients, graft rejection remains problematic. One strategy to decrease rejection is to protect donor activated T cells in the graft from methotrexate (MTX) by genetically modifying the cells to express MTX-resistant dihydrofolate reductase (Tyr22-DHFR), leaving the immunosuppressive effects of MTX to act solely on activated host T lymphocytes, shifting the balance to favor allogeneic engraftment. Methods To evaluate MTX resistance of Tyr22-DHFR+ T lymphocytes in vivo, we transplanted dogs with autologous CD34+ cells modified with YFP and DHFR-GFP lentivirus vectors. Dogs were then treated with a standard MTX regimen (days 1, 3, 6, and 11) following immune activation with a foreign antigen as a surrogate assay to mimic early transplantation. Results DHFR-GFP+ gene marking was maintained in CD3+CD25+ and CD4+ T lymphocytes after MTX treatment while the level of T lymphocytes that expressed only a fluorescent reporter (YFP+) decreased. These data show that Tyr22-DHFR expression protects T lymphocytes from MTX toxicity in dogs, highlighting a clinically relevant application for preserving donor T lymphocytes during post transplantation immunosuppression. Conclusions These findings have implications for clinical translation of MTX-resistant T cells to facilitate engraftment of allogeneic cells following nonmyeloablative conditioning and minimize the risk of rejection. In summary, Tyr22-DHFR expression in T lymphocytes provides chemoprotection from MTX-mediated elimination in the context of immune activation in vivo. PMID:23666780

  18. Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo.

    PubMed Central

    Blume, S W; Snyder, R C; Ray, R; Thomas, S; Koller, C A; Miller, D M

    1991-01-01

    The promoter of the human dihydrofolate reductase (DHFR) gene contains two consensus binding sites for the DNA binding protein Sp1. DNAse protection and gel mobility shift assays demonstrate binding of recombinant Sp1 to both decanucleotide Sp1 binding sequences which are located 49 and 14 base pairs upstream of the transcription start site. The more distal of the two binding sites exhibits a somewhat higher affinity for Sp1. The G-C specific DNA binding drug, mithramycin, binds to both consensus sequences and prevents subsequent Sp1 binding. Promoter-dependent in vitro transcription of a DHFR template is selectively inhibited by mithramycin when compared to the human H2b histone gene. A similar effect is also noted in vivo. Mithramycin treatment of MCF-7 human breast carcinoma cells containing an amplified DHFR gene induces selective inhibition of DHFR transcription initiation, resulting in a decline in DHFR mRNA level and enzyme activity. This selective inhibition of DHFR expression suggests that it is possible to modulate the overexpression of the DHFR gene in methotrexate resistant cells. Images PMID:1834700

  19. IN VITRO INHIBITION OF GLUTATHIONE REDUCTASE BY ARSENOTRI-GLUTATHIONE

    EPA Science Inventory

    Arsenotriglutathione, a product of the reduction of arsenate and the complexation of arsenite by glutathione, is a mixed type inhibitor of the reduction of glutathione disulfide by purified yeast glutathione reductase or the glutathione reductase activity in rabbit erythrocyte ly...

  20. Pharmacokinetics of sodium nitrite-induced methemoglobinemia in the rat.

    PubMed

    Kohn, Michael C; Melnick, Ronald L; Ye, Frank; Portier, Christopher J

    2002-06-01

    A biologically based mathematical model was created to characterize time and dose-dependent relationships between exposure to nitrite and induction of methemoglobinemia. The model includes mass action equations for processes known to occur: oral absorption of nitrite, elimination from the plasma, partitioning between plasma and erythrocytes, binding of nitrite to hemoglobin and methemoglobin, and the free radical chain reaction for hemoglobin oxidation. The model also includes Michaelis-Menten kinetics for methemoglobin reductase-catalyzed regeneration of hemoglobin. Body weight-scaled rate constants for absorption (k(a)) and elimination (k(e)), the effective erythrocyte/plasma partition coefficient (P), and the apparent K(m) for methemoglobin reductase were the only parameters estimated by formal optimization to reproduce the observed time course data. Time courses of plasma nitrite concentrations and blood levels of hemoglobin and methemoglobin in male and female rats that had received single intravenous or oral doses of sodium nitrite were measured. Peak plasma levels of nitrite were achieved in both sexes approximately 30 min after oral exposure, and peak methemoglobin levels were achieved after 100 min. The model predicts that 10% of the hemoglobin is oxidized to the ferric form after oral doses of 15.9 mg/kg in male rats and 11.0 mg/kg in female rats and after intravenous doses of 8.9 and 7.1 mg/kg in male and female rats, respectively. The t(1/2) for recovery from methemoglobinemia was 60 to 120 min depending on dose and route of administration. A sensitivity analysis of the model was performed to identify to which parameters the predictions of the model were most sensitive and guide attempts to simplify the model. Replacement of the V(max) of methemoglobin reductase with a value representative of humans predicted a 10% methemoglobinemia following an intravenous dose of 5.8 mg/kg, in close agreement with an observed value of 5.7 mg/kg for humans. PMID

  1. Using sludge fermentation liquid to improve wastewater short-cut nitrification-denitrification and denitrifying phosphorus removal via nitrite.

    PubMed

    Ji, Zhouying; Chen, Yinguang

    2010-12-01

    Wastewater biological nutrient removal (BNR) by short-cut nitrification-denitrification (SCND) and denitrifying phosphorus removal via nitrite (DPRN) has several advantages, such as organic carbon source saving. In this paper, a new method, i.e., by using waste activated sludge alkaline fermentation liquid as BNR carbon source, for simultaneously improving SCND and DPRN was reported. First, the performance of SCND and DPRN with sludge fermentation liquid as carbon source was compared with acetic acid, which was commonly used in literatures. Sludge fermentation liquid showed much higher nitrite accumulation during aerobic nitrification than acetic acid (81.8% versus 40.9%), and the former had significant anoxic denitrification and phosphorus uptake. The soluble phosphorus and total nitrogen removal efficiencies with sludge fermentation liquid were much higher than with acetic acid (97.6% against 73.4% and 98.7% versus 79.2%). Then the mechanisms for sludge fermentation liquid showed higher SCND and DPRN than acetic acid were investigated from the aspects of wastewater composition, microorganisms assayed by 16S rRNA gene clone library, and fluorescence in situ hybridization. More NO(2)(-)-N accumulated by the use of sludge fermentation liquid was attributed to be more humic acids in the influent, which inhibited nitrite oxidizing bacteria (NOB) more serious than ammonia oxidizing bacteria (AOB), and more AOB but less NOB were observed in the BNR system. The reasons for sludge fermentation liquid BNR system exhibiting greater short-cut denitrifying phosphorus removal were that there were less glycogen accumulating organisms and more phosphorus accumulating organisms and anoxic denitrifying phosphorus removal bacteria with higher nitrite reductase activity. PMID:21053972

  2. Nitrite inhibition of denitrification by Pseudomonas fluorescens

    SciTech Connect

    Almeida, J.S.; Julio, S.M.; Reis, M.A.M. |

    1995-05-05

    Using a pure culture of Pseudomonas fluorescens as a model system nitrite inhibition of denitrification was studied. A mineral media with acetate and nitrate as sole electron donor and acceptor, respectively, was used. Results obtained in continuous stirred-tank reactors (CSTR) operated at pH values between 6.6 and 7.8 showed that growth inhibition depended only on the nitrite undissociated fraction concentration (nitrous acid). A mathematical model to describe this dependence is put forward. The maximum nitrous acid concentration compatible with cell growth and denitrification activity was found to be 66 {mu}g N/L. Denitrification activity was partially associated with growth, as described by the Luedeking-Piret equation. However, when the freshly inoculated reactor was operated discontinuously, nitrite accumulation caused growth uncoupling from denitrification activity. The authors suggest that these results can be interpreted considering that (a) nitrous acid acts as a proton uncoupler; and (b) cultures continuously exposed to nitrous acid prevent the uncoupling effect but not the growth inhibition. Examination of the growth dependence on nitrite concentration at pH 7.0 showed that adapted cultures (growth on CSTR) are less sensitive to nitrous acid inhibition than the ones cultivated in batch.

  3. Inhibition of Rat 5α-Reductase Activity and Testosterone-Induced Sebum Synthesis in Hamster Sebocytes by an Extract of Quercus acutissima Cortex

    PubMed Central

    Koseki, Junichi; Matsumoto, Takashi; Matsubara, Yosuke; Tsuchiya, Kazuaki; Mizuhara, Yasuharu; Sekiguchi, Kyoji; Nishimura, Hiroaki; Watanabe, Junko; Kaneko, Atsushi; Hattori, Tomohisa; Maemura, Kazuya; Kase, Yoshio

    2015-01-01

    Objective. Bokusoku (BK) is an extract from the Quercus cortex used in folk medicine for treatment of skin disorders and convergence, and is present in jumihaidokuto, a traditional Japanese medicine that is prescribed for purulent skin diseases like acne vulgaris. The excess of sebum production induced by androgen is involved in the development of acne. Our aim is to examine whether BK and its constituents inhibit testosterone metabolism and testosterone-induced sebum synthesis. Methods. Measurements of 5α-reductase activity and lipogenesis were performed using rat liver microsomes and hamster sebocytes, respectively. Results. BK dose-dependently reduced the conversion of testosterone to a more active androgen, dihydrotestosterone in a 5α-reductase enzymatic reaction. Twenty polyphenols in BK categorized as gallotannin, ellagitannin, and flavonoid were identified by LC-MS/MS. Nine polyphenols with gallate group, tetragalloyl glucose, pentagalloyl glucose, eugeniin, 1-desgalloyl eugeniin, casuarinin, castalagin, stenophyllanin C, (−)-epicatechin gallate, and (−)-epigallocatechin gallate, inhibited testosterone metabolism. In particular, pentagalloyl glucose showed the strongest activity. BK and pentagalloyl glucose suppressed testosterone-induced lipogenesis, whereas they weakly inhibited the lipogenic action of insulin. Conclusions. BK inhibited androgen-related pathogenesis of acne, testosterone conversion, and sebum synthesis, partially through 5α-reductase inhibition, and has potential to be a useful agent in the therapeutic strategy of acne. PMID:25709710

  4. Nitrate Reduction to Nitrite, a Possible Source of Nitrite for Growth of Nitrite-Oxidizing Bacteria

    PubMed Central

    Belser, L. W.

    1977-01-01

    Growth yields and other parameters characterizing the kinetics of growth of nitrite-oxidizing bacteria are presented. These parameters were measured during laboratory enrichments of soil samples with added nitrite. They were then used to reanalyze data for nitrite oxidizer growth in a previously reported field study (M. G. Volz, L. W. Belser, M. S. Ardakani, and A. D. McLaren, J. Environ. Qual. 4:179-182, 1975), where nitrate, but not nitrite or ammonium, was added. In that report, analysis of the field data indicated that in unsaturated soils, the reduction of nitrate to nitrite may be a significant source of nitrite for the growth of nitrite oxidizers. A yield of 1.23 × 104 cells per μg of N was determined to be most appropriate for application to the field. It was determined that if nitrite came only from mineralized organic nitrogen via ammonium oxidation, 35 to 90% of the organic nitrogen would have had to have been mineralized to produce the growth observed. However, it is estimated that only about 2% of the organic nitrogen could have been mineralized during the growth period. Thus, it appears that another source of nitrite is required, the most likely being the reduction of nitrate to nitrite coupled to the oxidation of organic matter. PMID:921264

  5. Mainstream partial nitritation and anammox in a 200,000 m3/day activated sludge process in Singapore: scale-down by using laboratory fed-batch reactor.

    PubMed

    Yeshi, Cao; Hong, Kwok Bee; van Loosdrecht, Mark C M; Daigger, Glen T; Yi, Png Hui; Wah, Yuen Long; Chye, Chua Seng; Ghani, Yahya Abd

    2016-01-01

    A laboratory fed-batch reactor has been used to study under controlled conditions the performance of partial nitritation/anammox for the 200,000 m(3)/day step-feed activated sludge process at the Changi Water Reclamation Plant, Singapore. The similarity of the concentrations of NH(4), NO(2), NO(3), PO(4), suspended chemical oxygen demand (sCOD), pH, and alkalinity (ALK) between the on-site process and laboratory reactor illustrates that the laboratory fed-batch reactor can be used to simulate the site performance. The performance of the reactor fed by primary effluent illustrated the existence of anammox and heterotrophic denitrification and apparent excessive biological phosphorus removal as observed from the site. The performance of the reactor fed by final effluent proved the presence of anammox process on site. Both the laboratory reactor and on-site process showed that higher influent 5-day biochemical oxygen demand/total nitrogen (BOD(5)/TN) (COD/TN) ratio increases the nitrogen removal efficiency of the process. PMID:27386982

  6. Detoxification of superoxide without production of H2O2: Antioxidant activity of superoxide reductase complexed with ferrocyanide

    PubMed Central

    Molina-Heredia, Fernando P.; Houée-Levin, Chantal; Berthomieu, Catherine; Touati, Danièle; Tremey, Emilie; Favaudon, Vincent; Adam, Virgile; Nivière, Vincent

    2006-01-01

    The superoxide radical O2·̅ is a toxic by-product of oxygen metabolism. Two O2·̅ detoxifying enzymes have been described so far, superoxide dismutase and superoxide reductase (SOR), both forming H2O2 as a reaction product. Recently, the SOR active site, a ferrous iron in a [Fe2+ (N-His)4 (S-Cys)] pentacoordination, was shown to have the ability to form a complex with the organometallic compound ferrocyanide. Here, we have investigated in detail the reactivity of the SOR–ferrocyanide complex with O2·̅ by pulse and γ-ray radiolysis, infrared, and UV-visible spectroscopies. The complex reacts very efficiently with O2·̅. However, the presence of the ferrocyanide adduct markedly modifies the reaction mechanism of SOR, with the formation of transient intermediates different from those observed for SOR alone. A one-electron redox chemistry appears to be carried out by the ferrocyanide moiety of the complex, whereas the SOR iron site remains in the reduced state. Surprisingly, the toxic H2O2 species is no longer the reaction product. Accordingly, in vivoexperiments showed that formation of the SOR–ferrocyanide complex increased the antioxidant capabilities of SOR expressed in an Escherichia coli sodA sodB recA mutant strain. Altogether, these data describe an unprecedented O2·̅ detoxification activity, catalyzed by the SOR–ferrocyanide complex, which does not conduct to the production of the toxic H2O2 species. PMID:17001016

  7. Electron transport to periplasmic nitrate reductase (NapA) of Wolinella succinogenes is independent of a NapC protein.

    PubMed

    Simon, Jörg; Sänger, Monica; Schuster, Stephan C; Gross, Roland

    2003-07-01

    The rumen bacterium Wolinella succinogenes grows by respiratory nitrate ammonification with formate as electron donor. Whereas the enzymology and coupling mechanism of nitrite respiration is well known, nitrate reduction to nitrite has not yet been examined. We report here that intact cells and cell fractions catalyse nitrate and chlorate reduction by reduced viologen dyes with high specific activities. A gene cluster encoding components of a putative periplasmic nitrate reductase system (napA, G, H, B, F, L, D) was sequenced. The napA gene was inactivated by inserting a kanamycin resistance gene cassette. The resulting mutant did not grow by nitrate respiration and did not reduce nitrate during growth by fumarate respiration, in contrast to the wild type. An antigen was detected in wild-type cells using an antiserum raised against the periplasmic nitrate reductase (NapA) from Paracoccus pantotrophus. This antigen was absent in the W. succinogenes napA mutant. It is concluded that the periplasmic nitrate reductase NapA is the only respiratory nitrate reductase in W. succinogenes, although a second nitrate-reducing enzyme is apparently induced in the napA mutant. The nap cluster of W. succinogenes lacks a napC gene whose product is thought to function in quinol oxidation and electron transfer to NapA in other bacteria. The W. succinogenes genome encodes two members of the NapC/NirT family, NrfH and FccC. Characterization of corresponding deletion mutants indicates that neither of these two proteins is required for nitrate respiration. A mutant lacking the genes encoding respiratory nitrite reductase (nrfHA) had wild-type properties with respect to nitrate respiration. A model of the electron transport chain of nitrate respiration is proposed in which one or more of the napF, G, H and L gene products mediate electron transport from menaquinol to the periplasmic NapAB complex. Inspection of the W. succinogenes genome sequence suggests that ammonia formation from

  8. Comparative bioavailability of ammonium, nitrate, nitrite and urea to typically harmful cyanobacterium Microcystis aeruginosa.

    PubMed

    Li, Jihua; Zhang, Jibiao; Huang, Wei; Kong, Fanlong; Li, Yue; Xi, Min; Zheng, Zheng

    2016-09-15

    Phosphorus is generally considered as the prime limiting nutrient responsible for cyanobacterial blooms. However, recent research is drawing attention to the importance of bioavailable nitrogen (N) in freshwater eutrophication. This study investigated the bioavailability of NO3(-)-N, NO2(-)-N, NH4(+)-N and Urea-N under different concentrations of 1.2, 3.6 and 6.0mgL(-1) to Microcystis aeruginosa. Overall, Urea-N ranked the first in promoting M. aeruginosa growth, followed by NO3(-)-N and NO2(-)-N. However, the algal growth cultured in NH4(+)-N was depressed under test N levels. The bioavailability of N to M. aeruginosa was seriously influenced by both N forms and N concentrations (p<0.01). Total N concentrations in Urea-N treatment decreased the fastest, which were corresponding with the μ values of M. aeruginosa. The high enzymic activities of nitrate reductase, nitrite reductase and glutamine synthetase indicated that the decomposition process for urea is effective, which contributed in N assimilation and utilization in M. aeruginosa cells. PMID:27357916

  9. Effect of short term external perturbations on bacterial ecology and activities in a partial nitritation and anammox reactor.

    PubMed

    Wu, Sha; Bhattacharjee, Ananda S; Weissbrodt, David G; Morgenroth, Eberhard; Goel, Ramesh

    2016-11-01

    This research investigated the short term effects of temperature changes (lasting 2-4weeks each) from 35±2°C to 21±2°C and 13±2°C and sulfide toxicity on partial nitrification-anammox (PN/A) system. Temperatures below 20°C and sulfide content as low as 5mgSL(-1) affected both aerobic and anaerobic catabolic activities of ammonia oxidation and the expression of related functional gene markers. The activity of AOB was inversely correlated with ammonium monooxygenase (amoA) gene expression. In contrast, the activity of AMX bacteria was positively correlated with the expression of their hydrazine synthase (hzsA) gene. Although the overall activities of AMX bacteria decreased at lower temperatures, the AMX bacteria were still active at the low temperatures. The inverse correlation between amoA gene expressions and the corresponding AOB activities was surprising. 16S rDNA based high throughput amplicon sequencing revealed the dominance of Chloroflexi, Planctomycetes and Proteobacteria phyla the distribution of which changed with temperature changes. PMID:27522119

  10. An efficient non-mediated amperometric biosensor for nitrite determination.

    PubMed

    Silveira, Célia M; Gomes, Sofia P; Araújo, Alberto N; Montenegro, M Conceição B S M; Todorovic, Smilja; Viana, Ana S; Silva, Rui J C; Moura, José J G; Almeida, M Gabriela

    2010-05-15

    In this paper we propose the construction of a new non-mediated electrochemical biosensor for nitrite determination in complex samples. The device is based on the stable and selective cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans, which has both high turnover and heterogeneous electron transfer rates. In opposition to previous efforts making use of several redox mediators, in this work we exploited the capacity of ccNiR to display a direct electrochemical response when interacting with pyrolytic graphite (PG) surfaces. To enable the analytical application of such bioelectrode the protein was successfully incorporated within a porous silica glass made by the sol-gel process. In the presence of nitrite, the ccNiR/sol-gel/PG electrode promptly displays catalytic currents indicating that the entrapped ccNiR molecules are reduced via direct electron transfer. This result is noteworthy since the protein molecules are caged inside a non-conductive silica network, in the absence of any mediator species or electron relay. At optimal conditions, the minimum detectable concentration is 120 nM. The biosensor sensitivity is 430 mA M(-1) cm(-2) within a linear range of 0.25-50 microM, keeping a stable response up to two weeks. The analysis of nitrites in freshwaters using the method of standard addition was highly accurated. PMID:20188536

  11. Dissecting the Structural Elements for the Activation of β-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae

    PubMed Central

    Hou, Jing; Zheng, Heping; Chruszcz, Maksymilian; Zimmerman, Matthew D.; Shumilin, Igor A.; Osinski, Tomasz; Demas, Matt; Grimshaw, Sarah

    2015-01-01

    ABSTRACT β-Ketoacyl-(acyl carrier protein) reductase (FabG) catalyzes the key reductive reaction in the elongation cycle of fatty acid synthesis (FAS), which is a vital metabolic pathway in bacteria and a promising target for new antibiotic development. The activation of the enzyme is usually linked to the formation of a catalytic triad and cofactor binding, and crystal structures of FabG from different organisms have been captured in either the active or inactive conformation. However, the structural elements which enable activation of FabG require further exploration. Here we report the findings of structural, enzymatic, and binding studies of the FabG protein found in the causative agent of cholera, Vibrio cholerae (vcFabG). vcFabG exists predominantly as a dimer in solution and is able to self-associate to form tetramers, which is the state seen in the crystal structure. The formation of the tetramer may be promoted by the presence of the cofactor NADP(H). The transition between the dimeric and tetrameric states of vcFabG is related to changes in the conformations of the α4/α5 helices on the dimer-dimer interface. Two glycine residues adjacent to the dimer interface (G92 and G141) are identified to be the hinge for the conformational changes, while the catalytic tyrosine (Y155) and a glutamine residue that forms hydrogen bonds to both loop β4-α4 and loop β5-α5 (Q152) stabilize the active conformation. The functions of the aforementioned residues were confirmed by binding and enzymatic assays for the corresponding mutants. IMPORTANCE This paper describes the results of structural, enzymatic, and binding studies of FabG from Vibrio cholerae (vcFabG). In this work, we dissected the structural elements responsible for the activation of vcFabG. The structural information provided here is essential for the development of antibiotics specifically targeting bacterial FabG, especially for the multidrug-resistant strains of V. cholerae. PMID:26553852

  12. A new CuZ active form in the catalytic reduction of N(2)O by nitrous oxide reductase from Pseudomonas nautica.

    PubMed

    Dell'Acqua, Simone; Pauleta, Sofia R; Paes de Sousa, Patrícia M; Monzani, Enrico; Casella, Luigi; Moura, José J G; Moura, Isabel

    2010-08-01

    The final step of bacterial denitrification, the two-electron reduction of N(2)O to N(2), is catalyzed by a multi-copper enzyme named nitrous oxide reductase. The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The in vitro reconstruction of the activity requires a slow activation in the presence of the artificial electron donor, reduced methyl viologen, necessary to reduce CuZ from the resting non-active state (1Cu(II)/3Cu(I)) to the fully reduced state (4Cu(I)), in contrast to the turnover cycle, which is very fast. In the present work, the direct reaction of the activated form of Pseudomonas nautica nitrous oxide reductase with stoichiometric amounts of N(2)O allowed the identification of a new reactive intermediate of the catalytic centre, CuZ degrees , in the turnover cycle, characterized by an intense absorption band at 680 nm. Moreover, the first mediated electrochemical study of Ps. nautica nitrous oxide reductase with its physiological electron donor, cytochrome c-552, was performed. The intermolecular electron transfer was analysed by cyclic voltammetry, under catalytic conditions, and a second-order rate constant of (5.5 +/- 0.9) x 10(5) M(-1 )s(-1) was determined. Both the reaction of stoichiometric amounts of substrate and the electrochemical studies show that the active CuZ degrees species, generated in the absence of reductants, can rearrange to the resting non-active CuZ state. In this light, new aspects of the catalytic and activation/inactivation mechanism of the enzyme are discussed. PMID:20422435

  13. Evaluation of chromate reductase activity in the cell-free culture filtrate of Arthrobacter sp. SUK 1201 isolated from chromite mine overburden.

    PubMed

    Dey, Satarupa; Paul, A K

    2016-08-01

    Arthrobacter sp. SUK 1201, a chromate resistant and reducing bacterium isolated from chromite mine overburden of Sukinda valley, Odisha, India has been evaluated for its hexavalent chromium [Cr(VI)] reduction potential using cell-free culture filtrate as extracellular chromate reductase enzyme. Production of the enzyme was enhanced in presence of Cr(VI) and its reducing efficiency was increased with increasing concentration of Cr(VI). The Michaelis-Menten constant (Km) and the maximum specific velocity (Vmax) of the extracellular Cr(VI) reductase were calculated to be 54.03 μM Cr(VI) and 5.803 U mg(-1) of protein respectively showing high affinity towards Cr(VI). The reducing activity of the enzyme was maximum at pH 6.5-7.5 and at a temperature of 35 °C and was dependent on NADH. The enzyme was tolerant to different metals such as Mn(II), Mg(II) and Fe(III) and was able to reduce Cr(VI) present in chromite mine seepage. These findings suggest that the extracellular chromate reductase of Arthrobacter sp. SUK 1201 has a great promise for use in Cr(VI) detoxification under different environmental conditions, particularly in the mining waste water treatment systems. PMID:27176938

  14. Details in the catalytic mechanism of mammalian thioredoxin reductase 1 revealed using point mutations and juglone-coupled enzyme activities.

    PubMed

    Xu, Jianqiang; Cheng, Qing; Arnér, Elias S J

    2016-05-01

    The mammalian selenoprotein thioredoxin reductase 1 (TrxR1) is a key enzyme in redox regulation, antioxidant defense, and cellular growth. TrxR1 can catalyze efficient reduction of juglone (5-hydroxy-1,4-naphthoquinone; walnut toxin) in a reaction which, in contrast to reduction of most other substrates of TrxR1, is not dependent upon an intact selenocysteine (Sec, U) residue of the enzyme. Using a number of TrxR1 mutant variants, we here found that a sole Cys residue at the C-terminal tail of TrxR1 is required for high-efficiency juglone-coupled NADPH oxidase activity of Sec-deficient enzyme, occurring with mixed one- and two-electron reactions producing superoxide. The activity also utilizes the FAD and the N-terminal redox active disulfide/dithiol motif of TrxR1. If a sole Cys residue at the C-terminal tail of TrxR1, in the absence of Sec, was moved further towards the C-terminal end of the protein compared to its natural position at residue 497, juglone reduction was, surprisingly, further increased. Ala substitutions of Trp407, Asn418 and Asn419 in a previously described "guiding bar", thought to mediate interactions of the C-terminal tail of TrxR1 with the FAD/dithiol site at the N-terminal domain of the other subunit in the dimeric enzyme, lowered turnover with juglone about 4.5-fold. Four residues of Sec-deficient TrxR1 were found to be easily arylated by juglone, including the Cys residue at position 497. Based upon our observations we suggest a model for involvement of the juglone-arylated C-terminal motif of TrxR1 to explain its high activity with juglone. This study thus provides novel insights into the catalytic mechanisms of TrxR1. One-electron juglone reduction by TrxR1 producing superoxide should furthermore contribute to the well-known prooxidant cytotoxicity of juglone. PMID:26898501

  15. The activation of electrophile, nucleophile and leaving group during the reaction catalysed by pI258 arsenate reductase.

    PubMed

    Roos, Goedele; Loverix, Stefan; Brosens, Elke; Van Belle, Karolien; Wyns, Lode; Geerlings, Paul; Messens, Joris

    2006-06-01

    The reduction of arsenate to arsenite by pI258 arsenate reductase (ArsC) combines a nucleophilic displacement reaction with a unique intramolecular disulfide cascade. Within this reaction mechanism, the oxidative equivalents are translocated from the active site to the surface of ArsC. The first reaction step in the reduction of arsenate by pI258 ArsC consists of a nucleophilic displacement reaction carried out by Cys10 on dianionic arsenate. The second step involves the nucleophilic attack of Cys82 on the Cys10-arseno intermediate formed during the first reaction step. The onset of the second step is studied here by using quantum chemical calculations in a density functional theory context. The optimised geometry of the Cys10-arseno adduct in the ArsC catalytic site (sequence motif: Cys10-Thr11-Gly12-Asn13-Ser14-Cys15-Arg16-Ser17) forms the starting point for all subsequent calculations. Thermodynamic data and a hard and soft acids and bases (HSAB) reactivity analysis show a preferential nucleophilic attack on a monoanionic Cys10-arseno adduct, which is stabilised by Ser17. The P-loop active site of pI258 ArsC activates first a hydroxy group and subsequently arsenite as the leaving group, as is clear from an increase in the calculated nucleofugality of these groups upon going from the gas phase to the solvent phase to the enzymatic environment. Furthermore, the enzymatic environment stabilises the thiolate form of the nucleophile Cys82 by 3.3 pH units through the presence of the eight-residue alpha helix flanked by Cys82 and Cys89 (redox helix) and through a hydrogen bond with Thr11. The importance of Thr11 in the pKa regulation of Cys82 was confirmed by the observed decrease in the kcat value of the Thr11Ala mutant as compared to that of wild-type ArsC. During the final reaction step, Cys89 is activated as a nucleophile by structural alterations of the redox helix that functions as a pKa control switch for Cys89; this final step is necessary to expose a Cys82-Cys

  16. Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms

    SciTech Connect

    Yagi, T.

    1987-05-19

    The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition correlated with the presence of an energy-transducing site in this segment of the respiratory chain. Where the NADH-quinone reductase segment involved an energy-coupling site (e.g., in bovine heart and rat liver mitochondria, and in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes), DCCD acted as an inhibitor of ubiquinone reduction by NADH. By contrast, where energy-coupling site 1 was absent (e.g., in Saccharomyces cerevisiae mitochondria and BacilLus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by DCCD. In the bovine and P. denitrificans systems, DCCD inhibition was pseudo first order with respect to incubation time, and reaction order with respect to inhibitor concentration was close to unity, indicating that inhibition resulted from the binding of one inhibitor molecule per active unit of NADH-ubiquinone reductase. In the bovine NADH-ubiquinone reductase complex (complex I), (/sup 14/C)DCCD was preferentially incorporated into two subunits of molecular weight 49,000 and 29,000. The time course of labeling of the 29,000 molecular weight subunit with (/sup 14/C)DCCD paralleled the time course of inhibition of NADH-ubiquinone reductase activity.

  17. Determination of the active form of the tetranuclear copper sulfur cluster in nitrous oxide reductase.

    PubMed

    Johnston, Esther M; Dell'Acqua, Simone; Ramos, Susana; Pauleta, Sofia R; Moura, Isabel; Solomon, Edward I

    2014-01-15

    N2OR has been found to have two structural forms of its tetranuclear copper active site, the 4CuS Cu(Z)* form and the 4Cu2S Cu(Z) form. EPR, resonance Raman, and MCD spectroscopies have been used to determine the redox states of these sites under different reductant conditions, showing that the Cu(Z)* site accesses the 1-hole and fully reduced redox states, while the Cu(Z) site accesses the 2-hole and 1-hole redox states. Single-turnover reactions of N2OR for Cu(Z) and Cu(Z)* poised in these redox states and steady-state turnover assays with different proportions of Cu(Z) and Cu(Z)* show that only fully reduced Cu(Z)* is catalytically competent in rapid turnover with N2O. PMID:24364717

  18. Comparison of the relative propensities of isoamyl nitrite and sodium nitrite to ameliorate acute cyanide poisoning in mice and a novel antidotal effect arising from anesthetics.

    PubMed

    Cambal, Leah K; Weitz, Andrew C; Li, Hui-Hua; Zhang, Yang; Zheng, Xi; Pearce, Linda L; Peterson, Jim

    2013-05-20

    Isoamyl nitrite has previously been considered acceptable as an inhaled cyanide antidote; therefore, the antidotal utility of this organic nitrite compared with sodium nitrite was investigated. To facilitate a quantitative comparison, doses of both sodium nitrite and isoamyl nitrite were given intraperitoneally in equimolar amounts to sublethally cyanide-challenged mice. Righting recovery from the knockdown state was clearly compromised in the isoamyl nitrite-treated animals, the effect being attributable to the toxicity of the isoamyl alchol produced during hydrolysis of the isoamyl nitrite to release nitrite anion. Subsequently, inhaled aqueous sodium nitrite aerosol was demonstrated to ameliorate sublethal cyanide toxicity, when provided to mice after the toxic dose, by the more rapid recovery of righting ability compared to that of the control animals given only the toxicant. Aerosolized sodium nitrite has thus been shown by these experiments to have promise as a better alternative to organic nitrites for development as an inhaled cyanide antidote. The inhaled sodium nitrite led to the production of NO in the bloodstream as determined by the appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. The aerosol delivery was performed in an unmetered inhalation chamber, and in this study, no attempt was made to optimize the procedure. It is argued that administration of an effective inhaled aqueous sodium nitrite dose in humans is possible, though just beyond the capability of current individual metered-dose inhaler designs, such as those used for asthma. Finally, working at slightly greater than LD50 NaCN doses, it was fortuitously discovered that (i) anesthesia leads to significantly prolonged survival compared to that of unanesthetized animals and that (ii) the antidotal activity of nitrite anion was completely abolished under anesthesia. Plausible explanations for these effects in mice and their practical consequences in relation to

  19. Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A.

    PubMed

    Giovanella, Patricia; Cabral, Lucélia; Bento, Fátima Menezes; Gianello, Clesio; Camargo, Flávio Anastácio Oliveira

    2016-01-25

    This study aimed to isolate mercury resistant bacteria, determine the minimum inhibitory concentration for Hg, estimate mercury removal by selected isolates, explore the mer genes, and detect and characterize the activity of the enzyme mercuric (II) reductase produced by a new strain of Pseudomonas sp. B50A. The Hg removal capacity of the isolates was determined by incubating the isolates in Luria Bertani broth and the remaining mercury quantified by atomic absorption spectrophotometry. A PCR reaction was carried out to detect the merA gene and the mercury (II) reductase activity was determined in a spectrophotometer at 340 nm. Eight Gram-negative bacterial isolates were resistant to high mercury concentrations and capable of removing mercury, and of these, five were positive for the gene merA. The isolate Pseudomonas sp. B50A removed 86% of the mercury present in the culture medium and was chosen for further analysis of its enzyme activity. Mercuric (II) reductase activity was detected in the crude extract of this strain. This enzyme showed optimal activity at pH 8 and at temperatures between 37 °C and 45 °C. The ions NH4(+), Ba(2+), Sn(2+), Ni(2+) and Cd(2+) neither inhibited nor stimulated the enzyme activity but it decreased in the presence of the ions Ca(2+), Cu(+) and K(+). The isolate and the enzyme detected were effective in reducing Hg(II) to Hg(0), showing the potential to develop bioremediation technologies and processes to clean-up the environment and waste contaminated with mercury. PMID:26051077

  20. Synthesis and activity of novel 16-dehydropregnenolone acetate derivatives as inhibitors of type 1 5α-reductase and on cancer cell line SK-LU-1.

    PubMed

    Silva-Ortiz, Aylin Viviana; Bratoeff, Eugene; Ramírez-Apan, Teresa; Heuze, Yvonne; Sánchez, Araceli; Soriano, Juan; Cabeza, Marisa

    2015-12-15

    Testosterone (T) plays a crucial role in prostate growth. In androgen-dependent tissues T is reduced to dihydrotestosterone (DHT) because of the presence of the 5α-reductase enzyme. This androgen is more active than T, since it has a higher affinity for the androgen receptor (AR). When this mechanism is altered, androgen-dependent diseases, including prostate cancer, could result. The aim of this study was to synthesize several 16-dehydropregnenolone acetate derivatives containing a triazole ring at C-21 and a linear or alicyclic ester moiety at C-3 of the steroidal skeleton. These steroids were designed as potential inhibitors of the activity of both types (1 and 2) of 5α-reductase. The cytotoxic activity of these compounds was also evaluated on a panel of PC-3, MCF7, and SK-LU-1 human cancer cell lines. The results from this study showed that with the exception of steroids 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-propionate and 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-pentanoate, the compounds exhibit a lower inhibitory activity for both isoenzymes of 5α-reductase than finasteride. Furthermore the 3β-hydroxy-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-20-one and 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-acetate derivatives display 80% cytotoxic activity on the SK-LU-1 cell line. These results also indicated that the triazole derivatives, which have a hydroxyl or acetoxy group at C-3, could have an anticancer effect, whereas the derivatives with a alicyclic ester group at C-3 do not show biological activity. PMID:26631442

  1. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    NASA Astrophysics Data System (ADS)

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils.

  2. Zeatin reductase in Phaseolus embryos

    SciTech Connect

    Martin, R.C.; Mok, David, W.S.; Mok, M.C. )

    1989-04-01

    Zeatin was converted to O-xylosylzeatin in embryos of Phaseolus vulgaris . O-xylosyldihydrozeatin was also identified as a zeatin metabolite. Incubation of embryo extracts with {sup 14}C-zeatin and {sup 14}C-O-xylosylzeatin revealed that reduction preceeds the O-xylosylation of zeatin. An enzyme responsible for reducing the N{sup 6}-side chain was isolated and partially purified using ammonium sulfate fractionation and affinity, gel filtration and anion exchange chromatography. The NADPH dependent reductase was zeatin specific and did not recognize cis-zeatin, ribosylzeatin, i{sup 6}Ade or i{sup 6}Ado. Two forms of the reductase could be separated by either gel filtration or anion exchange HPLC. The HMW isozyme (Mr. 55,000) eluted from the anion exchange column later than the LMW isozyme (Mr. 25,000). Interspecific differences in zeatin reductase activity were also detected.

  3. Microbial community and metabolism activity in a bioelectrochemical denitrification system under long-term presence of p-nitrophenol.

    PubMed

    Chen, Dan; Yang, Kai; Wei, Li; Wang, Hongyu

    2016-10-01

    Bioelectrochemical denitrification system (BEDS) is a promising technology for nitrate removal from wastewaters. The hazards and effects concerning p-nitrophenol (PNP) towards BEDS lack enough investigations and possess great research prospects. This study investigated how PNP affected the nitrate removal efficiency, microbial communities, functional denitrifying genes abundances, nitrate and nitrite reductase activities, diffusible signal factors (DSF) release, and extracellular polymeric substances (EPS) production in the BEDS. Results indicated that nitrate removal efficiency decreased with initial PNP concentration increased from 0 to 100mg/L. Phylum Firmicutes and class Clostridia were the main contributors for denitrification process in this BEDS. The abundances of the denitrifying genes nirS, nirK, napA, and narG all presented decreased trends with increasing PNP. In addition, the concentrations of nitrate reductase (NR), nitrite reductase (NIR), and EPS obviously decreased, while the concentration of DSF increased with increasing PNP, which demonstrated that higher PNP would inhibit the biofilm formation. PMID:27367815

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

    PubMed

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

    2016-07-01

    Multiple glutaredoxins (Grx) and glutathione reductase (Glr) are vital for the thiol-disulfide redox system in budding yeast but generally unexplored in filamentous fungi. Here we characterized the Beauveria bassiana redox system comprising dithiol Grx1, monothiol Grx2-4, Grx-like Grx5, and Glr orthologue. Each grx or glr deletion was compensated by increased transcripts of some other grx genes in normal cultures. Particularly, grx3 compensated the absence of grx1, grx2, grx5, or glr under oxidative stress while its absence was compensated only by undeletable grx4 under normal conditions but by most of other undeleted grx and glr genes in response to menadione. Consequently, the redox state was disturbed in Δglr more than in Δgrx3 but not in Δgrx1/2/5. Superoxide dismutases were more active in normal Δgrx1-3 cultures but less in Δgrx5 or Δglr response to menadione. Total catalase activity increased differentially in all the mutant cultures stressed with or without H2O2 while total peroxidase activity decreased more in the normal or H2O2-stressed culture of Δglr than of Δgrx3. Among the mutants, Δgrx3 showed slightly increased sensitivity to menadione or H2O2; Δglr exhibited greater sensitivity to thiol-oxidizing diamide than thiol-reducing 1-chloro-2,4-dinitrobenzene as well as increased sensitivity to the two oxidants. Intriguingly, all the mutants grew slower in a Fe(3+)-inclusive medium perhaps due to elevated transcripts of two Fe(3+) transporter genes. More or fewer phenotypes linked with biocontrol potential were altered in four deletion mutants excluding Δgrx5. All the changes were restored by targeted gene complementation. Overall, Grx3 played more critical role than other Grx homologues in the Glr-dependent redox system of the fungal entomopathogen. PMID:26969041

  5. The effect of salts on the activity and stability of Escherichia coli and Haloferax volcanii dihydrofolate reductases.

    PubMed

    Wright, Donna B; Banks, Douglas D; Lohman, Jeremy R; Hilsenbeck, Jacqueline L; Gloss, Lisa M

    2002-10-18

    The extremely halophilic Archae require near-saturating concentrations of salt in the external environment and in their cytoplasm, potassium being the predominant intracellular cation. The proteins of these organisms have evolved to function in concentrations of salt that inactivate or precipitate homologous proteins from non-halophilic species. It has been proposed that haloadaptation is primarily due to clustering of acidic residues on the surface of the protein, and that these clusters bind networks of hydrated ions. The dihydrofolate reductases from Escherichia coli (ecDHFR) and two DHFR isozymes from Haloferax volcanii (hvDHFR1 and hvDHFR2) have been used as a model system to compare the effect of salts on a mesophilic and halophilic enzyme. The KCl-dependence of the activity and substrate affinity was investigated. ecDHFR is largely inactivated above 1M KCl, with no major effect on substrate affinity. hvDHFR1 and hvDHFR2 unfold at KCl concentrations below approximately 0.5M. Above approximately 1M, the KCl dependence of the hvDHFR activities can be attributed to the effect of salt on substrate affinity. The abilities of NaCl, KCl, and CsCl to enhance the stability to urea denaturation were determined, and similar efficacies of stabilization were observed for all three DHFR variants. The DeltaG degrees (H(2)O) values increased linearly with increasing KCl and CsCl concentrations. The increase of DeltaG degrees (H(2)O) as a function of the smallest cation, NaCl, is slightly curved, suggesting a minor stabilization from cation binding or screening of electrostatic repulsion. At their respective physiological ionic strengths, the DHFR variants exhibit similar stabilities. Salts stabilize ecDHFR and the hvDHFRs by a common mechanism, not a halophile-specific mechanism, such as the binding of hydrated salt networks. The primary mode of salt stabilization of the mesophilic and halophilic DHFRs appears to be through preferential hydration and the Hofmeister effect of

  6. Reduction of aliphatic nitroesters and N-nitramines by Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase: quantitative structure-activity relationships.

    PubMed

    Nivinskas, Henrikas; Sarlauskas, Jonas; Anusevicius, Zilvinas; Toogood, Helen S; Scrutton, Nigel S; Cenas, Narimantas

    2008-12-01

    Enterobacter cloacae PB2 NADPH:pentaerythritol tetranitrate reductase (PETNR) performs the biodegradation of explosive organic nitrate esters via their reductive denitration. In order to understand the enzyme substrate specificity, we have examined the reactions of PETNR with organic nitrates (n = 15) and their nitrogen analogues, N-nitramines (n = 4). The reactions of these compounds with PETNR were accompanied by the release of 1-2 mol of nitrite per mole of compound, but were not accompanied by their redox cycling and superoxide formation. The reduction rate constants (k(cat)/K(m)) of inositol hexanitrate, diglycerol tetranitrate, erythritol tetranitrate, mannitol hexanitrate and xylitol pentanitrate were similar to those of the established PETNR substrates, PETN and glycerol trinitrate, whereas the reactivities of hexahydro-1,3,5-trinitro-1,3,5-triazine and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine were three orders of magnitude lower. The log k(cat)/K(m) value of the compounds increased with a decrease in the enthalpy of formation of the hydride adducts [DeltaH(f)(R-O-N(OH)O(-)) or DeltaH(f)(R(1),R(2) > N-N(OH)O(-))], and with an increase in their lipophilicity (octanol/water partition coefficient, log P(ow)), and did not depend on their van der Waals' volumes. Hydrophobic organic nitroesters and hydrophilic N-nitramines compete for the same binding site in the reduced enzyme form. The role of the hydrophobic interaction of PETNR with glycerol trinitrate was supported by the positive dependence of glycerol trinitrate reactivity on the solution ionic strength. The discrimination of nitroesters and N-nitramines according to their log P(ow) values seems to be a specific feature of the Old Yellow Enzyme family of flavoenzymes. PMID:19016851

  7. Unraveling the Redox Properties of the Global Regulator FurA from Anabaena sp. PCC 7120: Disulfide Reductase Activity Based on Its CXXC Motifs

    PubMed Central

    Botello-Morte, Laura; Bes, M. Teresa; Heras, Begoña; Fernández-Otal, Ángela; Peleato, M. Luisa

    2014-01-01

    Abstract Cyanobacterial FurA works as a global regulator linking iron homeostasis to photosynthetic metabolism and the responses to different environmental stresses. Additionally, FurA modulates several genes involved in redox homeostasis and fulfills the characteristics of a heme-sensor protein whose interaction with this cofactor negatively affects its DNA binding ability. FurA from Anabaena PCC 7120 contains five cysteine residues, four of them arranged in two redox CXXC motifs. Aims: Our goals were to analyze in depth the putative contribution of these CXXC motifs in the redox properties of FurA and to identify potential interacting partners of this regulator. Results: Insulin reduction assays unravel that FurA exhibits disulfide reductase activity. Simultaneous presence of both CXXC signatures greatly enhances the reduction rate, although the redox motif containing Cys101 and Cys104 seems a major contributor to this activity. Disulfide reductase activity was not detected in other ferric uptake regulator (Fur) proteins isolated from heterotrophic bacteria. In vivo, FurA presents different redox states involving intramolecular disulfide bonds when is partially oxidized. Redox potential values for CXXC motifs, −235 and −238 mV, are consistent with those reported for other proteins displaying disulfide reductase activity. Pull-down and two-hybrid assays unveil potential FurA interacting partners, namely phosphoribulokinase Alr4123, the hypothetical amidase-containing domain All1140 and the DNA-binding protein HU. Innovation: A novel biochemical activity of cyanobacterial FurA based on its cysteine arrangements and the identification of novel interacting partners are reported. Conclusion: The present study discloses a putative connection of FurA with the cyanobacterial redox-signaling pathway. Antioxid. Redox Signal. 20, 1396–1406. PMID:24093463

  8. Antimicrobial activity of Satureja montana L. essential oil against Clostridium perfringens type A inoculated in mortadella-type sausages formulated with different levels of sodium nitrite.

    PubMed

    de Oliveira, Thales Leandro Coutinho; de Araújo Soares, Rodrigo; Ramos, Eduardo Mendes; das Graças Cardoso, Maria; Alves, Eduardo; Piccoli, Roberta Hilsdorf

    2011-01-01

    This research evaluated the antimicrobial effect of the winter savory (Satureja montana L.) essential oil (EO) against Clostridium perfringens type A (ATCC 3624) inoculated in mortadella-type sausages formulated with different levels of sodium nitrite (NaNO₂: 0 ppm, 100 ppm and 200 ppm) in addition to EO at concentrations of 0.0%, 0.78%, 1.56% and 3.125% stored at 25°C for 30 days. The EO extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometry (CG-MS) was tested in vitro using an agar well diffusion method for determination of minimum inhibitory concentration (MIC) on C. perfringens. According to compositional analysis of the winter savory EO, 26 chemical compounds were identified, and the major constituents were thymol (28.99%), p-cymene (12.00%), linalool (11.00%) and carvacrol (10.71%). The results obtained showed that EO applied at a concentration of 1.56%, which was defined as the MIC, exhibited antimicrobial activity against C. perfringens in the in vitro assays, and the transmission electron microscopy (TEM) revealed structural damage and cell lysis of C. perfringens caused by EO treatment. A synergistic effect between NaNO₂ and EO was observed. In mortadella-type sausages formulated with 100 ppm of NaNO₂ and EO at all concentrations tested, the population of target microorganisms was reduced (p≤0.05) compared to control samples during all storage period. This data suggests the potential combined use of savory EO and minimal amounts of the synthetic additive, NaNO₂ to control C. perfringens in mortadella, which goes according to current market trends, where consumers are requesting natural products. PMID:21131083

  9. Cell survival of glioblastoma grown in medium containing hydrogen peroxide and/or nitrite, or in plasma-activated medium.

    PubMed

    Kurake, Naoyuki; Tanaka, Hiromasa; Ishikawa, Kenji; Kondo, Takashi; Sekine, Makoto; Nakamura, Kae; Kajiyama, Hiroaki; Kikkawa, Fumitaka; Mizuno, Masaaki; Hori, Masaru

    2016-09-01

    Non-equilibrium atmospheric pressure plasmas generate a high electron density (on the order of 10(16) electrons per cm(-3)) using Ar gas. Culture medium in air at room temperature was plasma-irradiated for several hundred seconds. Tens of micromolar hydrogen peroxide (H2O2) and millimolar levels of nitrous ion (NO2(-)) were detected in the plasma-irradiated culture medium (plasma activated medium; PAM) and selectively induced the apoptotic death of glioblastoma tumor cells, but did not kill normal mammary epithelial cells. A similar antitumor effect was induced by spiking the medium with comparable concentrations of H2O2 and NO2(-). The PAM remained still a somewhat difference that it should also be assessed for understanding other latent mechanisms. PMID:26820218

  10. Effect of warfarin on plasma and liver vitamin K levels and vitamin K epoxide reductase activity in relation to plasma clotting factor levels in rats.

    PubMed

    Yamanaka, Y; Yamano, M; Yasunaga, K; Shike, T; Uchida, K

    1990-01-15

    Changes in plasma and liver vitamin K1 and vitamin K1 epoxide levels, liver microsomal vitamin K epoxide reductase activity, and plasma clotting factor II and VII levels were determined in rats after a single injection of warfarin (2.5 mg/kg, s.c.). The plasma and liver vitamin K1 levels gradually decreased after warfarin injection, attaining the lowest values at 2-3 hrs and remaining low for 48 hrs. They then returned to the control levels at 72 hrs. The changes in vitamin K1 epoxide levels were opposite, with an increase being seen soon after the warfarin injection, the highest values at 3 hrs and a gradual decrease to the initial levels occurring subsequently. The combined levels of vitamin K1 plus vitamin K1 epoxide, however, remained almost constant in both plasma and liver after the warfarin injection. The liver vitamin K epoxide reductase activity decreased to its lowest level soon after the injection and then gradually increased after 12 hrs, but the activity at 72 hrs was only about 30% of the initial activity. The plasma clotting factor levels gradually decreased after the injection, bottomed at 24 hrs and then began to increase, recovering almost to the initial levels at 72 hrs. A positive correlation was found between plasma and liver levels for both vitamin K1 and vitamin K1 epoxide, and the slope of the vitamin K1 epoxide curve was steeper than that for vitamin K1 in the warfarin-treated rats. A similar positive correlation was found for both vitamin K1 and vitamin K1 epoxide after vitamin K1 injection in normal untreated rats, but the slope of the vitamin K1 epoxide curve was much shallower. These results suggest that warfarin inhibits vitamin K epoxide reductase and decreases blood clotting factor synthesis, thus increasing plasma and liver vitamin K1 epoxide levels. A vitamin K epoxide reductase activity one third of that in normal rats is sufficient to maintain normal reduction of vitamin K1 epoxide and synthesis of blood clotting factors. PMID

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

    PubMed Central

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

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions. PMID:8597660

  12. Retention and leaching of nitrite by municipal solid waste incinerator bottom ash under the landfill circumstance.

    PubMed

    Yao, Jun; Kong, Qingna; Zhu, Huayue; Long, Yuyang; Shen, Dongsheng

    2015-01-01

    The retention and leaching of nitrite by municipal solid waste incinerator (MSWI) bottom ash could affect its migration in the landfill. In this study, the effect of the dosage of MSWI bottom ash as well as the variation of the landfill environmental parameters including pH, anions and organic matter on the nitrite retention and leaching behavior was investigated by batch experiments. The highest removal percentage (73.0%) of nitrite was observed when the dosage of MSWI bottom ash was 10 g L(-1) in 2 mg L(-1) nitrite solution. Further increase of the dosage would retard the retention, as the nitrite leaching from MSWI bottom ash was enhanced. The optimum retention of nitrite was observed when the pH was 5.0, while the leaching of nitrite showed a consistent reduction with the increase of pH. Besides, the presence of Cl(-), SO4(2)(-) and acetic acid could enhance the leaching of nitrite and mitigate the retention process. However, the retention of nitrite was enhanced by PO4(3)(-), which was probably due to the formation of the apatite, an active material for the adsorption of the nitrite. These results suggested that MSWI bottom ash could affect the migration of nitrite in the landfill, which was related to the variation of the landfill circumstance. PMID:25033242

  13. Oxygen as Intermediate in Anoxic Environments: Nitrite-Dependent Methane Oxidation and Beyond

    NASA Astrophysics Data System (ADS)

    Ettwig, K. F.

    2014-12-01

    In recent years the known diversity of hydrocarbon activation mechanisms under anaerobic conditions has been extended by intra-aerobic denitrification, a process in which oxygen is derived from NO and used for substrate activation. For two phylogenetically unrelated bacterial species, the freshwater NC10 phylum bacterium Methylomirabilis oxyfera [1] and the marine γ-proteobacterial strain HdN1 [2] it has been shown that, under anoxic conditions with nitrate and/or nitrite, mono-oxygenases are used for methane and hexadecane oxidation, respectively. No degradation was observed with nitrous oxide (N2O) only. In the anaerobic methanotroph M. oxyfera, which lacks apparent nitrous oxide reductase in its genome, substrate activation in the presence of nitrite was directly associated with both O2 and N2 formation. These findings strongly argue for the role of nitric oxide (NO), or an oxygen species derived from it, in the activation reaction of methane. Although intracellular oxygen generation has been experimentally documented and elegantly explains the utilization of 'aerobic' pathways under anoxic conditions, research about the underlying molecular mechanism has just started. The proposed candidate enzymes for oxygen (or possibly another another reactive intermediate) production from NO, an NO dismutase (NOD) [3], related to quinol-dependent NO reductases (qNORs), is present and highly expressed in both M. oxyfera and strain HdN1. Besides that, several recently sequenced species from the Cytophaga-Flavobacterium-Bacteroides group harbor Nod/Nor genes, but experimential evidence is needed to show if these have NOD activity, are unusual but functional qNORs, or represent transition states between the two. Additionally, for several anaerobic hydrocarbon-degrading organisms the biochemical mechanism of substrate activation has not been elucidated yet: whereas signature genes of anaerobic degradation are missing, monooxygenase genes are present. Also these microorganisms

  14. 21 CFR 573.700 - Sodium nitrite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Sodium nitrite. 573.700 Section 573.700 Food and... Listing § 573.700 Sodium nitrite. Sodium nitrite may be safely used in canned pet food containing meat and... byproducts so that the level of sodium nitrite does not exceed 20 parts per million. (b) To assure safe...

  15. 21 CFR 573.700 - Sodium nitrite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Sodium nitrite. 573.700 Section 573.700 Food and... Listing § 573.700 Sodium nitrite. Sodium nitrite may be safely used in canned pet food containing meat and... byproducts so that the level of sodium nitrite does not exceed 20 parts per million. (b) To assure safe...

  16. 21 CFR 573.700 - Sodium nitrite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Sodium nitrite. 573.700 Section 573.700 Food and... Listing § 573.700 Sodium nitrite. Sodium nitrite may be safely used in canned pet food containing meat and... byproducts so that the level of sodium nitrite does not exceed 20 parts per million. (b) To assure safe...