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Sample records for nitrite reductase activities

  1. Nitrate decreases xanthine oxidoreductase-mediated nitrite reductase activity and attenuates vascular and blood pressure responses to nitrite

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    Célio Damacena-Angelis

    2017-08-01

    Full Text Available Nitrite and nitrate restore deficient endogenous nitric oxide (NO production as they are converted back to NO, and therefore complement the classic enzymatic NO synthesis. Circulating nitrate and nitrite must cross membrane barriers to produce their effects and increased nitrate concentrations may attenuate the nitrite influx into cells, decreasing NO generation from nitrite. Moreover, xanthine oxidoreductase (XOR mediates NO formation from nitrite and nitrate. However, no study has examined whether nitrate attenuates XOR-mediated NO generation from nitrite. We hypothesized that nitrate attenuates the vascular and blood pressure responses to nitrite either by interfering with nitrite influx into vascular tissue, or by competing with nitrite for XOR, thus inhibiting XOR-mediated NO generation. We used two independent vascular function assays in rats (aortic ring preparations and isolated mesenteric arterial bed perfusion to examine the effects of sodium nitrate on the concentration-dependent responses to sodium nitrite. Both assays showed that nitrate attenuated the vascular responses to nitrite. Conversely, the aortic responses to the NO donor DETANONOate were not affected by sodium nitrate. Further confirming these results, we found that nitrate attenuated the acute blood pressure lowering effects of increasing doses of nitrite infused intravenously in freely moving rats. The possibility that nitrate could compete with nitrite and decrease nitrite influx into cells was tested by measuring the accumulation of nitrogen-15-labeled nitrite (15N-nitrite by aortic rings using ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS. Nitrate exerted no effect on aortic accumulation of 15N-nitrite. Next, we used chemiluminescence-based NO detection to examine whether nitrate attenuates XOR-mediated nitrite reductase activity. Nitrate significantly shifted the Michaelis Menten saturation curve to the right, with a 3-fold increase in

  2. Nitrite reductase activity and inhibition of H₂S biogenesis by human cystathionine ß-synthase.

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    Carmen Gherasim

    Full Text Available Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe(II-NO CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR and nitrite. Formation of Fe(II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.

  3. Variant Cell Lines of Haplopappus gracilis with Disturbed Activities of Nitrate Reductase and Nitrite Reductase.

    Science.gov (United States)

    Gilissen, L J; Barneix, A J; van Staveren, M; Breteler, H

    1985-07-01

    Selected variant cell lines of Haplopappus gracilis (Nutt) Gray that showed disturbed growth after transfer from an alanine medium to NO(3) (-) medium were characterized. The in vivo NO(3) (-) reductase activity (NRA) was lower in these lines than in the wild type. In vitro NRA assays suggest that decreased in vivo NRA was not caused by a lower amount of active enzyme. Cells of the variant lines revealed up to 75% lower extractable activity of NO(2) (-) reductase as compared with the wild type. This coincided with higher accumulation of NO(2) (-) by the variant than by the wild type cells after transfer from alanine medium to NO(3) (-) medium. NO(2) (-) accumulation was transient or continuous, depending on cell line, metabolic state of the cells, and light conditions.

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

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    Józef Buczek

    2014-01-01

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

  5. Extreme nitrite tolerance in the clown knifefish Chitala ornata is linked to up-regulation of methaemoglobin reductase activity.

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    Gam, Le Thi Hong; Jensen, Frank Bo; Damsgaard, Christian; Huong, Do Thi Thanh; Phuong, Nguyen Thanh; Bayley, Mark

    2017-06-01

    The clown knifefish is a facultative air breather, which is widely farmed in freshwater ponds in Vietnam. Here we report a very high nitrite tolerance (96h LC 50 of 7.82mM) in this species and examine the effects of 1mM (LC 5 ) and 2.5mM (LC 10 ) ambient nitrite on haemoglobin (Hb) derivatives, electrolyte levels, acid-base status, and total body water content during 7days of exposure. Furthermore, we tested the hypothesis that erythrocyte methaemoglobin (metHb) reductase activity is upregulated by nitrite exposure. Plasma nitrite levels increased for 2-3days but stayed below environmental levels and fell towards control values during the last half of the exposure period. Plasma nitrate, in contrast, rose continuously, reflecting detoxification of nitrite to nitrate. MetHb generated from the reaction between nitrite and erythrocyte Hb reached 38% at day 2, but then decreased to 17% by the end of experiment. The first order rate constant for metHb reduction by erythrocyte metHb reductase increased from 0.01 in controls to 0.046min -1 after 6days of nitrite exposure, showing up-regulation of this enzyme. While such upregulation has been suggested in nitrite-exposed fish species, this study provides the first experimental evidence. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. NITRITE REDUCTASE ACTIVITY OF NON-SYMBIOTIC HEMOGLOBINS FROM ARABIDOPSIS THALIANA†

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    Tiso, Mauro; Tejero, Jesús; Kenney, Claire; Frizzell, Sheila; Gladwin, Mark T.

    2013-01-01

    Plant non-symbiotic hemoglobins possess hexa-coordinate heme geometry similar to the heme protein neuroglobin. We recently discovered that deoxygenated neuroglobin converts nitrite to nitric oxide (NO), an important signaling molecule involved in many processes in plants. We sought to determine whether Arabidopsis thaliana non-symbiotic hemoglobins class 1 and 2 (AHb1 and AHb2) might function as nitrite reductases. We found that the reaction of nitrite with deoxygenated AHb1 and AHb2 generates NO gas and iron-nitrosyl-hemoglobin species. The bimolecular rate constants for nitrite reduction to NO are 19.8 ± 3.2 and 4.9 ± 0.2 M−1s−1, at pH = 7.4 and 25°C, respectively. We determined the pH dependence of these bimolecular rate constants and found a linear correlation with the concentration of protons, indicating the requirement for one proton in the reaction. Release of free NO gas during reaction in anoxic and hypoxic (2% oxygen) conditions was confirmed by chemiluminescence detection. These results demonstrate that deoxygenated AHb1 and AHb2 reduce nitrite to form NO via a mechanism analogous to that observed for hemoglobin, myoglobin and neuroglobin. Our findings suggest that during severe hypoxia and in the anaerobic plant roots, especially in water submerged species, non-symbiotic hemoglobins provide a viable pathway for NO generation via nitrite reduction. PMID:22620259

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

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    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

  8. Nitrite-reductase and peroxynitrite isomerization activities of Methanosarcina acetivorans protoglobin.

    Directory of Open Access Journals (Sweden)

    Paolo Ascenzi

    Full Text Available 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 k(app1= 9.6 ± 0.2 M(-1 s(-1 and k(app2 = 1.2 ± 0.1 M(-1 s(-1 (at pH 7.4 and 20 °C. The k(app1 and k(app2 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 h(app = 3.8 × 10(4 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.

  9. Extreme nitrite tolerance in the clown knifefish Chitala ornata is linked to up-regulation of methaemoglobin reductase activity

    DEFF Research Database (Denmark)

    Gam, Le Thi Hong; Jensen, Frank Bo; Damsgaard, Christian

    2017-01-01

    and fell towards control values during the last half of the exposure period. Plasma nitrate, in contrast, rose continuously, reflecting detoxification of nitrite to nitrate. MetHb generated from the reaction between nitrite and erythrocyte Hb reached 38% at day 2, but then decreased to 17% by the end......The clown knifefish is a facultative air breather, which is widely farmed in freshwater ponds in Vietnam. Here we report a very high nitrite tolerance (96h LC50 of 7.82mM) in this species and examine the effects of 1mM (LC5) and 2.5mM (LC10) ambient nitrite on haemoglobin (Hb) derivatives......, electrolyte levels, acid-base status, and total body water content during 7days of exposure. Furthermore, we tested the hypothesis that erythrocyte methaemoglobin (metHb) reductase activity is upregulated by nitrite exposure. Plasma nitrite levels increased for 2-3days but stayed below environmental levels...

  10. Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities

    DEFF Research Database (Denmark)

    Dalsgaard, Thomas; Simonsen, Ulf; Fago, Angela

    2007-01-01

    The reduction of circulating nitrite to nitric oxide (NO) has emerged as an important physiological reaction aimed to increase vasodilation during tissue hypoxia. Although hemoglobin, xanthine oxidase, endothelial NO synthase, and the bc(1) complex of the mitochondria are known to reduce nitrite...... effect of nitrite during hypoxia was abolished on inhibition of soluble guanylate cyclase and was unaffected by removal of the endothelium or by inhibition of xanthine oxidase and of the mitochondrial bc(1) complex. In the presence of hemoglobin and inositol hexaphosphate (which increases the fraction...

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

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

    2015-10-08

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

  12. Role of a conserved glutamine residue in tuning the catalytic activity of Escherichia coli cytochrome c nitrite reductase.

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    Clarke, Thomas A; Kemp, Gemma L; Van Wonderen, Jessica H; Doyle, Rose-Marie A S; Cole, Jeffrey A; Tovell, Nick; Cheesman, Myles R; Butt, Julea N; Richardson, David J; Hemmings, Andrew M

    2008-03-25

    The pentaheme cytochrome c nitrite reductase (NrfA) of Escherichia coli is responsible for nitrite reduction during anaerobic respiration when nitrate is scarce. The NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a glutamine (Q263) positioned 8 A from the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion. Mutation of this glutamine to the more usual calcium ligand, glutamate, results in an increase in the K m for nitrite by around 10-fold, while V max is unaltered. Protein film voltammetry showed that lower potentials were required to detect activity from NrfA Q263E when compared with native enzyme, consistent with the introduction of a negative charge into the vicinity of the active site heme. EPR and MCD spectroscopic studies revealed the high spin state of the active site to be preserved, indicating that a water/hydroxide molecule is still coordinated to the heme in the resting state of the enzyme. Comparison of the X-ray crystal structures of the as-prepared, oxidized native and mutant enzymes showed an increased bond distance between the active site heme Fe(III) iron and the distal ligand in the latter as well as changes to the structure and mobility of the active site water molecule network. These results suggest that an important function of the unusual Q263-calcium ion pair is to increase substrate affinity through its role in supporting a network of hydrogen bonded water molecules stabilizing the active site heme distal ligand.

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

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    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.

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

    African Journals Online (AJOL)

    STORAGESEVER

    2010-03-01

    Mar 1, 2010 ... BL21 (DE3) strain with the recombinant expression vector pET-28A-GhNiR. NiR activity assay showed that the crude GhNiR protein had obvious activity to NaNO2 substrate. Key words: Cotton, nitrite reductase, prokaryotic expression, semi-quantitative RT-PCR, GenBank Accession. No: GQ389691.

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

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    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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    DEFF Research Database (Denmark)

    Gøtterup, Jacob; Olsen, Karsten; Knøchel, Susanne

    2008-01-01

    nitrate depended on the specific Staphylococcus strain. Strains with high nitrate-reductase activity showed a significantly faster rate of pigment formation, but other factors were of influence as well. Product stability for the sliced, packaged sausage was evaluated as surface colour and oxidation...

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

    DEFF Research Database (Denmark)

    Farver, Ole; Brunori, Maurizio; Cutruzzolà, Francesca

    2009-01-01

    The cd(1) nitrite reductases, which catalyze the reduction of nitrite to nitric oxide, are homodimers of 60 kDa subunits, each containing one heme-c and one heme-d(1). Heme-c is the electron entry site, whereas heme-d(1) constitutes the catalytic center. The 3D structure of Pseudomonas aeruginosa...... nitrite reductase has been determined in both fully oxidized and reduced states. Intramolecular electron transfer (ET), between c and d(1) hemes is an essential step in the catalytic cycle. In earlier studies of the Pseudomonas stutzeri enzyme, we observed that a marked negative cooperativity...... is controlling this internal ET step. In this study we have investigated the internal ET in the wild-type and His369Ala mutant of P. aeruginosa nitrite reductases and have observed similar cooperativity to that of the Pseudomonas stutzeri enzyme. Heme-c was initially reduced, in an essentially diffusion...

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

    DEFF Research Database (Denmark)

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

    1998-01-01

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

  19. Recent structural insights into the function of copper nitrite reductases.

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    Horrell, Sam; Kekilli, Demet; Strange, Richard W; Hough, Michael A

    2017-11-15

    Copper nitrite reductases (CuNiR) carry out the first committed step of the denitrification pathway of the global nitrogen cycle, the reduction of nitrite (NO 2 - ) to nitric oxide (NO). As such, they are of major agronomic and environmental importance. CuNiRs occur primarily in denitrifying soil bacteria which carry out the overall reduction of nitrate to dinitrogen. In this article, we review the insights gained into copper nitrite reductase (CuNiR) function from three dimensional structures. We particularly focus on developments over the last decade, including insights from serial femtosecond crystallography using X-ray free electron lasers (XFELs) and from the recently discovered 3-domain CuNiRs.

  20. Interspecific variation and plasticity in hemoglobin nitrite reductase activity and its correlation with oxygen affinity in vertebrates

    DEFF Research Database (Denmark)

    Jensen, Frank B; Højer Kolind, Rasmus Aleksander; Skaarup Jensen, Natashia

    2017-01-01

    and O2 affinity. We also tested plastic changes in Hb properties via addition of T-structure-stabilizing organic phosphates (ATP and GTP). The decay in deoxyHb during its reaction with nitrite was exponential-like in ectotherms (Atlantic hagfish, carp, crucian carp, brown trout, rainbow trout, cane toad......-dependent manner. The initial second order rate constant of the deoxyHb-mediated nitrite reduction showed a strong curvilinear correlation with oxygen affinity among all ectothermic vertebrates, and the relationship also applied to plastic variations of Hb properties via organic phosphates. The relationship...

  1. Why Orange Guaymas Basin Beggiatoa spp. Are Orange: Single-Filament-Genome-Enabled Identification of an Abundant Octaheme Cytochrome with Hydroxylamine Oxidase, Hydrazine Oxidase, and Nitrite Reductase Activities

    Science.gov (United States)

    Biddle, Jennifer F.; Siebert, Jason R.; Staunton, Eric; Hegg, Eric L.; Matthysse, Ann G.; Teske, Andreas

    2013-01-01

    Orange, white, and yellow vacuolated Beggiatoaceae filaments are visually dominant members of microbial mats found near sea floor hydrothermal vents and cold seeps, with orange filaments typically concentrated toward the mat centers. No marine vacuolate Beggiatoaceae are yet in pure culture, but evidence to date suggests they are nitrate-reducing, sulfide-oxidizing bacteria. The nearly complete genome sequence of a single orange Beggiatoa (“Candidatus Maribeggiatoa”) filament from a microbial mat sample collected in 2008 at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) was recently obtained. From this sequence, the gene encoding an abundant soluble orange-pigmented protein in Guaymas Basin mat samples (collected in 2009) was identified by microcapillary reverse-phase high-performance liquid chromatography (HPLC) nano-electrospray tandem mass spectrometry (μLC–MS-MS) of a pigmented band excised from a denaturing polyacrylamide gel. The predicted protein sequence is related to a large group of octaheme cytochromes whose few characterized representatives are hydroxylamine or hydrazine oxidases. The protein was partially purified and shown by in vitro assays to have hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities. From what is known of Beggiatoaceae physiology, nitrite reduction is the most likely in vivo role of the octaheme protein, but future experiments are required to confirm this tentative conclusion. Thus, while present-day genomic and proteomic techniques have allowed precise identification of an abundant mat protein, and its potential activities could be assayed, proof of its physiological role remains elusive in the absence of a pure culture that can be genetically manipulated. PMID:23220958

  2. Why orange Guaymas Basin Beggiatoa spp. are orange: single-filament-genome-enabled identification of an abundant octaheme cytochrome with hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities.

    Science.gov (United States)

    MacGregor, Barbara J; Biddle, Jennifer F; Siebert, Jason R; Staunton, Eric; Hegg, Eric L; Matthysse, Ann G; Teske, Andreas

    2013-02-01

    Orange, white, and yellow vacuolated Beggiatoaceae filaments are visually dominant members of microbial mats found near sea floor hydrothermal vents and cold seeps, with orange filaments typically concentrated toward the mat centers. No marine vacuolate Beggiatoaceae are yet in pure culture, but evidence to date suggests they are nitrate-reducing, sulfide-oxidizing bacteria. The nearly complete genome sequence of a single orange Beggiatoa ("Candidatus Maribeggiatoa") filament from a microbial mat sample collected in 2008 at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) was recently obtained. From this sequence, the gene encoding an abundant soluble orange-pigmented protein in Guaymas Basin mat samples (collected in 2009) was identified by microcapillary reverse-phase high-performance liquid chromatography (HPLC) nano-electrospray tandem mass spectrometry (μLC-MS-MS) of a pigmented band excised from a denaturing polyacrylamide gel. The predicted protein sequence is related to a large group of octaheme cytochromes whose few characterized representatives are hydroxylamine or hydrazine oxidases. The protein was partially purified and shown by in vitro assays to have hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities. From what is known of Beggiatoaceae physiology, nitrite reduction is the most likely in vivo role of the octaheme protein, but future experiments are required to confirm this tentative conclusion. Thus, while present-day genomic and proteomic techniques have allowed precise identification of an abundant mat protein, and its potential activities could be assayed, proof of its physiological role remains elusive in the absence of a pure culture that can be genetically manipulated.

  3. Cloning, purification and characterization of novel Cu-containing nitrite reductase from the Bacillus firmus GY-49.

    Science.gov (United States)

    Gao, Haofeng; Li, Caiqing; Ramesh, Bandikari; Hu, Nan

    2017-12-18

    Nitrite is generated from the nitrogen cycle and its accumulation is harmful to environment and it can be reduced to nitric oxid by nitrite reductase. A novel gene from Bacillus firmus GY-49 is identified as a nirK gene encoding Cu-containing nitrite reductase by genome sequence. The full-length protein included a putative signal peptide of 26 amino acids and shown 72.73% similarity with other Cu-containing nitrite reductase whose function was verified. The 993-bp fragment encoding the mature peptide of NirK was cloned into pET-28a (+) vector and overexpressed as an active protein of 36.41 kDa in the E.coli system. The purified enzyme was green in the oxidized state and displayed double gentle peaks at 456 and 608 nm. The specific activity of purified enzyme was 98.4 U/mg toward sodium nitrite around pH 6.5 and 35 °C. The K m and K cat of NirK on sodium nitrite were 0.27 mM and 0.36 × 10 3  s -1 , respectively. Finally, homology model analysis of NirK indicated that the enzyme was a homotrimer structure and well conserved in Cu-binding sites for enzymatic functions. This is a first report for nitrite reductase from Bacillus firmus, which augment the acquaintance of nitrite reductase.

  4. Structural study of the X-ray-induced enzymatic reaction of octahaem cytochrome C nitrite reductase.

    Science.gov (United States)

    Trofimov, A A; Polyakov, K M; Lazarenko, V A; Popov, A N; Tikhonova, T V; Tikhonov, A V; Popov, V O

    2015-05-01

    Octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens catalyzes the reduction of nitrite to ammonium and of sulfite to sulfide. The reducing properties of X-ray radiation and the high quality of the enzyme crystals allow study of the catalytic reaction of cytochrome c nitrite reductase directly in a crystal of the enzyme, with the reaction being induced by X-rays. Series of diffraction data sets with increasing absorbed dose were collected from crystals of the free form of the enzyme and its complexes with nitrite and sulfite. The corresponding structures revealed gradual changes associated with the reduction of the catalytic haems by X-rays. In the case of the nitrite complex the conversion of the nitrite ions bound in the active sites to NO species was observed, which is the beginning of the catalytic reaction. For the free form, an increase in the distance between the oxygen ligand bound to the catalytic haem and the iron ion of the haem took place. In the case of the sulfite complex no enzymatic reaction was detected, but there were changes in the arrangement of the active-site water molecules that were presumably associated with a change in the protonation state of the sulfite ions.

  5. Nitrite reductase activity of rat and human xanthine oxidase, xanthine dehydrogenase, and aldehyde oxidase: evaluation of their contribution to NO formation in vivo.

    Science.gov (United States)

    Maia, Luisa B; Pereira, Vânia; Mira, Lurdes; Moura, José J G

    2015-01-27

    Nitrite is presently considered a NO "storage form" that can be made available, through its one-electron reduction, to maintain NO formation under hypoxia/anoxia. The molybdoenzymes xanthine oxidase/dehydrogenase (XO/XD) and aldehyde oxidase (AO) are two of the most promising mammalian nitrite reductases, and in this work, we characterized NO formation by rat and human XO/XD and AO. This is the first characterization of human enzymes, and our results support the employment of rat liver enzymes as suitable models of the human counterparts. A comprehensive kinetic characterization of the effect of pH on XO and AO-catalyzed nitrite reduction showed that the enzyme's specificity constant for nitrite increase 8-fold, while the Km(NO2(-)) decrease 6-fold, when the pH decreases from 7.4 to 6.3. These results demonstrate that the ability of XO/AO to trigger NO formation would be greatly enhanced under the acidic conditions characteristic of ischemia. The dioxygen inhibition was quantified, and the Ki(O2) values found (24.3-48.8 μM) suggest that in vivo NO formation would be fine-tuned by dioxygen availability. The potential in vivo relative physiological relevance of XO/XD/AO-dependent pathways of NO formation was evaluated using HepG2 and HMEC cell lines subjected to hypoxia. NO formation by the cells was found to be pH-, nitrite-, and dioxygen-dependent, and the relative contribution of XO/XD plus AO was found to be as high as 50%. Collectively, our results supported the possibility that XO/XD and AO can contribute to NO generation under hypoxia inside a living human cell. Furthermore, the molecular mechanism of XO/AO-catalyzed nitrite reduction was revised.

  6. Intramolecular electron transfer in cytochrome cd(1) nitrite reductase from Pseudomonas stutzeri; kinetics and thermodynamics

    DEFF Research Database (Denmark)

    Farver, Ole; Kroneck, Peter M H; Zumft, Walter G

    2002-01-01

    Cytochrome cd(1) nitrite reductase from Pseudomonas stutzeri catalyzes the one electron reduction of nitrite to nitric oxide. It is a homodimer, each monomer containing one heme-c and one heme-d(1), the former being the electron uptake site while the latter is the nitrite reduction site. Hence, i...

  7. Differential nitrate accumulation, nitrate reduction, nitrate reductase ...

    African Journals Online (AJOL)

    use

    2011-12-07

    Dec 7, 2011 ... reductase activity and nitrite accumulation depend on the exogenous nitrate. Nitrite itself is reduced to ammonium by palstidic nitrite reductase. Nitrite reductase is activated by both nitrate and nitrite ions by positive feed forward, whereas nitrate metabolites, most likely ammonium and glutamine; down.

  8. Characterization of a heme c nitrite reductase from a non-ammonifying microorganism, Desulfovibrio vulgaris Hildenborough.

    Science.gov (United States)

    Pereira, I A; LeGall, J; Xavier, A V; Teixeira, M

    2000-08-31

    A cytochrome c nitrite reductase (NiR) was purified for the first time from a microorganism not capable of growing on nitrate, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. It was isolated from the membranes as a large heterooligomeric complex of 760 kDa, containing two cytochrome c subunits of 56 and 18 kDa. This complex has nitrite and sulfite reductase activities of 685 micromol NH(4)(+)/min/mg and 1.0 micromol H(2)/min/mg. The enzyme was studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies. The overall redox behavior was determined through a visible redox titration. The data were analyzed with a set of four redox transitions, with an E(0)' of +160 mV (12% of total absorption), -5 mV (38% of total absorption), -110 mV (38% of total absorption) and -210 mV (12% of total absorption) at pH 7.6. The EPR spectra of oxidized and partially reduced NiR show a complex pattern, indicative of multiple heme-heme magnetic interactions. It was found that D. vulgaris Hildenborough is not capable of using nitrite as a terminal electron acceptor. These results indicate that in this organism the NiR is not involved in the dissimilative reduction of nitrite, as is the case with the other similar enzymes isolated so far. The possible role of this enzyme in the detoxification of nitrite and/or in the reduction of sulfite is discussed.

  9. Methemoglobin reductase activity in intact fish red blood cells

    DEFF Research Database (Denmark)

    Jensen, Frank B; Nielsen, Karsten

    2018-01-01

    Red blood cells (RBCs) possess methemoglobin reductase activity that counters the ongoing oxidation of hemoglobin (Hb) to methemoglobin (metHb), which in circulating blood is caused by Hb autoxidation or reactions with nitrite. We describe an assay for determining metHb reductase activity in intact...

  10. Electrochemical Single‐Molecule AFM of the Redox Metalloenzyme Copper Nitrite Reductase in Action

    DEFF Research Database (Denmark)

    Hao, Xian; Zhang, Jingdong; Christensen, Hans Erik Mølager

    2012-01-01

    We studied the electrochemical behavior of the redox metalloenzyme copper nitrite reductase (CNiR, Achromobacter xylosoxidans) immobilized on a Au(111)‐electrode surface modified by a self‐assembled cysteamine molecular monolayer (SAM) using a combination of cyclic voltammetry and electrochemically......‐controlled atomic force microscopy (in situ AFM). The enzyme showed no voltammetric signals in the absence of nitrite substrate, whereas a strong reductive electrocatalytic signal appeared in the presence of nitrite. Such a pattern is common in protein film and monolayer voltammetry and points to conformational...... in the presence of nitrite. No change in size was observed in the absence of nitrite over the same potential range. The enzyme size variation is suggested to offer clues to the broadly observed substrate triggering in metalloenzyme monolayer voltammetry....

  11. Diversity of nitrite reductase genes (nirS) in the denitrifying water column of the coastal Arabian Sea

    Digital Repository Service at National Institute of Oceanography (India)

    Jayakumar, D.A.; Francis, C.A.; Naqvi, S.W.A.; Ward, B.B.

    are investigated. Nitrite reduction to nitric oxide is the key step in the denitrification pathway, and is catalyzed by the enzyme nitrite reductase, which is encoded by the genes nirS and nirK. The diversity and distribution of nirS genes in relation to nitrite...

  12. Voltammetry and Electrocatalysis of Achrornobacter Xylosoxidans Copper Nitrite Reductase on Functionalized Au(111)-Electrode Surfaces

    DEFF Research Database (Denmark)

    Welinder, Anna C.; Zhang, Jingdong; Hansen, Allan G.

    2007-01-01

    A long-standing issue in protein film voltammetry (PFV), particularly electrocatalytic voltammetry of redox enzyme monolayers, is the variability of protein adsorption modes, reflected in distributions of catalytic activity of the adsorbed protein/enzyme molecules. Use of well-defined, atomically...... planar electrode surfaces is a step towards the resolution of this central issue. We report here the voltammetry of copper nitrite reductase (CNiR, Achromobacter xylosoxidons) on Au(111)-electrode surfaces modified by monolayers of a broad variety of thiol-based linker molecules. These represent......NiR thus shows highly efficient, close to ideal reversible electrocatalytic voltammetry on cysteamine-covered Au(111)-electrode surfaces, most likely due to two cysteamine orientations previously disclosed by in situ scanning tunnelling microscopy. Such a dual orientation exposes both a hydrophobic...

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

    Directory of Open Access Journals (Sweden)

    Sam Horrell

    2016-07-01

    Full Text Available 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.

  14. A Nitrite Biosensor Based on Co-immobilization of Nitrite Reductase and Viologen-modified Chitosan on a Glassy Carbon Electrode

    Directory of Open Access Journals (Sweden)

    De Quan

    2010-06-01

    Full Text Available 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 (t90% were 14.9 nA/mM, 0.04−11 mM (r2 = 0.999 and 15 s, respectively. The corresponding Lineweaver-Burk plot showed that the apparent Michaelis-Menten constant (KMapp was 65 mM. 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 mM of nitrite. An interference study showed that anions commonlyfound 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.

  15. Allosteric control of internal electron transfer in cytochrome cd1 nitrite reductase

    DEFF Research Database (Denmark)

    Farver, Ole; Kroneck, Peter M H; Zumft, Walter G

    2003-01-01

    Cytochrome cd1 nitrite reductase is a bifunctional multiheme enzyme catalyzing the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of dioxygen to water. Kinetics and thermodynamics of the internal electron transfer process in the Pseudomonas stutzeri enzyme have...... been studied and found to be dominated by pronounced interactions between the c and the d1 hemes. The interactions are expressed both in dramatic changes in the internal electron-transfer rates between these sites and in marked cooperativity in their electron affinity. The results constitute a prime...... example of intraprotein control of the electron-transfer rates by allosteric interactions....

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. An unprecedented dioxygen species revealed by serial femtosecond rotation crystallography in copper nitrite reductase

    Directory of Open Access Journals (Sweden)

    Thomas P. Halsted

    2018-01-01

    Full Text Available Synchrotron-based X-ray structural studies of ligand-bound enzymes are powerful tools to further our understanding of reaction mechanisms. For redox enzymes, it is necessary to study both the oxidized and reduced active sites to fully elucidate the reaction, an objective that is complicated by potential X-ray photoreduction. In the presence of the substrate, this can be exploited to construct a structural movie of the events associated with catalysis. Using the newly developed approach of serial femtosecond rotation crystallography (SF-ROX, an X-ray damage-free structure of the as-isolated copper nitrite reductase (CuNiR was visualized. The sub-10 fs X-ray pulse length from the SACLA X-ray free-electron laser allowed diffraction data to be collected to 1.6 Å resolution in a `time-frozen' state. The extremely short duration of the X-ray pulses ensures the capture of data prior to the onset of radiation-induced changes, including radiolysis. Unexpectedly, an O2 ligand was identified bound to the T2Cu in a brand-new binding mode for a diatomic ligand in CuNiRs. The observation of O2 in a time-frozen structure of the as-isolated oxidized enzyme provides long-awaited clear-cut evidence for the mode of O2 binding in CuNiRs. This provides an insight into how CuNiR from Alcaligenes xylosoxidans can function as an oxidase, reducing O2 to H2O2, or as a superoxide dismutase (SOD since it was shown to have ∼56% of the dismutase activity of the bovine SOD enzyme some two decades ago.

  18. Impact of hemoglobin nitrite to nitric oxide reductase on blood transfusion for resuscitation from hemorrhagic shock

    Directory of Open Access Journals (Sweden)

    Chad Brouse

    2015-01-01

    Full Text Available Background: Transfusion of blood remains the gold standard for fluid resuscitation from hemorrhagic shock. Hemoglobin (Hb within the red blood cell transports oxygen and modulates nitric oxide (NO through NO scavenging and nitrite reductase. Aims: This study was designed to examine the effects of incorporating a novel NO modulator, RRx-001, on systemic and microvascular hemodynamic response after blood transfusion for resuscitation from hemorrhagic shock in a hamster window chamber model. In addition, to RRx-001 the role of low dose of nitrite (1 × 10−9 moles per animal supplementation after resuscitation was studied. Materials and Methods: Severe hemorrhage was induced by arterial controlled bleeding of 50% of the blood volume (BV and the hypovolemic state was maintained for 1 h. The animals received volume resuscitation by an infusion of 25% of BV using fresh blood alone or with added nitrite, or fresh blood treated with RRx-001 (140 mg/kg or RRx-001 (140 mg/kg with added nitrite. Systemic and microvascular hemodynamics were followed at baseline and at different time points during the entire study. Tissue apoptosis and necrosis were measured 8 h after resuscitation to correlate hemodynamic changes with tissue viability. Results: Compared to resuscitation with blood alone, blood treated with RRx-001 decreased vascular resistance, increased blood flow and functional capillary density immediately after resuscitation and preserved tissue viability. Furthermore, in RRx-001 treated animals, both mean arterial pressure (MAP and met Hb were maintained within normal levels after resuscitation (MAP >90 mmHg and metHb <2%. The addition of nitrite to RRx-001 did not significantly improve the effects of RRx-001, as it increased methemoglobinemia and lower MAP. Conclusion: RRx-001 alone enhanced perfusion and reduced tissue damage as compared to blood; it may serve as an adjunct therapy to the current gold standard treatment for resuscitation from

  19. Heterologous expression and biochemical characterization of assimilatory nitrate and nitrite reductase reveals adaption and potential of Bacillus megaterium NCT-2 in secondary salinization soil.

    Science.gov (United States)

    Chu, Shaohua; Zhang, Dan; Wang, Daxin; Zhi, Yuee; Zhou, Pei

    2017-08-01

    Large accumulation of nitrate in soil has resulted in "salt stress" and soil secondary salinization. Bacillus megaterium NCT-2 which was isolated from secondary salinization soil showed high capability of nitrate reduction. The genes encoding assimilatory nitrate and nitrite reductase from NCT-2 were cloned and over-expressed in Escherichia coli. The optimum co-expression condition was obtained with E. coli BL21 (DE3) and 0.1mM IPTG for 10h when expression was carried out at 20°C and 120rpm in Luria-Bertani (LB) medium. The molecular mass of nitrate reductase was 87.3kDa and 80.5kDa for electron transfer and catalytic subunit, respectively. The large and small subunit of nitrite reductase was 88kDa and 11.7kDa, respectively. The purified recombinant enzymes showed broad activity range of temperature and pH. The maximum activities were obtained at 35°C and 30°C, pH 6.2 and 6.5, which was similar to the condition of greenhouse soils. Maximum stimulation of the enzymes occurred with addition of Fe 3+ , while Cu 2+ caused the maximum inhibition. The optimum electron donor was MV+Na 2 S 2 O 4 +EDTA and MV+Na 2 S 2 O 4 , respectively. Kinetic parameters of K m and V max were determined to be 670μM and 58U/mg for nitrate reductase, and 3100μM and 5.2U/mg for nitrite reductase. Results of quantitative real-time PCR showed that the maximum expression levels of nitrate and nitrite reductase were obtained at 50mM nitrate for 8h and 12h, respectively. These results provided information on novel assimilatory nitrate and nitrite reductase and their properties presumably revealed adaption of B. megaterium NCT-2 to secondary salinization condition. This study also shed light on the role played by the nitrate assimilatory pathway in B. megaterium NCT-2. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Effect of the methionine ligand on the reorganization energy of the type-1 copper site of nitrite Reductase

    DEFF Research Database (Denmark)

    Farver, Ole; Wijma, Hein J.; MacPherson, Iain

    2007-01-01

    Copper-containing nitrite reductase harbors a type-1 and a type-2 Cu site. The former acts as the electron acceptor site of the enzyme, and the latter is the site of catalytic action. The effect of the methionine ligand on the reorganization energy of the type-1 site was explored by studying...

  1. Catalytic monolayer voltammetry and in situ scanning tunneling microscopy of copper nitrite reductase on cysteamine-modified Au(111) electrodes

    DEFF Research Database (Denmark)

    Zhang, Jingdong; Welinder, A.C.; Hansen, Allan Glargaard

    2003-01-01

    We have studied the adsorption and electrocatalysis of the redox metalloenzyme blue copper nitrite reductase from Achromobacter xylosoxidans (AxCuNiR) on single-crystal Au(111)-electrode surfaces modified by a self-assembled monolayer of cysteamine. A combination of cyclic voltammetry and in situ...

  2. Mycobacterium tuberculosis expresses methionine sulphoxide reductases A and B that protect from killing by nitrite and hypochlorite.

    Science.gov (United States)

    Lee, Warren L; Gold, Benjamin; Darby, Crystal; Brot, Nathan; Jiang, Xiuju; de Carvalho, Luiz Pedro S; Wellner, Daniel; St John, Gregory; Jacobs, William R; Nathan, Carl

    2009-02-01

    Methionine sulphoxide reductases (Msr) reduce methionine sulphoxide to methionine and protect bacteria against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Many organisms express both MsrA, active against methionine-(S)-sulphoxide, and MsrB, active against methionine-(R)-sulphoxide. Mycobacterium tuberculosis (Mtb) expresses MsrA, which protects DeltamsrA-Escherichia coli from ROI and RNI. However, the function of MsrA in Mtb has not been defined, and it is unknown whether Mtb expresses MsrB. We identified MsrB as the protein encoded by Rv2674 in Mtb and confirmed the distinct stereospecificities of recombinant Mtb MsrA and MsrB. We generated strains of Mtb deficient in MsrA, MsrB or both and complemented the mutants. Lysates of singly deficient strains displayed half as much Msr activity as wild type against N-acetyl methionine sulphoxide. However, in contrast to other bacteria, single mutants were no more vulnerable than wild type to killing by ROI/RNI. Only Mtb lacking both MsrA and MsrB was more readily killed by nitrite or hypochlorite. Thus, MsrA and MsrB contribute to the enzymatic defences of Mtb against ROI and RNI.

  3. 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.

    Science.gov (United States)

    Frías, José E; Flores, Enrique

    2015-07-01

    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. 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 cyanobacteria

  4. Structural adaptations of octaheme nitrite reductases from haloalkaliphilic Thioalkalivibrio bacteria to alkaline pH and high salinity.

    Directory of Open Access Journals (Sweden)

    Anna Popinako

    Full Text Available Bacteria Tv. nitratireducens and Tv. paradoxus from soda lakes grow optimally in sodium carbonate/NaCl brines at pH range from 9.5 to 10 and salinity from 0.5 to 1.5 M Na+. Octaheme nitrite reductases (ONRs from haloalkaliphilic bacteria of genus Thioalkalivibrio are stable and active in a wide range of pH (up to 11 and salinity (up to 1 M NaCl. To establish adaptation mechanisms of ONRs from haloalkaliphilic bacteria a comparative analysis of amino acid sequences and structures of ONRs from haloalkaliphilic bacteria and their homologues from non-halophilic neutrophilic bacteria was performed. The following adaptation strategies were observed: (1 strategies specific for halophilic and alkaliphilic proteins (an increase in the number of aspartate and glutamate residues and a decrease in the number of lysine residues on the protein surface, (2 strategies specific for halophilic proteins (an increase in the arginine content and a decrease in the number of hydrophobic residues on the solvent-accessible protein surface, (3 strategies specific for alkaliphilic proteins (an increase in the area of intersubunit hydrophobic contacts. Unique adaptation mechanism inherent in the ONRs from bacteria of genus Thioalkalivibrio was revealed (an increase in the core in the number of tryptophan and phenylalanine residues, and an increase in the number of small side chain residues, such as alanine and valine, in the core.

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

    Directory of Open Access Journals (Sweden)

    Célia M Silveira

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  7. Copper-Containing Nitrite Reductase Employing Proton-Coupled Spin-Exchanged Electron-Transfer and Multiproton Synchronized Transfer to Reduce Nitrite.

    Science.gov (United States)

    Qin, Xin; Deng, Li; Hu, Caihong; Li, Li; Chen, Xiaohua

    2017-10-20

    The possible catalytic mechanism of the reduction of nitrite by copper-containing nitrite reductases (CuNiRs) is examined by using the M06 function according to two copper models, which include type-one copper (T1Cu) and type-two copper (T2Cu) sites. Examinations confirm that the protonation of two residues, His255 and Asp98, near the T2Cu site, can modulate the redox states of T1Cu and T2Cu, but cannot directly cause electron transfer from T1Cu to T2Cu. The electron hole remains at the T2Cu site when only one residue, His255 or Asp98, is protonated. However, the hole resides at the T1Cu site when both His255 and Asp98 are protonated. Then, the first protonation of nitrite takes place through indirect proton transfer from protonated His255 through the bridging H 2 O and Asp98 with three protons moving together, which cannot cause the cleavage of the HO-NO bond. Subsequently, the substrate is required to obtain another proton from reprotonated His255 through the bridging H 2 O. The reprotonation of nitrite induces the generation of nitric oxide (NO) and H 2 O at the T2Cu site through a special double-proton-coupled spin-exchanged electron-transfer mechanism with indirect proton transfer from His255 to the substrate, a beta-electron of T2Cu I shift to the NO cation, and the remaining alpha-electron changing spin direction at the same time. These results may provide useful information to better understand detailed proton-/electron-transfer reactions for the catalytic processes of CuNiR. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Oxygen and xenobiotic reductase activities of cytochrome P450.

    NARCIS (Netherlands)

    Goeptar, A.R.; Scheerens, H.; Vermeulen, N.P.E.

    1995-01-01

    The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (O

  9. Ferrisiderophore reductase activity in Bacillus megaterium.

    Science.gov (United States)

    Arceneaux, J E; Byers, B R

    1980-01-01

    The release of iron from ferrisiderophores (microbial ferric-chelating iron transport cofactors) by cell-free extracts of Bacillus megaterium was demonstrated. Reductive transfer of iron from ferrisiderophores to the ferrous-chelating agent ferrozine was measured spectrophotometrically. This ferrisiderophore reductase activity (reduced nicotinamide adenine dinucleotide phosphate:ferrisiderophore oxidoreductase) was associated primarily with the cell soluble rather than particulate (membrane) fraction. Ferrisiderophore reductase was inhibited by oxygen and required the addition of a reductant (reduced nicotinamide adenine dinucleotide phosphate was most effective) for maximal activity. The activity was destroyed by both heat and protease treatments and was inhibited by iodoacetamide treatment. Ferrisiderophore reductase activity for several microbial ferrisiderophores was measured; highest activity was displayed for ferrischizokinen, the ferrisiderophore produced by this organism. The Km and Vmax values of the reductase for ferrischizokinen were 2.5 x 10(-4) M and 35.7 nmol/min per mg of the ferrisiderophore reductase reaction. Preliminary fractionation of the cell soluble material by gel filtration chromatography resulted in the demonstration of ferrisiderophore reductase activity in three peaks of different molecular weight. Ferrisiderophore reductase probably mediates entrance of iron into cellular metabolism. PMID:6444944

  10. Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa▿

    Science.gov (United States)

    Van Alst, Nadine E.; Wellington, Melanie; Clark, Virginia L.; Haidaris, Constantine G.; Iglewski, Barbara H.

    2009-01-01

    The nitrate dissimilation pathway is important for anaerobic growth in Pseudomonas aeruginosa. In addition, this pathway contributes to P. aeruginosa virulence by using the nematode Caenorhabditis elegans as a model host, as well as biofilm formation and motility. We used a set of nitrate dissimilation pathway mutants to evaluate the virulence of P. aeruginosa PA14 in a model of P. aeruginosa-phagocyte interaction by using the human monocytic cell line THP-1. Both membrane nitrate reductase and nitrite reductase enzyme complexes were important for cytotoxicity during the interaction of P. aeruginosa PA14 with THP-1 cells. Furthermore, deletion mutations in genes encoding membrane nitrate reductase (ΔnarGH) and nitrite reductase (ΔnirS) produced defects in the expression of type III secretion system (T3SS) components, extracellular protease, and elastase. Interestingly, exotoxin A expression was unaffected in these mutants. Addition of exogenous nitric oxide (NO)-generating compounds to ΔnirS mutant cultures restored the production of T3SS phospholipase ExoU, whereas nitrite addition had no effect. These data suggest that NO generated via nitrite reductase NirS contributes to the regulation of expression of selected virulence factors in P. aeruginosa PA14. PMID:19651860

  11. Overexpression, purification, and biochemical and spectroscopic characterization of copper-containing nitrite reductase from Sinorhizobium meliloti 2011. Study of the interaction of the catalytic copper center with nitrite and NO.

    Science.gov (United States)

    Ferroni, Félix M; Guerrero, Sergio A; Rizzi, Alberto C; Brondino, Carlos D

    2012-09-01

    The entire nirK gene coding for a putative copper-nitrite reductase (Nir) from Sinorhizobium meliloti 2011 (Sm) was cloned and overexpressed heterologously in Escherichia coli for the first time. The spectroscopic and molecular properties of the enzyme indicate that SmNir is a green Nir with homotrimeric structure (42.5 kDa/subunit) containing two copper atoms per monomer, one of type 1 and the other of type 2. SmNir follows a Michaelis-Menten mechanism and is inhibited by cyanide. EPR spectra of the as-purified enzyme exhibit two magnetically different components associated with type 1 and type 2 copper centers in a 1:1 ratio. EPR characterization of the copper species obtained upon interaction of SmNir with nitrite, and catalytically-generated and exogenous NO reveals the formation of a Cu-NO EPR active species not detected before in closely related Nirs. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. An investigation into the unusual linkage isomerization and nitrite reduction activity of a novel tris(2-pyridyl) copper complex

    Science.gov (United States)

    Roger, Isolda; Wilson, Claire; Senn, Hans M.; Sproules, Stephen; Symes, Mark D.

    2017-08-01

    The copper-containing nitrite reductases (CuNIRs) are a class of enzymes that mediate the reduction of nitrite to nitric oxide in biological systems. Metal-ligand complexes that reproduce the salient features of the active site of CuNIRs are therefore of fundamental interest, both for elucidating the possible mode of action of the enzymes and for developing biomimetic catalysts for nitrite reduction. Herein, we describe the synthesis and characterization of a new tris(2-pyridyl) copper complex ([Cu1(NO2)2]) that binds two molecules of nitrite, and displays all three of the common binding modes for NO2-, with one nitrite bound in an asymmetric quasi-bidentate κ2-ONO manner and the other bound in a monodentate fashion with a linkage isomerism between the κ1-ONO and κ1-NO2 binding modes. We use density functional theory to help rationalize the presence of all three of these linkage isomers in one compound, before assessing the redox activity of [Cu1(NO2)2]. These latter studies show that the complex is not a competent nitrite reduction electrocatalyst in non-aqueous solvent, even in the presence of additional proton donors, a finding which may have implications for the design of biomimetic catalysts for nitrite reduction.

  13. Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling

    DEFF Research Database (Denmark)

    Treusch, Alexander H; Leininger, Sven; Kletzin, Arnulf

    2005-01-01

    at higher levels when the soil was incubated with ammonia (measured by quantitative PCR). Further variants of both genes were amplified from soil samples and were found in the environmental database from the Sargasso Sea plankton. Taken together, our findings suggest that mesophilic terrestrial and marine......Mesophilic crenarchaeota are frequently found in terrestrial and marine habitats worldwide, but despite their considerable abundance the physiology of these as yet uncultivated archaea has remained unknown. From a 1.2 Gb large-insert environmental fosmid library of a calcareous grassland soil, a 43...... kb genomic fragment was isolated with a ribosomal RNA that shows its affiliation to group 1.1b of crenarchaeota repeatedly found in soils. The insert encoded a homologue of a copper-containing nitrite reductase with an unusual C-terminus that encoded a potential amicyanin-like electron transfer...

  14. Diversity of nitrite reductase (nirK and nirS) gene fragments in forested upland and wetland soils

    DEFF Research Database (Denmark)

    Priemé, Anders; Braker, Gesche; Tiedje, James M.

    2002-01-01

    The genetic heterogeneity of nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods. nirK gene fragments could be amplified from both soils, whereas nirS gene fragments could be amplified only from...... the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of nirK clones was lower than the diversity of nirS clones. Among the 54 distinct nirK RFLP patterns identified in the two soils, only one...... marsh clones and all upland clones. Only a few of the nirK clone sequences branched with those of known denitrifying bacteria. The nirS clones formed two major clusters with several subclusters, but all nirS clones showed less than 80% identity to nirS sequences from known denitrifying bacteria. Overall...

  15. The roles of tissue nitrate reductase activity and myoglobin in securing nitric oxide availability in deeply hypoxic crucian carp

    DEFF Research Database (Denmark)

    Hansen, Marie N; Lundberg, Jon O; Filice, Mariacristina

    2016-01-01

    . We also tested whether liver, muscle and heart tissue possess nitrate reductase activity that supplies nitrite to the tissues during severe hypoxia. Crucian carp exposed to deep hypoxia (1nitrite in red musculature to more than double the value in normoxic fish......In mammals, treatment with low doses of nitrite has a cytoprotective effect in ischemia/reperfusion events, as a result of nitric oxide formation and S-nitrosation of proteins. Interestingly, anoxia-tolerant lower vertebrates possess an intrinsic ability to increase intracellular nitrite...... concentration during anoxia in tissues with high myoglobin and mitochondria content, such as the heart. Here, we tested the hypothesis that red and white skeletal muscles develop different nitrite levels in crucian carp exposed to deep hypoxia and assessed whether this correlates with myoglobin concentration...

  16. Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods.

    Science.gov (United States)

    Cristaldi, Julio C; Gómez, María C; González, Pablo J; Ferroni, Felix M; Dalosto, Sergio D; Rizzi, Alberto C; Rivas, María G; Brondino, Carlos D

    2018-03-01

    The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (k cat =3.9(4) s -1 ) lower than that of wt SmNir (k cat =240(50) s -1 ). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the N δ1 H…OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the N δ1 H…OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Effect of ruminal administration of Escherichia coli wild type or a genetically modified strain with enhanced high nitrite reductase activity on methane emission and nitrate toxicity in nitrate-infused sheep.

    Science.gov (United States)

    Sar, C; Mwenya, B; Pen, B; Takaura, K; Morikawa, R; Tsujimoto, A; Kuwaki, K; Isogai, N; Shinzato, I; Asakura, Y; Toride, Y; Takahashi, J

    2005-11-01

    The effects of two kinds of Escherichia coli (E. coli) strain, wild-type E. coli W3110 and E. coli nir-Ptac, which has enhanced NO(2) reduction activity, on oral CH(4) emission and NO(3) toxicity in NO(3)-treated sheep were assessed in a respiratory hood system in a 4 x 6 Youden square design. NO(3) (1.3 g NaNO(3)/kg(0.75) body weight) and/or E. coli strains were delivered into the rumen through a fistula as a single dose 30 min after the morning meal. Escherichia coli cells were inoculated for sheep to provide an initial E. coli cell density of optical density at 660 nm of 2, which corresponded to 2 x 10(10) cells/ml. The six treatments consisted of saline, E. coli W3110, E. coli nir-Ptac, NO(3), NO(3) plus E. coli W3110, and NO(3) plus E. coli nir-Ptac. CH(4) emission from sheep was reduced by the inoculation of E. coli W3110 or E. coli nir-Ptac by 6 % and 12 %, respectively. NO(3) markedly inhibited CH(4) emission from sheep. Compared with sheep given NO(3) alone, the inoculation of E. coli W3110 to NO(3)-infused sheep lessened ruminal and plasma toxic NO(2) accumulation and blood methaemoglobin production, while keeping ruminal methanogenesis low. Ruminal and plasma toxic NO(2) accumulation and blood methaemoglobin production in sheep were unaffected by the inoculation of E. coli nir-Ptac. These results suggest that ruminal methanogenesis may be reduced by the inoculation of E. coli W3110 or E. coli nir-Ptac. The inoculation of E. coli W3110 may abate NO(3) toxicity when NO(3) is used to inhibit CH(4) emission from ruminants.

  18. Electronic structure of the perturbed blue copper site in nitrite reductase: Spectroscopic properties, bonding, and implications for the entatic/rack state

    Energy Technology Data Exchange (ETDEWEB)

    LaCroix, L.B.; Shadle, S.E.; Hedman, B.; Hodgson, K.O.; Solomon, E.I. [Stanford Univ., CA (United States); Wang, Y.; Averill, B.A. [Univ. of Virginia, Charlottesville, VA (United States)

    1996-08-21

    Low-temperature optical absorption, circular dichroism, magnetic circular dichroism, and sulfur K-edge X-ray absorption spectra have been measured for the green `blue` copper center (type 1) in Achromobacter cycloclastes nitrite reductase. Combined with density functional calculations, the results of these spectroscopies have been used to define the extremely `perturbed` electronic structure of this site relative to that of the prototypical `classic` site found in plastocyanin. Experimentally calibrated density functional calculations have been further used to determine the specific geometric distortions which generate the perturbed electronic structure. These studies indicate that the principal electronic structure changes in nitrite reductase, relative to plastocyanin, are a rotation of the Cu d{sub x(2)-y(2)} half-filled, highest occupied molecular orbital (HOMO) and an increase in the ligand field strength at the Cu center. These changes in Cu-ligand interactions result in the redistribution of absorption intensity in the charge transfer and ligand field transitions. Additionally, the new S(Met)-Cu interaction accounts for the unexpectedly high sulfur covalency in the HOMO. The increase in ligand field strength shifts all the d {yields} d transitions in nitrite reductase to nearly 1000 cm{sup -1} higher energy than their counterparts in plastocyanin, which accounts for the EPR spectral differences between the type 1 sites in these complexes. 97 refs., 7 figs., 3 tabs.

  19. Voltammetry and Electrocatalysis of Achrornobacter Xylosoxidans Copper Nitrite Reductase on Functionalized Au(111)-Electrode Surfaces

    DEFF Research Database (Denmark)

    Welinder, Anna C.; Zhang, Jingdong; Hansen, Allan G.

    2007-01-01

    positively charged and electrostatically neutral, hydrophobic and hydrophilic, aliphatic and aromatic, and variable-length micro-environments, as well as their combinations. Optimal conditions for enzyme function seems to be a combination of hydrophobic and hydrophilic surface linker properties, which can...... lead to close to complete non-catalytic monolayer interfacial electron transfer function and electrocatalysis with activity approaching enzyme activity in homogeneous solution. Thiophenol (combined hydrophobic stacking and interdispersed water molecules), 4-methyl-thiophenol (hydrophobic and water...

  20. Unraveling the origin of the nitrite-mediated hypoxic vasodilation

    DEFF Research Database (Denmark)

    Fago, Angela; Dalsgaard, T.; Simonsen, U.

    2007-01-01

    are sufficient to induce NO-mediated vasodilation independently of the nitrite reductase activities here investigated. These results further indicate that the vasoactive effect of nitrite is intrinsic to the vessel and may be due to S-nitrosothiols formed within the arterial smooth muscle....

  1. Redox properties of lysine- and methionine-coordinated hemes ensure downhill electron transfer in NrfH2A4 nitrite reductase.

    Science.gov (United States)

    Todorovic, Smilja; Rodrigues, Maria Luísa; Matos, Daniela; Pereira, Inês A C

    2012-05-17

    The multiheme NrfHA nitrite reductase is a menaquinol:nitrite oxidoreductase that catalyzes the 6-electron reduction of nitrite to ammonia in a reaction that involves eight protons. X-ray crystallography of the enzyme from Desulfovibrio vulgaris revealed that the biological unit, NrfH2A4, houses 28 c-type heme groups, 22 of them with low spin and 6 with pentacoordinated high spin configuration. The high spin hemes, which are the electron entry and exit points of the complex, carry a highly unusual coordination for c-type hemes, lysine and methionine as proximal ligands in NrfA and NrfH, respectively. Employing redox titrations followed by X-band EPR spectroscopy and surface-enhanced resonance Raman spectroelectrochemistry, we provide the first experimental evidence for the midpoint redox potential of the NrfH menaquinol-interacting methionine-coordinated heme (-270 ± 10 mV, z = 0.96), identified by the use of the inhibitor HQNO, a structural analogue of the physiological electron donor. The redox potential of the catalytic lysine-coordinated high spin heme of NrfA is -50 ± 10 mV, z = 0.9. These values determined for the integral NrfH2A4 complex indicate that a driving force for a downhill electron transfer is ensured in this complex.

  2. Aldose reductase mediates retinal microglia activation

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J. Mark, E-mail: mark.petrash@ucdenver.edu

    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{sup GFP} mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR{sup 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. - Highlights: • AR inhibition prevents retinal microglial activation. • Endotoxin-induced ocular cytokine production is reduced in AR null mice. • Overexpression of AR spontaneously induces retinal microglial activation.

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

    Science.gov (United States)

    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.

  4. A mutant of Paracoccus denitrificans with disrupted genes coding for cytochrome c550 and pseudoazurin establishes these two proteins as the in vivo electron donors to cytochrome cd1 nitrite reductase

    NARCIS (Netherlands)

    Pearson, Isobel V; Page, M Dudley; van Spanning, Rob J M; Ferguson, Stuart J

    2003-01-01

    In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc(1) complex to the periplasmic nitrite reductase, cytochrome cd(1). The periplasmic protein cytochrome c(550) has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has

  5. Methylenetetrahydrofolate Reductase Activity and Folate Metabolism

    Directory of Open Access Journals (Sweden)

    Nursen Keser

    2014-04-01

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

  6. Pseudoazurin from Sinorhizobium meliloti as an electron donor to copper-containing nitrite reductase: influence of the redox partner on the reduction potentials of the enzyme copper centers.

    Science.gov (United States)

    Ferroni, Félix M; Marangon, Jacopo; Neuman, Nicolás I; Cristaldi, Julio C; Brambilla, Silvina M; Guerrero, Sergio A; Rivas, María G; Rizzi, Alberto C; Brondino, Carlos D

    2014-08-01

    Pseudoazurin (Paz) is the physiological electron donor to copper-containing nitrite reductase (Nir), which catalyzes the reduction of NO2 (-) to NO. The Nir reaction mechanism involves the reduction of the type 1 (T1) copper electron transfer center by the external physiological electron donor, intramolecular electron transfer from the T1 copper center to the T2 copper center, and nitrite reduction at the type 2 (T2) copper catalytic center. We report the cloning, expression, and characterization of Paz from Sinorhizobium meliloti 2011 (SmPaz), the ability of SmPaz to act as an electron donor partner of S. meliloti 2011 Nir (SmNir), and the redox properties of the metal centers involved in the electron transfer chain. Gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis together with UV-vis and EPR spectroscopies revealed that as-purified SmPaz is a mononuclear copper-containing protein that has a T1 copper site in a highly distorted tetrahedral geometry. The SmPaz/SmNir interaction investigated electrochemically showed that SmPaz serves as an efficient electron donor to SmNir. The formal reduction potentials of the T1 copper center in SmPaz and the T1 and T2 copper centers in SmNir, evaluated by cyclic voltammetry and by UV-vis- and EPR-mediated potentiometric titrations, are against an efficient Paz T1 center to Nir T1 center to Nir T2 center electron transfer. EPR experiments proved that as a result of the SmPaz/SmNir interaction in the presence of nitrite, the order of the reduction potentials of SmNir reversed, in line with T1 center to T2 center electron transfer being thermodynamically more favorable.

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

    DEFF Research Database (Denmark)

    Vlaeminck, S.E.; Terada, Akihiko; Smets, Barth F.

    2010-01-01

    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,

  8. Dietary nitrite attenuates oxidative stress and activates antioxidant genes in rat heart during hypobaric hypoxia.

    Science.gov (United States)

    Singh, Manjulata; Arya, Aditya; Kumar, Rajesh; Bhargava, Kalpana; Sethy, Niroj Kumar

    2012-01-01

    The nitrite anion represents the circulatory and tissue storage form of nitric oxide (NO) and a signaling molecule, capable of conferring cardioprotection and many other health benefits. However, molecular mechanisms for observed cardioprotective properties of nitrite remain largely unknown. We have evaluated the NO-like bioactivity and cardioprotective efficacies of sodium nitrite supplemented in drinking water in rats exposed to short-term chronic hypobaric hypoxia. We observed that, nitrite significantly attenuates hypoxia-induced oxidative stress, modulates HIF-1α stability and promotes NO-cGMP signaling in hypoxic heart. To elucidate potential downstream targets of nitrite during hypoxia, we performed a microarray analysis of nitrite supplemented hypoxic hearts and compared with both hypoxic and nitrite supplemented normoxic hearts respectively. The analysis revealed a significant increase in the expression of many antioxidant genes, transcription factors and cardioprotective signaling pathways which was subsequently confirmed by qRT-PCR and Western blotting. Conversely, hypoxia exposure increased oxidative stress, activated inflammatory cytokines, downregulated ion channels and altered expression of both pro- and anti-oxidant genes. Our results illustrate the physiological function of nitrite as an eNOS-independent source of NO in heart profoundly modulating the oxidative status and cardiac transcriptome during hypoxia. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Analysis of nitrate reductase mRNA expression and nitrate reductase activity in response to nitrogen supply

    OpenAIRE

    Gholamreza Kavoosi; Sadegh Balotf; Homeira Eshghi; Hasan Hasani

    2014-01-01

    Nitrate is one of the major sources of nitrogen for the growth of plants. It is taken up by plant roots and transported to the leaves where it is reduced to nitrite in the. The main objective of this research was to investigate stimulatory effects of sodium nitrate, potassium nitrate, ammonia and urea on the production/generation of the nitrate reductase mRNA in Triticum aestivum plants. The plants were grown in standard nutrient solution for 21 days and then starved in a media without nitrat...

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

    Science.gov (United States)

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

    2009-01-01

    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. 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 role of the two dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expression were examined by using a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 hr. In contrast, nitrate sensor-response regulator mutant ΔnarXL displayed growth arrest initially, but resumed growth after 72 hr and reached early stationary phase in liquid culture after 120 hr. 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, nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase. PMID:19935885

  11. Enhanced Activity and Selectivity of Carbon Nanofiber Supported Pd Catalysts for Nitrite Reduction

    KAUST Repository

    Shuai, Danmeng

    2012-03-06

    Pd-based catalyst treatment represents an emerging technology that shows promise to remove nitrate and nitrite from drinking water. In this work we use vapor-grown carbon nanofiber (CNF) supports in order to explore the effects of Pd nanoparticle size and interior versus exterior loading on nitrite reduction activity and selectivity (i.e., dinitrogen over ammonia production). Results show that nitrite reduction activity increases by 3.1-fold and selectivity decreases by 8.0-fold, with decreasing Pd nanoparticle size from 1.4 to 9.6 nm. Both activity and selectivity are not significantly influenced by Pd interior versus exterior CNF loading. Consequently, turnover frequencies (TOFs) among all CNF catalysts are similar, suggesting nitrite reduction is not sensitive to Pd location on CNFs nor Pd structure. CNF-based catalysts compare favorably to conventional Pd catalysts (i.e., Pd on activated carbon or alumina) with respect to nitrite reduction activity and selectivity, and they maintain activity over multiple reduction cycles. Hence, our results suggest new insights that an optimum Pd nanoparticle size on CNFs balances faster kinetics with lower ammonia production, that catalysts can be tailored at the nanoscale to improve catalytic performance for nitrite, and that CNFs hold promise as highly effective catalyst supports in drinking water treatment. © 2012 American Chemical Society.

  12. Nitrite-Mediated Hypoxic Vasodilation Predicted from Mathematical Modeling and Quantified from in Vivo Studies in Rat Mesentery

    Directory of Open Access Journals (Sweden)

    Donald G. Buerk

    2017-12-01

    Full Text Available Nitric oxide (NO generated from nitrite through nitrite reductase activity in red blood cells has been proposed to play a major role in hypoxic vasodilation. However, we have previously predicted from mathematical modeling that much more NO can be derived from tissue nitrite reductase activity than from red blood cell nitrite reductase activity. Evidence in the literature suggests that tissue nitrite reductase activity is associated with xanthine oxidoreductase (XOR and/or aldehyde oxidoreductase (AOR. We investigated the role of XOR and AOR in nitrite-mediated vasodilation from computer simulations and from in vivo exteriorized rat mesentery experiments. Vasodilation responses to nitrite in the superfusion medium bathing the mesentery equilibrated with 5% O2 (normoxia or zero O2 (hypoxia at either normal or acidic pH were quantified. Experiments were also conducted following intraperitoneal (IP injection of nitrite before and after inhibiting XOR with allopurinol or inhibiting AOR with raloxifene. Computer simulations for NO and O2 transport using reaction parameters reported in the literature were also conducted to predict nitrite-dependent NO production from XOR and AOR activity as a function of nitrite concentration, PO2 and pH. Experimentally, the largest arteriolar responses were found with nitrite >10 mM in the superfusate, but no statistically significant differences were found with hypoxic and acidic conditions in the superfusate. Nitrite-mediated vasodilation with IP nitrite injections was reduced or abolished after inhibiting XOR with allopurinol (p < 0.001. Responses to IP nitrite before and after inhibiting AOR with raloxifene were not as consistent. Our mathematical model predicts that under certain conditions, XOR and AOR nitrite reductase activity in tissue can significantly elevate smooth muscle cell NO and can serve as a compensatory pathway when endothelial NO production is limited by hypoxic conditions. Our theoretical and

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

    OpenAIRE

    Pandey, K N; Sabharwal, P S

    1982-01-01

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

  14. Aggregate size and architecture determine biomass activity for one-stage partial nitritation and anammox

    DEFF Research Database (Denmark)

    Vlaeminck, S.; Terada, Akihiko; Smets, Barth F.

    2010-01-01

    to the inoculation and operation of the reactors. Fluorescent in-situ hybridization (FISH) was applied on aggregate sections to quantify AerAOB and AnAOB, as well as to visualize the aggregate architecture. The activity balance of the aggregates was calculated as the nitrite accumulation rate ratio (NARR), i.......e. the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate, with all rates determined in aerobic and anoxic batch tests. The space occupied by extracellular polymeric substances (EPS) was calculated from transmission electron micrographs. All smallest aggregates were flocs...... and nitrite sources (NARR, > 1.7). Large 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 EPS. Large B aggregates were...

  15. Aldose reductase inhibitory activity and antioxidant capacity of pomegranate extracts.

    Science.gov (United States)

    Karasu, Cimen; Cumaoğlu, Ahmet; Gürpinar, Ali Rifat; Kartal, Murat; Kovacikova, Lucia; Milackova, Ivana; Stefek, Milan

    2012-03-01

    The pomegranate, Punica granatum L., has been the subject of current interest as a medicinal agent with wide-ranging therapeutic indications. In the present study, pomegranate ethanolic seed and hull extracts were tested, in comparison with a commercial sample, for the inhibition of aldose reductase, an enzyme involved in the etiology of diabetic complications. In vitro inhibition of rat lens aldose reductase was determined by a conventional method. Pomegranate ethanolic hull extract and commercial pomegranate hull extract exhibited similar aldose reductase inhibitory activity characterized by IC(50) values ranging from 3 to 33.3 μg/ml. They were more effective than pomegranate ethanolic seed extract with IC(50) ranging from 33.3 to 333 μg/ml. Antioxidant action of the novel compounds was documented in a DPPH test and in a liposomal membrane model, oxidatively stressed by peroxyl radicals. All the plant extracts showed considerable antioxidant potential in the DPPH assay. Pomegranate ethanolic hull extract and commercial pomegranate hull extract executed similar protective effects on peroxidatively damaged liposomal membranes characterized by 10ethanolic seed extract showed significantly lower antioxidant activity compared to both hull extracts studied. Pomegranate extracts are thus presented as bifunctional agents combining aldose reductase inhibitory action with antioxidant activity and with potential therapeutic use in prevention of diabetic complications.

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

    OpenAIRE

    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 cd 1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d 1. Whereas the haem?c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d 1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d 1 insertion protein NirN in?vivo. This NirS?NirN inter...

  17. Structural Basis of Eukaryotic Nitrate Reduction: Crystal Structures of the Nitrate Reductase Active Site

    Science.gov (United States)

    Fischer, Katrin; Barbier, Guillaume G.; Hecht, Hans-Juergen; Mendel, Ralf R.; Campbell, Wilbur H.; Schwarz, Guenter

    2005-01-01

    Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 Å. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis. PMID:15772287

  18. Evaluation of the nitrite and leukocyte esterase activity tests for the diagnosis of acute symptomatic urinary tract infection in men.

    NARCIS (Netherlands)

    Koeijers, J.J.; Kessels, A.G.H.; Nys, S.; Bartelds, A.; Donker, G.; Stobberingh, E.; Verbon, A.

    2007-01-01

    For 422 male patients with symptoms indicative of a urinary tract infection, nitrite and leukocyte esterase activity dipstick test results were compared with results of culture of urine samples. The positive predictive value of a positive nitrite test result was 96%. Addition of results of the

  19. The reduction of nitrate, nitrite and hydroxylamine to ammonia by enzymes from Cucurbita pepo L. in the presence of reduced benzyl viologen as electron donor

    Science.gov (United States)

    Cresswell, C. F.; Hageman, R. H.; Hewitt, E. J.; Hucklesby, D. P.

    1965-01-01

    1. Enzyme systems from Cucurbita pepo have been shown to catalyse the reduction of nitrite and hydroxylamine to ammonia in yields about 90–100%. 2. Reduced benzyl viologen serves as an efficient electron donor for both systems. Activity of the nitrite-reductase system is directly related to degree of dye reduction when expressed in terms of the function for oxidation–reduction potentials, but appears to decrease to negligible activity below about 9% dye reduction. 3. NADH and NADPH alone produce negligible nitrite loss, but NADPH can be linked to an endogenous diaphorase system to reduce nitrite to ammonia in the presence of catalytic amounts of benzyl viologen. 4. The NADH– or NADPH–nitrate-reductase system that is also present can accept electrons from reduced benzyl viologen, but shows relationships opposite to that for the nitrite-reductase system with regard to effect of degree of dye reduction on activity. The product of nitrate reduction may be nitrite alone, or nitrite and ammonia, or ammonia alone, according only to the degree of dye reduction. 5. The relative activities of nitrite-reductase and hydroxylamine-reductase systems show different relationships with degree of dye reduction and may become reversed in magnitude when effects of degree of dye reduction are tested over a suitable range. 6. Nitrite severely inhibits the rate of reduction of hydroxylamine without affecting the yield of ammonia as a percentage of total substrate loss, but hydroxylamine has a negligible effect on the activity of the nitrite-reductase system. 7. The apparent Km for nitrite (1 μm) is substantially less than that for hydroxylamine, for which variable values between 0·05 and 0·9mm (mean 0·51 mm) have been observed. 8. The apparent Km values for reduced benzyl viologen differ for the nitrite-reductase and hydroxylamine-reductase systems: 60 and 7·5 μm respectively. 9. It is concluded that free hydroxylamine may not be an intermediate in the reduction of nitrite

  20. Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes

    Science.gov (United States)

    Braker, Gesche; Ayala-del-Río, Héctor L.; Devol, Allan H.; Fesefeldt, Andreas; Tiedje, James M.

    2001-01-01

    Steep vertical gradients of oxidants (O2 and NO3−) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers, Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs of nirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis of nirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of Archaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities. PMID:11282647

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

    Science.gov (United States)

    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.

  2. Triterpenes and meroterpenes from Ganoderma lucidum with inhibitory activity against HMGs reductase, aldose reductase and α-glucosidase.

    Science.gov (United States)

    Chen, Baosong; Tian, Jin; Zhang, Jinjin; Wang, Kai; Liu, Li; Yang, Bo; Bao, Li; Liu, Hongwei

    2017-07-01

    Seven new compounds including four lanostane triterpenoids, lucidenic acids Q-S (1-3) and methyl ganoderate P (4), and three triterpene-farnesyl hydroquinone conjugates, ganolucinins A-C (5-7), one new natural product ganomycin J (8), and 73 known compounds (9-81) were isolated from fruiting bodies of Ganoderma lucidum. The structures of the compounds 1-8 were determined by spectroscopic methods. Bioactivities of compounds isolated were assayed against HMG-CoA reductase, aldose reductase, α-glucosidase, and PTP1B. Ganolucidic acid η (39), ganoderenic acid K (44), ganomycin J (8), and ganomycin B (61) showed strong inhibitory activity against HMG-CoA reductase with IC 50 of 29.8, 16.5, 30.3 and 14.3μM, respectively. Lucidumol A (67) had relatively good effect against aldose reductase with IC 50 of 19.1μM. Farnesyl hydroquinones ganomycin J (8), ganomycin B (61), ganomycin I (62), and triterpene-farnesyl hydroquinone conjugates ganoleuconin M (76) and ganoleuconin O (79) possessed good inhibitory activity against α-glucosidase with IC 50 in the range of 7.8 to 21.5μM. This work provides chemical and biological evidence for the usage of extracts of G. lucidum as herbal medicine and food supplements for the control of hyperglycemic and hyperlipidemic symptoms. Copyright © 2017. Published by Elsevier B.V.

  3. Siroheme- and [Fe4-S4]-dependent NirA from Mycobacterium tuberculosis is a sulfite reductase with a covalent Cys-Tyr bond in the active site.

    Science.gov (United States)

    Schnell, Robert; Sandalova, Tatyana; Hellman, Ulf; Lindqvist, Ylva; Schneider, Gunter

    2005-07-22

    The nirA gene of Mycobacterium tuberculosis is up-regulated in the persistent state of the bacteria, suggesting that it is a potential target for the development of antituberculosis agents particularly active against the pathogen in its dormant phase. This gene encodes a ferredoxin-dependent sulfite reductase, and the structure of the enzyme has been determined using x-ray crystallography. The enzyme is a monomer comprising 555 amino acids and contains a [Fe4-S4] cluster and a siroheme cofactor. The molecule is built up of three domains with an alpha/beta fold. The first domain consists of two ferredoxin-like subdomains, related by a pseudo-2-fold symmetry axis passing through the whole molecule. The other two domains, which provide much of the binding interactions with the cofactors, have a common fold that is unique to the sulfite/nitrite reductase family. The domains form a trilobal structure, with the cofactors and the active site located at the interface of all three domains in the center of the molecule. NirA contains an unusual covalent bond between the side chains of Tyr69 and Cys161 in the active site, in close proximity to the siroheme cofactor. Removal of this covalent bond by site-directed mutagenesis impairs catalytic activity, suggesting that it is important for the enzymatic reaction. These residues are part of a sequence fingerprint, able to distinguish between ferredoxin-dependent sulfite and nitrite reductases. Comparison of NirA with the structure of the truncated NADPH-dependent sulfite reductase from Escherichia coli suggests a binding site for the external electron donor ferredoxin close to the [Fe4-S4] cluster.

  4. Diterpenoids with thioredoxin reductase inhibitory activities from Jatropha multifida.

    Science.gov (United States)

    Zhu, Jian-Yong; Zhang, Chun-Yang; Dai, Jing-Jing; Rahman, Khalid; Zhang, Hong

    2017-12-01

    Chemical investigation of the Jatropha multifida has led to the isolation of nine diterpenoids (1-9), including a new jatromulone A, four podocarpane diterpenoids (2-5), two lathyrane-type diterpenoids (6 and 7) and two dinorditerpenoids (8 and 9). Their structures were elucidated by spectroscopic analysis, and the absolute configurations of 1 were determined by CD analysis. All of the diterpenoids were screened for inhibitory activity against thioredoxin reductase (TrxR), which is a potential target for cancer chemotherapy with redox balance and antioxidant functions. Compounds 6 and 7 exhibited stronger activity (IC 50 : 23.4 and 10.6 μM, respectively) than the positive control, curcumin (IC 50  = 25.0 μM). Compounds 2-9 were isolated from J. multifida for the first time.

  5. Mechanisms underlying erythrocyte and endothelial nitrite reduction to NO in hypoxia: role for Xanthine Oxidoreductase and eNOS

    Science.gov (United States)

    Webb, Andrew J.; Milsom, Alexandra B.; Rathod, Krishnaraj S.; Chu, Wai Lum; Qureshi, Shehla; Lovell, Matthew J.; Lecomte, Florence M.J.; Perrett, David; Raimondo, Carmello; Khoshbin, Espeed; Ahmed, Zubair; Uppal, Rakesh; Benjamin, Nigel; Hobbs, Adrian J.; Ahluwalia, Amrita

    2010-01-01

    Reduction of nitrite (NO2−) provides a major source of nitric oxide (NO) in the circulation, especially in hypoxemic conditions. Our previous studies suggest that xanthine oxidoreductase (XOR) is an important nitrite reductase in the heart and kidney. Herein, we have demonstrated that conversion of nitrite to NO by blood vessels and RBCs was enhanced in the presence of the XOR substrate, xanthine (10μM), and attenuated by the XOR inhibitor, allopurinol (100μM) in acidic and hypoxic conditions only. Whilst endothelial nitric oxide synthase (eNOS) inhibition had no effect on vascular nitrite reductase activity, in RBCs L-NAME, L-NMMA and L-arginine inhibited nitrite-derived NO production by >50% (p<0.01), at pH 7.4 and 6.8 under hypoxic conditions. Western blot and immunohistochemical analysis of RBC membranes confirmed the presence of eNOS and abundant XOR on whole RBCs. Thus, XOR and eNOS are ideally situated on the membranes of RBCs and blood vessels to generate intravascular vasodilator NO from nitrite during ischemic episodes. In addition to the proposed role of deoxyhemoglobin, our findings suggest that the nitrite reductase activity within the circulation, under hypoxic conditions (at physiological pH), is mediated by eNOS; however, as acidosis develops a substantial role for XOR becomes evident. PMID:18818408

  6. Arabidopsis Root-Type Ferredoxin:NADP(H) Oxidoreductase 2 is Involved in Detoxification of Nitrite in Roots.

    Science.gov (United States)

    Hachiya, Takushi; Ueda, Nanae; Kitagawa, Munenori; Hanke, Guy; Suzuki, Akira; Hase, Toshiharu; Sakakibara, Hitoshi

    2016-11-01

    Ferredoxin:NADP(H) oxidoreductase (FNR) plays a key role in redox metabolism in plastids. Whereas leaf FNR (LFNR) is required for photosynthesis, root FNR (RFNR) is believed to provide electrons to ferredoxin (Fd)-dependent enzymes, including nitrite reductase (NiR) and Fd-glutamine-oxoglutarate aminotransferase (Fd-GOGAT) in non-photosynthetic conditions. In some herbal species, however, most nitrate reductase activity is located in photosynthetic organs, and ammonium in roots is assimilated mainly by Fd-independent NADH-GOGAT. Therefore, RFNR might have a limited impact on N assimilation in roots grown with nitrate or ammonium nitrogen sources. AtRFNR genes are rapidly induced by application of toxic nitrite. Thus, we tested the hypothesis that RFNR could contribute to nitrite reduction in roots by comparing Arabidopsis thaliana seedlings of the wild type with loss-of-function mutants of RFNR2 When these seedlings were grown under nitrate, nitrite or ammonium, only nitrite nutrition caused impaired growth and nitrite accumulation in roots of rfnr2 Supplementation of nitrite with nitrate or ammonium as N sources did not restore the root growth in rfnr2 Also, a scavenger for nitric oxide (NO) could not effectively rescue the growth impairment. Thus, nitrite toxicity, rather than N depletion or nitrite-dependent NO production, probably causes the rfnr2 root growth defect. Our results strongly suggest that RFNR2 has a major role in reduction of toxic nitrite in roots. A specific set of genes related to nitrite reduction and the supply of reducing power responded to nitrite concomitantly, suggesting that the products of these genes act co-operatively with RFNR2 to reduce nitrite in roots. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  7. Antiproliferative and quinone reductase-inducing activities of withanolides derivatives.

    Science.gov (United States)

    García, Manuela E; Nicotra, Viviana E; Oberti, Juan C; Ríos-Luci, Carla; León, Leticia G; Marler, Laura; Li, Guannan; Pezzuto, John M; van Breemen, Richard B; Padrón, José M; Hueso-Falcón, Idaira; Estévez-Braun, Ana

    2014-07-23

    Two new and five known withanolides (jaborosalactones 2, 3, 4, 5, and 24) were isolated from the leaves of Jaborosa runcinata Lam. We also obtained some derivatives from jaborosalactone 5, which resulted to be the major isolated metabolite. The natural compounds as well as derivatives were evaluated for their antiproliferative activity and the induction of quinone reductase 1 (QR1; NQ01) activity. Structure-activity relationships revealed valuable information on the pharmacophore of withanolide-type compounds. Three compounds of this series showed significantly higher antiproliferative activity than jaborosalactone 5. The effect of these compounds on the cell cycle was determined. Furthermore, the ability of major compounds to induce QR1 was evaluated. It was found that all the active test compounds are monofunctional inducers that interact with Keap1. The most promising derivatives prepared from jaborosalactone 5 include (23R)-4β,12β,21-trihydroxy-1,22-dioxo-12,23-cycloergostan-2,5,17,24-tetraen-26,23-olide (18) and (23R)-21-acetoxy-12β-hydroxy-1,22-dioxo-12,23-cycloergostan-2,5,17,24-tetraen-26,23-lactame (20). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  8. Low activity of superoxide dismutase and high activity of glutathione reductase in erythrocytes from centenarians

    DEFF Research Database (Denmark)

    Andersen, Helle Raun; Jeune, B; Nybo, H

    1998-01-01

    aged between 60 and 79 years. MEASUREMENTS: enzyme activities of superoxide dismutase (CuZn-SOD), glutathione peroxidase, catalase and glutathione reductase (GR) in erythrocytes. Functional capacity among the centenarians was evaluated by Katz' index of activities of daily living, the Physical...

  9. Nitrite Reductase (nirK) as an Alternative Molecular Marker for Ammonia-oxidizing Archaea: Quantification and Characterization of Thaumarchaeal nirK Genes in Estuarine Sediments from San Francisco Bay

    Science.gov (United States)

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

    2016-02-01

    Nitrification, the microbially-mediated oxidation of ammonia to nitrate via nitrite, is a key component of the biogeochemical nitrogen cycle. The first and rate-limiting step of nitrification is the aerobic oxidation of ammonia to nitrite. Recent molecular ecological studies targeting ammonia monooxygenase (amoA) genes have identified ammonia-oxidizing archaea (AOA) as key players in the process. In addition to the ammonia oxidation machinery that includes the amoA gene, AOA also possess a gene for copper-containing nitrite reductase (nirK). Despite the notably high abundance and expression of archaeal nirK in various environments, the distribution patterns and functional role of this gene in AOA remain mostly unknown. In the present study, the diversity and abundance of nirK genes in estuarine sediments were investigated using quantitative polymerase chain reaction, cloning and sequencing approaches. The abundance and phylogeny were compared with that of the conventional marker gene, amoA. Overall, nirK was observed to be significantly more abundant than amoA in the sediment samples, with variation in copy numbers spanning several orders of magnitude between sampling sites. The substantial difference in relative abundance suggests that the conventionally used amoA primers could be underestimating AOA diversity in marine/estuarine sediments. Phylogenetic analysis targeting archaeal nirK revealed a number of unique sequence types, as well as many that clustered with sequences from previous estuarine studies and cultured AOA isolates, including Nitrosopumilus maritimus and Nitrosoarchaeaum limnia. The nirK phylogeny was mostly congruent with that of archaeal amoA. Overall, this study provides 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.

  10. Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue

    NARCIS (Netherlands)

    Lea, US; ten Hoopen, F; Provan, F; Kaiser, WM; Meyer, C; Lillo, C

    In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine

  11. Aldose reductase inhibitory activity and antioxidant capacity of pomegranate extracts

    OpenAIRE

    Karasu, Çimen; Cumaoğlu, Ahmet; Gürpinar, Ali Rifat; Kartal, Murat; Kovacikova, Lucia; Milackova, Ivana; Stefek, Milan

    2012-01-01

    The pomegranate, Punica granatum L., has been the subject of current interest as a medicinal agent with wide-ranging therapeutic indications. In the present study, pomegranate ethanolic seed and hull extracts were tested, in comparison with a commercial sample, for the inhibition of aldose reductase, an enzyme involved in the etiology of diabetic complications. In vitro inhibition of rat lens aldose reductase was determined by a conventional method. Pomegranate ethanolic hull extract and comm...

  12. Increased 5α-reductase activity in idiopathic hirsutism

    International Nuclear Information System (INIS)

    Serafini, P.; Lobo, R.A.

    1985-01-01

    In vitro, genital skin 5α-reductase activity (5α-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 5α-RA. In vitro 5α-RA was assessed by incubations of skin with 14 C-testosterone (T) for 2 hours, after which steroids were separated and the radioactivity of dihydrotestosterone (DHT) and 5α-androstane 3α-17β-estradiol (3α-diol) in specific eluates were determined. All androgens were normal in IH with the exception of higher levels of 3α-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 3α-diol in IH and PCO were similar, yet higher than in control subjects. Serum androgens showed no correlation with 5α-RA, while the CR of T to DHT showed a significant positive correlation with the Ferriman and Gallwey score. The increased 5α-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 5α-RA

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

    Science.gov (United States)

    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.

  14. Linking the Effect of Antibiotics on Partial-Nitritation Biofilters: Performance, Microbial Communities and Microbial Activities

    Directory of Open Access Journals (Sweden)

    Alejandro Gonzalez-Martinez

    2018-02-01

    Full Text Available The emergence and spread of antibiotics resistance in wastewater treatment systems have been pointed as a major environmental health problem. Nevertheless, research about adaptation and antibiotics resistance gain in wastewater treatment systems subjected to antibiotics has not been successfully developed considering bioreactor performance, microbial community dynamics and microbial activity dynamics at the same time. To observe this in autotrophic nitrogen removal systems, a partial-nitritation biofilter was subjected to a continuous loading of antibiotics mix of azithromycin, norfloxacin, trimethoprim, and sulfamethoxazole. The effect of the antibiotics mix over the performance, bacterial communities and bacterial activity in the system was evaluated. The addition of antibiotics caused a drop of ammonium oxidation efficiency (from 50 to 5% and of biomass concentration in the bioreactor, which was coupled to the loss of ammonium oxidizing bacteria Nitrosomonas in the bacterial community from 40 to 3%. Biomass in the partial nitritation biofilter experienced a sharp decrease of about 80% due to antibiotics loading, but the biomass adapted and experienced a growth by stabilization under antibiotics feeding. During the experiment several bacterial genera appeared, such as Alcaligenes, Paracoccus, and Acidovorax, clearly dominating the bacterial community with >20% relative abundance. The system reached around 30% ammonium oxidation efficiency after adaptation to antibiotics, but no effluent nitrite was found, suggesting that dominant antibiotics-resistant phylotypes could be involved in nitrification–denitrification metabolisms. The activity of ammonium oxidation measured as amoA and hao gene expression dropped a 98.25% and 99.21%, respectively, comparing the system before and after the addition of antibiotics. On the other hand, denitrifying activity increased as observed by higher expression of nir and nos genes (83.14% and 252

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Nitrate reductase activity in the diatom Biddulphia longicruris: characterization and daily oscillation

    Directory of Open Access Journals (Sweden)

    Cristina S Asano

    1995-01-01

    Full Text Available Nitrate reductase (NR activity was studied in the marine diatom Biddulphia longicruris. During 24 hours of sampling, NR activity was found during day time and in the transition day-night. Nitrite anions, the product of nitrate reduction, was released by the cells at the times NR was active, and accumulated in the culture medium. Whenever the cultures of B. longicmris were submitted to nitrogen deprivation, NR activity could not be detected. In vitro determination of KM values for NR using nitrate or NADH were respectively 50//M and 80 /*M. Temperature and pH dependence of NR activity were also determined for this organism.A atividade de nitrato redutase (NR foi estudada na diatomácea marinha Biddulphia longicruris. A NR é a enzima responsável pelo processo de assimilação de nitrato. O nitrato é reduzido no interior da célula a nitrito pela ação da NR. Esta enzima é normalmente oligomérica e utiliza o NADH como substrato doador de elétrons. Em alguns organismos também são encontradas NRs capazes de utilizarem o NADPH como doador eletrônico para a redução do nitrato a nitrito. Para a diatomácea B. longicmris foram apresentadas evidências de que a sua NR é específica para NADH e que a redução de nitrato em presença de NADPH não acontece. Determinações in vitro dos valores das constantes de Michaelis-Menten (KM usando nitrato e NADH como substratos, são respectivamente 50µ e 80µM. A temperatura ótima de reação enzimática e a sua dependência ao pH também foram estudadas. Cultivos de B. longicmris foram acompanhados por períodos de 24 horas e foi mostrado que a atividade de NR é encontrada em maiores níveis durante os períodos de transição de luz/escuro. Os anions nitrito, produtos da redução de nitrato, são eliminados pelas células nos períodos de maior atividade de NR e se acumulam no meio de cultura. Células submetidas à ausência de nitrato apresentam uma repressão da expressão de NR, sendo

  17. A new cotton SDR family gene encodes a polypeptide possessing aldehyde reductase and 3-ketoacyl-CoA reductase activities.

    Science.gov (United States)

    Pang, Yu; Song, Wen-Qiang; Chen, Fang-Yuan; Qin, Yong-Mei

    2010-03-01

    To understand regulatory mechanisms of cotton fiber development, microarray analysis has been performed for upland cotton (Gossypium hirsutum). Based on this, a cDNA (GhKCR3) encoding a polypeptide belonging to short-chain alcohol dehydrogenase/reductase family was isolated and cloned. It contains an open reading frame of 987 bp encoding a polypeptide of 328 amino acid residues. Following its overexpression in bacterial cells, the purified recombinant protein specifically uses NADPH to reduce a variety of short-chain aldehydes. A fragment between Gly180 and Gly191 was found to be essential for its catalytic activity. Though the GhKCR3 gene shares low sequence similarities to the ortholog of Saccharomyces cerevisiae YBR159w that encodes 3-ketoacyl-CoA reductase (KCR) catalyzing the second step of fatty acid elongation, it was surprisingly able to complement the yeast ybr159wDelta mutant. Gas chromatography-mass spectrometry analysis showed that very long-chain fatty acids, especially C26:0, were produced in the ybr159wDelta mutant cells expressing GhKCR3. Applying palmitoyl-CoA and malonyl-CoA as substrates, GhKCR3 showed KCR activity in vitro. Quantitative real time-PCR analysis indicated GhKCR3 transcripts accumulated in rapidly elongating fibers, roots, and stems. Our results suggest that GhKCR3 is probably a novel KCR contributing to very long-chain fatty acid biosynthesis in plants.

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

    Science.gov (United States)

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

    2014-06-23

    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.

  19. A diaper-embedded disposable nitrite sensor with integrated on-board urine-activated battery for UTI screening.

    Science.gov (United States)

    Yu, W; Seo, W; Tan, T; Jung, B; Ziaie, B

    2016-08-01

    This paper reports a low-cost solution to the early detection of urinary nitrite, a common surrogate for urinary tract infection (UTI). We present a facile method to fabricate a disposable and flexible colorimetric [1] nitrite sensor and its urine-activated power source [2] on a hydrophobic (wax) paper through laser-assisted patterning and lamination. Such device, integrated with interface circuitry and a Bluetooth low energy (BLE) module can be embedded onto a diaper, and transmit semi-quantitative UTI monitoring information in a point-of-care and autonomous fashion. The proposed nitrite sensing platform achieves a sensitivity of 1.35 ms/(mg/L) and a detection limit of 4 mg/L.

  20. Antimicrobial activity of essential oils and fruits supplement in reduced nitrite salts condition

    Directory of Open Access Journals (Sweden)

    Vujadinović Dragan P.

    2017-01-01

    Full Text Available Because of the growing negative perception of consumers related to the use of meat products produced by conventional curing methods, organic and natural products are increasingly accepted by consumers. Such products contain a large number of natural products derived from plants, spices, as well as their derivatives in form of essential oils, extracts, concentrates, and so on. These derivatives contain large number of active substances which are known to inhibit the metabolic processes of bacteria, yeasts and molds. Therefore, the goal of this paper was to investigate the synergistic antimicrobial activity of the models with a reduced presence of nitrite salt in aqueous solution, emulsions of essential oils in varying concentrations in vivo via antibiogram tests on pathogenic microorganisms. The effect of the six model groups was analyzed. Two groups were fruit powder solutions in concentrations of 0.2% to 1.2% (Acerola powder and fruit powder mix, while the other four groups were models of aqueous emulsion of essential oil in concentrations ranging from 0.05% to 1.2% (tea tree, clove, oregano, and cinnamon essential oils. In all models reduced amount of the sodium salt of 1.80%, 0.0075% nitrite salt and the liquid derivative as a natural source of the nitrate salt of 3% were used. Antibiogram tests were performed on five pathogenic bacteria (C. perfringens, E. coli, S. enterica, L. monocytogenes, and S. aureus. All antibiogram tests were performed according to Kirby-Bauer disk diffusion protocol. Results of antibiograms showed that without the presence of additional antimicrobial agents, in model systems with reduced content of salts, inhibition zones were not detected. Additionally, models with essential oils of tea tree oil and oregano had the widest inhibition zone diameters, ranging from 17.76±0.48mm for E. coli up to 42.50±0.13mm for S. aureus.

  1. INHIBITORY ACTIVITY OF FLAVONOIDS ON THE LENS ALDOSE REDUCTASE OF HEALTHY AND DIABETIC RATS

    Directory of Open Access Journals (Sweden)

    M. T. Goodarzi

    2006-05-01

    Full Text Available Aldose reductase is a critical enzyme in the polyol pathway that plays an important role in diabetes mellitus. Inhibition of the activity of this enzyme can prevent cataract in diabetic patients’lenses. In this study the inhibitory effect of two flavonoids, quercetin and naringin, in the activity of aldose reductase in streptozotocin-induced diabetic and healthy rats were investigated. Thirty male rats were divided in six groups. The first, second and third group were healthy rats that received water,quercetin and naringin, respectively. The fourth, fifth and sixth groups were streptozocin-induced diabetic rats that received water, quercetin and naringin, respectively. These rats were fed orally in a definite dose from each substance for 12 days. After this period rats were scarified and their lenses were separated and homogenized. The activity of aldose reductase was measured in each homogenized sample separately. The effect of feeding of these substances in blood sugar was also determined. Aldose reductase activity was reduced 73 and 69 percent in diabetic rats fed by quercetin and naringin, respectively, and the difference compared to control group was significant. In healthy rats this reduction was 63 and 59 percent, respectively, and the difference was significant compared to those who did not receive flavonoids. It was concluded that these substances were effective in reduction of aldose reductase activity in vivo and consequently could delay the progress of cataract.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. A Single Mutation Increases the Activity and Stability of Pectobacterium carotovorum Nitrile Reductase.

    Science.gov (United States)

    Zhou, Zheng; Li, Min; Xu, Jian-He; Zhang, Zhi-Jun

    2018-03-02

    Nitrile reductases are considered to be promising and environmentally benign nitrile-reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526 U g prot -1 by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2-fold), showing half-lives of 140 and 32 h at 30 and 40 °C, respectively. In the bioreduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ 0 ) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ 1 ), the variant was advantageous over the wild-type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. 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

    Science.gov (United States)

    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

  5. Low activity of superoxide dismutase and high activity of glutathione reductase in erythrocytes from centenarians

    DEFF Research Database (Denmark)

    Andersen, Helle Raun; Jeune, B; Nybo, H

    1998-01-01

    Performance Test and Mini-Mental State Examination. RESULTS: the mean CuZn-SOD activity was significantly lower and the mean GR activity was significantly higher in centenarians than in the group of elderly people. The centenarians with the lowest cognitive and physical functional capacity and who did...... aged between 60 and 79 years. MEASUREMENTS: enzyme activities of superoxide dismutase (CuZn-SOD), glutathione peroxidase, catalase and glutathione reductase (GR) in erythrocytes. Functional capacity among the centenarians was evaluated by Katz' index of activities of daily living, the Physical......OBJECTIVE: to compare the activities of antioxidative enzymes in erythrocytes between centenarians and a younger group of elderly subjects. DESIGN: cross-sectional study. SETTING: county of Funen, Denmark. SUBJECTS: 41 centenarians aged between 100 and 105 years and 52 community control subjects...

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

    International Nuclear Information System (INIS)

    Simpson, Philippa J.L.; McKinzie, Audra A.; Codd, Rachel

    2010-01-01

    Research highlights: → Two monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina. → Sequence of napA from napEDABC-type operon and napA from NapDAGHB-type operon. → 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.

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

    Science.gov (United States)

    Hofmann, Laurie C; Straub, Sandra; Bischof, Kai

    2013-02-01

    The concentration of CO(2) in global surface ocean waters is increasing due to rising atmospheric CO(2) 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 CO(2) 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 CO(2) concentrations. The activity of external carbonic anhydrase, an important enzyme functioning in macroalgal carbon-concentrating mechanisms, was inversely related to CO(2) concentration after long-term exposure (12 weeks). Nitrate reductase, the enzyme responsible for reduction of nitrate to nitrite, was stimulated by CO(2) and was highest in algae grown at 665 µatm CO(2). Nitrate and phosphate uptake rates were inversely related to CO(2), while ammonium uptake was unaffected, and the percentage of inorganic carbon in the algal skeleton decreased with increasing CO(2). The results indicate that the processes of inorganic carbon and nutrient uptake and assimilation are affected by elevated CO(2) 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 CO(2) are discussed.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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...

  10. The Activities of NADH-MethB Reductase and Glucose-6 ...

    African Journals Online (AJOL)

    The activities of NADH-MetHB reductase and G-6-PD were investigated in malaria patients in Calabar, Nigeria. Seventy malaria patients were selected for this study. Sixty-two age, sex – matched apparently healthy children were used as controls. Ages of subjects ranged from 6 months to 12 years (Mean = 5±1.3 years).

  11. Aldose reductase activity and glucose-related opacities in incubated lenses from dogs and cats.

    Science.gov (United States)

    Richter, Marianne; Guscetti, Franco; Spiess, Bernhard

    2002-11-01

    To determine responses of canine and feline lenses to incubation in a medium with a high glucose concentration. Lenses from 35 dogs and 26 cats. Glucose concentrations were measured in paired lenses from 25 dogs and 17 cats after incubation for 14 days in high-glucose (30 mmol of glucose/L) or control (6 mmol of glucose/L) medium. Aldose reductase activity was measured spectrophotometrically in the incubated lenses and in freshly frozen lenses from 10 dogs and 9 cats. Two lenses of each group were studied histologically. Canine and feline lenses in high-glucose medium developed glucose-specific opacities of variable localization and extent. Canine lenses developed equatorial vacuoles, but severity of the lesions was not associated with the age of the dog. Lenses from young cats (cats (> 4 years old) did not. Glucose concentrations were similar in all lenses incubated in high-glucose medium; however aldose reductase activity was significantly lower in lenses from older cats, compared with lenses from young cats and from dogs. High aldose reductase activity and glucose-related opacities suggest a central role for this enzyme in the pathogenesis of diabetic cataracts in dogs and cats. Because onset of diabetes mellitus usually occurs in cats > 7 years of age, low activity of aldose reductase in lenses of older cats may explain why diabetic cataracts are rare in this species despite hyperglycemia.

  12. Sequence diversity and enzyme activity of ferric-chelate reductase LeFRO1 in tomato.

    Science.gov (United States)

    Kong, Danyu; Chen, Chunlin; Wu, Huilan; Li, Ye; Li, Junming; Ling, Hong-Qing

    2013-11-20

    Ferric-chelate reductase which functions in the reduction of ferric to ferrous iron on root surface is a critical protein for iron homeostasis in strategy I plants. LeFRO1 is a major ferric-chelate reductase involved in iron uptake in tomato. To identify the natural variations of LeFRO1 and to assess their effect on the ferric-chelate reductase activity, we cloned the coding sequences of LeFRO1 from 16 tomato varieties collected from different regions, and detected three types of LeFRO1 (LeFRO1(MM), LeFRO1(Ailsa) and LeFRO1(Monita)) with five amino acid variations at the positions 21, 24, 112, 195 and 582. Enzyme activity assay revealed that the three types of LeFRO1 possessed different ferric-chelate reductase activity (LeFRO1(Ailsa) > LeFRO1(MM) > LeFRO1(Monita)). The 112th amino acid residue Ala of LeFRO1 is critical for maintaining the high activity of ferric-chelate reductase, because modification of this amino acid resulted in a significant reduction of enzyme activity. Further, we showed that the combination of the amino acid residue Ile at the site 24 with Lys at the site 582 played a positive role in the enzyme activity of LeFRO1. In conclusion, the findings are helpful to understand the natural adaptation mechanisms of plants to iron-limiting stress, and may provide new knowledge to select and manipulate LeFRO1 for improving the iron deficiency tolerance in tomato. Copyright © 2013. Published by Elsevier Ltd.

  13. 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.

    Science.gov (United States)

    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

  14. YLL056C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity.

    Science.gov (United States)

    Wang, Han-Yu; Xiao, Di-Fan; Zhou, Chang; Wang, Lin-Lu; Wu, Lan; Lu, Ya-Ting; Xiang, Quan-Ju; Zhao, Ke; Li, Xi; Ma, Meng -Gen

    2017-06-01

    The short-chain dehydrogenase/reductase (SDR) family, the largest family in dehydrogenase/reductase superfamily, is divided into "classical," "extended," "intermediate," "divergent," "complex," and "atypical" groups. Recently, several open reading frames (ORFs) were characterized as intermediate SDR aldehyde reductase genes in Saccharomyces cerevisiae. However, no functional protein in the atypical group has been characterized in S. cerevisiae till now. Herein, we report that an uncharacterized ORF YLL056C from S. cerevisiae was significantly upregulated under high furfural (2-furaldehyde) or 5-(hydroxymethyl)-2-furaldehyde concentrations, and transcription factors Yap1p, Hsf1p, Pdr1/3p, Yrr1p, and Stb5p likely controlled its upregulated transcription. This ORF indeed encoded a protein (Yll056cp), which was grouped into the atypical subgroup 7 in the SDR family and localized to the cytoplasm. Enzyme activity assays showed that Yll056cp is not a quinone or ketone reductase but an NADH-dependent aldehyde reductase, which can reduce at least seven aldehyde compounds. This enzyme showed the best Vmax, Kcat, and Kcat/Km to glycolaldehyde, but the highest affinity (Km) to formaldehyde. The optimum pH and temperature of this enzyme was pH 6.5 for reduction of glycolaldehyde, furfural, formaldehyde, butyraldehyde, and propylaldehyde, and 30 °C for reduction of formaldehyde or 35 °C for reduction of glycolaldehyde, furfural, butyraldehyde, and propylaldehyde. Temperature and pH affected stability of this enzyme and this influence varied with aldehyde substrate. Metal ions, salts, and chemical protective additives, especially at high concentrations, had different influence on enzyme activities for reduction of different aldehydes. This research provided guidelines for study of more uncharacterized atypical SDR enzymes from S. cerevisiae and other organisms.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-11-01

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

  16. Ferrisiderophore reductase activity associated with an aromatic biosynthetic enzyme complex in Bacillus subtilis.

    OpenAIRE

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

    1981-01-01

    The cytoplasmic fractions obtained from Bacillus subtilis strains W168 and WB2802 catalyzed reductive release of iron from the ferric chelate of 2,3-dihydroxybenzoic acid (ferri-DHB), the ferrisiderophore produced by B. subtilis. Ferrisiderophore reductase activity may insert iron into metabolism. This activity required a reductant (reduced nicotinamide adenine dinucleotide phosphate was preferred), was oxygen sensitive, and was stimulated by flavin mononucleotide plus certain divalent cation...

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

    Science.gov (United States)

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

    2018-02-07

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

  18. Preparing electrochemical active hierarchically porous carbons for detecting nitrite in drinkable water

    KAUST Repository

    Ding, Baojun

    2016-01-13

    A class of hierarchically porous carbons were prepared by a facile dual-templating approach. The obtained samples were characterized by scanning electron microscopy, X-ray diffraction, Raman spectroscopy, Brunaner-Emmett-Teller measurement and electrochemical work station, respectively. The porous carbons could possess large specific surface area, interconnected pore structures, high conductivity and graphitizing degree. The resulting materials were used to prepare integrated modified electrodes. Based on the experimental results, the as-prepared hierarchically porous graphite (HPG) modified electrode showed the best electroactive performances toward the detection of nitrite with a detection limit of 8.1 × 10-3 mM. This HPG electrode was also repeatable and stable for 6 weeks. Moreover, this electrode was used for the determination of nitrite in drinkable water, and had acceptable recoveries. © The Royal Society of Chemistry 2016.

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

    Directory of Open Access Journals (Sweden)

    Shao-Jie Wang

    2007-04-01

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

  20. Azospirillum Inoculation Alters Nitrate Reductase Activity and Nitrogen Uptake in Wheat Plant Under Water Deficit Conditions

    OpenAIRE

    N. Aliasgharzad, N. Aliasgharzad; Heydaryan, Zahra; Sarikhani, M.R

    2014-01-01

    Water deficit stress usually diminishes nitrogen uptake by plants. There are evidences that some nitrogen fixing bacteria can alleviate this stress by supplying nitrogen and improving its metabolism in plants. Four Azospirillum strains, A. lipoferum AC45-II, A. brasilense AC46-I, A. irakense AC49-VII and A. irakense AC51-VI were tested for nitrate reductase activity (NRA). In a pot culture experiment using a sandy loam soil, wheat plants (Triticum aestivum L. cv. Sardari) were inoculated with...

  1. Aldose Reductase Inhibitory and Antiglycation Activities of Four ...

    African Journals Online (AJOL)

    AR inhibitory activity and inhibition of AGEs formation was done by using ultraviolet-visible (UV-Vis) and fluorescence spectroscopy with aminoguanidine as a standard. In vivo AR inhibitory activity, which involves determination of rat lens galactitol levels in galactosemic condition by using reverse phase high pressure liquid ...

  2. ECTO-NOX (ENOX) proteins of the cell surface lack thioredoxin reductase activity.

    Science.gov (United States)

    Bosneaga, Elena; Kim, Chinpal; Shen, Bernard; Watanabe, Takahiro; Morre, Dorothy M; Morré, D James

    2008-01-01

    This study was to determine if ENOX proteins of the cell surface act as cell surface thioredoxin reductases. To measure formation of thiols a turbimetric insulin assay was used. No turbidity was observed with insulin alone or with insulin plus DTT. However, the combination of insulin +DTT + recombinant his-tagged ENOX2 (tNOX) did result in increased turbidity. An ENOX1 (CNOX) preparation also resulted in turbidity changes. In contrast, we were unable to demonstrate ENOX2-dependent insulin reduction by high density SDS-PAGE. Inclusion of reduced serum albumin as a source of free thiols for the protein disulfide interchange activity catalyzed by ENOX2 failed to result in insulin reduction in the presence of ENOX2. A direct effect of ENOX2 on thioredoxin reduction in the presence of NADPH also was not observed. The DTNB assay for thioredoxin reductase activity also failed to reveal activity. Thus, ENOX proteins appear not to function as thioredoxin reductases at the cell surface nor do they appear to recognize reduced insulin as a substrate for protein disulfide-thiol interchange. The enhanced turbidity of insulin solutions resulting from ENOX presence was traced to ENOX-catalyzed insulin fibrillation either through nucleation enhancement or some other mechanism. Fibrillation was determined using Thioflavin T fluorescence which paralleled the turbimetric results and the formation of multimers (polymerization) observed on SDS-PAGE.

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

    Science.gov (United States)

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

    2013-06-04

    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.

  4. Testosterone 5alpha-reductase inhibitory active constituents of Piper nigrum leaf.

    Science.gov (United States)

    Hirata, Noriko; Tokunaga, Masashi; Naruto, Shunsuke; Iinuma, Munekazu; Matsuda, Hideaki

    2007-12-01

    Previously we reported that Piper nigrum leaf extract showed a potent stimulation effect on melanogenesis and that (-)-cubebin (1) and (-)-3,4-dimethoxy-3,4-desmethylenedioxycubebin (2) were isolated as active constituents. As a part of our continuous studies on Piper species for the development of cosmetic hair-care agents, testosterone 5alpha-reductase inhibitory activity of aqueous ethanolic extracts obtained from several different parts of six Piper species, namely Piper nigrum, P. methysticum, P. betle, P. kadsura, P. longum, and P. cubeba, were examined. Among them, the extracts of P. nigrum leaf, P. nigrum fruit and P. cubeba fruit showed potent inhibitory activity. Activity-guided fractionation of P. nigrum leaf extract led to the isolation of 1 and 2. Fruits of P. cubeba contain 1 as a major lignan, thus inhibitory activity of the fruit may be attributable to 1. As a result of further assay on other known constituents of the cited Piper species, it was found that piperine, a major alkaloid amide of P. nigrum fruit, showed potent inhibitory activity, thus a part of the inhibitory activity of P. nigrum fruit may depend on piperine. The 5alpha-reductase inhibitory activities of 1 and piperine were found for the first time. In addition, the P. nigrum leaf extract showed in vivo anti-androgenic activity using the hair regrowth assay in testosterone sensitive male C57Black/6CrSlc strain mice.

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

    Science.gov (United States)

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

    2013-01-01

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

  6. Chloroplast Fe(III) chelate reductase activity is essential for seedling viability under iron limiting conditions.

    Science.gov (United States)

    Jeong, Jeeyon; Cohu, Christopher; Kerkeb, Loubna; Pilon, Marinus; Connolly, Erin L; Guerinot, Mary Lou

    2008-07-29

    Photosynthesis, heme biosynthesis, and Fe-S cluster assembly all take place in the chloroplast, and all require iron. Reduction of iron via a membrane-bound Fe(III) chelate reductase is required before iron transport across membranes in a variety of systems, but to date there has been no definitive genetic proof that chloroplasts have such a reduction system. Here we report that one of the eight members of the Arabidopsis ferric reductase oxidase (FRO) family, FRO7, localizes to the chloroplast. Chloroplasts prepared from fro7 loss-of-function mutants have 75% less Fe(III) chelate reductase activity and contain 33% less iron per microgram of chlorophyll than wild-type chloroplasts. This decreased iron content is presumably responsible for the observed defects in photosynthetic electron transport. When germinated in alkaline soil, fro7 seedlings show severe chlorosis and die without setting seed unless watered with high levels of soluble iron. Overall, our results provide molecular evidence that FRO7 plays a role in chloroplast iron acquisition and is required for efficient photosynthesis in young seedlings and for survival under iron-limiting conditions.

  7. Genetic evidence that induction of root Fe(III) chelate reductase activity is necessary for iron uptake under iron deficiency.

    Science.gov (United States)

    Yi, Y; Guerinot, M L

    1996-11-01

    Reduction of Fe(III) to Fe(II) by Fe(III) chelate reductase is thought to be an obligatory step in iron uptake as well as the primary factor in making iron available for absorption by all plants except grasses. Fe(III) chelate reductase has also been suggested to play a more general role in the regulation of cation absorption. In order to experimentally address the importance of Fe(III) chelate reductase activity in the mineral nutrition of plants, three Arabidopsis thaliana mutans (frd1-1, frd1-2 and frd1-3), that do not show induction of Fe(III) chelate reductase activity under iron-deficient growth conditions, have been isolated and characterized. These mutants are still capable of acidifying the rhizosphere under iron-deficiency and accumulate more Zn and Mn in their shoots relative to wild-type plants regardless of iron status. frd1 mutants do not translocate radiolabeled iron to the shoots when roots are presented with a tightly chelated form of Fe(III). These results: (1) confirm that iron must be reduced before it can be transported, (2) show that Fe(III) reduction can be uncoupled from proton release, the other major iron-deficiency response, and (3) demonstrate that Fe(III) chelate reductase activity per se is not necessarily responsible for accumulation of cations previously observed in pea and tomato mutants with constitutively high levels of Fe(III) chelate reductase activity.

  8. Selective activity of Mucor plumbeus reductase towards (-)-camphorquinone.

    Science.gov (United States)

    de Souza, Giovanni Gontijo; Anconi, Cleber Paulo Andrada; Cornelissen, Sjef; De Almeida, Wagner Batista; Dos Santos, Hélio Ferreira; Fortes, Isabel Cristina Pereira; Takahashi, Jacqueline Aparecida

    2009-08-01

    The biotransformation of 1R-(-)-camphorquinone, achieved by growing cells of four fungi species isolated from soil (Mucor plumbeus, Lecanicillium muscarium, Thamnostylum sp. and Syncephalastrum racemosum), was investigated in optimized culture media for each species. Fungi were grown aerobically under shaking and their activities with respect to camphorquinone were monitored for 20 days by gas chromatography coupled to mass spectrometry (GCMS). Camphorquinone was found to be stable in control flasks throughout the experiment. The most interesting results were found for M. plumbeus, which was only able to perform monoreduction of camphorquinone when cultivated on a glucose-peptone-yeast extract medium. Large-scale experiments were set up and the camphorquinone biotransformation products formed by M. plumbeus were purified by column chromatography and identified by (1)H and (13)C nuclear magnetic resonance (NMR). Theoretical calculations were employed as a complementary technique to unambiguously identify the biotransformation products. These findings suggest that M. plumbeus could be of great use for the selective reduction of camphorquinone and related compounds.

  9. Identification of HMG-CoA Reductase Inhibitor Active Compound in Medicinal Forest Plants

    Directory of Open Access Journals (Sweden)

    Shelly Rahmania

    2017-08-01

    Full Text Available Cardiovascular disease is a leading cause of death worldwide, hypercholesterolemia is one of the causes. Three medicinal forest plants are potential natural resources to be developed as cholesterol-reducing herbal product, but scientific informations on their mechanism is still limited. The objective of this research is to explore the potency of the leaf of Jati Belanda (Guazuma ulmifolia, Jabon (Antocephalus macrophyllus, and Mindi (Melia azedarach as inhibitor of HMG-CoA reductase (HMGR, a key enzyme in the regulation of cholesterol biosynthesis. Samples were macerated in ethanol 96% and the filtrate was partitioned using n-hexane and chloroform to obtain the ethanolic flavonoid extract. The effect of each extracts on the HMG-CoA reductase activity were analyzed using HMGR assay kit. At concentration of 10 ppm the G.ulmifolia ethanolic extract showed the highest inhibitory activity as well as pravastatin control inhibitor.  The phenolic content of the ethanolic extracts of G.ulmifolia, A.macrophyllus, and M.azedarach were: 11.00, 34.83, and 13.67 mg gallic acid AE/g dried leaves, respectively. The flavonoid content of the ethanolic extracts of G.ulmifolia, A.macrophyllus, and M.azedarach were: 0.22, 0.64, and 0.78 mg QE/g dried leaves, respectively. Interestingly, G.ulmifolia extract the lowest concentration of phenolic and flavonoid content. HPLC analysis showed that all samples contain quercetin at similiar small concentrations (6.7%, 6.6%, and 7.0% for G.ulmifolia, A.macrophyllus, and M.azedarach, respectively. This indicating other active compounds may play some roles in this inhibitory action on HMG-CoA reductase activity. Further identification using LC-MS/MS showed that G.ulmifolia flavonoid extract contained an unidetified coumpound with molecural weight of 380.0723 Da.  

  10. Seasonal Patterns of Nitrate Reductase and Nitrogenase Activities in Phaseolus vulgaris L. 1

    Science.gov (United States)

    Franco, Alvilio A.; Pereira, Joao C.; Neyra, Carlos A.

    1979-01-01

    The patterns of nitrate reductase activity (NRA) in the leaves (in vivo assay) and root nodule nitrogenase activity (C2H2 reduction) were investigated throughout the season in field-grown Phaseolus vulgaris plants. Maximal NRA (per g fresh weight) occurred at early stages of leaf development but total activity (per leaf) was maximal when the leaf reached full size. In mature plants, most NRA was associated with the upper leaves. Nitrogenase activity was initiated about 2 weeks after sowing, reached a maximum at flowering (5 weeks after sowing) and declined rapidly thereafter. Nitrogenase activity followed the pattern of nodule development. After flowering, P. vulgaris was apparently able to take up and assimilate NO−3 as evidenced by the increase in NO−3 content of the stem and the high levels of NRA in the leaves. Total plant NRA was maximal after flowering and addition of NH4NO3 to the soil at flowering resulted in even higher levels of NRA through most of the pod-filling period, thus resulting in higher seed yields (59% over control). It is proposed that P. vulgaris can benefit from both N2 fixation and NO−3 assimilation and that nitrate reductase plays an important role in the assimilation of nitrogen after flowering. PMID:16660740

  11. Activity profiles of prostaglandin 15- and 9-hydroxydehydrogenase and 13-reductase in the developing rat kidney.

    Science.gov (United States)

    Pace-Asciak, C

    1975-04-25

    Three prostaglandin F2alpha-catabolizing enzyme activities have been demonstrated in kidneys from adult rats. Activity of each of the enzymes varied with animal age. Whereas 15-hydroxydehydrogenase and delta13-reductase appeared important to the early developing kidney (prior to 4 weeks of age), 9-hydroxydehydrogenase appeared to be characteristic of the adult kidney. Prostaglandin 15-hydroxydehydrogenase rose sharply after birth to a maximal value at 19 days (59-fold relative to the adult) decreasing to adult values by Day 40. Prostaglandin delta13-reductase followed a similar pattern rising about 20-fold at Day 19. Prostaglandin 9-hydroxydehydrogenase, on the other hand, was undetectable up to Day 19, rising gradually to adult values by Day 50. Prostaglandin biosynthesis in whole kidney and renal papilla at the peak period of 15-hydroxydehydrogenase activity, i.e. 19, 22, and 24 days, did not vary significally from adult values. The dramatic rise in 15-hydroxydehydrogenase activity, reflecting an important requirement for prostaglandin inactivation during the first 3 weeks after birth, appears to correlate well with the increase during this period in the number of glomeruli, cortical tubules, and redistribution of blood flow to the cortex. These results suggest for the first time an important relationship between prostaglandin catabolizing activites and nephrogenesis.

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

    Science.gov (United States)

    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

  13. Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

    Directory of Open Access Journals (Sweden)

    Benedito Barraviera

    1988-10-01

    Full Text Available The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05 for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s., respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01 for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01 before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1

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

    Energy Technology Data Exchange (ETDEWEB)

    Cockburn, Andrew [Institute for Research on Environment and Sustainability, Devonshire Building, University of Newcastle upon Tyne, Newcastle upon Tyne, NE17RU (United Kingdom); Brambilla, Gianfranco [Istituto Superiore di Sanità, Toxicological chemistry unit, Viale Regina Elena 299, 00161 Rome (Italy); Fernández, Maria-Luisa [Departamento de Medio Ambiente, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Ministerio de Ciencia e Innovación, Carretera de la Coruña, 28040 Madrid (Spain); Arcella, Davide [Unit on Data Collection and Exposure, European Food Safety Authority, Largo N. Palli 5/A43100 Parma (Italy); Bordajandi, Luisa R. [Unit on Contaminants in the Food chain, European Food Safety Authority, Largo N. Palli 5/A, 43100 Parma (Italy); Cottrill, Bruce [Policy Delivery Group, Animal Health and Welfare, ADAS, Wolverhampton (United Kingdom); Peteghem, Carlos van [University of Gent, Harelbekestraat 72, 9000 Gent (Belgium); Dorne, Jean-Lou, E-mail: jean-lou.dorne@efsa.europa.eu [Unit on Contaminants in the Food chain, European Food Safety Authority, Largo N. Palli 5/A, 43100 Parma (Italy)

    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

  15. Determination of low level nitrate/nitrite contamination using SERS-active Ag/ITO substrates coupled to a self-designed Raman spectroscopy system

    Directory of Open Access Journals (Sweden)

    Chi T.K. Tran

    2017-06-01

    Full Text Available A portable and simple Raman scattering and photoluminescence spectroscopy system was set up for sensitive and rapid determination of nitrate/nitrite at low concentrations in water samples. The SERS (Surface Enhanced Raman Scattering – active Ag/ITO substrates were prepared and employed to obtain the enhanced Raman scattering light from the sample. Concentrations as low as 1 ppm and 0.1 ppm were detectable for nitrate and nitrite, respectively. The obtained results confirmed the usefulness of the designed system in actual environmental measurements and analysis.

  16. Transcerebral exchange kinetics of nitrite and calcitonin gene-related peptide in acute mountain sickness: evidence against trigeminovascular activation?

    DEFF Research Database (Denmark)

    Bailey, Damian M; Taudorf, Sarah; Berg, Ronan M G

    2009-01-01

    BACKGROUND AND PURPOSE: High-altitude headache is the primary symptom associated with acute mountain sickness, which may be caused by nitric oxide-mediated activation of the trigeminovascular system. Therefore, the present study examined the effects of inspiratory hypoxia on the transcerebral...... exchange kinetics of the vasoactive molecules, nitrite (NO(2)(*)), and calcitonin gene-related peptide (CGRP). METHODS: Ten males were examined in normoxia and after 9-hour exposure to hypoxia (12.9% O(2)). Global cerebral blood flow was measured by the Kety-Schmidt technique with paired samples obtained...... from the radial artery and jugular venous bulb. Plasma CGRP and NO(2)(*) were analyzed via radioimmunoassay and ozone-based chemiluminescence. Net cerebral exchange was calculated by the Fick principle and acute mountain sickness/headache scores assessed via clinically validated questionnaires. RESULTS...

  17. Antimicrobial activity and physical characterization of silver nanoparticles green synthesized using nitrate reductase from Fusarium oxysporum.

    Science.gov (United States)

    Gholami-Shabani, Mohammadhassan; Akbarzadeh, Azim; Norouzian, Dariush; Amini, Abdolhossein; Gholami-Shabani, Zeynab; Imani, Afshin; Chiani, Mohsen; Riazi, Gholamhossein; Shams-Ghahfarokhi, Masoomeh; Razzaghi-Abyaneh, Mehdi

    2014-04-01

    Nanostructures from natural sources have received major attention due to wide array of biological activities and less toxicity for humans, animals, and the environment. In the present study, silver nanoparticles were successfully synthesized using a fungal nitrate reductase, and their biological activity was assessed against human pathogenic fungi and bacteria. The enzyme was isolated from Fusarium oxysporum IRAN 31C after culturing on malt extract-glucose-yeast extract-peptone (MGYP) medium. The enzyme was purified by a combination of ultrafiltration and ion exchange chromatography on DEAE Sephadex and its molecular weight was estimated by gel filtration on Sephacryl S-300. The purified enzyme had a maximum yield of 50.84 % with a final purification of 70 folds. With a molecular weight of 214 KDa, it is composed of three subunits of 125, 60, and 25 KDa. The purified enzyme was successfully used for synthesis of silver nanoparticles in a way dependent upon NADPH using gelatin as a capping agent. The synthesized silver nanoparticles were characterized by X-ray diffraction, dynamic light scattering spectroscopy, and transmission and scanning electron microscopy. These stable nonaggregating nanoparticles were spherical in shape with an average size of 50 nm and a zeta potential of -34.3. Evaluation of the antimicrobial effects of synthesized nanoparticles by disk diffusion method showed strong growth inhibitory activity against all tested human pathogenic fungi and bacteria as evident from inhibition zones that ranged from 14 to 25 mm. Successful green synthesis of biologically active silver nanoparticles by a nitrate reductase from F. oxysporum in the present work not only reduces laborious downstream steps such as purification of nanoparticle from interfering cellular components, but also provides a constant source of safe biologically-active nanomaterials with potential application in agriculture and medicine.

  18. Comparative inhibition of tetrameric carbonyl reductase activity in pig heart cytosol by alkyl 4-pyridyl ketones.

    Science.gov (United States)

    Shimada, Hideaki; Tanigawa, Takahiro; Matayoshi, Kazunori; Katakura, Kazufumi; Babazono, Ken; Takayama, Hiroyuki; Murahashi, Tsuyoshi; Akita, Hiroyuki; Higuchi, Toshiyuki; Eto, Masashi; Imamura, Yorishige

    2014-06-01

    The present study is to elucidate the comparative inhibition of tetrameric carbonyl reductase (TCBR) activity by alkyl 4-pyridyl ketones, and to characterize its substrate-binding domain. The inhibitory effects of alkyl 4-pyridyl ketones on the stereoselective reduction of 4-benzoylpyridine (4-BP) catalyzed by TCBR were examined in the cytosolic fraction of pig heart. Of alkyl 4-pyridyl ketones, 4-hexanoylpyridine, which has a straight-chain alkyl group of five carbon atoms, inhibited most potently TCBR activity and was a competitive inhibitor. Furthermore, cyclohexyl pentyl ketone, which is substituted by cyclohexyl group instead of phenyl group of hexanophenone, had much lower ability to be reduced than hexanophenone. These results suggest that in addition to a hydrophobic cleft corresponding to a straight-chain alkyl group of five carbon atoms, a hydrophobic pocket with affinity for an aromatic group is located in the substrate-binding domain of TCBR.

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

    Directory of Open Access Journals (Sweden)

    Baskaran G

    2015-01-01

    Full Text Available Gunasekaran Baskaran,1 Shamala Salvamani,1 Siti Aqlima Ahmad,1 Noor Azmi Shaharuddin,1 Parveen Devi Pattiram,2 Mohd Yunus Shukor1 1Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, 2Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, Selangor, Malaysia Abstract: The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA reductase is the key enzyme of the mevalonate pathway that produces cholesterol. Inhibition of HMG-CoA reductase reduces cholesterol biosynthesis in the liver. Synthetic drugs, statins, are commonly used for the treatment of hypercholesterolemia. Due to the side effects of statins, natural HMG-CoA reductase inhibitors of plant origin are needed. In this study, 25 medicinal plant methanol extracts were screened for anti-HMG-CoA reductase activity. Basella alba leaf extract showed the highest inhibitory effect at about 74%. Thus, B. alba was examined in order to investigate its phytochemical components. Gas chromatography with tandem mass spectrometry and reversed phase high-performance liquid chromatography analysis revealed the presence of phenol 2,6-bis(1,1-dimethylethyl, 1-heptatriacotanol, oleic acid, eicosyl ester, naringin, apigenin, luteolin, ascorbic acid, and a-tocopherol, which have been reported to possess antihypercholesterolemic effects. Further investigation of in vivo models should be performed in order to confirm its potential as an alternative treatment for hypercholesterolemia and related cardiovascular diseases. Keywords: HMG-CoA reductase, Basella alba, phytochemical, GC-MS/MS, RP-HPLC, hypercholesterolemia

  20. In situ assay of nitrate reductase activity using portable water bath.

    Science.gov (United States)

    Rajsz, Adam; Wojtuń, Bronisław; Bytnerowicz, Andrzej

    2017-07-01

    In environmental research (i.e., plant ecophysiology, environmental microbiology, and environmental chemistry), some assays require incubation of samples at controlled temperature and darkness. Until now, due to a lack of equipment providing such possibility in situ, researchers had to move collected samples to the laboratory for incubation. Obviously, a delayed incubation and the ex situ conditions could seriously affect the assays' results. A good example of analysis where water bath use is needed is the nitrate reductase activity (NRA) in vivo assay where plant tissue samples are incubated in buffer solution at a predetermined temperature. We designed a transportable water bath with a temperature control which enables in situ measurements in many types of environmental studies. The presented device is small in size featuring a thermally insulated chamber and an electronically controlled thermostat system powered by a 12-V battery. Due to its modular design, it can be transported comfortably in difficult terrain. The incubation process can be carried out continuously in stable temperature and darkness. In order to examine the field usability of the presented device, we conducted measurements of plant nitrate reductase activity in difficult field conditions. The in situ assays were carried out at high altitudes in the Karkonosze mountains, SW Poland. The NRA was studied in two alpine species (Deschampsia caespitosa and Homogyne alpina). Our results showed low NR activity in H. alpina (mean 0.31 μM NO 2 g -1 DW h -1 ) and higher NRA in D. caespitosa (mean 2.7 μM NO 2 g -1 DW h -1 ). The obtained results were highly reproducible and had small variability (low standard error values).

  1. Effect of pharmaceutical potential endocrine disruptor compounds on protein disulfide isomerase reductase activity using di-eosin-oxidized-glutathione.

    Directory of Open Access Journals (Sweden)

    Danièle Klett

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

  2. Constituents from the fruiting bodies of Ganoderma applanatum and their aldose reductase inhibitory activity.

    Science.gov (United States)

    Lee, Sang Hyun; Shim, Sang Hee; Kim, Ju Sun; Kang, Sam Sik

    2006-06-01

    Eight compounds were isolated from the fruiting bodies of Ganoderma applanatum, and were identified as 2-methoxyfatty acids (1), 5-dihydroergosterol (2), ergosterol peroxide (3) 3beta,7beta, 20,23xi-tetrahydroxy-11,15-dioxolanosta-8-en-26-oic acid (4), 7beta,20,23xi-trihydroxy-3,11,15-trioxolanosta-8-en-26-oic acid (5), cerevisterol (6), 7beta,23xi-dihydroxy-3,11,15-trioxolanosta-8,20E (22)-dien-26-oic acid (7), and 7beta-hydroxy-3,11,15,23-tetraoxolanosta-8,20E(22)-dien-26-oic acid methyl ester (8) by spectral analysis. All compounds were isolated for the first time from this fruiting bodies, and their effect on rat lens aldose reductase (RLAR) activity was tested. Among these eight compounds, ergosterol peroxide (3) was found to exhibit potent RLAR inhibition, its IC50 value being 15.4 microg/mL.

  3. Alkene hydrogenation activity of enoate reductases for an environmentally benign biosynthesis of adipic acid.

    Science.gov (United States)

    Joo, Jeong Chan; Khusnutdinova, Anna N; Flick, Robert; Kim, Taeho; Bornscheuer, Uwe T; Yakunin, Alexander F; Mahadevan, Radhakrishnan

    2017-02-01

    Adipic acid, a precursor for Nylon-6,6 polymer, is one of the most important commodity chemicals, which is currently produced from petroleum. The biosynthesis of adipic acid from glucose still remains challenging due to the absence of biocatalysts required for the hydrogenation of unsaturated six-carbon dicarboxylic acids to adipic acid. Here, we demonstrate the first enzymatic hydrogenation of 2-hexenedioic acid and muconic acid to adipic acid using enoate reductases (ERs). ERs can hydrogenate 2-hexenedioic acid and muconic acid producing adipic acid with a high conversion rate and yield in vivo and in vitro . Purified ERs exhibit a broad substrate spectrum including aromatic and aliphatic 2-enoates and a significant oxygen tolerance. The discovery of the hydrogenation activity of ERs contributes to an understanding of the catalytic mechanism of these poorly characterized enzymes and enables the environmentally benign biosynthesis of adipic acid and other chemicals from renewable resources.

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

    Science.gov (United States)

    Sánchez Mainar, María; Leroy, Frédéric

    2015-11-06

    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

  5. Antitumor activity of C-methyl-beta-D-ribofuranosyladenine nucleoside ribonucleotide reductase inhibitors.

    Science.gov (United States)

    Franchetti, Palmarisa; Cappellacci, Loredana; Pasqualini, Michela; Petrelli, Riccardo; Vita, Patrizia; Jayaram, Hiremagalur N; Horvath, Zsuzsanna; Szekeres, Thomas; Grifantini, Mario

    2005-07-28

    A series of adenosine derivatives substituted at the 1'-, 2'-, or 3'-position of the ribose ring with a methyl group was synthesized and evaluated for antitumor activity. From this study 3'-C-methyladenosine (3'-Me-Ado) emerged as the most active compound, showing activity against human myelogenous leukemia K562, multidrug resistant human leukemia K562IU, human promyelocytic leukemia HL-60, human colon carcinoma HT-29, and human breast carcinoma MCF-7 cell lines with IC(50) values ranging from 11 to 38 muM. Structure-activity relationship studies showed that the structure of 3'-Me-Ado is crucial for the activity. Substitution of a hydrogen atom of the N(6)-amino group with a small alkyl or cycloalkyl group, the introduction of a chlorine atom in the 2-position of the purine ring, or the moving of the methyl group from the 3'-position to other ribose positions brought about a decrease or loss of antitumor activity. The antiproliferative activity of 3'-Me-Ado appears to be related to its ability to deplete both intracellular purine and pyrimidine deoxynucleotides through ribonucleotide reductase inhibition.

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

    Science.gov (United States)

    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.

  7. Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation.

    Directory of Open Access Journals (Sweden)

    Sirada Srihirun

    Full Text Available 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.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.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.

  8. Molecular assessment of ammonia- and nitrite-oxidizing bacteria in full-scale activated sludge wastewater treatment plants.

    Science.gov (United States)

    Robinson, K G; Dionisi, H M; Harms, G; Layton, A C; Gregory, I R; Sayler, G S

    2003-01-01

    Nitrification was assessed in two full-scale wastewater treatment plants (WWTPs) over time using molecular methods. Both WWTPs employed a complete-mix suspended growth, aerobic activated sludge process (with biomass recycle) for combined carbon and nitrogen treatment. However, one facility treated primarily municipal wastewater while the other only industrial wastewater. Real time PCR assays were developed to determine copy numbers for total 16S rDNA (a measure of biomass content), the amoA gene (a measure of ammonia-oxidizers), and the Nitrospira 16S rDNA gene (a measure of nitrite-oxidizers) in mixed liquor samples. In both the municipal and industrial WWTP samples, total 16S rDNA values were approximately 2-9 x 10(13) copies/L and Nitrospira 16S rDNA values were 2-4 x 10(10) copies/L. amoA gene concentrations averaged 1.73 x 10(9) copies/L (municipal) and 1.06 x 10(10) copies/L (industrial), however, assays for two distinct ammonia oxidizing bacteria were required.

  9. Anthraquinones from the seeds of Cassia tora with inhibitory activity on protein glycation and aldose reductase.

    Science.gov (United States)

    Jang, Dae Sik; Lee, Ga Young; Kim, Young Sook; Lee, Yun Mi; Kim, Chan-Sik; Yoo, Jeong Lim; Kim, Jin Sook

    2007-11-01

    Nine anthraquinones, aurantio-obtusin (1), chryso-obtusin (2), obtusin (3), chryso-obtusin-2-O-beta-D-glucoside (4), physcion (5), emodin (6), chrysophanol (7), obtusifolin (8), and obtusifolin-2-O-beta-D-glucoside (9), isolated from an EtOAc-soluble extract of the seeds of Cassia tora, were subjected to in vitro bioassays to evaluate their inhibitory activity against advanced glycation end products (AGEs) formation and rat lens aldose reductase (RLAR). Among the isolates, compounds 6 and 8 exhibited a significant inhibitory activity on AGEs formation with observed IC(50) values of 118 and 28.9 microM, respectively, in an AGEs-bovine serum albumin (BSA) assay by specific fluorescence. Furthermore, compounds 6 and 8 inhibited AGEs-BSA formation more effectively than aminoguanidine, an AGEs inhibitor, by indirect AGEs-ELISA. N(epsilon)-Carboxymethyllysine (CML)-BSA formation was also inhibited by compounds 6 and 8. Whereas compounds 1, 4, and 6 showed a significant inhibitory activity on RLAR with IC(50) values of 13.6, 8.8, and 15.9 microM, respectively.

  10. Aldose Reductase Inhibitor Protects against Hyperglycemic Stress by Activating Nrf2-Dependent Antioxidant Proteins

    Directory of Open Access Journals (Sweden)

    Kirtikar Shukla

    2017-01-01

    Full Text Available We have shown earlier that pretreatment of cultured cells with aldose reductase (AR inhibitors prevents hyperglycemia-induced mitogenic and proinflammatory responses. However, the effects of AR inhibitors on Nrf2-mediated anti-inflammatory responses have not been elucidated yet. We have investigated how AR inhibitor fidarestat protects high glucose- (HG- induced cell viability changes by increasing the expression of Nrf2 and its dependent phase II antioxidant enzymes. Fidarestat pretreatment prevents HG (25 mM-induced Thp1 monocyte viability. Further, treatment of Thp1 monocytes with fidarestat caused a time-dependent increase in the expression as well as the DNA-binding activity of Nrf2. In addition, fidarestat augmented the HG-induced Nrf2 expression and activity and also upregulated the expression of Nrf2-dependent proteins such as hemeoxygenase-1 (HO1 and NQO1 in Thp1 cells. Similarly, treatment with AR inhibitor also induced the expression of Nrf2 and HO1 in STZ-induced diabetic mice heart and kidney tissues. Further, AR inhibition increased the HG-induced expression of antioxidant enzymes such as SOD and catalase and activation of AMPK-α1 in Thp1 cells. Our results thus suggest that pretreatment with AR inhibitor prepares the monocytes against hyperglycemic stress by overexpressing the Nrf2-dependent antioxidative proteins.

  11. Aldose Reductase Inhibitor Protects against Hyperglycemic Stress by Activating Nrf2-Dependent Antioxidant Proteins.

    Science.gov (United States)

    Shukla, Kirtikar; Pal, Pabitra Bikash; Sonowal, Himangshu; Srivastava, Satish K; Ramana, Kota V

    2017-01-01

    We have shown earlier that pretreatment of cultured cells with aldose reductase (AR) inhibitors prevents hyperglycemia-induced mitogenic and proinflammatory responses. However, the effects of AR inhibitors on Nrf2-mediated anti-inflammatory responses have not been elucidated yet. We have investigated how AR inhibitor fidarestat protects high glucose- (HG-) induced cell viability changes by increasing the expression of Nrf2 and its dependent phase II antioxidant enzymes. Fidarestat pretreatment prevents HG (25 mM)-induced Thp1 monocyte viability. Further, treatment of Thp1 monocytes with fidarestat caused a time-dependent increase in the expression as well as the DNA-binding activity of Nrf2. In addition, fidarestat augmented the HG-induced Nrf2 expression and activity and also upregulated the expression of Nrf2-dependent proteins such as hemeoxygenase-1 (HO1) and NQO1 in Thp1 cells. Similarly, treatment with AR inhibitor also induced the expression of Nrf2 and HO1 in STZ-induced diabetic mice heart and kidney tissues. Further, AR inhibition increased the HG-induced expression of antioxidant enzymes such as SOD and catalase and activation of AMPK- α 1 in Thp1 cells. Our results thus suggest that pretreatment with AR inhibitor prepares the monocytes against hyperglycemic stress by overexpressing the Nrf2-dependent antioxidative proteins.

  12. 21 CFR 181.34 - Sodium nitrite and potassium nitrite.

    Science.gov (United States)

    2010-04-01

    ... fixatives and preservative agents, with or without sodium or potassium nitrate, in the curing of red meat... 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...

  13. The role of nitrite in nitric oxide homeostasis

    DEFF Research Database (Denmark)

    Jensen, Frank Bo

    2009-01-01

    Nitrite is endogenously produced as an oxidative metabolite of nitric oxide, but it also functions as a NO donor that can be activated by a number of cellular proteins under hypoxic conditions. This article discusses the physiological role of nitrite and nitrite-derived NO in blood flow regulatio...

  14. Aldose Reductase Inhibitory Activity of Compounds from??Zea mays L.

    OpenAIRE

    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 reducta...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

    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 effectively the effects of nitrite exposure.

  19. The catalytic mechanism of NADH-dependent reduction of 9,10-phenanthrenequinone by Candida tenuis xylose reductase reveals plasticity in an aldo-keto reductase active site.

    Science.gov (United States)

    Pival, Simone L; Klimacek, Mario; Nidetzky, Bernd

    2009-06-12

    Despite their widely varying physiological functions in carbonyl metabolism, AKR2B5 (Candida tenuis xylose reductase) and many related enzymes of the aldo-keto reductase protein superfamily utilise PQ (9,10-phenanthrenequinone) as a common in vitro substrate for NAD(P)H-dependent reduction. The catalytic roles of the conserved active-site residues (Tyr51, Lys80 and His113) of AKR2B5 in the conversion of the reactive alpha-dicarbonyl moiety of PQ are not well understood. Using wild-type and mutated (Tyr51, Lys80 and His113 individually replaced by alanine) forms of AKR2B5, we have conducted steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in coenzyme and solvent on the enzymatic rates of PQ reduction. Each mutation caused a 10(3)-10(4)-fold decrease in the rate constant for hydride transfer from NADH to PQ, whose value in the wild-type enzyme was determined as approximately 8 x 10(2) s(-1). The data presented support an enzymic mechanism in which a catalytic proton bridge from the protonated side chain of Lys80 (pK=8.6+/-0.1) to the carbonyl group adjacent to the hydride acceptor carbonyl facilitates the chemical reaction step. His113 contributes to positioning of the PQ substrate for catalysis. Contrasting its role as catalytic general acid for conversion of the physiological substrate xylose, Tyr51 controls release of the hydroquinone product. The proposed chemistry of AKR2B5 action involves delivery of both hydrogens required for reduction of the alpha-dicarbonyl substrate to the carbonyl group undergoing (stereoselective) transformation. Hydride transfer from NADH probably precedes the transfer of a proton from Tyr51 whose pK of 7.3+/-0.3 in the NAD+-bound enzyme appears suitable for protonation of a hydroquinone anion (pK=8.8). These results show that the mechanism of AKR2B5 is unusually plastic in the exploitation of the active-site residues, for the catalytic assistance provided to carbonyl group

  20. The nitrate reductase activity of some root and stem parasites and their hosts

    International Nuclear Information System (INIS)

    Hunter, J.J.

    1984-12-01

    This investigation surveyed the nitrate reductase activity (NRA) of some South African root and stem parasites, as well as their hosts. Fourteen species - five stem and nine root parasites, representative of seven families - and eleven different hosts from eight families, were studied. Two methods were applied in the determination of the NRA of parasite and host, namely the in vivo and in vitro methods. Because of the limited literature on the NRA of parasitic flowering plants both the in vivo and in vitro methods were developed for the host species and subsequently applied to that specific species of parasite as well. Parasites and hosts were also investigated in their natural habitat. The NRA of the roots could, however, only be increased providing phorsynthetic products as a source of NADH, were available. By using [U- 14 C]-Sucrose it was confirmed that the parasite could have fulfilled this need. Generally, the investigation showed that the parasites that were studied, have not altogether lost their ability to reduce nitrate. However, it would appear that the host is used as a source of reduced nitrogen, rather than nitrate, under natural conditions

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

    Science.gov (United States)

    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.

  2. Inverse relationship between age-dependent erythrocyte activity of methaemoglobin reductase and prilocaine-induced methaemoglobinaemia during infancy

    DEFF Research Database (Denmark)

    Nilsson, A; Engberg, G; Henneberg, S

    1990-01-01

    concentrations of local anaesthetics were low (maximum values: prilocaine 78 ng ml-1, lignocaine 412 ng ml-1). The activity of erythrocyte MetHb reductase (cytochrome b5 reductase) was analysed. Data from a previously studied group of infants aged 3-12 months were included also. Enzyme activity did not reach...... adult levels until after the age of 3 months. It showed a good inverse correlation with the maximum MetHb values after application of EMLA. Although the MetHb concentrations in the infants younger than 3 months were small, the enzyme capacity may be overloaded when EMLA is administered at the same time...... as other MetHb-inducing agents. It is concluded that the use of EMLA should be restricted in this age group....

  3. Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase.

    Science.gov (United States)

    Liu, C Tony; Layfield, Joshua P; Stewart, Robert J; French, Jarrod B; Hanoian, Philip; Asbury, John B; Hammes-Schiffer, Sharon; Benkovic, Stephen J

    2014-07-23

    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 (13)C 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

  6. Location of the redox-active thiols of ribonucleotide reductase: sequences similarity between the Escherichia coli and Lactobacillus leichmannii enzymes

    International Nuclear Information System (INIS)

    Lin, A.N.I.; Ashley, G.W.; Stubbe, J.

    1987-01-01

    The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with [1- 14 C]iodoacetamide. The dithiothreitol-reduce E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of 14 C. Sequencing of tryptic peptides shows that 2.8 equiv of 14 C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of 14 C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of 14 C. Sequencing of tryptic peptides shows that 1.4 equiv of 14 C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I

  7. H-rev107 Regulates Cytochrome P450 Reductase Activity and Increases Lipid Accumulation.

    Directory of Open Access Journals (Sweden)

    Fu-Ming Tsai

    Full Text Available H-rev107 is a member of the HREV107 type II tumor suppressor gene family and acts as a phospholipase to catalyze the release of fatty acids from glycerophospholipid. H-rev107 has been shown to play an important role in fat metabolism in adipocytes through the PGE2/cAMP pathway, but the detailed molecular mechanism underlying H-rev107-mediated lipid degradation has not been studied. In this study, the interaction between H-rev107 and cytochrome P450 reductase (POR, which is involved in hepatic lipid content regulation, was determined by yeast two-hybrid screen and confirmed by using in vitro pull down assays and immunofluorescent staining. The expression of POR in H-rev107-expressing cells enhanced the H-rev107-mediated release of arachidonic acid. However, H-rev107 inhibited POR activity and relieved POR-mediated decreased triglyceride content in HtTA and HeLa cervical cells. The inhibitory effect of H-rev107 will be abolished when POR-expressing cells transfected with PLA2-lacking pH-rev107 or treated with PLA2 inhibitor. Silencing of H-rev107 using siRNA resulted in increased glycerol production and reversion of free fatty acid-mediated growth suppression in Huh7 hepatic cells. In summary, our results revealed that H-rev107 is also involved in lipid accumulation in liver cells through the POR pathway via its PLA2 activity.

  8. Mercury resistance and mercuric reductase activities and expression among chemotrophic thermophilic Aquificae.

    Science.gov (United States)

    Freedman, Zachary; Zhu, Chengsheng; Barkay, Tamar

    2012-09-01

    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.

  9. Dissection of malonyl-coenzyme A reductase of Chloroflexus aurantiacus results in enzyme activity improvement.

    Directory of Open Access Journals (Sweden)

    Changshui Liu

    Full Text Available 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(AX(1-2G 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.

  10. Exposure to silver nanoparticles inhibits selenoprotein synthesis and the activity of thioredoxin reductase.

    Science.gov (United States)

    Srivastava, Milan; Singh, Sanjay; Self, William T

    2012-01-01

    Silver nanoparticles (AgNPs) and silver (Ag)-based materials are increasingly being incorporated into consumer products, and although humans have been exposed to colloidal Ag in many forms for decades, this rise in the use of Ag materials has spurred interest into their toxicology. Recent reports have shown that exposure to AgNPs or Ag ions leads to oxidative stress, endoplasmic reticulum stress, and reduced cell proliferation. Previous studies have shown that Ag accumulates in tissues as silver sulfides (Ag2S) and silver selenide (Ag2Se). In this study we investigated whether exposure of cells in culture to AgNPs or Ag ions at subtoxic doses would alter the effective metabolism of selenium, that is, the incorporation of selenium into selenoproteins. For these studies we used a keratinocyte cell model (HaCat) and a lung cell model (A549). We also tested (in vitro, both cellular and chemical) whether Ag ions could inhibit the activity of a key selenoenzyme, thioredoxin reductase (TrxR). We found that exposure to AgNPs or far lower levels of Ag ions led to a dose-dependent inhibition of selenium metabolism in both cell models. The synthesis of protein was not altered under these conditions. Exposure to nanomolar levels of Ag ions effectively blocked selenium metabolism, suggesting that Ag ion leaching was likely the mechanism underlying observed changes during AgNP exposure. Exposure likewise inhibited TrxR activity in cultured cells, and Ag ions were potent inhibitors of purified rat TrxR isoform 1 (cytosolic) (TrxR1) enzyme. Exposure to AgNPs leads to the inhibition of selenoprotein synthesis and inhibition of TrxR1. Further, we propose these two sites of action comprise the likely mechanism underlying increases in oxidative stress, increases endoplasmic reticulum stress, and reduced cell proliferation during exposure to Ag.

  11. Chloroplast Fe(III) chelate reductase activity is essential for seedling viability under iron limiting conditions

    OpenAIRE

    Jeong, Jeeyon; Cohu, Christopher; Kerkeb, Loubna; Pilon, Marinus; Connolly, Erin L.; Guerinot, Mary Lou

    2008-01-01

    Photosynthesis, heme biosynthesis, and Fe-S cluster assembly all take place in the chloroplast, and all require iron. Reduction of iron via a membrane-bound Fe(III) chelate reductase is required before iron transport across membranes in a variety of systems, but to date there has been no definitive genetic proof that chloroplasts have such a reduction system. Here we report that one of the eight members of the Arabidopsis ferric reductase oxidase (FRO) family, FRO7, localizes to the chloropla...

  12. Properties of latent and thiol-activated rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and regulation of enzyme activity.

    Science.gov (United States)

    Dotan, I; Shechter, I

    1983-10-15

    The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.

  13. Modification of membrane sulfhydryl groups in bacteriostatic action of nitrite

    International Nuclear Information System (INIS)

    Buchman, G.W. III; Hansen, J.N.

    1987-01-01

    The mechanism by which nitrite inhibits outgrowing spores of bacillus cereus T was examined by using techniques developed earlier for nitrite analogs. The morphological stage of inhibition, cooperativity effects, effect of pH on inhibition, kinetics of protection against tritiated iodoacetate incorporation into membrane sulfhydryl groups, and protection against the bacteriocidal effect of carboxymethylation of iodoacetate indicate that nitrite acts as a membrane-directed sulfhydryl agent. The mechanism by which nitrite modifies the chemical reactivity of the sulfhyrdyl group could be either direct covalent modification or inactivation through communication with another modified membrane component. Profiles of pH effects suggest that the active agent is the protonated form of nitrite. The nitrite concentrations which modify membrane sulfhydryl activity coincide with those which have a bacteriostatic effect. These results are consistent with membrane sulfhydryl modification as a component of the mechanism of nitrite-induced bacteriostasis in this aerobic sporeformer

  14. Evaluation of the impact on food safety of a Lactobacillus coryniformis strain from pickled vegetables with degradation activity against nitrite and other undesirable compounds.

    Science.gov (United States)

    Fang, Fang; Feng, Tingting; Du, Guocheng; Chen, Jian

    2016-01-01

    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.

  15. Isolation and characterization of a novel Rhodococcus strain with switchable carbonyl reductase and para-acetylphenol hydroxylase activities.

    Science.gov (United States)

    Zhang, Rui; Ren, Jie; Wang, Yu; Wu, Qiaqing; Wang, Min; Zhu, Dunming

    2013-01-01

    In the search for an effective biocatalyst for the reduction of acetophenones with unprotected hydroxy group on the benzene ring, a microorganism, which reduced para-acetylphenol to S-(-)-1-(para-hydroxyphenyl)ethanol under anaerobic conditions, was isolated from soil samples and the 16S rDNA study showed that it was phylogenetically affiliated with species of the genus Rhodococcus and was most similar to Rhodococcus pyridinivorans. Unexpectedly, this strain also hydroxylated para-acetylphenol to give 4-acetylcatechol in presence of oxygen, possessing para-acetylphenol hydroxylase activity. While the reduction of para-acetylphenol had an optimal reaction pH at 7 and a broad optimal temperature range (35-45 °C), the hydroxylation reached the maximum conversion at the pH range of 7-8 and 35 °C. This study identified for the first time a Rhodococcus strain with para-acetylphenol hydroxylase activity, which also contains highly enantioselective carbonyl reductase activity with potential applications for the asymmetric reduction of these less-explored but important ketones such as α-aminoacetophenone, 3'-hydroxyacetophenone and 4'-hydroxyacetophenone. The para-acetylphenol hydroxylase and carbonyl reductase activity are switchable by the reaction conditions.

  16. Reduced glutathione concentration and glutathione reductase activity in various rat tissues after the administration of some radioprotective agents

    International Nuclear Information System (INIS)

    Pulpanova, J.; Kovarova, H.; Ledvina, M.

    1982-01-01

    The concentrations of reduced glutathione (GSH) and activity of glutathione reductase were investigated in rat liver, kidney and spleen after intraperitoneal administration of cystamine (50 mg/kg), mexamine (10 mg/kg), or a mixture of cystamine with mexamine (20 + 10 mg/kg). The GSH concentration increased after the administration of cystamine in the liver (maximum between the 20th and 30th min), in the kidney and spleen (maximum after 60 min). The cystamine + mexamine mixture also caused a significant increase of the GSH concentration in all the organs investigated; however, the values increased at earlier intervals as after the cystamine administration. No substantial effect was shown in the case of the mexamine administration, only 30 min after the administration the values were higher. The activity of glutathione reductase was significantly lower over the entire period examined. This was found in the liver and kidney as after the administration of cystamine, as after the radioprotective mixture. There was also a less pronounced inhibition of the enzyme activity in the spleen. Mexamine as a single radioprotector had practically no influence on the activity. (author)

  17. Roles of nitric oxide, nitrite and myoglobin on myocardial efficiency in trout (Oncorthynchus mykiss) and goldfish (Carassius auratus): implications for hypoxia tolerance

    DEFF Research Database (Denmark)

    Pedersen, Claus Lunde; Faggiano, Serena; Helbo, Signe

    2010-01-01

    that mitochondrial respiration is under a tone of NOS-produced NO. When trout myocardial twitch force and O2 consumption are enhanced by adrenaline, this NO tone disappears. Consistent with its conversion to NO, nitrite reduced O2 consumption and increased myocardial efficiency in trout but not in goldfish...... and nitrite on the O2 consumption rate and isometric twitch force development in electrically paced ventricular preparations during hypoxia, and measured O2 affinity and nitrite reductase activity of the purified heart Mbs of both species. Upon hypoxia (9% O2), O2 consumption and developed force decreased...... in both trout and goldfish myocardium, with trout showing a significant increase in the O2 utilization efficiency, i.e. the ratio of twitch force to O2 consumption, suggesting an increased anaerobic metabolism. NOS inhibition enhanced myocardial O2 consumption and decreased efficiency, indicating...

  18. Supercritical fluid extraction of grape seeds: extract chemical composition, antioxidant activity and inhibition of nitrite production in LPS-stimulated Raw 264.7 cells.

    Science.gov (United States)

    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.

  19. Inactivation of Yersinia enterocolitica by nitrite and nitrate in food.

    Science.gov (United States)

    de Giusti, M; de Vito, E

    1992-01-01

    The antimicrobial effects of sodium nitrite and sodium and potassium nitrate against Yersinia enterocolitica were investigated in solution and in treated pork meat. Potassium nitrate and sodium nitrate showed only feeble antimicrobial activity in cultures; no antimicrobial activity was detected with sodium nitrite. Conversely, all three salts displayed apparent antimicrobial activity in pork meat, possibly due to selective effects on competitive flora.

  20. Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

    International Nuclear Information System (INIS)

    Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

    1987-01-01

    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 [ 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

  1. Substrate-dependent modulation of the enzymatic catalytic activity: reduction of nitrate, chlorate and perchlorate by respiratory nitrate reductase from Marinobacter hydrocarbonoclasticus 617.

    Science.gov (United States)

    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. Copyright © 2012. Published by Elsevier B.V.

  2. Construction of effective disposable biosensors for point of care testing of nitrite.

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Nitrate reductase and photosynthetic activities of wheat and their relationship with plant productivity under soil water deficit conditions (abstract)

    International Nuclear Information System (INIS)

    Ashraf, M.Y.; Sarwar, G.; Hussain, F.

    2005-01-01

    Experiments were conducted in lysimeters with wheat during two consecutive years. The first year experiment comprised of eight wheat genotypes with four water stress treatments, i.e. normal irrigation, pre-anthesis drought, post-anthesis drought and terminal drought, with four replications. The results showed that yield and yield parameters reduced with the severity of drought in all wheat lines. However, wheat lines Chakwal-86, DS-4 and Barani-83 had comparatively higher yield and yield components than others. The maximum reduction in all parameters was under terminal drought. The difference between pre- and post-anthesis drought was nonsignificant, particularly for grain yield. The second experiment was conducted with four wheat lines: two were tolerant (Chakwal-86 and DS-4) and two susceptible (Pavon and DS-17) under similar environments with same treatments to study the photosynthetic efficiency, nitrogen metabolism and their relationship with plant productivity (yield). The results showed that leaf area, transpiration, dry matter accumulation and nitrate reductase activity were reduced while diffusive resistance and total amino acids increased in all the wheat lines under water deficit conditions. The relationship between yield and leaf area, transpiration, dry matter accumulation and nitrate reductase activity was positive. The overall results showed that wheat lines Chakwal-86 and DS-4 showed better performance than others. (author)

  4. Overexpression of AtFRO6 in transgenic tobacco enhances ferric chelate reductase activity in leaves and increases tolerance to iron-deficiency chlorosis.

    Science.gov (United States)

    Li, Li-Ya; Cai, Qiu-Yi; Yu, Dian-Si; Guo, Chang-Hong

    2011-08-01

    The Arabidopsis gene FRO6(AtFRO6) encodes ferric chelate reductase and highly expressed in green tissues of plants. We have expressed the gene AtFRO6 under the control of a 35S promoter in transgenic tobacco plants. High-level expression of AtFRO6 in transgenic plants was confirmed by northern blot analysis. Ferric reductase activity in leaves of transgenic plants grown under iron-sufficient or iron-deficient conditions is 2.13 and 1.26 fold higher than in control plants respectively. The enhanced ferric reductase activity led to increased concentrations of ferrous iron and chlorophyll, and reduced the iron deficiency chlorosis in the transgenic plants, compared to the control plants. In roots, the concentration of ferrous iron and ferric reductase activity were not significantly different in the transgenic plants compared to the control plants. These results suggest that FRO6 functions as a ferric chelate reductase for iron uptake by leaf cells, and overexpression of AtFRO6 in transgenic plants can reduce iron deficiency chlorosis.

  5. Determination of the crystal structure and active residues of FabV, the enoyl-ACP reductase from Xanthomonas oryzae.

    Directory of Open Access Journals (Sweden)

    He Li

    Full Text Available Enoyl-ACP reductase (ENR catalyses the last reduction reaction in the fatty acid elongation cycle in bacteria and is a good antimicrobial target candidate. FabV is the most recently discovered class of ENR, but we lack information about the atomic structure and the key residues involved in reductase activity except for the known conserved tyrosine and lysine residues in the Y-X(8-K active site motif.Here we report the crystal structure of FabV from Xanthomonas oryzae (xoFabV. The crystal structure of this enzyme has been solved to 1.6 Å resolution in space group P2(12(12(1. The model of xoFabV consists of one monomer in the asymmetric unit which is composed of 13 α-helices and 11 β-strands, representing a canonical Rossmann fold architecture. Structural comparison presents that the locations of the conserved tyrosine (Y236 and lysine (K245 residues in the Y-X(8-K active site motif of xoFabV and the Y-X(6-K motif of ecFabI are notably similar. However, the conformations of Y236 in xoFabV and Y156 in ecFabI are distinct. Structure-based site-directed mutagenesis and enzymatic activity assays reveal that in addition to the conserved Y236 and K245 in the Y-X(8-K motif, Y53, D111 and Y226 are key residues implicated in the reductase activity, and F113 and T276 are also important for enzyme function. Moreover, a proposed active lysine located immediately after the Y-X(8-K motif in FabV from Burkholderia mallei (bmFabV is altered to an inactive V246 in xoFabV.We determine the first crystal structure of the FabV enzyme and identify several residues important for its enzymatic activity. These findings lay a solid foundation for the development of specific antibacterial inhibitors of the pathogenic bacteria, such as Vibrio cholerae, Burkholderia species and Xanthomonas species.

  6. Effects of methodological variation on assessment of riboflavin status using the erythrocyte glutathione reductase activation coefficient assay.

    Science.gov (United States)

    Hill, Marilyn H E; Bradley, Angela; Mushtaq, Sohail; Williams, Elizabeth A; Powers, Hilary J

    2009-07-01

    Riboflavin status is usually measured as the in vitro stimulation with flavin adenine dinucleotide of the erythrocyte enzyme glutathione reductase, and expressed as an erythrocyte glutathione reductase activation coefficient (EGRAC). This method is used for the National Diet and Nutrition Surveys (NDNS) of the UK. In the period between the 1990 and 2003 surveys of UK adults, the estimated prevalence of riboflavin deficiency, expressed as an EGRAC value > or = 1.30, increased from 2 to 46 % in males and from 1 to 34 % in females. We hypothesised that subtle but important differences in the detail of the methodology between the two NDNS accounted for this difference. We carried out an evaluation of the performance of the methods used in the two NDNS and compared against an 'in-house' method, using blood samples collected from a riboflavin intervention study. Results indicated that the method used for the 1990 NDNS gave a significantly lower mean EGRAC value than both the 2003 NDNS method and the 'in-house' method (P FAD used in the assay and the duration of the period of incubation of FAD with enzyme. The details of the EGRAC method should be standardised for use in different laboratories and over time. Additionally, it is proposed that consideration be given to re-evaluating the basis of the EGRAC threshold for riboflavin deficiency.

  7. Chemical Constituents of Smilax china L. Stems and Their Inhibitory Activities against Glycation, Aldose Reductase, α-Glucosidase, and Lipase.

    Science.gov (United States)

    Lee, Hee Eun; Kim, Jin Ah; Whang, Wan Kyunn

    2017-03-11

    The search for natural inhibitors with anti-diabetes properties has gained increasing attention. Among four selected Smilacaceae family plants, Smilax china L. stems (SCS) showed significant in vitro anti-glycation and rat lens aldose reductase inhibitory activities. Bioactivity-guided isolation was performed with SCS and four solvent fractions were obtained, which in turn yielded 10 compounds, including one phenolic acid, three chlorogenic acids, four flavonoids, one stilbene, and one phenylpropanoid glycoside; their structures were elucidated using nuclear magnetic resonance and mass spectrometry. All solvent fractions, isolated compounds, and stem extracts from plants sourced from six different provinces of South Korea were next tested for their inhibitory effects against advanced glycation end products, as well as aldose reductase. α-Glucosidase, and lipase assays were also performed on the fractions and compounds. Since compounds 3 , 4 , 6 , and 8 appeared to be the superior inhibitors among the tested compounds, a comparative study was performed via high-performance liquid chromatography with photodiode array detection using a self-developed analysis method to confirm the relationship between the quantity and bioactivity of the compounds in each extract. The findings of this study demonstrate the potent therapeutic efficacy of SCS and its potential use as a cost-effective natural alternative medicine against type 2 diabetes and its complications.

  8. Chemical Constituents of Smilax china L. Stems and Their Inhibitory Activities against Glycation, Aldose Reductase, α-Glucosidase, and Lipase

    Directory of Open Access Journals (Sweden)

    Hee Eun Lee

    2017-03-01

    Full Text Available The search for natural inhibitors with anti-diabetes properties has gained increasing attention. Among four selected Smilacaceae family plants, Smilax china L. stems (SCS showed significant in vitro anti-glycation and rat lens aldose reductase inhibitory activities. Bioactivity-guided isolation was performed with SCS and four solvent fractions were obtained, which in turn yielded 10 compounds, including one phenolic acid, three chlorogenic acids, four flavonoids, one stilbene, and one phenylpropanoid glycoside; their structures were elucidated using nuclear magnetic resonance and mass spectrometry. All solvent fractions, isolated compounds, and stem extracts from plants sourced from six different provinces of South Korea were next tested for their inhibitory effects against advanced glycation end products, as well as aldose reductase. α-Glucosidase, and lipase assays were also performed on the fractions and compounds. Since compounds 3, 4, 6, and 8 appeared to be the superior inhibitors among the tested compounds, a comparative study was performed via high-performance liquid chromatography with photodiode array detection using a self-developed analysis method to confirm the relationship between the quantity and bioactivity of the compounds in each extract. The findings of this study demonstrate the potent therapeutic efficacy of SCS and its potential use as a cost-effective natural alternative medicine against type 2 diabetes and its complications.

  9. Anti-HMG-CoA Reductase, Antioxidant, and Anti-Inflammatory Activities of Amaranthus viridis Leaf Extract as a Potential Treatment for Hypercholesterolemia

    Directory of Open Access Journals (Sweden)

    Shamala Salvamani

    2016-01-01

    Full Text Available 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.

  10. The ferredoxin-binding site of ferredoxin: Nitrite oxidoreductase. Differential chemical modification of the free enzyme and its complex with ferredoxin.

    Science.gov (United States)

    Dose, M M; Hirasawa, M; Kleis-SanFrancisco, S; Lew, E L; Knaff, D B

    1997-07-01

    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.

  11. Tetrathionate reductase of Salmonella thyphimurium: a molybdenum containing enzyme

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  12. Variation and inheritance of iron reductase activity in the roots of common bean (Phaseolus vulgaris L. and association with seed iron accumulation QTL

    Directory of Open Access Journals (Sweden)

    Fernandez Andrea C

    2010-10-01

    Full Text Available Abstract Background Iron deficiency anemia is a global problem which often affects women and children of developing countries. Strategy I plants, such as common bean (Phaseolus vulgaris L. take up iron through a process that involves an iron reduction mechanism in their roots; this reduction is required to convert ferric iron to ferrous iron. Root absorbed iron is critical for the iron nutrition of the plant, and for the delivery of iron to the shoot and ultimately the seeds. The objectives of this study were to determine the variability and inheritance for iron reductase activity in a range of genotypes and in a low × high seed iron cross (DOR364 × G19833, to identify quantitative trait loci (QTL for this trait, and to assess possible associations with seed iron levels. Results The experiments were carried out with hydroponically grown plants provided different amounts of iron varying between 0 and 20 μM Fe(III-EDDHA. The parents, DOR364 and G19833, plus 13 other cultivated or wild beans, were found to differ in iron reductase activity. Based on these initial experiments, two growth conditions (iron limited and iron sufficient were selected as treatments for evaluating the DOR364 × G19833 recombinant inbred lines. A single major QTL was found for iron reductase activity under iron-limited conditions (1 μM Fe on linkage group b02 and another major QTL was found under iron sufficient conditions (15 μM Fe on linkage group b11. Associations between the b11 QTL were found with several QTL for seed iron. Conclusions Genes conditioning iron reductase activity in iron sufficient bean plants appear to be associated with genes contributing to seed iron accumulation. Markers for bean iron reductase (FRO homologues were found with in silico mapping based on common bean synteny with soybean and Medicago truncatula on b06 and b07; however, neither locus aligned with the QTL for iron reductase activity. In summary, the QTL for iron reductase activity

  13. Antimicrobial activities of dihydrofolate reductase inhibitors, used singly or in combination with dapsone, against Mycobacterium ulcerans.

    Science.gov (United States)

    Dhople, A M

    2001-01-01

    Development of new treatments against Mycobacterium ulcerans infection has become crucial because of its wide-scale prevalence throughout the world. The effects of dihydrofolate reductase inhibitors, used either singly or in combination with dapsone against M. ulcerans were evaluated in vitro. When used singly, epiroprim was the most potent, with MICs between 0.5 and 1.0 mg/L, while trimethoprim was totally ineffective. The MICs of K-130 and brodimoprim ranged from 1.0-2.0 mg/L for the former to 2.0-16.0 mg/L for the latter. When combined with dapsone, synergic effects were observed with epiroprim. These results indicate the great potential of epiroprim in treating M. ulcerans infections.

  14. Iron reductases from Pseudomonas aeruginosa.

    Science.gov (United States)

    Cox, C D

    1980-01-01

    Cell-free extracts of Pseudomonas aeruginosa contain enzyme activities which reduce Fe(III) to Fe(II) when iron is provided in certain chelates, but not when the iron is uncomplexed. Iron reductase activities for two substrates, ferripyochelin and ferric citrate, appear to be separate enzymes because of differences in heat stabilities, in locations in fractions of cell-free extracts, in reductant specificity, and in apparent sizes during gel filtration chromatography. Ferric citrate iron reductase is an extremely labile activity found in the cytoplasmic fraction, and ferripyochelin iron reductase is a more stable activity found in the periplasmic as well as cytoplasmic fraction of extracts. A small amount of activity detectable in the membrane fraction seemed to be loosely associated with the membranes. Although both enzymes have highest activity reduced nicotinamide adenine dinucleotide, reduced glutathione also worked with ferripyochelin iron reductase. In addition, oxygen caused an irreversible loss of a percentage of the ferripyochelin iron reductase following sparge of reaction mixtures, whereas the reductase for ferric citrate was not appreciably affected by oxygen. PMID:6766439

  15. Kinetic assays for determining in vitro APS reductase activity in plants without the use of radioactive substances.

    Science.gov (United States)

    Brychkova, Galina; Yarmolinsky, Dmitry; Sagi, Moshe

    2012-09-01

    Adenosine 5'-phosphosulfate (APS) reductase (APR; EC 1.8.4.9) catalyzes the two-electron reduction of APS to sulfite and AMP, a key step in the sulfate assimilation pathway in higher plants. In spite of the importance of this enzyme, methods currently available for detection of APR activity rely on radioactive labeling and can only be performed in a very few specially equipped laboratories. Here we present two novel kinetic assays for detecting in vitro APR activity that do not require radioactive labeling. In the first assay, APS is used as substrate and reduced glutathione (GSH) as electron donor, while in the second assay APS is replaced by an APS-regenerating system in which ATP sulfurylase catalyzes APS in the reaction medium, which employs sulfate and ATP as substrates. Both kinetic assays rely on fuchsin colorimetric detection of sulfite, the final product of APR activity. Incubation of the desalted protein extract, prior to assay initiation, with tungstate that inhibits the oxidation of sulfite by sulfite oxidase activity, resulted in enhancement of the actual APR activity. The reliability of the two methods was confirmed by assaying leaf extract from Arabidopsis wild-type and APR mutants with impaired or overexpressed APR2 protein, the former lacking APR activity and the latter exhibiting much higher activity than the wild type. The assays were further tested on tomato leaves, which revealed a higher APR activity than Arabidopsis. The proposed APR assays are highly specific, technically simple and readily performed in any laboratory.

  16. Nitrogenase and nitrate reductase activities in young Alnus glutinosa, relationship and effect of light-dark treatments

    International Nuclear Information System (INIS)

    Benamar, S.; Thiery, G.; Pizelle, G.

    1995-01-01

    Relations between in vivo nitrogenase (N-2-ase; EC 1.18.6.1) and nitrate reductase (NR; EC 1.6.6.1) activities were studied in young nodulated Alnus glutinosa (L. ) Gaertn. Positive correlations linked N-2-ase activity, constitutive (non-inducible by nitrate) leaf NR activity and plant growth. Light/dark treatments applied to the whole shoot or, separately, to the upper and lower part of shoot led to the finding that(a) the constitutive leaf NR activity depended on direct illumination of the leaf and did not appear subordinate to the N-2-ase activity; (b) the N-2-ase activity was much more efficiently supported by the illumination of the upper (young) leaves than by that of the lower (mature) leaves; (c) the maintenance of nitrate-inducible root NR activity required leaf illumination. The variations of both N-2-ase and root NR activities in response to light/dark treatments emphasize the importance of the photosynthesis for the reduction of dinitrogen and nitrate in nodulated roots of Alnus glutinosa

  17. Towards a systematic analysis of human short-chain dehydrogenases/reductases (SDR): Ligand identification and structure-activity relationships.

    Science.gov (United States)

    Bhatia, Chitra; Oerum, Stephanie; Bray, James; Kavanagh, Kathryn L; Shafqat, Naeem; Yue, Wyatt; Oppermann, Udo

    2015-06-05

    Short-chain dehydrogenases/reductases (SDRs) constitute a large, functionally diverse branch of enzymes within the class of NAD(P)(H) dependent oxidoreductases. In humans, over 80 genes have been identified with distinct metabolic roles in carbohydrate, amino acid, lipid, retinoid and steroid hormone metabolism, frequently associated with inherited genetic defects. Besides metabolic functions, a subset of atypical SDR proteins appears to play critical roles in adapting to redox status or RNA processing, and thereby controlling metabolic pathways. Here we present an update on the human SDR superfamily and a ligand identification strategy using differential scanning fluorimetry (DSF) with a focused library of oxidoreductase and metabolic ligands to identify substrate classes and inhibitor chemotypes. This method is applicable to investigate structure-activity relationships of oxidoreductases and ultimately to better understand their physiological roles. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Dimerization and enzymatic activity of fungal 17β-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily

    Directory of Open Access Journals (Sweden)

    Kristan Katja

    2005-12-01

    Full Text Available Abstract Background 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl is a member of the short-chain dehydrogenase/reductase (SDR superfamily. SDR proteins usually function as dimers or tetramers and 17β-HSDcl is also a homodimer under native conditions. Results We have investigated here which secondary structure elements are involved in the dimerization of 17β-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the αE-helices interact with the Asp121, Glu117 and Asp187 residues from the αE and αF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17β-HSDcl monomeric, while the mutant 17β-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity. Conclusion We have shown by site-directed mutagenesis and structure/function analysis that 17β-HSDcl dimerization involves the αE and αF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor.

  19. Effect of NaCI and supplemental calcium on growth parameters and nitrate reductase activity in maize

    Directory of Open Access Journals (Sweden)

    Elżbieta Sacała

    2011-01-01

    Full Text Available In this study, investigated were the effects of NaCl (60 mmol/dm-3 and NaCl supplemented with different salts (5 mmol/dm-3 CaCl2, CaSO4, CaCO3, KCl, on growth of two maize varieties (Cyrkon and Limko. After 7 days of cultivation in nutrient solution the growth response to salinity of both maize varieties was similar. NaCl led to a dramatic decrease in growth of plants (approx. 50% reduction in fresh and dry weight of root, and 70% reduction in fresh weight of shoot. Addition of extra Ca2+ or K+ to nutrient solution containing NaCl did not definitely improve the growth parameters of maize. However, among the tested salts, CaCl2 had a beneficial visual effect on maize seedlings. In other cases the plants showed noticeable symptoms of salt damage. In long term exposure to salinity (two weeks growth of Cyrkon was more inhibited than Limko. Comparison of growth responses in short-term exposure to salinity (7 days with long-term (14 days showed that in Cyrkon variety the negative effects of NaCl were intensified and addition of CaCl2 to salinized solution had not positive effects on growth. On the contrary, in Limko variety, there was a significant improvement in growth (especially in root dry weight. This fact indicates that during longer exposure to salinity Limko was able to adapt to those conditions. Salinity caused a significant decrease in leaf nitrate reductase activity (60% and 30% reduction respectively in Limko and Cyrkon. Addition of CaCl2 to salinized nutrient solution resulted in greater enzyme inhibition in Cyrkon (50% decline in relation to plants grown under sole NaCl, and 30% increase in Limko. Inhibition of nitrate reductase activity did not cause a decrease in concentration of soluble protein in maize leaves.

  20. Nitrogen nutrition of Canna indica: Effects of ammonium versus nitrate on growth, biomass allocation, photosynthesis, nitrate reductase activity and N uptake rates

    DEFF Research Database (Denmark)

    Konnerup, Dennis; Brix, Hans

    2010-01-01

    The effects of inorganic nitrogen (N) source (NH4+, NO3- or both) on growth, biomass allocation, photosynthesis, N uptake rate, nitrate reductase activity and mineral composition of Canna indica were studied in hydroponic culture. The relative growth rates (0.05-0.06 g g-1 d-1), biomass allocation...

  1. Expression and activity of sulfate transporters and APS reductase in curly kale in response to sulfate deprivation and re-supply

    NARCIS (Netherlands)

    Koralewska, Aleksandra; Buchner, Peter; Stuiver, C. Elisabeth E.; Posthumus, Freek S.; Kopriva, Stanislav; Hawkesford, Malcolm J.; De Kok, Luit J.

    2009-01-01

    Both activity and expression of sulfate transporters and APS reductase in plants are modulated by the sulfur status of the plant. To examine the regulatory mechanisms in curly kale (Brossica olerracea L.), the sulfate supply was manipulated by the transfer of seedlings to sulfate-deprived

  2. NITRITE AND NITRATE DETERMINATIONS IN PLASMA - A CRITICAL-EVALUATION

    NARCIS (Netherlands)

    MOSHAGE, H; KOK, B; HUIZENGA, [No Value; JANSEN, PLM

    Plasma nitrite and nitrate determinations are increasingly being used in clinical chemistry as markers for the activity of nitric oxide synthase and the production of nitric oxide radicals. However, a systematic evaluation of the determination of nitrite and nitrate in plasma has not been performed.

  3. Nitrite and nitrate determinations in plasma: a critical evaluation

    NARCIS (Netherlands)

    Moshage, H.; Kok, B.; Huizenga, J. R.; Jansen, P. L.

    1995-01-01

    Plasma nitrite and nitrate determinations are increasingly being used in clinical chemistry as markers for the activity of nitric oxide synthase and the production of nitric oxide radicals. However, a systematic evaluation of the determination of nitrite and nitrate in plasma has not been performed.

  4. Nitrite as a stimulus for ammonia-starved Nitrosomonas europaea

    NARCIS (Netherlands)

    Laanbroek, H.J.; Bär-Gilissen, M.J.; Hoogveld, H.L.

    2002-01-01

    Ammonia-starved cells of Nitrosomonas europaea are able to preserve a high level of ammonia-oxidizing activity in the absence of ammonium. However, when the nitrite-oxidizing cells that form part of the natural nitrifying community do not keep pace with the ammonia-oxidizing cells, nitrite

  5. Vitamin E Improves Serum Paraoxonase-1 Activity and Some Metabolic Factors in Patients with Type 2 Diabetes: No Effects on Nitrite/Nitrate Levels.

    Science.gov (United States)

    Rafraf, Maryam; Bazyun, Behnaz; Sarabchian, Mohammad Ali; Safaeiyan, Abdolrasoul; Gargari, Bahram Pourghassem

    2016-08-01

    It is known that markers of oxidative stress and nitrite/nitrate anion (NO x ) increase and activity of antioxidative enzyme paraoxonase-1 decline in type 2 diabetes mellitus (DM). The effects of vitamin E on paraoxonase-1 activity and NO x in patients with type 2 diabetes are not known. The purpose of this study was to examine the hypothesis that vitamin E supplementation would affect paraoxonase-1 activity, metabolic factors, and NO x in patients with DM. This double-blind, randomized, controlled clinical trial was conducted on 83 patients with DM aged 30-60 years. Forty-two of the subjects had taken 400 IU/day vitamin E and 41 were given placebo over 8 weeks. Fasting blood samples, anthropometric measurements, and dietary intake data were collected at the baseline and at the end of the trial. Vitamin E significantly increased serum vitamin E level, paraoxonase-1 activity, and total antioxidant status (TAS) and decreased fasting blood sugar (FBS) compared to the control group (p 0.05). Vitamin E improved serum vitamin E level, paraoxonase-1 activity, TAS, and FBS in patients with type 2 diabetes. Longitudinal studies are warranted to assess the outcome of these results in reducing complications of diabetes in patients with type 2 diabetes.

  6. YNL134C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity for detoxification of furfural derived from lignocellulosic biomass.

    Science.gov (United States)

    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. Copyright © 2015 John Wiley & Sons, Ltd.

  7. Post-Transcriptional Regulation Prevents Accumulation of Glutathione Reductase Protein and Activity in the Bundle Sheath Cells of Maize1

    Science.gov (United States)

    Pastori, Gabriela M.; Mullineaux, Philip M.; Foyer, Christine H.

    2000-01-01

    Glutathione reductase (GR; EC 1.6.4.2) activity was assayed in bundle sheath and mesophyll cells of maize (Zea mays L. var H99) from plants grown at 20°C, 18°C, and 15°C. The purity of each fraction was determined by measuring the associated activity of the compartment-specific marker enzymes, Rubisco and phosphoenolpyruvate carboxylase, respectively. GR activity and the abundance of GR protein and mRNA increased in plants grown at 15°C and 18°C compared with those grown at 20°C. In all cases GR activity was found only in mesophyll fractions of the leaves, with no GR activity being detectable in bundle sheath extracts. Immunogold labeling with GR-specific antibodies showed that the GR protein was exclusively localized in the mesophyll cells of leaves at all growth temperatures, whereas GR transcripts (as determined by in situ hybridization techniques) were observed in both cell types. These results indicate that post-transcriptional regulation prevents GR accumulation in the bundle sheath cells of maize leaves. The resulting limitation on the capacity for regeneration of reduced glutathione in this compartment may contribute to the extreme chilling sensitivity of maize leaves. PMID:10712529

  8. HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via geranylgeranylation and RhoA activation

    International Nuclear Information System (INIS)

    Al-Haidari, Amr A.; Syk, Ingvar; Thorlacius, Henrik

    2014-01-01

    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

  9. Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. In Silico Screening, Structure-Activity Relationship, and Biologic Evaluation of Selective Pteridine Reductase Inhibitors Targeting Visceral Leishmaniasis▿ †

    Science.gov (United States)

    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

  11. E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Zejun [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China); Gong, Chaoju [Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058 (China); Liu, Hong [Zhejiang Normal University – Jinhua People' s Hospital Joint Center for Biomedical Research, Jinhua, Zhejiang, 321004 (China); Zhang, Xiaomin; Mei, Lingming [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China); Song, Mintao [Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS), School of Basic Medicine, Peking Union Medical College (PUMC), Beijing, 100005 (China); Qiu, Lanlan; Luo, Shuchai; Zhu, Zhihua; Zhang, Ronghui; Gu, Hongqian [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China); Chen, Xiang, E-mail: sychenxiang@126.com [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China)

    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

  12. Synthesis of Superparamagnetic Core-Shell Structure Supported Pd Nanocatalysts for Catalytic Nitrite Reduction with Enhanced Activity, No Detection of Undesirable Product of Ammonium, and Easy Magnetic Separation Capability.

    Science.gov (United States)

    Sun, Wuzhu; Yang, Weiyi; Xu, Zhengchao; Li, Qi; Shang, Jian Ku

    2016-01-27

    Superparamagnetic nanocatalysts could minimize both the external and internal mass transport limitations and neutralize OH(-) produced in the reaction more effectively to enhance the catalytic nitrite reduction efficiency with the depressed product selectivity to undesirable ammonium, while possess an easy magnetic separation capability. However, commonly used qusi-monodispersed superparamagnetic Fe3O4 nanosphere is not suitable as catalyst support for nitrite reduction because it could reduce the catalytic reaction efficiency and the product selectivity to N2, and the iron leakage could bring secondary contamination to the treated water. In this study, protective shells of SiO2, polymethylacrylic acid, and carbon were introduced to synthesize Fe3O4@SiO2/Pd, Fe3O4@PMAA/Pd, and Fe3O4@C/Pd catalysts for catalytic nitrite reduction. It was found that SiO2 shell could provide the complete protection to Fe3O4 nanosphere core among these shells. Because of its good dispersion, dense structure, and complete protection to Fe3O4, the Fe3O4@SiO2/Pd catalyst demonstrated the highest catalytic nitrite reduction activity without the detection of NH4(+) produced. Due to this unique structure, the activity of Fe3O4@SiO2/Pd catalysts for nitrite reduction was found to be independent of the Pd nanoparticle size or shape, and their product selectivity was independent of the Pd nanoparticle size, shape, and content. Furthermore, their superparamagnetic nature and high saturation magnetization allowed their easy magnetic separation from treated water, and they also demonstrated a good stability during the subsequent recycling experiment.

  13. A nanotherapy strategy significantly enhances anticryptosporidial activity of an inhibitor of bifunctional thymidylate synthase-dihydrofolate reductase from Cryptosporidium.

    Science.gov (United States)

    Mukerjee, Anindita; Iyidogan, Pinar; Castellanos-Gonzalez, Alejandro; Cisneros, José A; Czyzyk, Daniel; Ranjan, Amalendu Prakash; Jorgensen, William L; White, A Clinton; Vishwanatha, Jamboor K; Anderson, Karen S

    2015-01-01

    Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having μM antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. [Effects of nitrogen application rate on nitrate reductase activity, nitric oxide content and gas exchange in winter wheat leaves].

    Science.gov (United States)

    Shangguan, Zhou-Ping

    2007-07-01

    In this paper, the effects of different nitrogen application rates on the nitrate reductase (NR) activity, nitric oxide (NO) content and gas exchange parameters in winter wheat (Triticum aestivum L.) leaves from tillering stage to heading stage and on grain yield were studied. The results showed that the photosynthetic rate (P(n)), transpiration rate (T(r)) and instantaneous water use efficiency (IWUE) of leaves as well as the grain yield were increased with increasing nitrogen application rate first but decreased then, with the values of all these parameters reached the highest in treatment N180. The NR activity increased with increasing nitrogen application rate, and there was a significant linear correlation between NR activity and NO content at tillering and jointing stages (R2 > or = 0.68, n = 15). NO content had a quadratic positive correlation with stomatal conductance (G(s)) (R2 > or = 0.43, n = 15). The lower NO content produced by lower NR activity under lower nitrogen application rate promoted the stoma opened, while the higher NO content produced by higher NR activity under higher nitrogen application rate induced the stoma closed. Although the leaf NO content had a quadratic positive correlation with stomatal conductance (R2 > or = 0.36, n = 15), no remarkable correlation was observed between NR activity and NO content at heading stage, suggesting that nitrogen fertilization could not affect leaf NO content through promoting NR activity, and further more, regulate the stomatal action. Under appropriate nitrogen application the leaf NR activity and NO content were lower, G(s), T(r) and IWUE were higher, and thus, the crop had a better drought-resistant ability, higher P(n), and higher grain yield.

  15. S-nitrosoglutathione covalently modifies cysteine residues of human carbonyl reductase 1 and affects its activity.

    Science.gov (United States)

    Hartmanová, Tereza; Tambor, Vojtěch; Lenčo, Juraj; Staab-Weijnitz, Claudia A; Maser, Edmund; Wsól, Vladimír

    2013-02-25

    Carbonyl reductase 1 (CBR1 or SDR21C1) is a ubiquitously-expressed, cytosolic, monomeric, and NADPH-dependent enzyme. CBR1 participates in apoptosis, carcinogenesis and drug resistance, and has a protective role in oxidative stress, cancer and neurodegeneration. S-Nitrosoglutathione (GSNO) represents the newest addition to its diverse substrate spectrum, which includes a wide range of xenobiotics and endogenous substances. GSNO has also been shown to covalently modify and inhibit CBR1. The aim of the present study was to quantify and characterize the resulting modifications. Of five candidate cysteines for modification by 2 mM GSNO (positions 26, 122, 150, 226, 227), the last four were analyzed using MALDI-TOF/TOF mass spectrometry and then quantified using the Selected Reaction Monitoring Approach on hyphenated HPLC with a triple quadrupole mass spectrometer. The analysis confirmed GSNO concentration-dependent S-glutathionylation of cysteines at positions 122, 150, 226, 227 which was 2-700 times higher compared to wild-type CBR1 (WT-CBR1). Moreover, a disulfide bond between neighboring Cys-226 and Cys-227 was detected. We suggest a role of these two cysteines as a redox-sensitive cysteine pair. The catalytic properties of wild-type and enzyme modified with 2 mM GSNO were also investigated by steady state kinetic experiments with various substrates. GSNO treatment of CBR1 resulted in a 2-5-fold decrease in kcat with menadione, 4-benzoylpyridine, 2,3-hexanedione, daunorubicin and 1,4-naphthoquinone. In contrast, the same treatment increased kcat for substrates containing a 1,2-diketo group in a ring structure (1,2-naphthoquinone, 9,10-phenanthrenequinone, isatin). Except for 9,10-phenanthrenequinone, all changes in kcat were at least in part compensated for by a similar change in Km, overall yielding no drastic changes in catalytic efficiency. The findings indicate that GSNO-induced covalent modification of cysteine residues affects the kinetic mechanism of CBR1

  16. Nitrite disrupts multiple physiological functions in aquatic animals

    DEFF Research Database (Denmark)

    Jensen, Frank Bo

    2003-01-01

    is at the expense of chloride, leading to chloride depletion. Nitrite also activates efflux of potassium from skeletal muscle and erythrocytes, disturbing intracellular and extracellular K+ levels. Nitrite transfer across the erythrocytic membrane leads to oxidation of haemoglobin to methaemoglobin (met......Hb), compromising blood O2 transport. Other haem proteins are also oxidised. Hyperventilation is observed, and eventually tissue O2 shortage becomes reflected in elevated lactate concentrations. Heart rate increases rapidly, before any significant elevations in metHb or extracellular potassium occur. This suggests...... nitrite-induced vasodilation (possibly via nitric oxide generated from nitrite) that is countered by increased cardiac pumping to re-establish blood pressure. Nitrite can form and/or mimic nitric oxide and thereby interfere with processes regulated by this local hormone. Steroid hormone synthesis may...

  17. A novel in-gel assay and an improved kinetic assay for determining in vitro sulfite reductase activity in plants.

    Science.gov (United States)

    Brychkova, Galina; Yarmolinsky, Dmitry; Ventura, Yvonne; Sagi, Moshe

    2012-08-01

    Sulfite reductase (SiR; EC 1.8.7.1), an essential enzyme in the sulfate reduction pathway, catalyzes the reduction of sulfite to sulfide, as an intermediate for cysteine biosynthesis. The commonly used kinetic assay for the detection of in vitro SiR activity in plants is based on a coupled reaction, in which the sulfide produced is converted to cysteine through the presence, in the assay medium, of O-acetylserine sulfhydralase (EC 2.5.1.47) and its substrate, O-acetylserine. An improved kinetic assay for SiR activity in crude desalted protein extracts was developed. The improvement was based on pre-treatment of the protein with tungstate, which improved SiR activity in Arabidopsis and tomato leaf by 29 and 12%, respectively, and the addition of NADPH to the reaction medium, which increased SiR activity by 1.6- and 2.8-fold in Arabidopsis and tomato, respectively, in comparison with the current protocols. Despite the availability and reliability of the kinetic assay, there is currently no assay that enables the direct detection of SiR in relatively large numbers of samples. To meet this need, we developed a novel in-gel assay to detect SiR activity in crude extracts. The method is based on the detection of a brownish-black precipitated band of lead sulfide, formed by the reaction of lead acetate with sulfide. The in-gel assay for SiR activity is reliable, sensitive and technically simpler than the kinetic assay, and opens up the possibility for detecting active SiR isoenzymes and splice variants.

  18. Iron deficiency-induced increase of root branching contributes to the enhanced root ferric chelate reductase activity.

    Science.gov (United States)

    Jin, Chong-Wei; Chen, Wei-Wei; Meng, Zhi-Bin; Zheng, Shao-Jian

    2008-12-01

    In various plant species, Fe deficiency increases lateral root branching. However, whether this morphological alteration contributes to the Fe deficiency-induced physiological responses still remains to be demonstrated. In the present research, we demonstrated that the lateral root development of red clover (Trifolium pretense L.) was significantly enhanced by Fe deficient treatment, and the total lateral root number correlated well with the Fe deficiency-induced ferric chelate reductase (FCR) activity. By analyzing the results from Dasgan et al. (2002), we also found that although the two tomato genotypes line227/1 (P1) and Roza (P2) and their reciprocal F1 hybrid lines ("P1 x P2" and "P2 x P1") were cultured under two different lower Fe conditions (10(-6) and 10(-7) M FeEDDHA), their FCR activities are significantly correlated with the lateral root number. More interestingly, the -Fe chlorosis tolerant ability of these four tomato lines displays similar trends with the lateral root density. Taking these results together, it was proposed that the Fe deficiency-induced increases of the lateral root should play an important role in resistance to Fe deficiency, which may act as harnesses of a useful trait for the selection and breeding of more Fe-efficient crops among the genotypes that have evolved a Fe deficiency-induced Fe uptake system.

  19. Involvement of tristetraprolin in transcriptional activation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin

    Energy Technology Data Exchange (ETDEWEB)

    Ness, Gene C., E-mail: gness@hsc.usf.edu [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612 (United States); Edelman, Jeffrey L.; Brooks, Patricia A. [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612 (United States)

    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.

  20. Structure-activity relationships and docking studies of synthetic 2-arylindole derivatives determined with aromatase and quinone reductase 1.

    Science.gov (United States)

    Prior, Allan M; Yu, Xufen; Park, Eun-Jung; Kondratyuk, Tamara P; Lin, Yan; Pezzuto, John M; Sun, Dianqing

    2017-12-15

    In our ongoing effort of discovering anticancer and chemopreventive agents, a series of 2-arylindole derivatives were synthesized and evaluated toward aromatase and quinone reductase 1 (QR1). Biological evaluation revealed that several compounds (e.g., 2d, IC 50  = 1.61 μM; 21, IC 50  = 3.05 μM; and 27, IC 50  = 3.34 μM) showed aromatase inhibitory activity with half maximal inhibitory concentration (IC 50 ) values in the low micromolar concentrations. With regard to the QR1 induction activity, 11 exhibited the highest QR1 induction ratio (IR) with a low concentration to double activity (CD) value (IR = 8.34, CD = 2.75 μM), while 7 showed the most potent CD value of 1.12 μM. A dual acting compound 24 showed aromatase inhibition (IC 50  = 9.00 μM) as well as QR1 induction (CD = 5.76 μM) activities. Computational docking studies using CDOCKER (Discovery Studio 3.5) provided insight in regard to the potential binding modes of 2-arylindoles within the aromatase active site. Predominantly, the 2-arylindoles preferred binding with the 2-aryl group toward a small hydrophobic pocket within the active site. The C-5 electron withdrawing group on indole was predicted to have an important role and formed a hydrogen bond with Ser478 (OH). Alternatively, meta-pyridyl analogs may orient with the pyridyl 3'-nitrogen coordinating with the heme group. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Methionine sulfoxide reductase B3 deficiency stimulates heme oxygenase-1 expression via ROS-dependent and Nrf2 activation pathways

    Energy Technology Data Exchange (ETDEWEB)

    Kwak, Geun-Hee; Kim, Ki Young; Kim, Hwa-Young, E-mail: hykim@ynu.ac.kr

    2016-05-13

    Methionine sulfoxide reductase B3 (MsrB3), which is primarily found in the endoplasmic reticulum (ER), is an important protein repair enzyme that stereospecifically reduces methionine-R-sulfoxide residues. We previously found that MsrB3 deficiency arrests the cell cycle at the G{sub 1}/S stage through up-regulation of p21 and p27. In this study, we report a critical role of MsrB3 in gene expression of heme oxygenase-1 (HO-1), which has an anti-proliferative effect associated with p21 up-regulation. Depletion of MsrB3 elevated HO-1 expression in mammalian cells, whereas MsrB3 overexpression had no effect. MsrB3 deficiency increased cellular reactive oxygen species (ROS), particularly in the mitochondria. ER stress, which is associated with up-regulation of HO-1, was also induced by depletion of MsrB3. Treatment with N-acetylcysteine as an ROS scavenger reduced augmented HO-1 levels in MsrB3-depleted cells. MsrB3 deficiency activated Nrf2 transcription factor by enhancing its expression and nuclear import. The activation of Nrf2 induced by MsrB3 depletion was confirmed by increased expression levels of its other target genes, such as γ-glutamylcysteine ligase. Taken together, these data suggest that MsrB3 attenuates HO-1 induction by inhibiting ROS production, ER stress, and Nrf2 activation. -- Highlights: •MsrB3 depletion induces HO-1 expression. •MsrB3 deficiency increases cellular ROS and ER stress. •MsrB3 deficiency activates Nrf2 by increasing its expression and nuclear import. •MsrB3 attenuates HO-1 induction by inhibiting ROS production and Nrf2 activation.

  2. Achieve efficient nitrogen removal from real sewage in a plug-flow integrated fixed-film activated sludge (IFAS) reactor via partial nitritation/anammox pathway.

    Science.gov (United States)

    Yang, Yandong; Zhang, Liang; Cheng, Jun; Zhang, Shujun; Li, Baikun; Peng, Yongzhen

    2017-09-01

    This study tested the feasibility of plug-flow integrated fixed-film activated sludge (IFAS) reactor in applying sewage partial nitritation/anammox (PN/A) process. The IFAS reactor was fed with real pre-treated sewage (C/N ratio=1.3) and operated for 200days. High nitrogen removal efficiency of 82% was achieved with nitrogen removal rates of 0.097±0.019kgN/(m 3 ·d). Therefore, plug-flow IFAS reactor could be an alternative to applying sewage PN/A process. Besides, it was found that the stability of sewage PN/A process was significantly affected by residual ammonium. Nitrate accumulated in effluent and PN/A performance deteriorated when residual ammonium was below 1mg/L. On the contrary, long-term stable PN/A operation was achieved when residual ammonium was over 3mg/L. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774.

    Science.gov (United States)

    González, Pablo J; Rivas, María G; Brondino, Carlos D; Bursakov, Sergey A; Moura, Isabel; Moura, José J G

    2006-07-01

    Nitrate reductases are enzymes that catalyze the conversion of nitrate to nitrite. We report here electron paramagnetic resonance (EPR) studies in the periplasmic nitrate reductase isolated from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774. This protein, belonging to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes, comprises a single 80-kDa subunit and contains a Mo bis(molybdopterin guanosine dinucleotide) cofactor and a [4Fe-4S] cluster. EPR-monitored redox titrations, carried out with and without nitrate in the potential range from 200 to -500 mV, and EPR studies of the enzyme, in both catalytic and inhibited conditions, reveal distinct types of Mo(V) EPR-active species, which indicates that the Mo site presents high coordination flexibility. These studies show that nitrate modulates the redox properties of the Mo active site, but not those of the [4Fe-4S] center. The possible structures and the role in catalysis of the distinct Mo(V) species detected by EPR are discussed.

  4. The changes of glutation reductase activity in maize seedlings under heavy metals and herbicide frontjere influence

    Directory of Open Access Journals (Sweden)

    V. S. Bilchuk

    2005-01-01

    Full Text Available In modelling experiment joint action of heavy metal ions (lead, cadmium and cloroacetanilide herbicide frontjere on glutationreductase activity in maize seedlings at initial stages of ontogenesis was investigated. The increasing of enzyme activity in a sprouting grain at herbicide and ions of lead and cadmium presence and variation of enzyme activity in seedlings were established at joint action of toxicants.

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

    Directory of Open Access Journals (Sweden)

    Marek Burzyński

    2014-01-01

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

  6. ACCUMULATION OF NITROGEN COMPOUNDS AND NITRATE REDUCTASE ACTIVITY IN LETTUCE CULTIVATED IN DIFFERENT CROP SYSTEMS ACÚMULO DE COMPOSTOS NITROGENADOS E ATIVIDADE DA REDUTASE DO NITRATO EM ALFACE PRODUZIDA EM DIFERENTES SISTEMAS DE CULTIVO

    Directory of Open Access Journals (Sweden)

    Fernanda Nunes Ibrahim

    2008-09-01

    Full Text Available

    Nitrate content determination is important for food quality evaluation, therefore when ingested nitrate is reduced the nitrite, which can generate harmful compounds to the human organism. Ahead of this, the present work had as objective to study the transport and accumulation of nitrogen compounds and the nitrate reductase activity in lettuce cultivar 'Vera' produced in Registro (SP in different cropping systems. Were collected samples of the xylem sap, aerial part and root for quantification of nitrogen compounds and of the reductase activity in vivo. The nitrate concentration in the xylem sap, the nitrate and amino acids contents, as well as the nitrate reductase activity, demonstrated more intense transport, accumulation and assimilation in plants cultivated in in hydroponic solution, followed of the conventional system and finally, of the organic. The stem of the plant in the three systems of culture presented high capacity of accumulation and assimilation the nitrogen compounds. The nitrate reductase activity in leaves was superior of the root. The content nitrate, independent of the culture system, varied of 24.32 the 800.06 mg kg-1 of FW in the different parts of the plant. However, it did not exceed the maximum

  7. Changes of sodium nitrate, nitrite, and N-nitrosodiethylamine during in vitro human digestion.

    Science.gov (United States)

    Kim, Hyeong Sang; Hur, Sun Jin

    2017-06-15

    This study aimed to determine the changes in sodium nitrate, sodium nitrite, and N-nitrosodiethylamine (NDEA) during in vitro human digestion, and the effect of enterobacteria on the changes in these compounds. The concentrations of nitrate, nitrite, and NDEA were significantly reduced from 150, 150, and 1ppm to 42.8, 63.2, and 0.85ppm, respectively, during in vitro human digestion (pdigestion. This study is the first to report that E. coli can dramatically reduce the amount of nitrite during in vitro human digestion and this may be due to the effect of nitrite reductase present in E. coli. We therefore conclude that the amounts of potentially harmful substances and their toxicity can be decreased during human digestion. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. The Effect of Nitrate Levels and Harvest Times on Fe, Zn, Cu, and K, Concentrations and Nitrate Reductase Activity in Lettuce and Spinach

    Directory of Open Access Journals (Sweden)

    Z. Gheshlaghi

    2015-09-01

    Full Text Available Leafy vegetables are considered as the main sources of nitrate in the human diet. In order to investigate the effect of nitrate levels and harvest times on nitrate accumulation, nitrate reductase activity, concentrations of Fe, Zn, Cu and K in Lettuce and Spinach and their relation to nitrate accumulation in these leafy vegetables, two harvest times (29 and 46 days after transplanting, two vegetable species of lettuce and spinach and two concentrations of nitrate (10 and 20 mM were used in a hydroponics greenhouse experiment with a completely randomized design and 3 replications. Modified Hoagland and Arnon nutrient solutions were used for the experiment. The results indicated that by increasing nitrate concentration of solution, nitrate accumulation in roots and shoots of lettuce and spinach increased significantly (P ≤ 0.05, and the same trend was observed for the nitrate reductase activity in the shoots of the two species. Increasing the nitrate concentrations of solution, reduced the shoot dry weight and the concentration of Fe and Cu in both species, where as it increased the K and Zn concentrations in the shoots of the two species in each both harvest times, the nitrate accumulation increased, but the nitrate reductase activity decreased in the shoots of the two species over the course of the growth. The Concentration of Fe, Cu and K decreased in the shoots of lettuce and the spinach with the time, despite the increase in Zn concentration in the shoots. The results also indicated that increasing nitrate concentrations of solution to the levels greater than the plant capacity for reduction and net uptake of nitrate, leads to the nitrate accumulation in the plants. Nitrate accumulation in plant tissue led to decreases in fresh shoot yield and Fe and Cu concentrations and nitrate reductase activities in both lettuce and spinach.

  9. ROS-mediated inhibition of S-nitrosoglutathione reductase contributes to the activation of anti-oxidative mechanisms

    Directory of Open Access Journals (Sweden)

    Izabella Kovacs

    2016-11-01

    Full Text Available Nitric oxide (NO has emerged as a signaling molecule in plants being involved in diverse physiological processes like germination, root growth, stomata closing and response to biotic and abiotic stress. S-nitrosoglutathione (GSNO as a biological NO donor has a very important function in NO signaling since it can transfer its NO moiety to other proteins (trans-nitrosylation. Such trans-nitrosylation reactions are equilibrium reactions and depend on GSNO level. The breakdown of GSNO and thus the level of S-nitrosylated proteins are regulated by GSNO-reductase (GSNOR. In this way, this enzyme controls S-nitrosothiol levels and regulates NO signaling. Here we report that Arabidopsis thaliana GSNOR activity is reversibly inhibited by H2O2 in-vitro and by paraquat-induced oxidative stress in-vivo. Light scattering analyses of reduced and oxidized recombinant GSNOR demonstrated that GSNOR proteins form dimers under both reducing and oxidizing conditions. Moreover, mass spectrometric analyses revealed that H2O2-treatment increased the amount of oxidative modifications on Zn2+-coordinating Cys47 and Cys177. Inhibition of GSNOR results in enhanced levels of S-nitrosothiols followed by accumulation of glutathione. Moreover, transcript levels of redox-regulated genes and activities of glutathione-dependent enzymes are increased in gsnor-ko plants, which may contribute to the enhanced resistance against oxidative stress. In sum, our results demonstrate that ROS-dependent inhibition of GSNOR is playing an important role in activation of anti-oxidative mechanisms to damping oxidative damage and imply a direct crosstalk between ROS- and NO-signaling.

  10. ROS-Mediated Inhibition of S-nitrosoglutathione Reductase Contributes to the Activation of Anti-oxidative Mechanisms

    Science.gov (United States)

    Kovacs, Izabella; Holzmeister, Christian; Wirtz, Markus; Geerlof, Arie; Fröhlich, Thomas; Römling, Gaby; Kuruthukulangarakoola, Gitto T.; Linster, Eric; Hell, Rüdiger; Arnold, Georg J.; Durner, Jörg; Lindermayr, Christian

    2016-01-01

    Nitric oxide (NO) has emerged as a signaling molecule in plants being involved in diverse physiological processes like germination, root growth, stomata closing and response to biotic and abiotic stress. S-nitrosoglutathione (GSNO) as a biological NO donor has a very important function in NO signaling since it can transfer its NO moiety to other proteins (trans-nitrosylation). Such trans-nitrosylation reactions are equilibrium reactions and depend on GSNO level. The breakdown of GSNO and thus the level of S-nitrosylated proteins are regulated by GSNO-reductase (GSNOR). In this way, this enzyme controls S-nitrosothiol levels and regulates NO signaling. Here we report that Arabidopsis thaliana GSNOR activity is reversibly inhibited by H2O2 in vitro and by paraquat-induced oxidative stress in vivo. Light scattering analyses of reduced and oxidized recombinant GSNOR demonstrated that GSNOR proteins form dimers under both reducing and oxidizing conditions. Moreover, mass spectrometric analyses revealed that H2O2-treatment increased the amount of oxidative modifications on Zn2+-coordinating Cys47 and Cys177. Inhibition of GSNOR results in enhanced levels of S-nitrosothiols followed by accumulation of glutathione. Moreover, transcript levels of redox-regulated genes and activities of glutathione-dependent enzymes are increased in gsnor-ko plants, which may contribute to the enhanced resistance against oxidative stress. In sum, our results demonstrate that reactive oxygen species (ROS)-dependent inhibition of GSNOR is playing an important role in activation of anti-oxidative mechanisms to damping oxidative damage and imply a direct crosstalk between ROS- and NO-signaling. PMID:27891135

  11. Ferric reductase activity of low molecular weight human milk fraction is associated with enhanced iron solubility and uptake in Caco-2 cells.

    Science.gov (United States)

    Pullakhandam, Raghu; Nair, Madhavan Krishnapillai; Kasula, Sunanda; Kilari, Sreenivasulu; Thippande, Tippeswamy Gowda

    2008-09-19

    It is known that the fractional absorption of extrinsic iron from human milk is higher in infants and adults. A low molecular weight milk fraction has been proposed to increase the bioavailability of iron from human milk. Nevertheless, the mechanisms remained elusive. Here in we demonstrate ferric reductase activity (Km7.73x10(-6)M) in low molecular weight human milk fraction (10kF, filtrate derived from ultra filtration of milk whey through 10kDa cutoff membrane), which increased ferric iron solubility and iron uptake in Caco-2 cells. The 10kF fraction was as effective as ascorbic acid (1:20 iron to ascorbic acid) in increasing the ferric iron solubility and uptake in Caco-2 cells. Further, gel filtration chromatography on peptide column led to co-elution of ferric reductase and iron solubilization activities at an apparent molecular mass of iron in Caco-2 cells. Thus, it is concluded that human milk possesses ferric reductase activity and is associated with ferric iron solubilization and enhanced absorption.

  12. Inhibition of Rat 5α-Reductase Activity and Testosterone-Induced Sebum Synthesis in Hamster Sebocytes by an Extract of Quercus acutissima Cortex

    Directory of Open Access Journals (Sweden)

    Junichi Koseki

    2015-01-01

    Full Text Available 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.

  13. 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.

    Science.gov (United States)

    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

  14. Generation of nitric oxide from nitrite by carbonic anhydrase

    DEFF Research Database (Denmark)

    Aamand, Rasmus; Dalsgaard, Thomas; Jensen, Frank B

    2009-01-01

    In catalyzing the reversible hydration of CO2 to bicarbonate and protons, the ubiquitous enzyme carbonic anhydrase (CA) plays a crucial role in CO2 transport, in acid-base balance, and in linking local acidosis to O2 unloading from hemoglobin. Considering the structural similarity between...... bicarbonate and nitrite, we hypothesized that CA uses nitrite as a substrate to produce the potent vasodilator nitric oxide (NO) to increase local blood flow to metabolically active tissues. Here we show that CA readily reacts with nitrite to generate NO, particularly at low pH, and that the NO produced...

  15. Methyl jasmonate and 1-methylcyclopropene treatment effects on quinone reductase inducing activity and post-harvest quality of broccoli.

    Science.gov (United States)

    Ku, Kang Mo; Choi, Jeong Hee; Kim, Hyoung Seok; Kushad, Mosbah M; Jeffery, Elizabeth H; Juvik, John A

    2013-01-01

    Effect of pre-harvest methyl jasmonate (MeJA) and post-harvest 1-methylcyclopropene (1-MCP) treatments on broccoli floret glucosinolate (GS) concentrations and quinone reductase (QR, an in vitro anti-cancer biomarker) inducing activity were evaluated two days prior to harvest, at harvest and at 10, 20, and 30 days of post-harvest storage at 4 °C. MeJA treatments four days prior to harvest of broccoli heads was observed to significantly increase floret ethylene biosynthesis resulting in chlorophyll catabolism during post-harvest storage and reduced product quality. Post-harvest treatment with 1-methylcyclopropene (1-MCP), which competitively binds to protein ethylene receptors, maintained post-harvest floret chlorophyll concentrations and product visual quality in both control and MeJA-treated broccoli. Transcript abundance of BoPPH, a gene which is responsible for the synthesis of pheophytinase, the primary enzyme associated with chlorophyll catabolism in broccoli, was reduced by 1-MCP treatment and showed a significant, negative correlation with floret chlorophyll concentrations. The GS, glucobrassicin, neoglucobrassicin, and gluconasturtiin were significantly increased by MeJA treatments. The products of some of the GS from endogenous myrosinase hydrolysis [sulforaphane (SF), neoascorbigen (NeoASG), N-methoxyindole-3-carbinol (NI3C), and phenethyl isothiocyanate (PEITC)] were also quantified and found to be significantly correlated with QR. Sulforaphane, the isothiocyanate hydrolysis product of the GS glucoraphanin, was found to be the most potent QR induction agent. Increased sulforaphane formation from the hydrolysis of glucoraphanin was associated with up-regulated gene expression of myrosinase (BoMyo) and the myrosinase enzyme co-factor gene, epithiospecifier modifier1 (BoESM1). This study demonstrates the combined treatment of MeJA and 1-MCP increased QR activity without post-harvest quality loss.

  16. Methyl Jasmonate and 1-Methylcyclopropene Treatment Effects on Quinone Reductase Inducing Activity and Post-Harvest Quality of Broccoli

    Science.gov (United States)

    Ku, Kang Mo; Choi, Jeong Hee; Kim, Hyoung Seok; Kushad, Mosbah M.; Jeffery, Elizabeth H.; Juvik, John A.

    2013-01-01

    Effect of pre-harvest methyl jasmonate (MeJA) and post-harvest 1-methylcyclopropene (1-MCP) treatments on broccoli floret glucosinolate (GS) concentrations and quinone reductase (QR, an in vitro anti-cancer biomarker) inducing activity were evaluated two days prior to harvest, at harvest and at 10, 20, and 30 days of post-harvest storage at 4 °C. MeJA treatments four days prior to harvest of broccoli heads was observed to significantly increase floret ethylene biosynthesis resulting in chlorophyll catabolism during post-harvest storage and reduced product quality. Post-harvest treatment with 1-methylcyclopropene (1-MCP), which competitively binds to protein ethylene receptors, maintained post-harvest floret chlorophyll concentrations and product visual quality in both control and MeJA-treated broccoli. Transcript abundance of BoPPH, a gene which is responsible for the synthesis of pheophytinase, the primary enzyme associated with chlorophyll catabolism in broccoli, was reduced by 1-MCP treatment and showed a significant, negative correlation with floret chlorophyll concentrations. The GS, glucobrassicin, neoglucobrassicin, and gluconasturtiin were significantly increased by MeJA treatments. The products of some of the GS from endogenous myrosinase hydrolysis [sulforaphane (SF), neoascorbigen (NeoASG), N-methoxyindole-3-carbinol (NI3C), and phenethyl isothiocyanate (PEITC)] were also quantified and found to be significantly correlated with QR. Sulforaphane, the isothiocyanate hydrolysis product of the GS glucoraphanin, was found to be the most potent QR induction agent. Increased sulforaphane formation from the hydrolysis of glucoraphanin was associated with up-regulated gene expression of myrosinase (BoMyo) and the myrosinase enzyme co-factor gene, epithiospecifier modifier1 (BoESM1). This study demonstrates the combined treatment of MeJA and 1-MCP increased QR activity without post-harvest quality loss. PMID:24146962

  17. Effect of cephalandra indica against advanced glycation end products, sorbitol accumulation and aldose reductase activity in homoeopathic formulation

    Directory of Open Access Journals (Sweden)

    Lalit Kishore

    2018-01-01

    Full Text Available Background: Extreme generation of free radicals leads to oxidative stress which has been apprehensive in several disease processes such as diabetic complications and vascular and neurodegenerative diseases. Objective: The present study was designed to evaluate the potential of homoeopathic preparations of Cephalandra indica L. against oxidative stress. Materials and Methods: Potencies of Cephalandra indica (mother tincture, 6C and 30C were procured from Dr. Willmar Schwabe India Pvt. Ltd. The antioxidant activity of Cephalandra indica was evaluated by employing various in vitro antioxidant methods. Results: The total phenol content was found to be 1905, 849 and 495 mg/g gallic acid equivalents in mother tincture, 6C and 30C of Cephalandra indica and total antioxidant capacity was found to be 2710, 759 and 510 μM/g ascorbic acid equivalents, respectively. Mother tincture, 6C and 30C of Cephalandra indica was found to have strong reducing power, 2,2-diphenyl-1-picrylhydrazyl radical, hydrogen peroxide, nitric oxide and superoxide radical scavenging activity. Percentage inhibition of AGEs formation by mother tincture, 6C and 30C of Cephalandra indica (10–50 μl was found to be 30.34%–91.77%, 29.98%–65.71% and 33.05%–57.75%, respectively. Mother tincture, 6C and 30C of Cephalandra indica showed inhibitory effect against sorbitol accumulation with IC50value of 26.12 μl, 203.10 μl and 897.3 μl, respectively, whereas, in aldose reductase inhibition assay, the IC50value was 32.54 μl, 175.02 μl and 834.34 μl, respectively. Conclusion: The results revealed that homoeopathic preparations of Cephalandra indica exhibit protective effect against oxidative stress.

  18. Crosstalk between nitrite, myoglobin and reactive oxygen species to regulate vasodilation under hypoxia.

    Directory of Open Access Journals (Sweden)

    Matthias Totzeck

    Full Text Available The systemic response to decreasing oxygen levels is hypoxic vasodilation. While this mechanism has been known for more than a century, the underlying cellular events have remained incompletely understood. Nitrite signaling is critically involved in vessel relaxation under hypoxia. This can be attributed to the presence of myoglobin in the vessel wall together with other potential nitrite reductases, which generate nitric oxide, one of the most potent vasodilatory signaling molecules. Questions remain relating to the precise concentration of nitrite and the exact dose-response relations between nitrite and myoglobin under hypoxia. It is furthermore unclear whether regulatory mechanisms exist which balance this interaction. Nitrite tissue levels were similar across all species investigated. We then investigated the exact fractional myoglobin desaturation in an ex vivo approach when gassing with 1% oxygen. Within a short time frame myoglobin desaturated to 58±12%. Given that myoglobin significantly contributes to nitrite reduction under hypoxia, dose-response experiments using physiological to pharmacological nitrite concentrations were conducted. Along all concentrations, abrogation of myoglobin in mice impaired vasodilation. As reactive oxygen species may counteract the vasodilatory response, we used superoxide dismutase and its mimic tempol as well as catalase and ebselen to reduce the levels of reactive oxygen species during hypoxic vasodilation. Incubation of tempol in conjunction with catalase alone and catalase/ebselen increased the vasodilatory response to nitrite. Our study shows that modest hypoxia leads to a significant nitrite-dependent vessel relaxation. This requires the presence of vascular myoglobin for both physiological and pharmacological nitrite levels. Reactive oxygen species, in turn, modulate this vasodilation response.

  19. Efficient Reduction of Vertebrate Cytoglobins by the Cytochrome b5/Cytochrome b5Reductase/NADH System.

    Science.gov (United States)

    Amdahl, Matthew B; Sparacino-Watkins, Courtney E; Corti, Paola; Gladwin, Mark T; Tejero, Jesús

    2017-08-01

    Cytoglobin is a heme-containing protein ubiquitous in mammalian tissues. Unlike the evolutionarily related proteins hemoglobin and myoglobin, cytoglobin shows a six-coordinated heme binding, with the heme iron coordinated by two histidine side chains. Cytoglobin is involved in cytoprotection pathways through yet undefined mechanisms, and it has recently been demonstrated that cytoglobin has redox signaling properties via nitric oxide (NO) and nitrite metabolism. The reduced, ferrous cytoglobin can bind oxygen and will react with NO in a dioxygenation reaction to form nitrate, which dampens NO signaling. When deoxygenated, cytoglobin can bind nitrite and reduce it to NO. This oxidoreductase activity could be catalytic if an effective reduction system exists to regenerate the reduced heme species. The nature of the physiological cytoglobin reducing system is unknown, although it has been proposed that ascorbate and cytochrome b 5 could fulfill this role. Here we describe that physiological concentrations of cytochrome b 5 and cytochrome b 5 reductase can reduce human and fish cytoglobins at rates up to 250-fold higher than those reported for their known physiological substrates, hemoglobin and myoglobin, and up to 100-fold faster than 5 mM ascorbate. These data suggest that the cytochrome b 5 /cytochrome b 5 reductase system is a viable reductant for cytoglobin in vivo, allowing for catalytic oxidoreductase activity.

  20. Aldose reductase modulates acute activation of mesenchymal markers via the β-catenin pathway during cardiac ischemia-reperfusion.

    Directory of Open Access Journals (Sweden)

    Devi Thiagarajan

    Full Text Available Aldose reductase (AR: human, AKR1B1; mouse, AKR1B3, the first enzyme in the polyol pathway, plays a key role in mediating myocardial ischemia/reperfusion (I/R injury. In earlier studies, using transgenic mice broadly expressing human AKR1B1 to human-relevant levels, mice devoid of Akr1b3, and pharmacological inhibitors of AR, we demonstrated that AR is an important component of myocardial I/R injury and that inhibition of this enzyme protects the heart from I/R injury. In this study, our objective was to investigate if AR modulates the β-catenin pathway and consequent activation of mesenchymal markers during I/R in the heart. To test this premise, we used two different experimental models: in vivo, Akr1b3 null mice and wild type C57BL/6 mice (WT were exposed to acute occlusion of the left anterior descending coronary artery (LAD followed by recovery for 48 hours or 28 days, and ex-vivo, WT and Akr1b3 null murine hearts were perfused using the Langendorff technique (LT and subjected to 30 min of global (zero-flow ischemia followed by 60 min of reperfusion. Our in vivo results reveal reduced infarct size and improved functional recovery at 48 hours in mice devoid of Akr1b3 compared to WT mice. We demonstrate that the cardioprotection observed in Akr1b3 null mice was linked to acute activation of the β-catenin pathway and consequent activation of mesenchymal markers and genes linked to fibrotic remodeling. The increased activity of the β-catenin pathway at 48 hours of recovery post-LAD was not observed at 28 days post-infarction, thus indicating that the observed increase in β-catenin activity was transient in the mice hearts devoid of Akr1b3. In ex vivo studies, inhibition of β-catenin blocked the cardioprotection observed in Akr1b3 null mice hearts. Taken together, these data indicate that AR suppresses acute activation of β-catenin and, thereby, blocks consequent induction of mesenchymal markers during early reperfusion after myocardial

  1. Nitrification activity and community structure of nitrite-oxidizing bacteria in the bioreactors operated with addition of pharmaceuticals

    Energy Technology Data Exchange (ETDEWEB)

    Kraigher, Barbara, E-mail: barbara.kraigher@bf.uni-lj.si [University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, Chair of Microbiology, Vecna pot 111, 1000 Ljubljana (Slovenia); Mandic-Mulec, Ines [University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, Chair of Microbiology, Vecna pot 111, 1000 Ljubljana (Slovenia)

    2011-04-15

    Pharmaceuticals represent a group of the new emerging contaminants, which might influence microbial communities in the activated sludge. Nitrification activity and Nitrospira community structure in the small-scale reactors supplied with different concentrations (0, 50, 200, 500 {mu}g L{sup -1}) of the selected pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid) were evaluated. Ammonia removal was not influenced by selected pharmaceuticals. However, in the two reactors operated with 50 {mu}g L{sup -1} of pharmaceuticals (R50 and R50P), the effluent concentration of N-(NO{sub 2}{sup -} + NO{sub 3}{sup -}) was significantly higher than in the other reactors. Nitrospira community structure was assessed by terminal restriction fragment length polymorphism (T-RFLP) and by cloning and sequencing of the partial genes for 16S rRNA. Nitrospira spp. were detected in all reactors. The two dominant T-RFs represented the sublineages I and II of the genus Nitrospira. Main shifts were observed in the reactors R50 and R50P, where the T-RF representing sublineage II was much higher as compared to the other reactors. Consistent with this, the Nitrospira sublineage II was detected only in the clone libraries from the reactors R50 and R50P. Our results suggest that the relative abundance of Nitrospira sublineage II could be related to the effluent N-(NO{sub 2}{sup -} + NO{sub 3}{sup -}) concentration.

  2. Nitrification activity and community structure of nitrite-oxidizing bacteria in the bioreactors operated with addition of pharmaceuticals

    International Nuclear Information System (INIS)

    Kraigher, Barbara; Mandic-Mulec, Ines

    2011-01-01

    Pharmaceuticals represent a group of the new emerging contaminants, which might influence microbial communities in the activated sludge. Nitrification activity and Nitrospira community structure in the small-scale reactors supplied with different concentrations (0, 50, 200, 500 μg L -1 ) of the selected pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid) were evaluated. Ammonia removal was not influenced by selected pharmaceuticals. However, in the two reactors operated with 50 μg L -1 of pharmaceuticals (R50 and R50P), the effluent concentration of N-(NO 2 - + NO 3 - ) was significantly higher than in the other reactors. Nitrospira community structure was assessed by terminal restriction fragment length polymorphism (T-RFLP) and by cloning and sequencing of the partial genes for 16S rRNA. Nitrospira spp. were detected in all reactors. The two dominant T-RFs represented the sublineages I and II of the genus Nitrospira. Main shifts were observed in the reactors R50 and R50P, where the T-RF representing sublineage II was much higher as compared to the other reactors. Consistent with this, the Nitrospira sublineage II was detected only in the clone libraries from the reactors R50 and R50P. Our results suggest that the relative abundance of Nitrospira sublineage II could be related to the effluent N-(NO 2 - + NO 3 - ) concentration.

  3. Biological activity of Fe(III) aquo-complexes towards ferric chelate reductase (FCR).

    Science.gov (United States)

    Escudero, Rosa; Gómez-Gallego, Mar; Romano, Santiago; Fernández, Israel; Gutiérrez-Alonso, Ángel; Sierra, Miguel A; López-Rayo, Sandra; Nadal, Paloma; Lucena, Juan J

    2012-03-21

    In this study we have obtained experimental evidence that confirms the high activity of aquo complexes III and IV towards the enzyme FCR, responsible for the reduction of Fe(III) to Fe(II) in the process of iron acquisition by plants. The in vivo FCR assays in roots of stressed cucumber plants have shown a higher efficiency of the family of complexes III and a striking structure-activity relationship with the nature of the substituent placed in a phenyl group far away from the metal center. The results obtained in this work demonstrate that all the aquo compounds tested interact efficiently with the enzyme FCR and hence constitute a new concept of iron chelates that could be of great use in agronomy.

  4. Dissecting the Structural Elements for the Activation of β-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae.

    Science.gov (United States)

    Hou, Jing; Zheng, Heping; Chruszcz, Maksymilian; Zimmerman, Matthew D; Shumilin, Igor A; Osinski, Tomasz; Demas, Matt; Grimshaw, Sarah; Minor, Wladek

    2016-02-01

    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. 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. Copyright © 2016, American Society for

  5. Kinetic and docking studies of the interaction of quinones with the quinone reductase active site.

    Science.gov (United States)

    Zhou, Zhigang; Fisher, Derek; Spidel, Jared; Greenfield, Jodi; Patson, Brian; Fazal, Aleem; Wigal, Carl; Moe, Owen A; Madura, Jeffry D

    2003-02-25

    NAD(P)H/quinone acceptor oxidoreductase type 1 (QR1) protects cells from cytotoxic and neoplastic effects of quinones though two-electron reduction. Kinetic experiments, docking, and binding affinity calculations were performed on a series of structurally varied quinone substrates. A good correlation between calculated and measured binding affinities from kinetic determinations was obtained. The experimental and theoretical studies independently support a model in which quinones (with one to three fused aromatic rings) bind in the QR1 active site utilizing a pi-stacking interaction with the isoalloxazine ring of the FAD cofactor.

  6. Practical Use of Nitrite and Basis for Dosage in the Manufacture of Meat Products

    DEFF Research Database (Denmark)

    Adler-Nissen, Jens; Ekgreen, Maria Helbo; Risum, Jørgen

    . Nitrite also has a desirable anti-oxidant activity and contributes to the formation of pleasant flavours. A systematic literature review on the function and use of nitrite in meat leads to a tentative first conclusion that if the level of nitrite added to meat products is sufficient to protect against......The use of nitrite (NaNO2) in the manufacture of salted (cured) meat products has a long tradition in the industry, dating back to the early twentieth century. Nitrite serves several technological purposes, primarily by the formation of a stable red colour in the meat and the inhibition...... of the growth of Clostridium botulinum. According to an assessment report by the European Food Safety Authority (The EFSA Journal, 14, p. 1-134, 2003) all evidence points to that it is the added amount of nitrite rather than the residual amount of nitrite in the product which exerts the antimicrobial effect...

  7. Syringic Acid Extracted from Herba dendrobii Prevents Diabetic Cataract Pathogenesis by Inhibiting Aldose Reductase Activity

    Directory of Open Access Journals (Sweden)

    Xiaoyong Wei

    2012-01-01

    Full Text Available Objective. Effects of Syringic acid (SA extracted from dendrobii on diabetic cataract (DC pathogenesis were explored. Methods. Both in vitro and in vivo DC lens models were established using D-gal, and proliferation of HLEC exposed to SA was determined by MMT assay. After 60-day treatment with SA, rat lens transparency was observed by anatomical microscopy using a slit lamp. SA protein targets were extracted and isolated using 2-DE and MALDI TOF/TOF. AR gene expression was investigated using qRT-PCR. Interaction sites and binding characteristics were determined by molecule-docking techniques and dynamic models. Results. Targeting AR, SA provided protection from D-gal-induced damage by consistently maintaining lens transparency and delaying lens turbidity development. Inhibition of AR gene expression by SA was confirmed by qRT-PCR. IC50 of SA for inhibition of AR activity was 213.17 μg/mL. AR-SA binding sites were Trp111, His110, Tyr48, Trp20, Trp79, Leu300, and Phe122. The main binding modes involved hydrophobic interactions and hydrogen bonding. The stoichiometric ratio of non-covalent bonding between SA and AR was 1.0 to 13.3. Conclusion. SA acts to prevent DC in rat lenses by inhibiting AR activity and gene expression, which has potential to be developed into a novel drug for therapeutic management of DC.

  8. Nitrate reductase, arginine deaminase, urease and dehydrogenase activities in natural soil (ridges with forest) and in cotton soil after acetamiprid treatments.

    Science.gov (United States)

    Singh, Dileep K; Kumar, Sunil

    2008-03-01

    Soil enzymes are indicators of microbial activities in soil and are often considered as an indicator of soil health and fertility. They are very sensitive to the agricultural practices, pH of the soil, nutrients, inhibitors and weather conditions. To understand the effect of an insecticide, acetamiprid (IUPAC Name: (E)-N1-[(6-chloro-3-pyridyl) methyl]-N2-cyano-N1-methyl acetamidine) on different soil enzyme activities, the experiments were conducted for three consecutive years (2003--2005) at control and cotton experimental fields of Indian Agricultural Research Institute (IARI) and natural area (ridges with forest) in Delhi. The combined results for all three years were presented here to understand the impact of acetamiprid on soil enzyme activities. Acetamiprid was applied three times in one crop season after 41, 48 and 73 days of sowing, to control the pest. Soil of treated fields was analyzed for insecticide residues immediately after first insecticide treatment and thereafter at definite period. The residues of acetamiprid in experimental soil was varied from 0.30+/-0.13 to 22.67+/-0.2 microg g(-1)d.wt. soil, during the crop period of 2003. The insecticide residues for 2004 ranged between 0.59+/-0.38 and 13.42+/-0.71 microg g(-1)d.wt. soil and for 2005 it ranged between 0.48+/-0.22 and 19.81+/-0.33 microg g(-1)d.wt. soil. An average half life of acetamiprid in our treated field was 11.2+/-1.7 days for all three years. Similarly, the soil from natural area and control were also tested for insecticide residues. No detectable insecticide residues had been found. Soil from three localities i.e. natural, control and experimental fields were tested for different enzyme activities. Nitrate reductase, arginine deaminase, urease and dehydrogenase activities were high in natural soil in comparison to control soil and insecticide treated soil in all three experimental years. At the same time, nitrate reductase activity was all time low in acetamiprid treated soil

  9. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1

    Directory of Open Access Journals (Sweden)

    Matthew J. Randall

    2013-01-01

    Full Text Available Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal. Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1, a critical enzyme involved in regulation of thioredoxin (Trx-mediated redox signaling, by alkylation at its selenocysteine (Sec residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1–30 μM resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases

  10. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1.

    Science.gov (United States)

    Randall, Matthew J; Spiess, Page C; Hristova, Milena; Hondal, Robert J; van der Vliet, Albert

    2013-01-01

    Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1-30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK, and

  11. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1☆☆☆

    Science.gov (United States)

    Randall, Matthew J.; Spiess, Page C.; Hristova, Milena; Hondal, Robert J.; van der Vliet, Albert

    2013-01-01

    Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1–30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated1 kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK

  12. Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms

    International Nuclear Information System (INIS)

    Yagi, T.

    1987-01-01

    The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition correlated with the presence of an energy-transducing site in this segment of the respiratory chain. Where the NADH-quinone reductase segment involved an energy-coupling site (e.g., in bovine heart and rat liver mitochondria, and in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes), DCCD acted as an inhibitor of ubiquinone reduction by NADH. By contrast, where energy-coupling site 1 was absent (e.g., in Saccharomyces cerevisiae mitochondria and BacilLus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by DCCD. In the bovine and P. denitrificans systems, DCCD inhibition was pseudo first order with respect to incubation time, and reaction order with respect to inhibitor concentration was close to unity, indicating that inhibition resulted from the binding of one inhibitor molecule per active unit of NADH-ubiquinone reductase. In the bovine NADH-ubiquinone reductase complex (complex I), [ 14 C]DCCD was preferentially incorporated into two subunits of molecular weight 49,000 and 29,000. The time course of labeling of the 29,000 molecular weight subunit with [ 14 C]DCCD paralleled the time course of inhibition of NADH-ubiquinone reductase activity

  13. Retention and leaching of nitrite by municipal solid waste incinerator bottom ash under the landfill circumstance.

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A.

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Mitochondria recycle nitrite back to the bioregulator nitric monoxide

    International Nuclear Information System (INIS)

    Nohl, H.; Staniek, K.; Sobhian, B.; Bahrami, S.; Redl, H.; Kozlov, A.V.

    2000-01-01

    Nitric monoxide (NO) exerts a great variety of physiological functions. L-Arginine supplies amino groups which are transformed to NO in various NO-synthase-active isoenzyme complexes. NO-synthesis is stimulated under various conditions increasing the tissue of stable NO-metabolites. The major oxidation product found is nitrite. Elevated nitrite levels were reported to exist in a variety of diseases including HIV, reperfusion injury and hypovolemic shock. Denitrifying bacteria such as Paracoccus denitrificans have a membrane bound set of cytochromes (cyt cd 1 , cyt bc) which were shown to be involved in nitrite reduction activities. Mammalian mitochondria have similar cytochromes which form part of the respiratory chain. Like in bacteria quinols are used as reductants of these types of cytochromes. The observation of one-e - divergence from this redox-couple to external dioxygen made us to study whether this site of the respiratory chain may also recycle nitrite back to its bioactive form NO. Thus, the aim of the present study was therefore to confirm the existence of a reductive pathway which reestablishes the existence of the bioregulator NO from its main metabolite NO 2 - . Our results show that respiring mitochondria readily reduce added nitrite to NO which was made visible by nitrosylation of deoxyhemoglobin. The adduct gives characteristic triplet-ESR-signals. Using inhibitors of the respiratory chain for chemical sequestration of respiratory segments we were able to identify the site where nitrite is reduced. The results confirm the ubiquinone/cyt bc 1 couple as the reductant site where nitrite is recycled. The high affinity of NO to the heme-iron of cytochrome oxidase will result in an impairment of mitochondrial energy-production. ''Nitrite tolerance'' of angina pectoris patients using NO-donors may be explained in that way. (author)

  16. Mutagenicity of some alkyl nitrites used as recreational drugs

    Energy Technology Data Exchange (ETDEWEB)

    Dunkel, V.C.; Cameron, T.P. (National Institute of Health, Bethesda (USA)); Rogers-Back, A.M.; Lawlor, T.E.; Harbell, J.W. (Microbiological Associates Inc., Rockville, MD (USA))

    1989-01-01

    When the AIDS epidemic was in its earliest stages, and prior to identification of HIV as the etiological factor, the use of volatile nitrites by the male homosexual community to enhance sexual activities appeared to have a significant role in this disease. Preliminary observations indicated that that portion of the male homosexual community which developed Kaposi's sarcoma were also heavy nitrite users. These nitrites had been demonstrated to be mutagenic in bacteria and thus it was postulated that they could be responsible for the appearance of the sarcoma. To evaluate further the genotoxic activity of these chemicals, six nitrites, including those most commonly used by homosexuals for sexual gratification, were selected for testing in the mouse lymphoma TK {plus minus} and Salmonell typhimurium mutagenicity assays. One chemical, n-amyl nitrite, was negative in the mouse lymphoma assay, while the other five chemicals, n-butyl, isobutyl, iso-amyl, sec-butyl, and n-propyl nitrite, were positive. All six compounds were positive in the Salmonella assay. The mutagenic and known toxic effects of these chemicals remain a concern because a large population of teenagers and young adults continue to abuse these substances.

  17. Reduced iron associated with secondary nitrite maxima in the Arabian Sea

    Digital Repository Service at National Institute of Oceanography (India)

    Moffetta, J.W.; Goepferta, T.J.; Naqvi, S.W.A.

    in large regions (de Baar and de Jong, 2001). Concentrations are low because of high biological demand and because the thermodynami- cally favorable oxidation state, Fe(III), is strongly hydrolyzed at seawater pH and is relatively in- soluble (Waite, 2001.... Denitrifiers, with their high Fe requirement associated with nitrate reductase and nitrite reductase would benefit from the presence of Fe(II). Moreover, Witter et al. Codispoti, L.A., Brandes, J.A., Christensen, J.P., Devol, H., ARTICLE IN PRESS J.W. Moffett...

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

    Science.gov (United States)

    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.

  19. Metabolic fates and effects of nitrite in brown trout under normoxic and hypoxic conditions: blood and tissue nitrite metabolism and interactions with branchial NOS, Na+/K+-ATPase and hsp70 expression

    DEFF Research Database (Denmark)

    Jensen, Frank Bo; Gerber, Lucie; Hansen, Marie Niemann

    2015-01-01

    Nitrite secures essential nitric oxide (NO) bioavailability in hypoxia at low endogenous concentrations, whereas it becomes toxic at high concentrations. We exposed brown trout to normoxic and hypoxic water in the absence and presence of added ambient nitrite to decipher the cellular metabolism...... and effects of nitrite at basal and elevated concentrations under different oxygen regimes. We also tested hypotheses concerning the influence of nitrite on branchial nitric oxide synthase (NOS), Na+/K+-ATPase (nka) and heat shock protein (hsp70) mRNA expression. Basal plasma and erythrocyte nitrite levels...... were higher in hypoxia than normoxia, suggesting increased NOS activity. Nitrite exposure strongly elevated nitrite concentrations in plasma, erythrocytes, heart tissue and white muscle, which was associated with an extensive metabolism of nitrite to nitrate and to iron-nitrosylated and S...

  20. Lens Aldose Reductase Inhibitory and Free Radical Scavenging Activity of Fractions of Chromolaena odorata (Siam Weed: Potential for Cataract Remediation

    Directory of Open Access Journals (Sweden)

    Emmanuel O. AJANI

    2016-09-01

    Full Text Available Searching for effective and safe aldose reductase (AR inhibitor agent is a major thrust area in the mainstream of anti-cataractogenic research. This study was set up to investigate the in vitro aldose reductase inhibitory (ARI activity of fractions of methanolic extract of Chromolaena odorata leaves, on partially purified AR from goat lens, for potential use in the development of anticataractogenic agent. The phyto-constituents of the leaves were screened in aqueous and methanolic extracts and the free radical scavenging activities of the fractions were evaluated. The kinetics of the enzyme in the presence of fractions of the leaves was then compared. Phenol, flavonoid, alkaloid, saponin, terpenoid, quinones and phlobatannins were detected in both extracts. All the fractions inhibited AR in an uncompetitive manner, showing a reduced V max and Km when compared with glyceraldehyde. ARI activity was found to be the highest with aqueous fraction (IC50, 0.22 ± 0.01 mg/ml. All other fractions showed mild to moderate AR inhibition capacity, while it was found to be the lowest within hexane fraction (IC50, 1.20 ± 0.10 mg/ml. All the fractions showed free radical scavenging activity and metal chelating activity. The study confirmed the ARI and antioxidant capacity of Chromolaena odorata which may be due to its phenolic constituents, indicating that the plant may serve as a base for the development of anticataract agent.

  1. The Effects of Dose Rhizoctonia Binucleat (BNR and Phosphorus to Nitrate Reductase Activity (NRA and Chlorophyll of Vanilla Seedling (Vanilla planifolia Andrews

    Directory of Open Access Journals (Sweden)

    Haryuni Haryuni

    2016-09-01

    Full Text Available Vanilla (Vanilla planifolia Andrews is one of the important exported commodities in Indonesia. Indonesia is one of top five major vanilla exporters in the world, that produce the high quality of Indonesian vanilla with high vanillin content (2.75%. The aims of this research were to determine the effects of dose binukleat Rhizoctonia (BNR and phosphorus as well as the interaction of the nitrate reductase activity (NRA and chlorophyll of the vanilla seedling (Vanilla planifolia Andrew. Method in this research used completely randomized factorial design, by involving two factors (dose of BNR inoculation and Phosphor. The first factor is without inoculation and inoculation BNR (M0, M1, M2, M3 wich consists of (0,5, 10, 15 g/polybag, the second factor is the dose of phosphorus fertilizer (P0, P1, P2, P3 which consists of (0, 3, 6, 9 g/polibag. The results showed that the inoculation dose of BNR and doses of phosphorus not significant and lower levels of NRA and chlorophyll while the interaction dose of BNR and phosphorus significantly and increase levels of NRA and chlorophyll of vanilla seedling. Nitrate Reductase Activity and chlorophyll has important role in metabolism process as a plant growth indicator.How to CiteHaryuni, H., & Dewi, T. S. K. (2016. The Effects of Dose Rhizoctonia Binucleat (BNR and Phosphorus to Nitrate Reductase Activity (NRA and Chlorophyll of Vanilla Seedling (Vanilla planifolia Andrews. Biosaintifika: Journal of Biology & Biology Education, 8(2, 141-147.

  2. Arabidopsis cpFtsY mutants exhibit pleiotropic defects including an inability to increase iron deficiency-inducible root Fe(III) chelate reductase activity.

    Science.gov (United States)

    Durrett, Timothy P; Connolly, Erin L; Rogers, Elizabeth E

    2006-08-01

    All plants, except for the grasses, must reduce Fe(III) to Fe(II) in order to acquire iron. In Arabidopsis, the enzyme responsible for this reductase activity in the roots is encoded by FRO2. Two Arabidopsis mutants, frd4-1 and frd4-2, were isolated in a screen for plants that do not induce Fe(III) chelate reductase activity in their roots in response to iron deficiency. frd4 mutant plants are chlorotic and grow more slowly than wild-type Col-0 plants. Additionally, frd4 chloroplasts are smaller in size and possess dramatically fewer thylakoid membranes and grana stacks when compared with wild-type chloroplasts. frd4 mutant plants express both FRO2 and IRT1 mRNA normally in their roots under iron deficiency, arguing against any defects in systemic iron-deficiency signaling. Further, transgenic frd4 plants accumulate FRO2-dHA fusion protein under iron-deficient conditions, suggesting that the frd4 mutation acts post-translationally in reducing Fe(III) chelate reductase activity. FRO2-dHA appears to localize to the plasma membrane of root epidermal cells in both Col-0 and frd4-1 transgenic plants when grown under iron-deficient conditions. Map-based cloning revealed that the frd4 mutations reside in cpFtsY, which encodes a component of one of the pathways responsible for the insertion of proteins into the thylakoid membranes of the chloroplast. The presence of cpFtsY mRNA and protein in the roots of wild-type plants suggests additional roles for this protein, in addition to its known function in targeting proteins to the thylakoid membrane in chloroplasts.

  3. Camphene, a plant-derived monoterpene, reduces plasma cholesterol and triglycerides in hyperlipidemic rats independently of HMG-CoA reductase activity.

    Directory of Open Access Journals (Sweden)

    Ioanna Vallianou

    Full Text Available Central to the pathology of coronary heart disease is the accumulation of lipids, cholesterol and triglycerides, within the intima of arterial blood vessels. The search for drugs to treat dislipidemia, remains a major pharmaceutical focus. In this study, we evaluated the hypolipidemic properties of the essential oil from Chios mastic gum (MGO.The hypolipidemic effect of MGO was investigated in naïve as well as in rats susceptible to detergent-induced hyperlipidemia. Serum cholesterol and triglycerides were determined using commercial kits. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A reductase activity was measured in HepG2 cell extracts using a radioactive assay; cellular cholesterol and cholesterol esters were assessed using gas chromatography. MGO administration into naïve rats resulted in a dose-dependent reduction in the constitutive synthesis of serum cholesterol and triglycerides. In hyperlipidemic rats, MGO treatment had also a strong hypolipidemic effect. By testing various components of MGO, we show for the first time that the hypolipidemic action is associated with camphene. Administration of camphene at a dose of 30 µg/gr of body weight in hyperlipidemic rats resulted in a 54.5% reduction of total cholesterol (p<0.001, 54% of Low Density Lipoprotein (LDL-cholesterol (p<0.001 and 34.5% of triglycerides (p<0.001. Treatment of HepG2 cells with camphene led to a decrease in cellular cholesterol content to the same extend as mevinolin, a known HMG-CoA reductase inhibitor. The hypolipidemic action of camphene is independent of HMG-CoA reductase activity, suggesting that its hypocholesterolemic and hypotriglyceridemic effects are associated with a mechanism of action different than that of statins.Given the critical role that the control of hyperlipidemia plays in cardiovascular disease, the results of our study provide insights into the use of camphene as an alternative lipid lowering agent and merits further evaluation.

  4. Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity.

    Science.gov (United States)

    Matsunaga, Toshiyuki; Endo, Satoshi; Maeda, Satoshi; Ishikura, Shuhei; Tajima, Kazuo; Tanaka, Nobutada; Nakamura, Kazuo T; Imamura, Yorishige; Hara, Akira

    2008-09-15

    Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and alpha-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C(19)/C(21)-steroids into 3beta-hydroxysteroids. The stereospecific conversion to 3beta-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor alpha ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3beta-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.

  5. Implications of Limited Thermophilicity of Nitrite Reduction for Control of Sulfide Production in Oil Reservoirs

    Science.gov (United States)

    Fida, Tekle Tafese; Chen, Chuan; Okpala, Gloria

    2016-01-01

    ABSTRACT Nitrate reduction to nitrite in oil fields appears to be more thermophilic than the subsequent reduction of nitrite. Concentrated microbial consortia from oil fields reduced both nitrate and nitrite at 40 and 45°C but only nitrate at and above 50°C. The abundance of the nirS gene correlated with mesophilic nitrite reduction activity. Thauera and Pseudomonas were the dominant mesophilic nitrate-reducing bacteria (mNRB), whereas Petrobacter and Geobacillus were the dominant thermophilic NRB (tNRB) in these consortia. The mNRB Thauera sp. strain TK001, isolated in this study, reduced nitrate and nitrite at 40 and 45°C but not at 50°C, whereas the tNRB Petrobacter sp. strain TK002 and Geobacillus sp. strain TK003 reduced nitrate to nitrite but did not reduce nitrite further from 50 to 70°C. Testing of 12 deposited pure cultures of tNRB with 4 electron donors indicated reduction of nitrate in 40 of 48 and reduction of nitrite in only 9 of 48 incubations. Nitrate is injected into high-temperature oil fields to prevent sulfide formation (souring) by sulfate-reducing bacteria (SRB), which are strongly inhibited by nitrite. Injection of cold seawater to produce oil creates mesothermic zones. Our results suggest that preventing the temperature of these zones from dropping below 50°C will limit the reduction of nitrite, allowing more effective souring control. IMPORTANCE Nitrite can accumulate at temperatures of 50 to 70°C, because nitrate reduction extends to higher temperatures than the subsequent reduction of nitrite. This is important for understanding the fundamentals of thermophilicity and for the control of souring in oil fields catalyzed by SRB, which are strongly inhibited by nitrite. PMID:27208132

  6. Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

    Science.gov (United States)

    Donnelly, Mark

    2006-08-01

    The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.

  7. Crystal structure and functional characterization of yeast YLR011wp, an enzyme with NAD(P)H-FMN and ferric iron reductase activities.

    Science.gov (United States)

    Liger, Dominique; Graille, Marc; Zhou, Cong-Zhao; Leulliot, Nicolas; Quevillon-Cheruel, Sophie; Blondeau, Karine; Janin, Joël; van Tilbeurgh, Herman

    2004-08-13

    Flavodoxins are involved in a variety of electron transfer reactions that are essential for life. Although FMN-binding proteins are well characterized in prokaryotic organisms, information is scarce for eukaryotic flavodoxins. We describe the 2.0-A resolution crystal structure of the Saccharomyces cerevisiae YLR011w gene product, a predicted flavoprotein. YLR011wp indeed adopts a flavodoxin fold, binds the FMN cofactor, and self-associates as a homodimer. Despite the absence of the flavodoxin key fingerprint motif involved in FMN binding, YLR011wp binds this cofactor in a manner very analogous to classical flavodoxins. YLR011wp closest structural homologue is the homodimeric Bacillus subtilis Yhda protein (25% sequence identity) whose homodimer perfectly superimposes onto the YLR011wp one. Yhda, whose function is not documented, has 53% sequence identity with the Bacillus sp. OY1-2 azoreductase. We show that YLR011wp has an NAD(P)H-dependent FMN reductase and a strong ferricyanide reductase activity. We further demonstrate a weak but specific reductive activity on azo dyes and nitrocompounds.

  8. Trypanothione Reductase and Superoxide Dismutase as Current Drug Targets for Trypanosoma cruzi: An Overview of Compounds with Activity against Chagas Disease.

    Science.gov (United States)

    Beltran-Hortelano, Ivan; Perez-Silanes, Silvia; Galiano, Silvia

    2017-05-31

    It has been over a century since Carlos Chagas discovered the Trypanosoma cruzi (T. cruzi) as the causative agent of Chagas disease (CD), a neglected tropical disease with several socioeconomic, epidemiological and human health repercussions. Currently, there are only two commercialized drugs to treat CD in acute phase, nifurtimox and benznidazol, with several adverse side effects. Thus, new orally available and safe drugs for this parasitic infection are urgently required. One strategy of great importance in new drug discovery programmes is based on the search of molecules enabling to interfere with enzymes involved in T. cruzi metabolism. This review will focus on two of the most promising targets for the therapy of CD: trypanothione reductase (TR) and the iron-containing superoxide dismutase (Fe- SOD), which protect the parasite against oxidative damage by reactive oxygen species. A brief comparison of the function, mechanism of action and the active sites between T. cruzi TR and Fe-SOD with their analogues enzymes in human, glutathione reductase (GR) and the corresponding SODs, will be discussed. This review will also summarize the recent development and structure-activity relationships of novel compounds reported for their ability to selectively inhibit these targets, aiming to define molecular bases in the search for new effective treatment of CD. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. A novel marine nitrite-oxidizing

    NARCIS (Netherlands)

    Haaijer, S.C.M.; Ji, K.; van Niftrik, L.; Hoischen, A.; Speth, D.R.; Jetten, M.S.M.; Sinninghe Damsté, J.S.; Op den Camp, H.J.M.

    2013-01-01

    Marine microorganisms are important for the global nitrogen cycle, but marine nitrifiers, especially aerobic nitrite oxidizers, remain largely unexplored. To increase the number of cultured representatives of marine nitrite-oxidizing bacteria (NOB), a bioreactor cultivation approach was adopted to

  10. Influence of beet sugar, calcium lactate, and Staphylococcus xylosus (with nitrate reductase activity) on the chemical, microbiological, and sensorial properties of Persian uncured frankfurters.

    Science.gov (United States)

    Tahmouzi, Saeed; Razavi, Seyed Hadi; Safari, Mohammad; Emam-Djomeh, Zahra

    2012-10-01

    This study investigated the effects of beet sugar (BS), calcium lactate (CL), and Staphylococcus xylosus (SX) on the general characteristics of uncured frankfurters. Minimum residual nitrite was observed in samples with a high level of BS and S. xylosus (8log(10) l0 viable cfu/g) after 2 mo. There was an increase in total aerobic counts in frankfurters after 4 wk. Samples containing higher BS and CL had no Clostridium perfringens at storage time. Histamine content was similar for all treatments except SX. No differences were observed in textural properties among the treatments. Water activity was decreased significantly in treatment CL1+BS2. The results indicated that samples treated with BS or CL had (P marketing these products. © 2012 Institute of Food Technologists®

  11. The Reaction of Oxy Hemoglobin with Nitrite

    DEFF Research Database (Denmark)

    Hathazi, Denisa; Scurtu, Florina; Bischin, Cristina

    2018-01-01

    -peroxynitrate. Density functional theory (DFT) calculations support this latter assignment. The reaction allows for differentiating between the reactivities of various chemically modified hemoglobins, including candidates for blood substitutes. Polymerization of hemoglobin slows the nitrite-induced oxidation, in sharp...... contrast to oxidative-stress type reactions which are generally accelerated, not inhibited. Sheep hemoglobin is found to be distinctly more resistant to reaction with nitrite compared to bovine Hb, at large nitrite concentrations (stopped-flow experiments directly observing the oxy + nitrite reaction...

  12. Implications of Limited Thermophilicity of Nitrite Reduction for Control of Sulfide Production in Oil Reservoirs

    OpenAIRE

    Fida, Tekle Tafese; Chen, Chuan; Okpala, Gloria; Voordouw, Gerrit

    2016-01-01

    Nitrate reduction to nitrite in oil fields appears to be more thermophilic than the subsequent reduction of nitrite. Concentrated microbial consortia from oil fields reduced both nitrate and nitrite at 40 and 45°C but only nitrate at and above 50°C. The abundance of the nirS gene correlated with mesophilic nitrite reduction activity. Thauera and Pseudomonas were the dominant mesophilic nitrate-reducing bacteria (mNRB), whereas Petrobacter and Geobacillus were the dominant thermophilic NRB (tN...

  13. 21 CFR 172.175 - Sodium nitrite.

    Science.gov (United States)

    2010-04-01

    ... Preservatives § 172.175 Sodium nitrite. The food additive sodium nitrite may be safely used in or on specified... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium nitrite. 172.175 Section 172.175 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN...

  14. 21 CFR 573.700 - Sodium nitrite.

    Science.gov (United States)

    2010-04-01

    ... Listing § 573.700 Sodium nitrite. Sodium nitrite may be safely used in canned pet food containing meat and... as a preservative and color fixative in canned pet food containing fish, meat, and fish and meat... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium nitrite. 573.700 Section 573.700 Food and...

  15. Effect of Electrolytes on the Adsorption of Nitrite and Nitrate from ...

    African Journals Online (AJOL)

    Nitrite and nitrate levels were quantitatively adsorbed to wood-derived activated carbon in aqueous system and the effects of electrolytes investigated in this study using batch sorption process. The data showed that nitrate adsorbed nearly 1.5 times higher than that of nitrite. The adsorption is adequately explained by ...

  16. Atividade da enzima nitrato redutase em milho cultivado sob diferentes níveis de adubação nitrogenada e potássica Activity of the enzyme reductase nitrate in corn cultivated under different levels of nitrogen and potassium fertilization

    Directory of Open Access Journals (Sweden)

    Sueli Maria da Silva

    2011-11-01

    Full Text Available Em função de seu uso na alimentação humana e animal e de um elevado potencial produtivo, o milho constitui-se num dos mais importantes cereais cultivados e comercializados no mundo. O objetivo deste trabalho foi avaliar o efeito de diferentes doses de nitrogênio e potássio na atividade da enzima redutase do nitrato na cultura do milho em casa de vegetação. Foram utilizadas cinco doses de nitrogênio (0; 50; 100; 150 e 200kg ha-1 e cinco doses de potássio (0; 40; 80; 120 e 160kg ha-1. A atividade da enzima foi estimada in vivo após as plantas atingirem quatro folhas totalmente desdobradas. Utilizou-se o método baseado no princípio de que a quantidade de nitrito liberada por fragmentos de tecidos vivos num tampão, na presença de uma agente permanente e do substrato, reflete a atividade potencial da enzima. A atividade da enzima nitrato redutase sofreu influência significativa da interação das doses de nitrogênio e potássio, já que a interação N=100kg ha-1 e K=40kg ha-1 proporcionou as melhores médias do experimento. A partir dessa dose, o aumento no fornecimento tanto de nitrogênio quanto de potássio promoveu redução da atividade enzimática.Because of the use in human feeding, animal, and high productive potential, corn is considered to be one of the most important cultivated and commercialized cereals in the world. The aim of this research was to evaluate the effect of different potassium doses and nitrogen in the activity of the enzyme nitrate reductase in the corn culture in a greenhouse environment. Five dosages of nitrogen (0; 50; 100; 150 and 200kg ha-1 and five potassium dosages (0; 40; 80; 120 and 160kg ha-1 were used. The activity of the nitrate reductase was esteemed, in vivo after the plants reached four leaves totally unfolded. The method that was used is based on the principle of the amount of nitrite liberated by fragments of alive tissue in a buffer in the presence of a permanent agent and substrate

  17. Drought-Induced Effects on Nitrate Reductase Activity and mRNA and on the Coordination of Nitrogen and Carbon Metabolism in Maize Leaves1

    Science.gov (United States)

    Foyer, Christine H.; Valadier, Marie-Hélène; Migge, Andrea; Becker, Thomas W.

    1998-01-01

    Maize (Zea mays L.) plants were grown to the nine-leaf stage. Despite a saturating N supply, the youngest mature leaves (seventh position on the stem) contained little NO3− reserve. Droughted plants (deprived of nutrient solution) showed changes in foliar enzyme activities, mRNA accumulation, photosynthesis, and carbohydrate and amino acid contents. Total leaf water potential and CO2 assimilation rates, measured 3 h into the photoperiod, decreased 3 d after the onset of drought. Starch, glucose, fructose, and amino acids, but not sucrose (Suc), accumulated in the leaves of droughted plants. Maximal extractable phosphoenolpyruvate carboxylase activities increased slightly during water deficit, whereas the sensitivity of this enzyme to the inhibitor malate decreased. Maximal extractable Suc phosphate synthase activities decreased as a result of water stress, and there was an increase in the sensitivity to the inhibitor orthophosphate. A correlation between maximal extractable foliar nitrate reductase (NR) activity and the rate of CO2 assimilation was observed. The NR activation state and maximal extractable NR activity declined rapidly in response to drought. Photosynthesis and NR activity recovered rapidly when nutrient solution was restored at this point. The decrease in maximal extractable NR activity was accompanied by a decrease in NR transcripts, whereas Suc phosphate synthase and phosphoenolpyruvate carboxylase mRNAs were much less affected. The coordination of N and C metabolism is retained during drought conditions via modulation of the activities of Suc phosphate synthase and NR commensurate with the prevailing rate of photosynthesis. PMID:9576798

  18. Overexpression of a GmCnx1 gene enhanced activity of nitrate reductase and aldehyde oxidase, and boosted mosaic virus resistance in soybean.

    Directory of Open Access Journals (Sweden)

    Zheng Zhou

    Full Text Available Molybdenum cofactor (Moco is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1 is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L. Cnx1 gene (GmCnx1 was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR and aldehydeoxidase (AO of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 μg g(-1 h(-1 and 30 pmol L(-1, respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants.In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV. DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.

  19. Use of [1,2-3 h] testosterone in 5 α- reductase enzymatic activity dosing in dermal fibroblast cultures from polycystic ovarian patients

    International Nuclear Information System (INIS)

    Matei, Lidia; Postolache, Cristian; Condac, Eduard

    2003-01-01

    Polycystic ovarian syndrome is an endocrine malady very frequent in women characterized by the presence of ovarian cysts, visible or not by ultrasonography, menstrual cycle deregulation and sometimes by high plasmatic concentrations of androgen hormones. Many cases of polycystic syndrome could not be easily diagnosed or had an erroneous diagnostic. Therefore, is useful to know the plasmatic androgen hormone profile. This profile could indicate the cause for observed clinical manifestations; this cause may be observed in ovarian, suprarenal glands or hypothalamo-hypophysis level. In vitro studies on dermal fibroblasts permit the detail determination of steroid hormones metabolism in target organs and offer important information regarding action mechanism. This study follows the identification of testosterone metabolites in fibroblasts and enzymatic activities of 5α-reductase using testosterone radioactively labeled with tritium. (authors)

  20. Properties of nitrogen fertilization are decisive in determining the effects of elevated atmospheric CO2 on the activity of nitrate reductase in plants.

    Science.gov (United States)

    Zhang, Ranran; Du, Shaoting

    2016-01-01

    The concentration of atmospheric CO2 is predicted to double by the end of this century. The response of higher plants to an increase in atmospheric CO2 often includes a change in nitrate reductase (NR) activity. In a recent study, we showed that, under elevated CO2 levels, NR induction in low-nitrate plants and NR inhibition in high-nitrate plants are regulated by nitric oxide (NO) generated via nitric oxide synthases. This finding provides an explanation for the diverse responses of plants to elevated CO2 levels, and suggests that the use of nitrogen fertilizers on soil will have a major influence on the nitrogen assimilation capacity of plants in response to CO2 elevation.

  1. DIETARY-CHOLESTEROL INDUCED DOWN-REGULATION OF INTESTINAL 3-HYDROXY-3-METHYLGLUTARYL COENZYME-A REDUCTASE-ACTIVITY IS DIMINISHED IN RABBITS WITH HYPERRESPONSE OF SERUM-CHOLESTEROL TO DIETARY-CHOLESTEROL

    NARCIS (Netherlands)

    MEIJER, GW; SMIT, MJ; VANDERPALEN, JGP; KUIPERS, F; VONK, RJ; VANZUTPHEN, BFM; BEYNEN, AC

    Key enzymes of cholesterol metabolism were studied in two inbred strains of rabbits with hyper- or hyporesponse of serum cholesterol to dietary cholesterol. Baseline 3-hydroxy-3-methylglutaryl (HMG)CoA reductase activity in liver was similar in hypo- and hyperresponders, but that in intestine was

  2. The enzymatic activities of brain catechol-O-methyltransferase (COMT) and methionine sulphoxide reductase are correlated in a COMT Val/Met allele-dependent fashion.

    Science.gov (United States)

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

    2015-12-01

    The enzyme catechol-O-methyltransferase (COMT) plays a primary role in the metabolism of catecholamine neurotransmitters and is implicated in the modulation of cognitive and emotional responses. The best characterized single nucleotide polymorphism (SNP) of the COMT gene consists of a valine (Val)-to-methionine (Met) substitution at codon 108/158. The Met-containing variant confers a marked reduction in COMT catalytic activity. We recently showed that the activity of recombinant COMT is positively regulated by the enzyme Met sulphoxide reductase (MSR), which counters the oxidation of Met residues of proteins. The current study was designed to assess whether brain COMT activity may be correlated to MSR in an allele-dependent fashion. COMT and MSR activities were measured from post-mortem samples of prefrontal cortices, striata and cerebella of 32 subjects by using catechol and dabsyl-Met sulphoxide as substrates, respectively. Allelic discrimination of COMT Val(108/185) Met SNP was performed using the Taqman 5'nuclease assay. Our studies revealed that, in homozygous carriers of Met, but not Val alleles, the activity of COMT and MSR was significantly correlated throughout all tested brain regions. These results suggest that the reduced enzymatic activity of Met-containing COMT may be secondary to Met sulphoxidation and point to MSR as a key molecular determinant for the modulation of COMT activity. © 2015 British Neuropathological Society.

  3. Cortisol metabolism in healthy young adults: sexual dimorphism in activities of A-ring reductases, but not 11beta-hydroxysteroid dehydrogenases.

    Science.gov (United States)

    Finken, M J; Andrews, R C; Andrew, R; Walker, B R

    1999-09-01

    Cortisol is metabolized irreversibly by A-ring reductases (5alpha- and 5beta-reductases) and reversibly (to cortisone) by 11beta-hydroxysteroid dehydrogenases (11betaHSDs). In rats, estradiol down-regulates 11betaHSD1 expression. In humans, ratios of urinary cortisol/cortisone metabolites differ in men and women. In this study, urinary cortisol metabolites and hepatic 11betaHSD1 activity were measured in healthy young men and women at different phases of the menstrual cycle. Ten men and 10 women with regular menstrual cycles collected a 24-h urine sample, took 250 microg oral dexamethasone at 2300 h, took 25 mg oral cortisone at 0900 h (after fasting), and had blood sampled for plasma cortisol estimation over the subsequent 150 min. Women repeated the tests in random order in menstrual, follicular, and luteal phases. Women excreted disproportionately less A-ring-reduced metabolites of cortisol [median 5alpha-tetrahydrocortisol, 1811 (interquartile range, 1391-2300) microg/day in menstrual phase vs. 2723 (interquartile range, 2454-3154) in men (P = 0.01); 5beta-tetrahydrocortisol, 1600 (interquartile range, 1419-1968) vs. 2197 (interquartile range, 1748-2995; P = 0.03)] but similar amounts of cortisol, cortisone, and tetrahydrocortisone. Analogous differences were observed in urinary excretion of androgen metabolites. Conversion of cortisone to cortisol on hepatic first pass metabolism was not different (peak plasma cortisol, 733 +/- 60 nmol/L in women vs. 684 +/- 53 nmol/L in men; mean +/- SEM; P = 0.55). There were no differences in cortisol or androgen metabolism between phases of the menstrual cycle. We conclude that sexual dimorphism in cortisol metabolite excretion is attributable to less A-ring reduction of cortisol in women, rather than less reactivation of cortisone to cortisol by 11betaHSD1. This difference is not influenced acutely by gonadal steroids. 11BetaHSD1 has been suggested to modulate insulin sensitivity and body fat distribution, but caution

  4. Dietary Nitrite: from menace to marvel

    Directory of Open Access Journals (Sweden)

    Nathan S. Bryan

    2016-11-01

    Full Text Available The health benefits of nitrite are now indisputable when administered in a clinical setting for specific diseases. Currently, most published reports identify the production of nitric oxide (NO as the mechanism of action for nitrite. Basic science, in addition to clinical studies, demonstrate that nitrite and/or nitrate cannot restore NO homeostasis as an endothelium independent source of NO that may be a redundant system for endogenous NO production. Nitrate must first be reduced to nitrite by oral commensal bacteria; nitrite can then be further reduced to NO along the physiological oxygen gradient. But despite decades of rigorous research on sodium nitrate’s safety and efficacy as a curing agent, sodium nitrite is still regarded by many as a toxic undesirable food additive. However, research within the biomedical science community has revealed enormous therapeutic benefits of nitrite which are being developed as novel therapies for conditions associated with nitric oxide insufficiency. Thus, this review will highlight the fundamental biochemistry of nitrite in human physiology and provide evidence that nitrite be considered an essential nutrient. Foods or diets enriched with nitrite can have profound positive health benefits.

  5. NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase.

    Science.gov (United States)

    Askerlund, P; Laurent, P; Nakagawa, H; Kader, J C

    1991-01-01

    Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b(5) reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber

  6. Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617.

    Science.gov (United States)

    Correia, Cristina; Besson, Stéphane; Brondino, Carlos D; González, Pablo J; Fauque, Guy; Lampreia, Jorge; Moura, Isabel; Moura, José J G

    2008-11-01

    Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Iota) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at Em=+197 mV (heme c) and -4.5 mV (heme b). Variable-temperature (4-120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe-4S]+ cluster and overlapping signals associated with at least three types of [4Fe-4S]+ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called "low-pH" and "high-pH," changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases.

  7. Seasonal Distribution of Nitrate and Nitrite Levels in Eleme Abattoir ...

    African Journals Online (AJOL)

    The study deals with the seasonal distribution of nitrate (NO3) and nitrite (NO2) levels in Eleme Abattoir environment. Samples of soil, surface water and groundwater were collected from areas unaffected and those affected by abattoir activities. For the soils from the affected area and control points respectively, nitrate levels ...

  8. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1 ? ??

    OpenAIRE

    Randall, Matthew J.; Spiess, Page C.; Hristova, Milena; Hondal, Robert J.; van der Vliet, Albert

    2013-01-01

    Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant impl...

  9. Controls of nitrite oxidation in ammonia-removing biological air filters

    DEFF Research Database (Denmark)

    Juhler, Susanne; Ottosen, Lars Ditlev Mørck; Nielsen, Lars Peter

    2008-01-01

    in accumulation of nitrate rather than nitrite and a significant decline in pH. As a consequence, ammonia is removed more efficiently, but heterotrophic oxidation of odorous compounds might be inhibited.  To identify the controlling mechanisms of nitrite oxidation, full-scale biological air filters were...... activity resulting in a lowered pH and thus a decreased FA concentration, promoting further growth of NOB. Yet, in some cases a situation with a nitrate-to-nitrite ratio of 1 and moderate pH remained stable even under varying air load and water supply, suggesting that additional mechanisms were involved......In biological air filters ammonia is removed due to the action of Ammonia Oxidizing Bacteria (AOB) resulting in nitrite accumulation exceeding 100 mM. Among filters treating exhaust air from pig facilities successful establishment of Nitrite Oxidizing Bacteria (NOB) sometimes occurs, resulting...

  10. Avicequinone C Isolated from Avicennia marina Exhibits 5α-Reductase-Type 1 Inhibitory Activity Using an Androgenic Alopecia Relevant Cell-Based Assay System

    Directory of Open Access Journals (Sweden)

    Ruchy Jain

    2014-05-01

    Full Text Available Avicennia marina (AM exhibits various biological activities and has been traditionally used in Egypt to cure skin diseases. In this study, the methanolic heartwood extract of AM was evaluated for inhibitory activity against 5α-reductase (5α-R [E.C.1.3.99.5], the enzyme responsible for the over-production of 5α-dihydrotestosterone (5α-DHT causing androgenic alopecia (AGA. An AGA-relevant cell-based assay was developed using human hair dermal papilla cells (HHDPCs, the main regulator of hair growth and the only cells within the hair follicle that are the direct site of 5α-DHT action, combined with a non-radioactive thin layer chromatography (TLC detection technique. The results revealed that AM is a potent 5α-R type 1 (5α-R1 inhibitor, reducing the 5α-DHT production by 52% at the final concentration of 10 µg/mL. Activity-guided fractionation has led to the identification of avicequinone C, a furanonaphthaquinone, as a 5α-R1 inhibitor with an IC50 of 9.94 ± 0.33 µg/mL or 38.8 ± 1.29 µM. This paper is the first to report anti-androgenic activity through 5α-R1 inhibition of AM and avicequinone C.

  11. Avicequinone C isolated from Avicennia marina exhibits 5α-reductase-type 1 inhibitory activity using an androgenic alopecia relevant cell-based assay system.

    Science.gov (United States)

    Jain, Ruchy; Monthakantirat, Orawan; Tengamnuay, Parkpoom; De-Eknamkul, Wanchai

    2014-05-23

    Avicennia marina (AM) exhibits various biological activities and has been traditionally used in Egypt to cure skin diseases. In this study, the methanolic heartwood extract of AM was evaluated for inhibitory activity against 5α-reductase (5α-R) [E.C.1.3.99.5], the enzyme responsible for the over-production of 5α-dihydrotestosterone (5α-DHT) causing androgenic alopecia (AGA). An AGA-relevant cell-based assay was developed using human hair dermal papilla cells (HHDPCs), the main regulator of hair growth and the only cells within the hair follicle that are the direct site of 5α-DHT action, combined with a non-radioactive thin layer chromatography (TLC) detection technique. The results revealed that AM is a potent 5α-R type 1 (5α-R1) inhibitor, reducing the 5α-DHT production by 52% at the final concentration of 10 µg/mL. Activity-guided fractionation has led to the identification of avicequinone C, a furanonaphthaquinone, as a 5α-R1 inhibitor with an IC50 of 9.94 ± 0.33 µg/mL or 38.8 ± 1.29 µM. This paper is the first to report anti-androgenic activity through 5α-R1 inhibition of AM and avicequinone C.

  12. Elevated CO2-induced production of nitric oxide (NO) by NO synthase differentially affects nitrate reductase activity in Arabidopsis plants under different nitrate supplies.

    Science.gov (United States)

    Du, Shaoting; Zhang, Ranran; Zhang, Peng; Liu, Huijun; Yan, Minggang; Chen, Ni; Xie, Huaqiang; Ke, Shouwei

    2016-02-01

    CO2 elevation often alters the plant's nitrate reductase (NR) activity, the first enzyme acting in the nitrate assimilation pathway. However, the mechanism underlying this process remains unknown. The association between elevated CO2-induced alterations of NR activity and nitric oxide (NO) was examined in Col-0 Arabidopsis fed with 0.2-10 mM nitrate, using NO donors, NO scavenger, and NO synthase (NOS) inhibitor. The noa1 mutant, in which most NOS activity was lost, and the NR activity-null mutant nia1 nia2 were also used to examine the above association. In response to CO2 elevation, NR activity increased in low-nitrate Col-0 plants but was inhibited in high-nitrate Col-0 plants. NO scavenger and NOS inhibitor could eliminate these two responses, whereas the application of NO donors mimicked these distinct responses in ambient CO2-grown Col-0 plants. Furthermore, in both low- and high-nitrate conditions, elevated CO2 increased NOS activity and NO levels in Col-0 and nia1 nia2 plants but had little effect on NO level and NR activity in noa1 plants. Considering all of these findings, this study concluded that, in response to CO2 elevation, either the NR activity induction in low-nitrate plants or the NR activity inhibition in high-nitrate plants is regulated by NOS-generated NO. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  13. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase fibrinolytic activity in rat aortic endothelial cells. Role of geranylgeranylation and Rho proteins.

    Science.gov (United States)

    Essig, M; Nguyen, G; Prié, D; Escoubet, B; Sraer, J D; Friedlander, G

    1998-10-05

    3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (HRIs) have been recently shown to prevent atherosclerosis progression. Clinical benefit results from combined actions on various components of the atherosclerotic lesion. This study was designed to identify the effects of HRI on one of these components, the endothelial fibrinolytic system. Aortas isolated from rats treated for 2 days with lovastatin (4 mg/kg body wt per day) showed a 3-fold increase in tissue plasminogen activator (tPA) activity. In a rat aortic endothelial cell line (SVARECs) and in human nontransformed endothelial cells (HUVECs), HRI induced an increase in tPA activity and antigen in a time- and concentration-dependent manner. In SVARECs, the maximal response was observed when cells were incubated for 48 hours with 50 micromol/L HRI. An increase of tPA mRNA was also in evidence. In contrast, HRI inhibited plasminogen activator inhibitor-1 activity and mRNA. The effects of HRI were reversed by mevalonate and geranylgeranyl pyrophosphate, but not by LDL cholesterol and farnesyl pyrophosphate, and were not induced by alpha-hydroxyfarnesyl phosphonic acid, an inhibitor of protein farnesyl transferase. C3 exoenzyme, an inhibitor of the geranylgeranylated-activated Rho protein, reproduced the effect of lovastatin on tPA and plasminogen activator inhibitor-1 activity and blocked its reversal by geranylgeranyl pyrophosphate. The effect of HRI was associated with a disruption of cellular actin filaments without modification of microtubules. A disrupter of actin filaments, cytochalasin D, induced the same effect as lovastatin on tPA, whereas a disrupter of microtubules, nocodazole, did not. In conclusion, HRI can modify the fibrinolytic potential of endothelial cells, likely via inhibition of geranylgeranylated Rho protein and disruption of the actin filaments. The resulting increase of fibrinolytic activity of endothelial cells may contribute to the beneficial effects of HRI in the

  14. Nitrite and nitroso compounds can serve as specific catalase inhibitors.

    Science.gov (United States)

    Titov, Vladimir Yu; Osipov, Anatoly N

    2017-03-01

    We present evidence that nitrite and nitrosothiols, nitrosoamines and non-heme dinitrosyl iron complexes can reversibly inhibit catalase with equal effectiveness. Catalase activity was evaluated by the permanganatometric and calorimetric assays. This inhibition is not the result of chemical transformations of these compounds to a single inhibitor, as well as it is not the result of NO release from these substances (as NO traps have no effect on the extent of inhibition). It was found that chloride and bromide in concentration above 80 mM and thiocyanate in concentration above 20 μM enhance catalase inhibition by nitrite and the nitroso compounds more than 100 times. The inhibition degree in this case is comparable with that induced by azide. We propose that the direct catalase inhibitor is a positively charged NO-group. This group acquires a positive charge in the active center of enzyme by interaction of nitrite or nitroso compounds with some enzyme groups. Halides and thiocyanate protect the NO + group from hydration and thus increase its inhibition effect. It is probable that a comparatively low chloride concentration in many cells is the main factor to protect catalase from inhibition by nitrite and nitroso compounds.

  15. Nitrate reductase activity (NRA in the invasive alien Fallopia japonica: seasonal variation, differences among habitats types, and comparison with native species

    Directory of Open Access Journals (Sweden)

    Damian Chmura

    2016-09-01

    Full Text Available Nitrate reductase activity (NRA was studied in the invasive alien plant F. japonica (Japanese knotweed during the vegetation season and among natural, semi-natural, and human-made habitats and compared with NRA in selected native species. NRA was measured directly in the field from the beginning of May until the beginning of October. NRA was much higher than in the plant’s native range, i.e., East Asia, and showed a high degree of variation over time with the highest values being reached at the stage of fast vegetative growth and at the beginning of fruiting. NRA was highest on dumping sites probably due to the high nitrogen input into soils and near traffic and the emission of NOx by vehicles. A comparison of the enzyme activity in four selected native plant species indicated that NRA in F. japonica was the highest with the exception of Urtica dioica, which exhibited a similar activity of the enzyme. A detailed comparison with this species showed that differences between these species on particular dates were influenced by differences in the phenology of both plants. The initial results that were obtained suggest that nitrogen pollution in an environment can contribute to habitat invasibility and a high level of NRA, which in addition to the many plant traits that are commonly accepted as characteristic of invasiveness features, may be an important factor that enhances invasion success.

  16. Ionic liquid mediated stereoselective synthesis of alanine linked hybrid quinazoline-4(3H)-one derivatives perturbing the malarial reductase activity in folate pathway.

    Science.gov (United States)

    Patel, Tarosh S; Bhatt, Jaimin D; Vanparia, Satish F; Patel, Urmila H; Dixit, Ritu B; Chudasama, Chaitanya J; Patel, Bhavesh D; Dixit, Bharat C

    2017-12-15

    Grimmel's method was optimized as well as modified leading to the cyclization and incorporation of alanine linked sulphonamide in 4-quinazolin-(3H)-ones. Further, the generation of heterocyclic motif at position-3 of 4-quinazolinones was explored by synthesis of imines, which unfortunately led to an isomeric mixture of stereoisomers. The hurdle of diastereomers encountered on the path was eminently rectified by development of new rapid and reproducible methodology involving the use of imidazolium based ionic liquid as solvents as well as catalyst for cyclization as well as synthesis of imines in situ at position-3 leading to procurement of single E-isomer as the target hybrid heterocyclic molecules. The purity and presence of single isomer was also confirmed by HPLC and spectroscopic techniques. Further, the synthesized sulphonamide linked 4-quinazolin-(3H)-ones hybrids were screened for their antimalarial potency rendering potent entities (4b, 4c, 4 l, 4 t and 4u). The active hybrids were progressively screened for enzyme inhibitory efficacy against presumed receptor Pf-DHFR and h-DHFR computationally as well as in vitro, proving their potency as dihydrofolate reductase inhibitors. The ADME properties of these active molecules were also predicted to enhance the knowhow of the oral bioavailability, indicating good bioavailability of the active entities. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. In Silico Screening, Synthesis and In Vitro Evaluation of Some Quinazolinone and Pyridine Derivatives as Dihydrofolate Reductase Inhibitors for Anticancer Activity

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    A. G. Nerkar

    2009-01-01

    Full Text Available Dihydrofolate reductase (DHFR is the important target for anticancer drugs belonging to the class of antimetabolites as the enzyme plays important role in the de novo purine synthesis. We here report the in silico screening to obtain best fit molecules as DHFR inhibitors, synthesis of some ʻbest fitʼ quinazolinone from 2-phenyl-3-(substituted-benzilidine-amino quinazolinones (Quinazolinone Shiff's bases QSB1-5 and pyridine-4-carbohydrazide Shiff's bases (ISB1-5 derivatives and their in vitro anticancer assay. Synthesis of the molecules was performed using microwave assisted synthesis. The structures of these molecules were elucidated by IR and 1H-NMR. These compounds were then subjected for in vitro anticancer evaluation against five human cancer cell-lines for anticancer cyto-toxicity assay. Methotrexate (MTX was used as standard for this evaluation to give a comparable inhibition of the cell proliferation by DHFR inhibition. Placlitaxel, adriamycin and 5-fluoro-uracil were also used as standard to give a comparable activity of these compounds with other mechanism of anticancer activity. ISB3 (4-(N, N-dimethyl-amino-phenyl Schiff''s base derivative of pyridine carbohydrazide showed equipotent activity with the standards used in in vitro anticancer assay as per the NCI (National Cancer Institute guidelines.

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

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    R. Seenivasagan

    2016-07-01

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

  19. Evaluation of antioxidant activity of Ginkgo biloba phytosomes in rat brain.

    Science.gov (United States)

    Naik, Suresh R; Pilgaonkar, Vinaya W; Panda, Vandana S

    2006-11-01

    Ginkgo biloba from the traditional Chinese system of medicine has been found to possess neurocognitive enhancing effects. The mechanism of action of Ginkgo seems to be related to its antioxidant properties. In the present study, Ginkgo biloba phytosomes were administered to Wistar rats at 50 mg/kg and 100 mg/kg for 7 and 14 days. Chemical hypoxia was induced by administration of sodium nitrite (75 mg/kg) 1 h after the last administration of treatment. Thirty minutes after sodium nitrite administration, the animals were killed and the cerebral cortex, cerebellum, hippocampus and striatum were isolated and homogenized. The supernatants were used for the estimation of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. Ginkgo biloba phytosome treatment was found to increase superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities in all the brain regions compared with those treated only with sodium nitrite. The prevention of depletion of the antioxidant enzymes by sodium nitrite in the presence of Ginkgo biloba phytosomes may be correlated to its antioxidant activity. Copyright (c) 2006 John Wiley & Sons, Ltd.

  20. The Effects of Physicochemical Factors and Cell Density on Nitrite Transformation in a Lipid-Rich Chlorella.

    Science.gov (United States)

    Liang, Fang; Du, Kui; Wen, Xiaobin; Luo, Liming; Geng, Yahong; Li, Yeguang

    2015-12-28

    To understand the effects of physicochemical factors on nitrite transformation by microalgae, a lipid-rich Chlorella with high nitrite tolerance was cultured with 8 mmol/l sodium nitrite as sole nitrogen source under different conditions. The results showed that nitrite transformation was mainly dependent on the metabolic activities of algal cells rather than oxidation of nitrite by dissolved oxygen. Light intensity, temperature, pH, NaHCO3 concentrations, and initial cell densities had significant effects on the rate of nitrite transformation. Single-factor experiments revealed that the optimum conditions for nitrite transformation were light intensity: 300 μmol/m(2); temperature: 30°C; pH: 7-8; NaHCO3 concentration: 2.0 g/l; and initial cell density: 0.15 g/l; and the highest nitrite transformation rate of 1.36 mmol/l/d was achieved. There was a positive correlation between nitrite transformation rate and the growth of Chlorella. The relationship between nitrite transformation rate (mg/l/d) and biomass productivity (g/l/d) could be described by the regression equation y = 61.3x (R(2) = 0.9665), meaning that 61.3 mg N element was assimilated by 1.0 g dry biomass on average, which indicated that the nitrite transformation is a process of consuming nitrite as nitrogen source by Chlorella. The results demonstrated that the Chlorella suspension was able to assimilate nitrite efficiently, which implied the feasibility of using flue gas for mass production of Chlorella without preliminary removal of NOX.

  1. Production of the R2 subunit of ribonucleotide reductase from herpes simplex virus with prokaryotic and eukaryotic expression systems: higher activity of R2 produced by eukaryotic cells related to higher iron-binding capacity.

    OpenAIRE

    Lamarche, N; Matton, G; Massie, B; Fontecave, M; Atta, M; Dumas, F; Gaudreau, P; Langelier, Y

    1996-01-01

    The R2 subunit of ribonucleotide reductase from herpes simplex virus type 2 was overproduced with prokaryotic and eukaryotic expression systems. The recombinant R2 purified by a two-step procedure exhibited a 3-fold higher activity when produced in eukaryotic cells. Precise quantification of the R2 concentration at each step of the purification indicated that the activity was not altered during the purification procedure. Moreover, we have observed that the level of R2 expression, in eukaryot...

  2. Nitrite-induced enhancement of toxicity of phenanthrene in fish and its implications for coastal waters

    Science.gov (United States)

    Shailaja, M. S.; Rodrigues, A.

    2003-04-01

    Coastal areas are prone to varying degrees of anthropogenic chemical contamination. In many coastal environments experiencing reducing conditions in the water column, nitrite is produced as a result of denitrification. With a view to determining the effect of a natural stress such as the presence of nitrite in water on the xenobiotic metabolism in fish, the euryhaline cichlid Oreochromis mossambicus was exposed for up to 9 days to environmentally relevant concentrations of water-borne nitrite and phenanthrene, a polycyclic aromatic hydrocarbon. Analyses of different biomarkers in the treated fish indicated significant increase in the metabolism of phenanthrene as a result of exposure to nitrite. For example, the activity of the biotransformation enzyme measured as 7-ethoxyresorufin- O-deethylase activity was, in the presence of 1 μM nitrite, nearly twice that produced by phenanthrene alone. Similarly, biliary fixed fluorescence values reflecting phenanthrene and its metabolites were rendered 1.7 times higher when exposed simultaneously to nitrite. Contact with nitrite and phenanthrene together also led to severe hepatic damage with possible cell death as inferred from the large enhancement in sorbitol dehydrogenase activity in the serum and reduced liver somatic index.

  3. Characterization of nitrite degradation by Lactobacillus casei subsp. rhamnosus LCR 6013.

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    Dong-mei Liu

    Full Text Available Nitrites are potential carcinogens. Therefore, limiting nitrites in food is critically important for food safety. The nitrite degradation capacity of Lactobacillus casei subsp. rhamnosus LCR 6013 was investigated in pickle fermentation. After LCR 6013 fermentation for 120 h at 37°C, the nitrite concentration in the fermentation system was significantly lower than that in the control sample without the LCR 6013 strain. The effects of NaCl and Vc on nitrite degradation by LCR 6013 in the De Man, Rogosa and Sharpe (MRS medium were also investigated. The highest nitrite degradations, 9.29 mg/L and 9.89 mg/L, were observed when NaCl and Vc concentrations were 0.75% and 0.02%, respectively in the MRS medium, which was significantly higher than the control group (p ≤ 0.01. Electron capture/gas chromatography and indophenol blue staining were used to study the nitrite degradation pathway of LCR 6013. The nitrite degradation products contained N2O, but no NH4(+. The LCR 6013 strain completely degraded all NaNO2 (50.00 mg/L after 16 h of fermentation. The enzyme activity of NiR in the periplasmic space was 2.5 times of that in the cytoplasm. Our results demonstrated that L. casei subsp. rhamnosus LCR 6013 can effectively degrade nitrites in both the pickle fermentation system and in MRS medium by NiR. Nitrites are degraded by the LCR 6013 strain, likely via the nitrate respiration pathway (NO2(->NO->N2O->N2, rather than the aammonium formation pathway (dissimilatory nitrate reduction to ammonium, DNRA, because the degradation products contain N2O, but not NH4(+.

  4. Isoniazid acetylating phenotype in patients with paracoccidioidomycosis and its relationship with serum sulfadoxin levels, glucose-6-phosphate dehydrogenase and glutathione reductase activities

    Directory of Open Access Journals (Sweden)

    Benedito Barraviera

    1991-06-01

    Full Text Available The authors evaluated the isoniazid acetylating phenotype and measured hematocrit, hemoglobin, glucose-6-phosphate dehydrogenase and glutathione reductase activities plus serum sulfadoxin levels in 39 patients with paracoccidioidomycosis (33 males and 6 females aged 17 to 58 years. Twenty one (53.84% of the patients presented a slow acetylatingphenotype and 18(46.16% a fast acetylating phenotype. Glucose-6-phosphate- dehydrogenase (G6PD acti vity was decreased in 5(23.80% slow acetylators and in 4(22.22% fast acetylators. Glutathione reductase activity was decreased in 14 (66.66% slow acetylators and in 12 (66.66% fast acetylators. Serum levels of free and total sulfadoxin Were higher in slow acetylator (p Os autores avaliaram o fenótipo acetilador da isoniazida, hematócrito, hemoglobina, atividade da glicose-6- fosfato desidrogenase, glutationa redutase e os níveis séricos de sulfadoxina de 39 doentes com paracoccidíoidomicose, senão 33 do sexo masculino e 6 do feminino, com idades compreendidas entre 17 e 58 anos. Vinte e um (53,84% doentes apresentaram fenótipo acetilador lento e 18 (46,16% rápido. A atividade da glicose-6-fosfato desidrogenase (G6PD esteve diminuída em 5 (23,80% acetiladores lentos e 4 (22,22% rápidos. A atividade da glutationa redutase esteve diminuída em 14 (66,66% acetiladores lentos e 12 (66,66% rápidos. Os níveis séricos de sulfadoxina livre e total foram maiores nos acetiladores lentos (p < 0,02. A análise dos resultados permite concluir que os níveis séricos de sulfadoxina relaciona-se com o fenótipo acetilador. Além disso, os níveis estiveram sempre acima de 50 µg/ml, níveis estes considerados terapêuticos. Por outro lado, a deficiência de glutationa redutase pode estar relacionada com a má absorção intestinal de nutrientes, entre eles riboflavina, vitamina precursora de FAD.

  5. Tempol improves xanthine oxidoreductase-mediated vascular responses to nitrite in experimental renovascular hypertension

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    Gustavo H. Oliveira-Paula

    2016-08-01

    Full Text Available Upregulation of xanthine oxidoreductase (XOR increases vascular reactive oxygen species (ROS levels and contributes to nitroso-redox imbalance. However, XOR can generate nitric oxide (NO from nitrite, and increased superoxide could inactivate NO formed from nitrite. This study tested the hypothesis that XOR contributes to the cardiovascular effects of nitrite in renovascular hypertension, and that treatment with the antioxidant tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl improves XOR-mediated effects of nitrite. Blood pressure was assessed weekly in two-kidney one-clip (2K1C and control rats. After six weeks of hypertension, the relaxing responses to nitrite were assessed in aortic rings in the presence of the XOR inhibitor oxypurinol (or vehicle, either in the absence or in the presence of tempol. Moreover, in vivo hypotensive responses to nitrite were also examined in the presence of oxypurinol (or vehicle and tempol (or vehicle. Aortic XOR activity and expression were evaluated by fluorescence and Western blot, respectively. Vascular ROS production was assessed by the dihydroethidium assay. 2K1C hypertensive rats showed increased aortic XOR activity and vascular ROS production compared with control rats. Oxypurinol shifted the nitrite concentration–response curve to the right in aortic rings from 2K1C rats (but not in controls. Oxypurinol also attenuated the hypotensive responses to nitrite in 2K1C rats (but not in controls. These functional findings agree with increased aortic and plasma XOR activity found in 2K1C rats. Tempol treatment enhanced oxypurinol-induced shift of the nitrite concentration–response curve to the right. However, antioxidant treatment did not affect XOR-mediated hypotensive effects of nitrite. Our results show that XOR is important to the cardiovascular responses to nitrite in 2K1C hypertension, and XOR inhibitors commonly used by patients may cancel this effect. This finding suggests that nitrite

  6. Tempol improves xanthine oxidoreductase-mediated vascular responses to nitrite in experimental renovascular hypertension.

    Science.gov (United States)

    Oliveira-Paula, Gustavo H; Pinheiro, Lucas C; Guimaraes, Danielle A; Tella, Sandra O Conde; Blanco, Ana L Furlan; Angelis, Celio D; Schechter, Alan N; Tanus-Santos, Jose E

    2016-08-01

    Upregulation of xanthine oxidoreductase (XOR) increases vascular reactive oxygen species (ROS) levels and contributes to nitroso-redox imbalance. However, XOR can generate nitric oxide (NO) from nitrite, and increased superoxide could inactivate NO formed from nitrite. This study tested the hypothesis that XOR contributes to the cardiovascular effects of nitrite in renovascular hypertension, and that treatment with the antioxidant tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) improves XOR-mediated effects of nitrite. Blood pressure was assessed weekly in two-kidney one-clip (2K1C) and control rats. After six weeks of hypertension, the relaxing responses to nitrite were assessed in aortic rings in the presence of the XOR inhibitor oxypurinol (or vehicle), either in the absence or in the presence of tempol. Moreover, in vivo hypotensive responses to nitrite were also examined in the presence of oxypurinol (or vehicle) and tempol (or vehicle). Aortic XOR activity and expression were evaluated by fluorescence and Western blot, respectively. Vascular ROS production was assessed by the dihydroethidium assay. 2K1C hypertensive rats showed increased aortic XOR activity and vascular ROS production compared with control rats. Oxypurinol shifted the nitrite concentration-response curve to the right in aortic rings from 2K1C rats (but not in controls). Oxypurinol also attenuated the hypotensive responses to nitrite in 2K1C rats (but not in controls). These functional findings agree with increased aortic and plasma XOR activity found in 2K1C rats. Tempol treatment enhanced oxypurinol-induced shift of the nitrite concentration-response curve to the right. However, antioxidant treatment did not affect XOR-mediated hypotensive effects of nitrite. Our results show that XOR is important to the cardiovascular responses to nitrite in 2K1C hypertension, and XOR inhibitors commonly used by patients may cancel this effect. This finding suggests that nitrite treatment may not be

  7. Effect of riboflavin supply on student body's provision in north-western Poland with riboflavin measured by activity of glutathione reductase considering daily intake of other nutrients.

    Science.gov (United States)

    Szczuko, Małgorzata; Seidler, Teresa; Mierzwa, Mariusz; Stachowska, Ewa; Chlubek, Dariusz

    2011-06-01

    The riboflavin nutritional status of 120 people, age 22-25, studying in Szczecin, Poland, together with contents of their daily food servings were studied. Body's provision with riboflavin was determined using the erythrocyte glutathione reductase activity coefficient (EGRAC) and was compared with a sample in which the enzyme activity was stimulated with flavin adenine dinucleotide. The information concerning diets was collected with the method of a 7-day food record prior to blood collection. Biochemical deficiency in riboflavin was observed in 33.7% of women and 25% of men. The resulting average EGRAC value was 1.02 for women and 0.88 for men. Assessment of significant differences in riboflavin provision between the sexes revealed better provision in the male group. The comparison of EGRAC values with riboflavin content in 7-day diets of the respondents showed that the average intake of this vitamin in the female group, in which biochemical deficiency was observed, amounted to 1.05 mg, whereas in the male group it was, on average, 1.39 mg. In the group of people in which the potential risk of riboflavin deficiency in the body was not observed, the level of this vitamin consumption was, on average, 1.43 mg and 1.8 mg in the female and male groups, respectively. Women with biochemical riboflavin deficiency consumed significantly less of all the analyzed nutrients in comparison with the people without riboflavin deficiency.

  8. Three-dimensional quantitative structure-activity relationships and docking studies of some structurally diverse flavonoids and design of new aldose reductase inhibitors

    Directory of Open Access Journals (Sweden)

    Utpal Chandra De

    2015-01-01

    Full Text Available Aldose reductase (AR plays an important role in the development of several long-term diabetic complications. Inhibition of AR activities is a strategy for controlling complications arising from chronic diabetes. Several AR inhibitors have been reported in the literature. Flavonoid type compounds are shown to have significant AR inhibition. The objective of this study was to perform a computational work to get an idea about structural insight of flavonoid type compounds for developing as well as for searching new flavonoid based AR inhibitors. The data-set comprising 68 flavones along with their pIC 50 values ranging from 0.44 to 4.59 have been collected from literature. Structure of all the flavonoids were drawn in Chembiodraw Ultra 11.0, converted into corresponding three-dimensional structure, saved as mole file and then imported to maestro project table. Imported ligands were prepared using LigPrep option of maestro 9.6 version. Three-dimensional quantitative structure-activity relationships and docking studies were performed with appropriate options of maestro 9.6 version installed in HP Z820 workstation with CentOS 6.3 (Linux. A model with partial least squares factor 5, standard deviation 0.2482, R 2 = 0.9502 and variance ratio of regression 122 has been found as the best statistical model.

  9. NADH:ubiquinone reductase and succinate dehydrogenase activity in the liver of rats with acetaminophen-induced toxic hepatitis on the background of alimentary protein deficiency

    Directory of Open Access Journals (Sweden)

    G. P. Kopylchuk

    2015-02-01

    Full Text Available The ratio between the redox forms of the nicotinamide coenzymes and key enzymatic activity of the I and II respiratory chain complexes in the liver cells mitochondria of rats with acetaminophen-induced hepatitis under the conditions of alimentary deprivation of protein was studied. It was estimated, that under the conditions of acute acetaminophen-induced hepatitis of rats kept on a low-protein diet during 4 weeks a significant decrease of the NADH:ubiquinone reductase and succinate dehydrogenase activity with simultaneous increase of the ratio between redox forms of the nicotinamide coenzymes (NAD+/NADН is observed compared to the same indices in the liver cells of animals with experimental hepatitis kept on the ration balanced by all nutrients. Results of research may become basic ones for the biochemical rationale for the approaches directed to the correction and elimination of the consequences­ of energy exchange in the toxic hepatitis, induced on the background of protein deficiency.

  10. Improved proteolytic stability and potent activity against Leishmania infantum trypanothione reductase of α/β-peptide foldamers conjugated to cell-penetrating peptides.

    Science.gov (United States)

    de Lucio, Héctor; Gamo, Ana María; Ruiz-Santaquiteria, Marta; de Castro, Sonia; Sánchez-Murcia, Pedro A; Toro, Miguel A; Gutiérrez, Kilian Jesús; Gago, Federico; Jiménez-Ruiz, Antonio; Camarasa, María-José; Velázquez, Sonsoles

    2017-11-10

    The objective of the current study was to enhance the proteolytic stability of peptide-based inhibitors that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR) using a backbone modification strategy. To achieve this goal we carried out the synthesis, proteolytic stability studies and biological evaluation of a small library of α/β 3 -peptide foldamers of different length (from 9-mers to 13-mers) and different α→β substitution patterns related to prototype linear α-peptides. We show that several 13-residue α/β 3 -peptide foldamers retain inhibitory potency against the enzyme (in both activity and dimerization assays) while they are far less susceptible to proteolytic degradation than an analogous α-peptide. The strong dependence of the binding affinities for Li-TryR on the length of the α,β-peptides is supported by theoretical calculations on conformational ensembles of the resulting complexes. The conjugation of the most proteolytically stable α/β-peptide with oligoarginines results in a molecule with potent activity against L. infantum promastigotes and amastigotes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  11. SPECTROPHOTOMETRIC DETERMINATION OF NITRITE BY ITS ...

    African Journals Online (AJOL)

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    The main sources of nitrite ion in unpolluted surface waters are the processes of organic matter mineralization and nitrification by Nitrosomonas bacteria. The content of nitrite in unpolluted surface and ground water is very low, generally < 0.01–0.02 mg L-1 NO2. - [2]. Similar concentrations are typically found in unpolluted ...

  12. Nitric oxide acts downstream of auxin to trigger root ferric-chelate reductase activity in response to iron deficiency in Arabidopsis.

    Science.gov (United States)

    Chen, Wei Wei; Yang, Jian Li; Qin, Cheng; Jin, Chong Wei; Mo, Ji Hao; Ye, Ting; Zheng, Shao Jian

    2010-10-01

    In response to iron (Fe) deficiency, dicots employ a reduction-based mechanism by inducing ferric-chelate reductase (FCR) at the root plasma membrane to enhance Fe uptake. However, the signal pathway leading to FCR induction is still unclear. Here, we found that the Fe-deficiency-induced increase of auxin and nitric oxide (NO) levels in wild-type Arabidopsis (Arabidopsis thaliana) was accompanied by up-regulation of root FCR activity and the expression of the basic helix-loop-helix transcription factor (FIT) and the ferric reduction oxidase 2 (FRO2) genes. This was further stimulated by application of exogenous auxin (α-naphthaleneacetic acid) or NO donor (S-nitrosoglutathione [GSNO]), but suppressed by either polar auxin transport inhibition with 1-naphthylphthalamic acid or NO scavenging with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, tungstate, or N(ω)-nitro-L-arginine methyl ester hydrochloride. On the other hand, the root FCR activity, NO level, and gene expression of FIT and FRO2 were higher in auxin-overproducing mutant yucca under Fe deficiency, which were sharply restrained by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide treatment. The opposite response was observed in a basipetal auxin transport impaired mutant aux1-7, which was slightly rescued by exogenous GSNO application. Furthermore, Fe deficiency or α-naphthaleneacetic acid application failed to induce Fe-deficiency responses in noa1 and nial nia2, two mutants with reduced NO synthesis, but root FCR activities in both mutants could be significantly elevated by GSNO. The inability to induce NO burst and FCR activity was further verified in a double mutant yucca noa1 with elevated auxin production and reduced NO accumulation. Therefore, we presented a novel signaling pathway where NO acts downstream of auxin to activate root FCR activity under Fe deficiency in Arabidopsis.

  13. Structure-based approach to pharmacophore identification, in silico screening, and three-dimensional quantitative structure-activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function

    Energy Technology Data Exchange (ETDEWEB)

    Schormann, N.; Senkovich, O.; Walker, K.; Wright, D.L.; Anderson, A.C.; Rosowsky, A.; Ananthan, S.; Shinkre, B.; Velu, S.; Chattopadhyay, D. (UAB); (Connecticut); (Southern Research); (DFCI)

    2009-07-10

    We have employed a structure-based three-dimensional quantitative structure-activity relationship (3D-QSAR) approach to predict the biochemical activity for inhibitors of T. cruzi dihydrofolate reductase-thymidylate synthase (DHFR-TS). Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate-free state (DHFR domain) were used to validate and refine docking poses of ligands that constitute likely active conformations. Structural information from these complexes formed the basis for the structure-based alignment used as input for the QSAR study. Contrary to indirect ligand-based approaches the strategy described here employs a direct receptor-based approach. The goal is to generate a library of selective lead inhibitors for further development as antiparasitic agents. 3D-QSAR models were obtained for T. cruzi DHFR-TS (30 inhibitors in learning set) and human DHFR (36 inhibitors in learning set) that show a very good agreement between experimental and predicted enzyme inhibition data. For crossvalidation of the QSAR model(s), we have used the 10% leave-one-out method. The derived 3D-QSAR models were tested against a few selected compounds (a small test set of six inhibitors for each enzyme) with known activity, which were not part of the learning set, and the quality of prediction of the initial 3D-QSAR models demonstrated that such studies are feasible. Further refinement of the models through integration of additional activity data and optimization of reliable docking poses is expected to lead to an improved predictive ability.

  14. Respiratory arsenate reductase as a bidirectional enzyme

    Science.gov (United States)

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

    2009-01-01

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

  15. Peroxynitrite formation from the simultaneous reduction of nitrite and oxygen by xanthine oxidase.

    Science.gov (United States)

    Millar, Timothy M

    2004-03-26

    One electron reductions of oxygen and nitrite by xanthine oxidase form peroxynitrite. The nitrite and oxygen reducing activities of xanthine oxidase are regulated by oxygen with K(oxygen) 26 and 100 microM and K(nitrite) 1.0 and 1.1 mM with xanthine and NADH as donor substrates. Optimal peroxynitrite formation occurs at 70 microM oxygen with purine substrates. Kinetic parameters: V(max) approximately 50 nmol/min/mg and K(m) of 22, 36 and 70 microM for hypoxanthine, pterin and nitrite respectively. Peroxynitrite generation is inhibited by allopurinol, superoxide dismutase and diphenylene iodonium. A role for this enzyme activity can be found in the antibacterial activity of milk and circulating xanthine oxidase activity.

  16. Sensitivity to anti-Fas is independent of increased cathepsin D activity and adrenodoxin reductase expression occurring in NOS-3 overexpressing HepG2 cells.

    Science.gov (United States)

    Linares, Clara I; Ferrín, Gustavo; Aguilar-Melero, Patricia; González-Rubio, Sandra; Rodríguez-Perálvarez, Manuel; Sánchez-Aragó, María; Chicano-Gálvez, Eduardo; Cuezva, José M; Montero-Álvarez, José L; Muntané, Jordi; de la Mata, Manuel

    2015-05-01

    Stable overexpression of endothelial nitric oxide synthase (NOS-3) in HepG2 cells (4TO-NOS) leads to increased nitro-oxidative stress and upregulation of the cell death mediators p53 and Fas. Thus, NOS-3 overexpression has been suggested as a useful antiproliferative mechanism in hepatocarcinoma cells. We aimed to identify the underlying mechanism of cell death induced by NOS-3 overexpression at basal conditions and with anti-Fas treatment. The intracellular localization of NOS-3, the nitro-oxidative stress and the mitochondrial activity were analysed. In addition, the protein expression profile in 4TO-NOS was screened for differentially expressed proteins potentially involved in the induction of apoptosis. NOS-3 localization in the mitochondrial outer membrane was not associated with changes in the respiratory cellular capacity, but was related to the mitochondrial biogenesis increase and with a higher protein expression of mitochondrial complex IV. Nitro-oxidative stress and cell death in NOS-3 overexpressing cells occurred with the expression increase of pro-apoptotic genes and a higher expression/activity of the enzymes adrenodoxin reductase mitochondrial (AR) and cathepsin D (CatD). CatD overexpression in 4TO-NOS was related to the apoptosis induction independently of its catalytic activity. In addition, CatD activity inhibition by pepstatin A was not effective in blocking apoptosis induced by anti-Fas. In summary, NOS-3 overexpression resulted in an increased sensitivity to anti-Fas induced cell death, independently of AR expression and CatD activity. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Crassiflorone derivatives that inhibit Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and Trypanosoma cruzi trypanothione reductase (TcTR) and display trypanocidal activity.

    Science.gov (United States)

    Uliassi, Elisa; Fiorani, Giulia; Krauth-Siegel, R Luise; Bergamini, Christian; Fato, Romana; Bianchini, Giulia; Carlos Menéndez, J; Molina, Maria Teresa; López-Montero, Eulogio; Falchi, Federico; Cavalli, Andrea; Gul, Sheraz; Kuzikov, Maria; Ellinger, Bernhard; Witt, Gesa; Moraes, Carolina B; Freitas-Junior, Lucio H; Borsari, Chiara; Costi, Maria Paola; Bolognesi, Maria Laura

    2017-12-01

    Crassiflorone is a natural product with anti-mycobacterial and anti-gonorrhoeal properties, isolated from the stem bark of the African ebony tree Diospyros crassiflora. We noticed that its pentacyclic core possesses structural resemblance to the quinone-coumarin hybrid 3, which we reported to exhibit a dual-targeted inhibitory profile towards Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and Trypanosoma cruzi trypanothione reductase (TcTR). Following this basic idea, we synthesized a small library of crassiflorone derivatives 15-23 and investigated their potential as anti-trypanosomatid agents. 19 is the only compound of the series showing a balanced dual profile at 10 μM (% inhibition TbGAPDH  = 64% and % inhibition TcTR  = 65%). In phenotypic assay, the most active compounds were 18 and 21, which at 5 μM inhibited Tb bloodstream-form growth by 29% and 38%, respectively. Notably, all the newly synthesized compounds at 10 μM did not affect viability and the status of mitochondria in human A549 and 786-O cell lines, respectively. However, further optimization that addresses metabolic liabilities including solubility, as well as cytochromes P450 (CYP1A2, CYP2C9, CYP2C19, and CYP2D6) inhibition, is required before this class of natural product-derived compounds can be further progressed. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  18. Effects of elevated CO2 on the photosynthesis and nitrate reductase activity of Pyropia haitanensis (Bangiales, Rhodophyta) grown at different nutrient levels

    Science.gov (United States)

    Liu, Chunxiang; Zou, Dinghui

    2015-03-01

    Pyropia haitanensis, a commercially important species, was cultured at two CO2 concentrations (390×10-6 and 700×10-6 (parts per million)) and at low and high nutrient levels, to explore the effect of elevated CO2 on the species under nutrient enrichment. Results show that in CO2-enriched thalli, relative growth rate (RGR) was enhanced under nutrient enrichment. Elevated CO2 decreased phycobiliprotein (PB) contents, but increased the contents of soluble carbohydrates. Nutrient enrichment increased the contents of chlorophyll a (Chl a) and PB, while soluble carbohydrate content decreased. CO2 enrichment enhanced the relative maximum electronic transport rate and light saturation point. In nutrient-enriched thalli the activity of nitrate reductase (NRA) increased under elevated CO2. An instantaneous pH change in seawater (from 8.1 to 9.6) resulted in reduction of NRA, and the thalli grown under both elevated CO2 and nutrient enrichment exhibited less pronounced reduction than in algae grown at the ambient CO2. The thermal optima of NRA under elevated CO2 and/or nutrient enrichment shifted to a lower temperature (10-15°C) compared to that in ambient conditions (20°C). We propose that accelerated photosynthesis could result in growth increment. N assimilation remained high in acidified seawater and reflected increased temperature sensitivity in response to elevated CO2 and eutrophication.

  19. Nitrate reductase and nitrogenase activities in relation to N-uptake from soil, 15N-fertilizer and symbiotic fixation in soybean (Glycine max)

    International Nuclear Information System (INIS)

    Ruschel, A.P.; Saito, S.M.T.; Vose, P.B.

    1980-01-01

    Nitrate reductase (NRA) and nitrogenase (ARA) activities were evaluated in relation to nitrogen in the plant from soil (NFS), fertilizer (NFF) and symbiotic fixation (NFN 2 ) to study the pattern of utilization of nitrogen in nodulated and non nodulated soybean, 35, 55 and 75 days after planting. Three levels of ( 15 NH 4 ) 2 SO 4 - added to soil were used (0 - 25 and 50 kg N/ha), being the experiment conducted in the greenhouse, with a split plot statistical design and 4 replications. Maximum levels of RNA and ARA occurred 55 days after planting. Addition of 50 kg N/ha decreased NRA at all harvesting time studied; and nodule ARA only 75 days after planting. By that time the nodulated isoline showed higher NRA than the non nodulated one, the NFS and NFF of the isolines were not different 35 and 55 days after planting, but decreased at the last harvest, especially in nodulated soybean. Symbiotic N 2 -fixation increased plant-N after 55 days growth, contribution about 65% of plant-N in the period between 55 and 75 days after planting. Nodulated plant showed higher N than non nodulated, a sinergistic effect of the three sources of N studied on N increase of nodulated plants was observed. (Author) [pt

  20. HMG CoA reductase inhibitor suppresses the expression of tissue factor and plasminogen activator inhibitor-1 induced by angiotensin II in cultured rat aortic endothelial cells.

    Science.gov (United States)

    Kunieda, Yasufumi; Nakagawa, Katsumi; Nishimura, Hiromi; Kato, Hisato; Ukimura, Naoki; Yano, Shingo; Kawano, Hidehiko; Kimura, Shinzo; Nakagawa, Masao; Tsuji, Hajime

    2003-06-01

    It has been demonstrated that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (HRIs) reduce the incidence of acute cardiovascular events in patients with hyperlipidemia. Recent reports have shown that the protective effects of these drugs against cardiovascular events are also observed in patients without hyperlipidemia, but the mechanism of this favorable effect still remains unclear. In this study, the effects of HRIs on the endothelial regulation of thrombus formation were elucidated. The mRNA and protein expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) induced by angiotensin II (Ang II) were evaluated in cultured rat aortic endothelial cells. Pretreatment with simvastatin (0.03-3 microg/ml) significantly inhibited TF and PAI-1 induction by Ang II in a dose- and time-dependent manner. These inhibitions were significantly attenuated by mevalonic acid or geranylgeranyl pyrophosphate. Both Rho inhibitor, C3 exoenzyme, and Rho kinase inhibitor, Y-27632, mimicked the inhibitory effects of simvastatin against TF and PAI-1 induced by Ang II. This result suggested that the Rho/Rho kinase pathway is related to the TF and PAI-1 induction by Ang II. It was indicated that simvastatin maintains endothelial cells to be antithrombotic by inhibiting TF and PAI-1 expression via the Rho/Rho kinase pathways in which AngII induces TF and PAI-1 expression. These observations explain, at least partly, the mechanism of the favorable effects of simvastatin in reducing the thrombotic events.

  1. Ribose-modified purine nucleosides as ribonucleotide reductase inhibitors. Synthesis, antitumor activity, and molecular modeling of N6-substituted 3'-C-methyladenosine derivatives.

    Science.gov (United States)

    Cappellacci, Loredana; Franchetti, Palmarisa; Vita, Patrizia; Petrelli, Riccardo; Lavecchia, Antonio; Jayaram, Hiremagalur N; Saiko, Philipp; Graser, Geraldine; Szekeres, Thomas; Grifantini, Mario

    2008-07-24

    A series of cycloalkyl, bicycloalkyl, aryl, and heteroaryl N (6)-substituted derivatives of the antitumor agent 3'- C-methyladenosine (3'-Me-Ado), an inhibitor of the alpha Rnr1 subunit of mammalian ribonucleotide reductase (RR), were synthesized. The cytotoxicity of these compounds was evaluated against a panel of human leukemia and carcinoma cell lines and compared to that of some corresponding N (6)-substituted adenosine analogues. N (6)-cycloalkyl-3'- C-methylribonucleosides 2- 7 and N (6)-phenyl analogue 8 were found to inhibit the proliferation of K562 leukemia cells. N (6)-(+/-)- endo-2-norbornyl-3'- C-methyladenosine ( 7) was found to be the most cytotoxic compound, with GI 50 values slightly higher than that of 3'-Me-Ado against K562 and carcinoma cell lines and 2.7 fold higher cytotoxicity against human promyelocytic leukemia HL-60 cells. The SAR study confirms that an unsubstituted N (6)-amino group is essential for optimal cytotoxicity of 3'-Me-Ado against both K562 and carcinoma cell lines. Computational studies, carried out on the eukaryotic alpha subunit (Rnr1) of RR from Saccharomyces cerevisiae were performed to rationalize the observed structure-activity relationships.

  2. In vivo identification of promoter elements and transcription factors mediating activation of hepatic HMG-CoA reductase by T{sub 3}

    Energy Technology Data Exchange (ETDEWEB)

    Boone, Lindsey R.; Niesen, Melissa I. [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL (United States); Jaroszeski, Mark [Department of Chemical and Biomedical Engineering, College of Engineering, University of South Florida, Tampa, FL (United States); Ness, Gene C., E-mail: gness@hsc.usf.edu [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL (United States)

    2009-07-31

    The promoter elements and transcription factors necessary for triiodothyronine (T{sub 3}) induction of hepatic HMG-CoA reductase (HMGR) were investigated by transfecting rat livers with wild type and mutant HMGR promoter-luciferase constructs using in vivo electroporation. Mutations in the sterol response element (SRE), nuclear factor-y (NF-Y) site, and the newly identified upstream transcription factor-2 (USF-2) site essentially abolished the T{sub 3} response. Chromatin immunoprecipitation (ChIP) analysis demonstrated that T{sub 3} treatment caused a 4-fold increase in in vivo binding of USF-2 to the HMGR promoter. Co-transfection of the wild type HMGR promoter with siRNAs to USF-2, SREBP-2, or NF-Y nearly abolished the T{sub 3} induction, as measured by promoter activity. These data provide in vivo evidence for functional roles for USF-2, SREBP-2, and NF-Y in mediating the T{sub 3}-induction of hepatic HMGR transcription.

  3. Differential nitrate accumulation, nitrate reduction, nitrate reductase ...

    African Journals Online (AJOL)

    use

    2011-12-07

    Dec 7, 2011 ... nitrate salts supply on nitrate accumulation, amino acid biosynthesis, total protein production, nitrate reductase activity and carbohydrate biosynthesis in the roots and leaves of the plants. The results indicate that both sodium and potassium nitrate supplementation had stimulatory effects on all of the.

  4. Inhibition of Cholesterol Synthesis in HepG2 Cells by GINST-Decreasing HMG-CoA Reductase Expression Via AMP-Activated Protein Kinase.

    Science.gov (United States)

    Han, Joon-Seung; Sung, Jong Hwan; Lee, Seung Kwon

    2017-11-01

    GINST, a hydrolyzed ginseng extract, has been reported to have antidiabetic effects and to reduce hyperglycemia and hyperlipidemia. Hypercholesterolemia is caused by diet or genetic factors and can lead to atherosclerosis and coronary heart disease. Thus, the purpose of this study is to determine whether GINST and the ginsenoside metabolite, IH-901 (compound K), reduce cholesterol synthesis in HepG2 cells and the signal transduction pathways involved. Concentrations of cholesterol were measured by using an enzymatic method. Expression levels of sterol regulatory element-binding protein 2 (SREBP2), HMG-CoA reductase (HMGCR), peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer-binding proteins α (C/EBPα), GAPDH, and phosphorylation of AMP-activated protein kinase α (AMPKα), protein kinase B (PKB, also known as Akt), and mechanistic target of rapamycin complex 1 (mTORC1) were measured using western blot. Total cholesterol concentration decreased after GINST treatment for 24 and 48 h. Expression of HMGCR decreased more with GINST than with the inhibitors, U18666A and atorvastatin, after 48 h in a dose-dependent manner. Phosphorylation of AMPKα increased 2.5x by GINST after 360 min of treatment, and phosphorylation of Akt decreased after 120 and 360 min. We separated compound K from GINST extracts flash chromatography. Compound K decreased cholesterol synthesis in HepG2 cells at 24 and 48 h. Therefore, we conclude that GINST inhibits cholesterol synthesis in HepG2 cells by decreasing HMGCR expression via AMPKα activation. GINST, a hydrolyzed ginseng extract, can inhibit cholesterol synthesis in liver cells via activation of AMPKα. IH-901 (compound K), which is the main component with bioactivity in GINST, also has anticholesterol effects. Thus, we suggest that GINST can be used to reduce hypercholesterolemia. © 2017 Institute of Food Technologists®.

  5. Fluoxetine elevates allopregnanolone in female rat brain but inhibits a steroid microsomal dehydrogenase rather than activating an aldo-keto reductase.

    Science.gov (United States)

    Fry, J P; Li, K Y; Devall, A J; Cockcroft, S; Honour, J W; Lovick, T A

    2014-12-01

    Fluoxetine, a selective serotonin reuptake inhibitor, elevates brain concentrations of the neuroactive progesterone metabolite allopregnanolone, an effect suggested to underlie its use in the treatment of premenstrual dysphoria. One report showed fluoxetine to activate the aldo-keto reductase (AKR) component of 3α-hydroxysteroid dehydrogenase (3α-HSD), which catalyses production of allopregnanolone from 5α-dihydroprogesterone. However, this action was not observed by others. The present study sought to clarify the site of action for fluoxetine in elevating brain allopregnanolone. Adult male rats and female rats in dioestrus were treated with fluoxetine and their brains assayed for allopregnanolone and its precursors, progesterone and 5α-dihydroprogesterone. Subcellular fractions of rat brain were also used to investigate the actions of fluoxetine on 3α-HSD activity in both the reductive direction, producing allopregnanolone from 5α-dihydroprogesterone, and the reverse oxidative direction. Fluoxetine was also tested on these recombinant enzyme activities expressed in HEK cells. Short-term treatment with fluoxetine increased brain allopregnanolone concentrations in female, but not male, rats. Enzyme assays on native rat brain fractions and on activities expressed in HEK cells showed fluoxetine did not affect the AKR producing allopregnanolone from 5α-dihydroprogesterone but did inhibit the microsomal dehydrogenase oxidizing allopregnanolone to 5α-dihydroprogesterone. Fluoxetine elevated allopregnanolone in female rat brain by inhibiting its oxidation to 5α-dihydroprogesterone by a microsomal dehydrogenase. This is a novel site of action for fluoxetine, with implications for the development of new agents and/or dosing regimens to raise brain allopregnanolone. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

  6. The effect of aluminium-stress and exogenous spermidine on chlorophyll degradation, glutathione reductase activity and the photosystem II D1 protein gene (psbA) transcript level in lichen Xanthoria parietina.

    Science.gov (United States)

    Sen, Gulseren; Eryilmaz, Isil Ezgi; Ozakca, Dilek

    2014-02-01

    In this study, the effects of short-term aluminium toxicity and the application of spermidine on the lichen Xanthoria parietina were investigated at the physiological and transcriptional levels. Our results suggest that aluminium stress leads to physiological processes in a dose-dependent manner through differences in lipid peroxidation rate, chlorophyll content and glutathione reductase (EC 1.6.4.2) activity in aluminium and spermidine treated samples. The expression of the photosystem II D1 protein (psbA) gene was quantified using semi-quantitative RT-PCR. Increased glutathione reductase activity and psbA mRNA transcript levels were observed in the X. parietina thalli that were treated with spermidine before aluminium-stress. The results showed that the application of spermidine could mitigate aluminium-induced lipid peroxidation and chlorophyll degradation on lichen X. parietina thalli through an increase in psbA transcript levels and activity of glutathione reductase (GR) enzymes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Nitrite reduction and methanogenesis in a single-stage UASB reactor.

    Science.gov (United States)

    Borges, L I; López-Vazquez, C M; García, H; van Lier, J B

    2015-01-01

    In this study, nitrite reduction and methanogenesis in a single-stage upflow anaerobic sludge blanket (UASB) reactor was investigated, using high-strength synthetic domestic wastewater as substrate. To assess long-term effects and evaluate the mechanisms that allow successful nitrite reduction and methanogenesis in a single-stage UASB, sludge was exposed to relatively high nitrite loading rates (315 ± 13 mgNO(2)(-)-N/(l.d)), using a chemical oxygen demand (COD) to nitrogen ratio of 18 gCOD/gNO(2)(-)-N, and an organic loading rate of 5.4 ± 0.2 gCOD/(l.d). In parallel, the effects of sludge morphology on methanogenesis inhibition were studied by performing short-term batch activity tests at different COD/NO(2)(-)-N ratios with anaerobic sludge samples. In long-term tests, denitrification was practically complete and COD removal efficiency did not change significantly after nitrite addition. Furthermore, methane production only decreased by 13%, agreeing with the reducing equivalents requirement for complete NO(2)(-) reduction to N₂. Apparently, the spatial separation of denitrification and methanogenesis zones inside the UASB reactor allowed nitrite reduction and methanogenesis to occur at the same moment. Batch tests showed that granules seem to protect methanogens from nitrite inhibition, probably due to transport limitations. Combined COD and N removal via nitrite in a single-stage UASB reactor could be a feasible technology to treat high-strength domestic wastewater.

  8. The protective potential of Yucca schidigera (Sarsaponin 30) against nitrite-induced oxidative stress in rats.

    Science.gov (United States)

    Cigerci, I Hakki; Fidan, A Fatih; Konuk, Muhsin; Yuksel, Hayati; Kucukkurt, Ismail; Eryavuz, Abdullah; Sozbilir, Nalan Baysu

    2009-07-01

    The present study was designed to determine the protective effects of Yucca schidigera (Ys) against oxidative damage induced by acute nitrite intoxication as well as the histopathological evaluation of Ys in rats. The rats were divided into three groups each containing 12 rats: control (C); nitrite intoxication (N); Ys + nitrite intoxication (NY). C and N groups were fed standard rat feed (SRF). The NY group was fed SRF + 100 ppm Ys powder for 4 weeks. Acute nitrite intoxication was induced by subcutaneous (s.c.) administration of sodium nitrite (60 mg/kg) 1 day after the feeding period. Fifty minutes after sodium nitrite administration, blood samples and tissues including lung, liver, and kidney were collected for clinical biochemistry and histopathological investigations. Ys treatment was found to decrease methemoglobin, blood and tissue malondialdehyde, and tissue nitric oxide concentrations, and to increase the glutathione in blood and various tissues. However, plasma nitric oxide, total antioxidant activity, beta-carotene, and vitamin A did not differ between N and NY groups. While the N group rats showed distinct pathology in various tissues (compared with controls), the NY group had similar lung and liver pathology to the control. Only moderate or mild hemorrhage and hyperemia were seen in kidneys of NY group rats. Consequently, the natural compounds found in Ys, such as polyphenols, steroidal saponins, and other phytonutrients, could be used to substantially protect the organism from nitrite-induced oxidative damage and its complications.

  9. Progesterone receptor membrane component 1 inhibits the activity of drug-metabolizing cytochromes P450 and binds to cytochrome P450 reductase.

    Science.gov (United States)

    Szczesna-Skorupa, Elzbieta; Kemper, Byron

    2011-03-01

    Progesterone receptor membrane component 1 (PGRMC1) has been shown to interact with several cytochromes P450 (P450s) and to activate enzymatic activity of P450s involved in sterol biosynthesis. We analyzed the interactions of PGRMC1 with the drug-metabolizing P450s, CYP2C2, CYP2C8, and CYP3A4, in transfected cells. Based on coimmunoprecipitation assays, PGRMC1 bound efficiently to all three P450s, and binding to the catalytic cytoplasmic domain of CYP2C2 was much more efficient than to a chimera containing only the N-terminal transmembrane domain. Down-regulation of PGRMC1 expression levels in human embryonic kidney 293 and HepG2 cell lines stably expressing PGRMC1-specific small interfering RNA had no effect on the endoplasmic reticulum localization and expression levels of P450s, whereas enzymatic activities of CYP2C2, CYP2C8, and CYP3A4 were slightly higher in PGRMC1-deficient cells. Cotransfection of cells with P450s and PGRMC1 resulted in PGRMC1 concentration-dependent inhibition of the P450 activities, and this inhibition was partially reversed by increased expression of the P450 reductase (CPR). In contrast, CYP51 activity was decreased by down-regulation of PGRMC1 and expression of PGRMC1 in the PGRMC1-deficient cells increased CYP51 activity. In cells cotransfected with CPR and PGRMC1, strong binding of CPR to PGRMC1 was observed; however, in the presence of CYP2C2, interaction of PGRMC1 with CPR was significantly reduced, suggesting that CYP2C2 competes with CPR for binding to PGRMC1. These data show that in contrast to sterol synthesizing P450, PGRMC1 is not required for the activities of several drug-metabolizing P450s, and its overexpression inhibits those P450 activities. Furthermore, PGRMC1 binds to CPR, which may influence P450 activity.

  10. Lagenaria siceraria ameliorates atheromatous lesions by modulating HMG–CoA reductase and lipoprotein lipase enzymes activity in hypercholesterolemic rats

    Directory of Open Access Journals (Sweden)

    Mithun Singh Rajput

    2014-01-01

    Conclusion: It can be concluded that ethanolic extract of fruits of L. siceraria contains active components which ameliorates the atheromatous lesions in rat aorta and lowers the risk of atherosclerosis in hypercholesterolemic rats.

  11. Fluorometric determination of nitrite with 4-hydroxycoumarin

    Energy Technology Data Exchange (ETDEWEB)

    Ohta, T.; Arai, Y.; Takitani, S.

    1986-12-01

    A simple, sensitive, and reproducible fluorometric method for determination of nitrite has been developed. This method is based on the nitrosation of 4-hydroxycoumarin in acidic medium and subsequent reduction to 3-amino-4-hydroxy-coumarin, which is fluorescent in alkaline medium. The fluorescence intensity is proportional to the nitrite concentration in the range of 3 ng/mL to 1 ..mu..g/mL in the sample solution, with a relative standard deviation of 0.5% (50 ng/mL). The method has been applied to the determination of nitrite in saliva.

  12. 2AF sodium nitrite monitor: interim report

    International Nuclear Information System (INIS)

    Dierks, R.D.; Russell, J.L.

    1970-01-01

    The feasibility of monitoring the sodium nitrite in the 2AF makeup tank in the Purex process by measuring the NO 2 concentration of the gas phase was investigated in simulation studies. It was concluded that the NO 2 concentration is a function of the nitrite concentration but is also dependent on the sample temperature, acid concentration of the sample, and air/liquid ratio maintained in the simulated conditions. However under proper conditions of constant air purging, the NO 2 content can be related to the nitrite concentration. (U.S.)

  13. Reduction of the explosive 2,4,6-trinitrophenylmethylnitramine (tetryl) catalyzed by oxygen sensitive nitro reductase enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Shah, M.M.; Spain, J.C. [Tyndall Air Force Base, FL (United States)

    1995-12-01

    Reduction of nitroaromatic compounds by nitroreductase enzymes generally leads to the formation of the corresponding amines. However, we recently found that the incubation of the explosive 2,4,6-trinitrophenylmethylnitramine (tetryl) with ferredoxin-NADP oxidoreductase, an oxygen sensitive nitroreductase from spinach in the presence of NADPH led to the elimination of the nitramine nitro group from tetryl and the formation of N-methylpicramide (NMP). Other oxygen sensitive nitroreductase enzymes including glutathione reductase, xanthine oxidase, and cytochrome c reductase were also able to release nitrite from tetryl. Nitrite was not eliminated from tetryl by an oxygen insensitive nitrobenzene reductase. For every mole of tetryl reduced, one mole each of nitrite and NMP were produced. The rate of nitrite elimination was inhibited under aerobic conditions. Subsequent oxygen uptake studies suggested that under aerobic conditions, molecular oxygen was reduced by FNR and tetryl served as the redox mediator. Our results suggest that under aerobic conditions; tetryl is reduced to the nitroanion radical by the enzyme and this radical is involved in the reduction of molecular oxygen.

  14. Identification of danthron as an isoform-specific inhibitor of HEME OXYGENASE-1/cytochrome P450 reductase interaction with anti-tumor activity.

    Science.gov (United States)

    Chou, Yi-Tai; Hsu, Fu-Fei; Hu, Dun-Yao; Chen, Ying-Chih; Hsu, Yuan-Hao; Hsu, John T-A; Chau, Lee-Young

    2018-01-23

    Heme oxygenase (HO) catalyzes NADPH-dependent degradation of heme to liberate iron, carbon monoxide and biliverdin. The interaction between HO and cytochrome P450 reductase (CPR), an electron donor, is essential for HO activity. HO-1 is a stress-inducible isoform whereas HO-2 is constitutively expressed. HO-1 induction is commonly seen in cancers and impacts disease progression, supporting the possibility of targeting HO-1 for cancer therapy. We employed a cell-based bioluminescence resonance energy transfer assay to screen compounds with ability to inhibit HO-1/CPR interaction. The effect of the identified compound on HO-1/CPR interaction was confirmed by pull down assay. Moreover, the anti-tumorigenic activity of the identified compound on HO-1-enhanced tumor growth and migration was assessed by trypan blue exclusion method and wound healing assay. Danthron was identified as an effective small molecule able to interfere with the interaction between HO-1 and CPR but not HO-2 and CPR. Additional experiments with structural analogues of danthron revealed that the positions of hydroxyl moieties significantly affected the potency of inhibition on HO-1/CPR interaction. Pull-down assay confirmed that danthron inhibited the interaction of CPR with HO-1 but not HO-2. Danthron suppressed growth and migration of HeLa cells with stable HO-1 overexpression but not mock cells. In contrast, anthrarufin, a structural analog with no ability to interfere HO-1/CPR interaction, exhibited no significant effect on HO-1-overexpressing HeLa cells. These findings demonstrate that danthron is an isoform-specific inhibitor for HO-1/CPR interaction and may serve as a lead compound for novel anticancer drug.

  15. Lapachol inhibition of vitamin K epoxide reductase and vitamin K quinone reductase.

    Science.gov (United States)

    Preusch, P C; Suttie, J W

    1984-11-01

    Lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone] has been shown to be a potent inhibitor of both vitamin K epoxide reductase and the dithiothreitol-dependent vitamin K quinone reductase of rat liver microsomes in vitro. These observations explain the anticoagulant activity of lapachol previously observed in both rats and humans. Lapachol inhibition of the vitamin K epoxide and quinone reductases resembled coumarin anticoagulant inhibition, and was observed in normal strain but not in warfarin-resistant strain rat liver microsomes. This similarity of action suggests that the lactone functionality of the coumarins is not critical for their activity. The initial-velocity steady-state inhibition patterns for lapachol inhibition of the solubilized vitamin K epoxide reductase were consistent with tight binding of lapachol to the oxidized form of the enzyme, and somewhat lower affinity for the reduced form. It is proposed that lapachol assumes a 4-enol tautomeric structure similar to that of the 4-hydroxy coumarins. These structures are analogs of the postulated hydroxyvitamin K enolate intermediate bound to the oxidized form of the enzyme in the chemical reaction mechanism of vitamin K epoxide reductase, thus explaining their high affinity.

  16. Consistent antioxidant and antihypertensive effects of oral sodium nitrite in DOCA-salt hypertension

    Directory of Open Access Journals (Sweden)

    Jefferson H. Amaral

    2015-08-01

    Full Text Available Hypertension is a common disease that includes oxidative stress as a major feature, and oxidative stress impairs physiological nitric oxide (NO activity promoting cardiovascular pathophysiological mechanisms. While inorganic nitrite and nitrate are now recognized as relevant sources of NO after their bioactivation by enzymatic and non-enzymatic pathways, thus lowering blood pressure, mounting evidence suggests that sodium nitrite also exerts antioxidant effects. Here we show for the first time that sodium nitrite exerts consistent systemic and vascular antioxidant and antihypertensive effects in the deoxycorticosterone-salt (DOCA-salt hypertension model. This is particularly important because increased oxidative stress plays a major role in the DOCA-salt hypertension model, which is less dependent on activation of the renin-angiotensin system than other hypertension models. Indeed, antihypertensive effects of oral nitrite were associated with increased plasma nitrite and nitrate concentrations, and completely blunted hypertension-induced increases in plasma 8-isoprostane and lipid peroxide levels, in vascular reactive oxygen species, in vascular NADPH oxidase activity, and in vascular xanthine oxidoreductase activity. Together, these findings provide evidence that the oral administration of sodium nitrite consistently decreases the blood pressure in association with major antioxidant effects in experimental hypertension.

  17. Inhibitory activities of some traditional Chinese herbs against testosterone 5α-reductase and effects of Cacumen platycladi on hair re-growth in testosterone-treated mice.

    Science.gov (United States)

    Zhang, Bei; Zhang, Rong-weng; Yin, Xi-quan; Lao, Zi-zhao; Zhang, Zhe; Wu, Qing-guang; Yu, Liang-wen; Lai, Xiao-ping; Wan, Yu-hua; Li, Geng

    2016-01-11

    Many traditional Chinese medicines (TCM) have been used for hundreds of years for hair blackening and hair nourishing, and now many of them are commonly used in Chinese herbal shampoo to nourish the hair and promote hair growth. The present study was performed to screen 5α-reductase (5αR) inhibitors from traditional Chinese medicines, evaluate its hair growth promoting activity in vivo, and further investigate its effects on androgen metabolism and the expression of 5αR II in hair follicles. Nine TCM which were dried, ground and extracted by maceration with 75% ethanol or distilled water were used for screening 5αR inhibitors, and enzymes were extracted from the rat epididymis. The leaves of Platycladus orientalis (L.) Franco was used to evaluate the in vivo anti-androgenic activity. Skin color was observed daily and the hair re-growth was assessed by assigning the hair growth score. The longitudinal sections of hair follicles were used for observing follicle morphology, classifying of distinct stages of hair follicle morphogenesis and calculate the average score. The transverse sections were used for determination of hair follicle counts. Testosterone (T), Dihydrotestosterone (DHT) and Estradiol (E2) levels in serum and skin tissue were detected by ELISA kits. The immunofluorescence assay was used to detect the influence of CP-ext on 5αR expression in dorsal skin. We found the extract of Ganoderma lucidum (GL-ext), Polygonum multiflori (PM-ext), Cacumen platycladi (CP-ext) and Cynomorium songaricum (CS-ext) showed stronger 5αR inhibitory activity. CP-ext (5mg and 2mg/mouse/day) could significantly shorten the time of the dorsal skin darkening and got longhaired (Phair re-growth promoting activity. Furthermore the histological data of hair follicles in each group showed that CP-ext could promote the growth of hair follicle and slowed down hair follicles enter the telogen. What's more CP-ext significantly reduced DHT levels and down-regulated the expression of

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

    OpenAIRE

    Hofmann, Laurie C.; Straub, Sandra; Bischof, Kai

    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, 6...

  19. Atomic scale determination of enzyme flexibility and active site stability through static modes: case of dihydrofolate reductase.

    Science.gov (United States)

    Brut, Marie; Estève, Alain; Landa, Georges; Renvez, Guillaume; Djafari Rouhani, Mehdi; Vaisset, Marc

    2011-02-24

    A Static Mode approach is used to screen the biomechanical properties of DHFR. In this approach, a specific external stimulus may be designed at the atomic scale granularity to arrive at a proper molecular mechanism. In this frame, we address the issues related to the overall molecular flexibility versus loop motions and versus enzymatic activity. We show that backbone motions are particularly important to ensure DHFR domain communication and notably highlight the role of a α-helix in Met20 loop motion. We also investigate the active site flexibility in different bound states. Whereas in the occluded conformation the Met20 loop is highly flexible, in the closed conformation backbone motions are no longer significant, the Met20 loop is rigidified by new intra- and intermolecular weak bonds, which stabilizes the complex and promotes the hydride transfer. Finally, while various simulations, including I14 V and I14A mutations, confirm that Ile14 is a key residue in catalytic activity, we isolate and characterize at the atomic scale how a specific intraresidue chemical group makes it possible to assist ligand positioning, to direct the nicotinamide ring toward the folate ring.

  20. Spectroscopic Evidence for a H Bond Network at Y356 Located at the Subunit Interface of Active E. coli Ribonucleotide Reductase.

    Science.gov (United States)

    Nick, Thomas U; Ravichandran, Kanchana R; Stubbe, JoAnne; Kasanmascheff, Müge; Bennati, Marina

    2017-07-18

    The reaction catalyzed by E. coli ribonucleotide reductase (RNR) composed of α and β subunits that form an active α2β2 complex is a paradigm for proton-coupled electron transfer (PCET) processes in biological transformations. β2 contains the diferric tyrosyl radical (Y 122 ·) cofactor that initiates radical transfer (RT) over 35 Å via a specific pathway of amino acids (Y 122 · ⇆ [W 48 ] ⇆ Y 356 in β2 to Y 731 ⇆ Y 730 ⇆ C 439 in α2). Experimental evidence exists for colinear and orthogonal PCET in α2 and β2, respectively. No mechanistic model yet exists for the PCET across the subunit (α/β) interface. Here, we report unique EPR spectroscopic features of Y 356 ·-β, the pathway intermediate generated by the reaction of 2,3,5-F 3 Y 122 ·-β2/CDP/ATP with wt-α2, Y 731 F-α2, or Y 730 F-α2. High field EPR (94 and 263 GHz) reveals a dramatically perturbed g tensor. [ 1 H] and [ 2 H]-ENDOR reveal two exchangeable H bonds to Y 356 ·: a moderate one almost in-plane with the π-system and a weak one. DFT calculation on small models of Y· indicates that two in-plane, moderate H bonds (r O-H ∼1.8-1.9 Å) are required to reproduce the g x value of Y 356 · (wt-α2). The results are consistent with a model, in which a cluster of two, almost symmetrically oriented, water molecules provide the two moderate H bonds to Y 356 · that likely form a hydrogen bond network of water molecules involved in either the reversible PCET across the subunit interface or in H + release to the solvent during Y 356 oxidation.

  1. The activity of the artemisinic aldehyde Δ11(13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L.

    Science.gov (United States)

    Yang, Ke; Monfared, Sajad Rashidi; Monafared, Rashidi Sajad; Wang, Hongzhen; Lundgren, Anneli; Brodelius, Peter E

    2015-07-01

    The artemisinic aldehyde double bond reductase (DBR2) plays an important role in the biosynthesis of the antimalarial artemisinin in Artemisia annua. Artemisinic aldehyde is reduced into dihydroartemisinic aldehyde by DBR2. Artemisinic aldehyde can also be oxidized by amorpha-4,11-diene 12-hydroxylase and/or aldehyde dehydrogenase 1 to artemisinic acid, a precursor of arteannuin B. In order to better understand the effects of DBR2 expression on the flow of artemisinic aldehyde into either artemisinin or arteannuin B, we determined the content of dihydroartemisinic aldehyde, artemisinin, artemisinic acid and arteannuin B content of A. annua varieties sorted into two chemotypes. The high artemisinin producers (HAPs), which includes the '2/39', 'Chongqing' and 'Anamed' varieties, produce more artemisinin than arteannuin B; the low artemisinin producers (LAPs), which include the 'Meise', 'Iran#8', 'Iran#14', 'Iran#24' and 'Iran#47' varieties, produce more arteannuin B than artemisinin. Quantitative PCR showed that the relative expression of DBR2 was significantly higher in the HAP varieties. We cloned and sequenced the promoter of the DBR2 gene from varieties of both the LAP and the HAP groups. There were deletions/insertions in the region just upstream of the ATG start codon in the LAP varities, which might be the reason for the different promoter activities of the HAP and LAP varieties. The relevance of promoter variation, DBR2 expression levels and artemisinin biosynthesis capabilities are discussed and a selection method for HAP varieties with a DNA marker is suggested. Furthermore, putative cis-acting regulatory elements differ between the HAP and LAP varieties.

  2. Nitrite exerts antioxidant effects, inhibits the mTOR pathway and reverses hypertension-induced cardiac hypertrophy.

    Science.gov (United States)

    Guimaraes, Danielle A; Dos Passos, Madla A; Rizzi, Elen; Pinheiro, Lucas C; Amaral, Jefferson H; Gerlach, Raquel F; Castro, Michele M; Tanus-Santos, Jose E

    2018-03-09

    Cardiac hypertrophy is a common consequence of chronic hypertension and leads to heart failure and premature death. The anion nitrite is now considered as a bioactive molecule able to exert beneficial cardiovascular effects. Previous results showed that nitrite attenuates hypertension-induced increases in reactive oxygen species (ROS) production in the vasculature. Whether antioxidant effects induced by nitrite block critical signaling pathways involved in cardiac hypertrophy induced by hypertension has not been determined yet. The Akt/mTOR signaling pathway is responsible to activate protein synthesis during cardiac remodeling and is activated by increased ROS production, which is commonly found in hypertension. Here, we investigated the effects of nitrite treatment on cardiac remodeling and activation of this hypertrophic signaling pathway in 2 kidney-1 clip (2K1C) hypertension. Sham and 2K1C rats were treated with oral nitrite at 1 or 15mg/kg for four weeks. Nitrite treatment (15mg/kg) reduced systolic blood pressure and decreased ROS production in the heart tissue from hypertensive rats. This nitrite dose also blunted hypertension-induced activation of mTOR pathway and cardiac hypertrophy. While the lower nitrite dose (1mg/kg) did not affect blood pressure, it exerted antioxidant effects and tended to attenuate mTOR pathway activation and cardiac hypertrophy induced by hypertension. Our findings provide strong evidence that nitrite treatment decreases cardiac remodeling induced by hypertension as a result of its antioxidants effects and downregulation of mTOR signaling pathway. This study may help to establish nitrite as an effective therapy in hypertension-induced cardiac hypertrophic remodeling. Copyright © 2018. Published by Elsevier Inc.

  3. Nitrite Biosensing via Selective Enzymes—A Long but Promising Route

    Directory of Open Access Journals (Sweden)

    M. Gabriela Almeida

    2010-12-01

    Full Text Available The last decades have witnessed a steady increase of the social and political awareness for the need of monitoring and controlling environmental and industrial processes. In the case of nitrite ion, due to its potential toxicity for human health, the European Union has recently implemented a number of rules to restrict its level in drinking waters and food products. Although several analytical protocols have been proposed for nitrite quantification, none of them enable a reliable and quick analysis of complex samples. An alternative approach relies on the construction of biosensing devices using stable enzymes, with both high activity and specificity for nitrite. In this paper we review the current state-of-the-art in the field of electrochemical and optical biosensors using nitrite reducing enzymes as biorecognition elements and discuss the opportunities and challenges in this emerging market.

  4. Evidence That the [beta] Subunit of Chlamydia trachomatis Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(IV)/Iron(III) Cofactor in Site 1

    Energy Technology Data Exchange (ETDEWEB)

    Dassama, Laura M.K.; Boal, Amie K.; Krebs, Carsten; Rosenzweig, Amy C.; Bollinger, Jr., J. Martin (NWU); (Penn)

    2014-10-02

    The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the {beta} subunit oxidizes a cysteine residue {approx}35 {angstrom} away in the {alpha} subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the Mn{sup IV} ion of a Mn{sup IV}/Fe{sup III} cluster, which assembles in a reaction between O{sub 2} and the Mn{sup II}/Fe{sup II} complex of {beta}. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn{sup IV} ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn{sup IV} ion most likely resides in this site (i.e., {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}), but a subsequent computational study favored its occupation of site 2 ({sup 1}Fe{sup III}/{sup 2}Mn{sup IV}). In this work, we have sought to resolve the location of the Mn{sup IV} ion in Ct RNR-{beta} by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily Mn{sup IV}/Fe{sup III} clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both {sup 1}Mn{sup II}/{sup 2}Fe{sup II} and {sup 1}Fe{sup II}/{sup 2}Mn{sup II} complexes are competent to react with O{sub 2} to produce the corresponding oxidized states. However, with diminished Mn{sup II} loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these 'low-Mn' samples on a per-Mn basis implies that the {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}-{beta} is at least the more active of the two oxidized forms and may be the only active form.

  5. Regeneration of NADPH Coupled with HMG-CoA Reductase Activity Increases Squalene Synthesis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Paramasivan, Kalaivani; Mutturi, Sarma

    2017-09-20

    Although overexpression of the tHMG1 gene is a well-known strategy for terpene synthesis in Saccharomyces cerevisiae, the optimal level for tHMG1p has not been established. In the present study, it was observed that two copies of the tHMG1 gene on a dual gene expression cassette improved squalene synthesis in laboratory strain by 16.8-fold in comparison to single-copy expression. It was also observed that tHMG1p is limited by its cofactor (NADPH), as the overexpression of NADPH regenerating genes', viz., ZWF1 and POS5 (full length and without mitochondrial presequence), has led to its increased enzyme activity. Further, it was demonstrated that overexpression of full-length POS5 has improved squalene synthesis in cytosol. Finally, when tHMG1 and full-length POS5 were co-overexpressed there was a net 27.5-fold increase in squalene when compared to control strain. These results suggest novel strategies to increase squalene accumulation in S. cerevisiae.

  6. Clone-Specific Response in Leaf Nitrate Reductase Activity among Unrelated Hybrid Poplars in relation to Soil Nitrate Availability

    Directory of Open Access Journals (Sweden)

    Julien Fortier

    2012-01-01

    Full Text Available In this field study, we used in vivo NRA activity in hybrid poplar leaves as an indicator of NO3- assimilation for five unrelated hybrid poplar clones. We also examined if leaf NRA of these clones is influenced to the same extent by different levels of soil NO3- availability in two riparian agroforestry systems located in pastures. Leaf NRA differences of more than one order of magnitude were observed between the clones, clearly showing their different abilities to reduce NO3- in leaves. Clone DxN-3570, a P. deltoides x P. nigra hybrid (Aigeiros intrasectional hybrid, always had the highest leaf NRA during the field assays. This clone was also the only one to increase its leaf NRA with increasing NO3- soil availability, which resulted in a significant Site x Clone interaction and a positive relationship between soil NO3- concentration and NRA. All of the four other clones studied had one or both parental species from the Tacamahaca section. They had relatively low leaf NRA and they did not increase their leaf NRA when grown on the NO3- rich site. These results provide evidence that NO3- assimilation in leaves varies widely among hybrid poplars of different parentages, suggesting potential preferences for N forms.

  7. On The Regulation of Spinach Nitrate Reductase 1

    Science.gov (United States)

    Sanchez, Juan; Heldt, Hans W.

    1990-01-01

    A coupled assay has been worked out to study spinach (Spinacea oleracea L.) nitrate reductase under low, more physiological concentrations of NADH. In this assay the reduction of nitrate is coupled to the oxidation of malate catalyzed by spinach NAD-malate dehydrogenase. The use of this coupled system allows the assay of nitrate reductase activity at steady-state concentrations of NADH below micromolar. We have used this coupled assay to study the kinetic parameters of spinach nitrate reductase and to reinvestigate the putative regulatory role of adenine nucleotides, inorganic phosphate, amino acids, and calcium and calmodulin. PMID:16667335

  8. The effects of elevated environmental CO2 on nitrite uptake in the air-breathing clown knifefish, Chitala ornata.

    Science.gov (United States)

    Gam, Le Thi Hong; Jensen, Frank Bo; Huong, Do Thi Thanh; Phuong, Nguyen Thanh; Bayley, Mark

    2018-03-01

    Nitrite and carbon dioxide are common environmental contaminants in the intensive aquaculture ponds used to farm clown knifefish (Chitala ornata) in the Mekong delta, Vietnam. Here we tested the hypothesis that hypercapnia reduces nitrite uptake across the gills, because pH regulation will reduce chloride uptake and hence nitrite uptake as the two ions compete for the same transport route via the branchial HCO 3 - /Cl - exchanger. Fish fitted with arterial catheters were exposed to normocapnic/normoxic water (control), nitrite (1 mM), hypercapnia (21 mmHg CO 2 ), or combined hypercapnia (acclimated hypercapnia) and nitrite for 96 h. Blood was sampled to measure acid-base status, haemoglobin derivatives and plasma ions. Plasma nitrite increased for 48 h, but levels stayed below the exposure concentration, and subsequently decreased as a result of nitrite detoxification to nitrate. The total uptake of nitrite (evaluated as [NO 2 - ] + [NO 3 - ]) was significantly decreased in hypercapnia, in accordance with the hypothesis. Methemoglobin and nitrosylhemoglobin levels were similarly lower during hypercapnic compared to normocapnic nitrite exposure. The respiratory acidosis induced by hypercapnia was half-compensated by bicarbonate accumulation in 96 h, which was mainly chloride-mediated (i.e. reduced Cl - influx via the branchial HCO 3 - /Cl - exchanger). Plasma osmolality and main ions (Na + , Cl - ) were significantly decreased by hypercapnia and by nitrite exposure, consistent with inhibition of active transport. We conclude that hypercapnia induces a long-lasting, and mainly chloride-mediated acid-base regulation that reduces the uptake of nitrite across the gills. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Glutathione reductase in leaves of cowpea: cloning of two cDNAs, expression and enzymatic activity under progressive drought stress, desiccation and abscisic acid treatment.

    Science.gov (United States)

    Contour-Ansel, Dominique; Torres-Franklin, Maria Lucia; Cruz DE Carvalho, Maria Helena; D'Arcy-Lameta, Agnès; Zuily-Fodil, Yasmine

    2006-12-01

    Reactive oxygen species are frequently produced when plants are exposed to abiotic stresses. Among the detoxication systems, two enzymes, ascorbate peroxidase and glutathione reductase (GR) play key roles. GR has also a central role in keeping the reduced glutathione pool during stress thus allowing the adjustments on the cellular redox reactions. The aim of this work was to study the variations in cytosolic and dual-targeted GR gene expression in the leaves of cowpea plants submitted to progressive drought, rapid desiccation and application of exogenous abscisic acid (ABA). Two cowpea (Vigna unguiculata) cultivars, one drought-resistant ('EPACE-1'), the other drought-sensitive ('1183') were submitted to progressive drought stress by withholding irrigation. Cut-off leaves were air-dried or treated with exogenous ABA. Two GR cDNAs, one cytosolic, the other dual-targeted to chloroplasts and mitochondria were isolated by PCR and cloned in plasmid vectors. Reverse-transcription PCR was used to study the variations in GR gene expression. Two new cDNAs encoding a putative dual-targeted and a cytosolic GR were cloned and sequenced from leaves of V. unguiculata. Drought stress induced an up-regulation of the expression of the cytosolic GR gene directly related to the intensity of the stress in both cultivars. The expression of dual-targeted GR was up-regulated by the drought treatment in the susceptible cultivar only. Under a fast desiccation, the '1183' cultivar responded later than the 'EPACE-1', although in 'EPACE-1' it was the cytosolic isoform which responded and in '1183' the dual-targeted one. Exogenous ABA enhanced significantly the activity and expression levels of GR in both cultivars after treatment for 24 h. These results demonstrate a noticeable activation in both cultivars of the antioxidant metabolism under a progressive water stress, which involves both GR genes in the case of the susceptible cultivar. Under a fast desiccation, the susceptible cultivar

  10. Determination of nitrite/nitrate in human biological material by the simple Griess reaction.

    Science.gov (United States)

    Guevara, I; Iwanejko, J; Dembińska-Kieć, A; Pankiewicz, J; Wanat, A; Anna, P; Gołabek, I; Bartuś, S; Malczewska-Malec, M; Szczudlik, A

    1998-06-22

    Since a number of pathological processes such as septic shock, inflammation, graft rejection, diabetes, etc. are associated with a release of nitric oxide (NO), rapid and accurate methods of monitoring of NO concentration are of interest. Various methods for measurement of nitrite and nitrate (NO2-, NO3- ) -- the stable metabolites of NO -- are commonly used for this purpose. In this paper we have shown that the proper Griess procedure for nitrite determination significantly increases the sensitivity of this method. This procedure, supplemented with deproteinization and reduction of nitrates to nitrites in the presence of NADPH-sensitive reductase, can be successfully applied for measurement of NOx levels in human body fluids (serum, urine and CSF). Deproteinization of samples with methanol/diethylether is required and does not influence the sensitivity of detection of NO metabolites. The recovery of the method is 88%+/-6% (n = 30). The NOx concentrations measured by this procedure ranged from 25.0 to 39.0 micromol/l in blood, 4.6 to 14.6 micromol/l in CSF and 0.37 to 2.52 mmol/l (adjusted to creatinine concentration) in urine. The coefficient of variation for this method was between 1.3-2.2%. This method can also be recommended for measurement of NOx produced by cells in tissue cell culture.

  11. Nitrate ammonification in mangrove soils: a hidden source of nitrite?

    Science.gov (United States)

    Balk, Melike; Laverman, Anniet M; Keuskamp, Joost A; Laanbroek, Hendrikus J

    2015-01-01

    Nitrate reduction is considered to be a minor microbial pathway in the oxidation of mangrove-derived organic matter due to a limited supply of nitrate in mangrove soils. At a limited availability of this electron acceptor compared to the supply of degradable carbon, nitrate ammonification is thought to be the preferential pathway of nitrate reduction. Mangrove forest mutually differ in their productivity, which may lead to different available carbon to nitrate ratios in their soil. Hence, nitrate ammonification is expected to be of more importance in high- compared to low-productive forests. The hypothesis was tested in flow-through reactors that contain undisturbed mangrove soils from high-productive Avicennia germinans and Rhizophora mangle forests in Florida and low-productive Avicennia marina forests in Saudi Arabia. Nitrate was undetectable in the soils from both regions. It was assumed that a legacy of nitrate ammonification would be reflected by a higher ammonium production from these soils upon the addition of nitrate. Unexpectedly, the soils from the low-productive forests in Saudi Arabia produced considerably more ammonium than the soils from the high-productive forests in Florida. Hence, other environmental factors than productivity must govern the selection of nitrate ammonification or denitrification. A rather intriguing observation was the 1:1 production of nitrite and ammonium during the consumption of nitrate, more or less independent from sampling region, location, sampling depth, mangrove species and from the absence or presence of additional degradable carbon. This 1:1 ratio points to a coupled production of ammonium and nitrite by one group of nitrate-reducing microorganisms. Such a production of nitrite will be hidden by the presence of active nitrite-reducing microorganisms under the nitrate-limited conditions of most mangrove forest soils.

  12. Nitrate ammonification in mangrove soils: a hidden source of nitrite?

    KAUST Repository

    Balk, Melike

    2015-03-02

    Nitrate reduction is considered to be a minor microbial pathway in the oxidation of mangrove-derived organic matter due to a limited supply of nitrate in mangrove soils. At a limited availability of this electron acceptor compared to the supply of degradable carbon, nitrate ammonification is thought to be the preferential pathway of nitrate reduction. Mangrove forest mutually differ in their productivity, which may lead to different available carbon to nitrate ratios in their soil. Hence, nitrate ammonification is expected to be of more importance in high- compared to low-productive forests. The hypothesis was tested in flow-through reactors that contain undisturbed mangrove soils from high-productive Avicennia germinans and Rhizophora mangle forests in Florida and low-productive Avicennia marina forests in Saudi Arabia. Nitrate was undetectable in the soils from both regions. It was assumed that a legacy of nitrate ammonification would be reflected by a higher ammonium production from these soils upon the addition of nitrate. Unexpectedly, the soils from the low-productive forests in Saudi Arabia produced considerably more ammonium than the soils from the high-productive forests in Florida. Hence, other environmental factors than productivity must govern the selection of nitrate ammonification or denitrification. A rather intriguing observation was the 1:1 production of nitrite and ammonium during the consumption of nitrate, more or less independent from sampling region, location, sampling depth, mangrove species and from the absence or presence of additional degradable carbon. This 1:1 ratio points to a coupled production of ammonium and nitrite by one group of nitrate-reducing microorganisms. Such a production of nitrite will be hidden by the presence of active nitrite-reducing microorganisms under the nitrate-limited conditions of most mangrove forest soils.

  13. Production and mitigation of N2O in sequentially membrane-aerated redox-stratified nitritation/anammox biofilms

    DEFF Research Database (Denmark)

    Smets, Barth F.; Pellicer i Nàcher, Carles; Thamdrup, Bo

    batch incubations with biofilm samples revealed a significant N2O assimilatory activity. Anoxic incubations with N-15 enriched nitrite, nitrate, or ammonium, in presence or absence of acetate revealed the following: a very high conversion of original nitrite or nitrate N to N2O over N2, no stimulatory......Combining partial nitritation with anaerobic ammonium oxidation maybe a cost- and energy-efficient alternative to remove reduced nitrogen from nitrogen rich waste streams. However, increased N2O emissions (upto several % of the incoming N flux) have been observed for reactors performing partial...... nitritation, which is likely due to the stimulatory effect of combined elevated nitrite and ammonium concentrations and reduced oxygen concentrations on nitrous oxide formation by ammonium oxidizing bacteria. Because increased N2O emission may be inherent to partial nitrification systems, we have explored how...

  14. Electro-oxidation nitrite based on copper calcined layered double hydroxide and gold nanoparticles modified glassy carbon electrode

    International Nuclear Information System (INIS)

    Cui Lin; Meng Xiaomeng; Xu Minrong; Shang Kun; Ai Shiyun; Liu Yinping

    2011-01-01

    Highlights: → A nitrite sensor fabricated based on copper calcined layered double hydroxides and gold nanoparticles modified electrode. → This sensor exhibited excellent electrocatalytic oxidation to nitrite. → This nitrite sensor exhibited very good analytical performance with low cost, convenient preparation and rapid detection. - Abstract: In this paper, a novel nitrite sensor was constructed based on electrodeposition of gold nanoparticles (AuNPs) on a copper calcined layered double hydroxide (Cu-CLDH) modified glassy carbon electrode. Electrochemical experiments showed that AuNPs/CLDH composite film exhibited excellent electrocatalytic oxidation activity with nitrite due to the synergistic effect of the Cu-CLDH with AuNPs. The fabricated sensor exhibited excellent performance for nitrite detection within a wide concentration interval of 1-191 μM and with a detection limit of 0.5 μM. The superior electrocatalytic response to nitrite was mainly attributed to the large surface area, minimized diffusion resistance, and enhanced electron transfer of the Cu-CLDH and AuNPs composition film. This platform offers a novel route for nitrite sensing with wide analytical applications and will supply the practical applications for a variety of simple, robust, and easy-to-manufacture analytical approaches in the future.

  15. Nitrite regulates hypoxic vasodilation via myoglobin-dependent nitric oxide generation.

    Science.gov (United States)

    Totzeck, Matthias; Hendgen-Cotta, Ulrike B; Luedike, Peter; Berenbrink, Michael; Klare, Johann P; Steinhoff, Heinz-Juergen; Semmler, Dominik; Shiva, Sruti; Williams, Daryl; Kipar, Anja; Gladwin, Mark T; Schrader, Juergen; Kelm, Malte; Cossins, Andrew R; Rassaf, Tienush

    2012-07-17

    Hypoxic vasodilation is a physiological response to low oxygen tension that increases blood supply to match metabolic demands. Although this response has been characterized for >100 years, the underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin in the heart can reduce nitrite to nitric oxide (NO·) and thereby contribute to cardiomyocyte NO· signaling during ischemia. On the basis of recent observations that myoglobin is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular myoglobin to form NO·. We show in the present study that myoglobin is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO· from nitrite reduction by deoxygenated myoglobin activates canonical soluble guanylate cyclase/cGMP signaling pathways. In vivo and ex vivo vasodilation responses, the reduction of nitrite to NO·, and the subsequent signal transduction mechanisms were all significantly impaired in mice without myoglobin. Hypoxic vasodilation studies in myoglobin and endothelial and inducible NO synthase knockout models suggest that only myoglobin contributes to systemic hypoxic vasodilatory responses in mice. Endogenous nitrite is a physiological effector of hypoxic vasodilation. Its reduction to NO· via the heme globin myoglobin enhances blood flow and matches O(2) supply to increased metabolic demands under hypoxic conditions.

  16. Use of gamma radiation on control of Clostridium botulinum in mortadella formulated with different nitrite levels

    Science.gov (United States)

    Dutra, Monalisa Pereira; Aleixo, Glécia de Cássia; Ramos, Alcinéia de Lemos Souza; Silva, Maurício Henriques Louzada; Pereira, Marcio Tadeu; Piccoli, Roberta Hilsdorf; Ramos, Eduardo Mendes

    2016-02-01

    This study investigated the effects of applying different doses of gamma radiation (0, 10 and 20 kGy) on Clostridium botulinum spores (107 spores/g) inoculated into mortadellas with different nitrite contents (0, 150 and 300 ppm). We also evaluated the order of application of heat (cooking) and irradiation processing. The products were evaluated for survival of C. botulinum, pH, water activity (Aw), redox potential (Eh) and residual nitrite content. In the non-irradiated raw batters, almost all spores could be recovered when no nitrite was added and only half was recovered with the addition of 150 ppm of nitrite. The use of 150 ppm of nitrite was able to inhibit the germination or growth of C. botulinum in non-irradiated cooked mortadellas after 48 h of processing. However, after 30 days of chilling storage (4 °C), it was possible to recover 105 UFC/g of this microorganism. The gamma irradiation (>10 kGy) had a positive effect on the inactivation of C. botulinum in mortadellas, independent of the sodium nitrite level used and the cooking/irradiation processing order.

  17. Energetic Consequences of nitrite stress in Desulfovibrio vulgarisHildenborough, inferred from global transcriptional analysis

    Energy Technology Data Exchange (ETDEWEB)

    He, Qiang; Huang, Katherine H.; He, Zhili; Alm, Eric J.; Fields,Matthew W.; Hazen, Terry C.; Arkin, Adam P.; Wall, Judy D.; Zhou, Jizhong

    2005-11-03

    Many of the proteins that are candidates for bioenergetic pathways involved with sulfate respiration in Desulfovibrio spp. have been studied, but complete pathways and overall cell physiology remain to be resolved for many environmentally relevant conditions. In order to understand the metabolism of these microorganisms under adverse environmental conditions for improved bioremediation efforts, Desulfovibrio vulgaris Hildenborough was used as a model organism to study stress response to nitrite, an important intermediate in the nitrogen cycle. Previous physiological studies demonstrated that growth was inhibited by nitrite and that nitrite reduction was observed to be the primary mechanism of detoxification. Global transcriptional profiling with whole-genome microarrays revealed coordinated cascades of responses to nitrite in pathways of energy metabolism, nitrogen metabolism, oxidative stress response, and iron homeostasis. In agreement with previous observations, nitrite-stressed cells showed a decrease in the expression of genes encoding sulfate reduction functions in addition to respiratory oxidative phosphorylation and ATP synthase activity. Consequently, the stressed cells had decreased expression of the genes encoding ATP-dependent amino acid transporters and proteins involved in translation. Other genes up-regulated in response to nitrite include the genes in the Fur regulon, which is suggested to be involved in iron homeostasis, and genes in the Per regulon, which is predicted to be responsible for oxidative stress response.

  18. Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira.

    Science.gov (United States)

    Koch, Hanna; Lücker, Sebastian; Albertsen, Mads; Kitzinger, Katharina; Herbold, Craig; Spieck, Eva; Nielsen, Per Halkjaer; Wagner, Michael; Daims, Holger

    2015-09-08

    Nitrospira are a diverse group of nitrite-oxidizing bacteria and among the environmentally most widespread nitrifiers. However, they remain scarcely studied and mostly uncultured. Based on genomic and experimental data from Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II, we identified ecophysiological traits that contribute to the ecological success of Nitrospira. Unexpectedly, N. moscoviensis possesses genes coding for a urease and cleaves urea to ammonia and CO2. Ureolysis was not observed yet in nitrite oxidizers and enables N. moscoviensis to supply ammonia oxidizers lacking urease with ammonia from urea, which is fully nitrified by this consortium through reciprocal feeding. The presence of highly similar urease genes in Nitrospira lenta from activated sludge, in metagenomes from soils and freshwater habitats, and of other ureases in marine nitrite oxidizers, suggests a wide distribution of this extended interaction between ammonia and nitrite oxidizers, which enables nitrite-oxidizing bacteria to indirectly use urea as a source of energy. A soluble formate dehydrogenase lends additional ecophysiological flexibility and allows N. moscoviensis to use formate, with or without concomitant nitrite oxidation, using oxygen, nitrate, or both compounds as terminal electron acceptors. Compared with Nitrospira defluvii from lineage I, N. moscoviensis shares the Nitrospira core metabolism but shows substantial genomic dissimilarity including genes for adaptations to elevated oxygen concentrations. Reciprocal feeding and metabolic versatility, including the participation in different nitrogen cycling processes, likely are key factors for the niche partitioning, the ubiquity, and the high diversity of Nitrospira in natural and engineered ecosystems.

  19. Role of nitrite, urate and pepsin in the gastroprotective effects of saliva

    Science.gov (United States)

    Rocha, Bárbara S.; Lundberg, Jon O; Radi, Rafael; Laranjinha, João

    2016-01-01

    Dietary nitrate is now recognized as an alternative substrate for nitric oxide (•NO) production in the gut. This novel pathway implies the sequential reduction of nitrate to nitrite, •NO and other bioactive nitrogen oxides but the physiological relevance of these oxidants has remained elusive. We have previously shown that dietary nitrite fuels an hitherto unrecognized nitrating pathway at acidic gastric pH, through which pepsinogen is nitrated in the gastric mucosa, yielding a less active form of pepsin in vitro. Here, we demonstrate that pepsin is nitrated in vivo and explore the functional impact of protein nitration by means of peptic ulcer development. Upon administration of pentagastrin and human nitrite-rich saliva or sodium nitrite to rats, nitrated pepsin was detected in the animal's stomach by immunoprecipitation. •NO was measured in the gastric headspace before and after nitrite instillation by chemiluminescence. At the end of each procedure, the stomach's lesions, ranging from gastric erosions to haemorrhagic ulcers, were scored. Nitrite increased gastric •NO by 200-fold (pgastric juice and the mucosa (pgastric ulcers, as the lesions were only apparent when pepsin nitration was inhibited by urate. In sum, this work unravels a novel dietary-dependent nitrating pathway in which pepsin is nitrated and inactivated in the stomach, preventing the progression of gastric ulcers. PMID:27156250

  20. Marked differences in drug-induced methemoglobinemia in sheep are not due to RBC glucose-6-phosphate dehydrogenase, reduced glutathione, or methemoglobin reductase activity

    Energy Technology Data Exchange (ETDEWEB)

    Martin, D.G.; Guertler, A.T.; Lagutchik, M.S.; Woodard, C.L.; Leonard, D.A.

    1993-05-13

    Benzocaine is a commonly used topical anesthetic that is structurally similar to current candidates for cyanide prophylaxis. Benzocaine induces profound methemoglobinemia in some sheep but not others. After topical benzocaine administration certain sheep respond to form MHb (elevated MHb 16-50% after a 56-280 mg dose, a 2-10 second spray with benzocine), while other phenotypically similar sheep fail to significantly form MHb (less than a 2% increase from baseline). Deficiencies in Glucose-6-phosphate dehydrogenase (G-6-PD), reduced glutathione (GSH), and MHb reductase increase the susceptibility to methemoglobinemia in man and animals. Sheep are used as a model for G-6-PD deficiency in man, and differences in this enzyme level could cause the variable response seen in these sheep. Similarly, differences in GSH and MHb reductase could be responsible for the observed differences in MHb formation.

  1. Acute, Sub-lethal Cyanide Poisoning in Mice is Ameliorated by Nitrite Alone: Complications Arising from Concomitant Administration of Nitrite and Thiosulfate as an Antidotal Combination

    Science.gov (United States)

    Cambal, Leah K.; Swanson, Megan R.; Yuan, Quan; Weitz, Andrew C.; Li, Hui-Hua; Pitt, Bruce R.; Pearce, Linda L.; Peterson, Jim

    2011-01-01

    Sodium nitrite alone is shown to ameliorate sub-lethal cyanide toxicity in mice when given from ~1 hour before until 20 minutes after the toxic dose as demonstrated by the recovery of righting ability. An optimum dose (12 mg/kg) was determined to significantly relieve cyanide toxicity (5.0 mg/kg) when administered to mice intraperitoneally. Nitrite so administered was shown to rapidly produce NO in the bloodsteam as judged by the dose dependent appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. It is argued that antagonism of cyanide inhibition of cytochrome c oxidase by NO is the crucial antidotal activity rather than the methemoglobin-forming action of nitrite. Concomitant addition of sodium thiosulfate to nitrite-treated blood resulted in the detection of sulfidomethemoblobin by EPR spectroscopy. Sulfide is a product of thiosulfate hydrolysis and, like cyanide, is known to be a potent inhibitor of cytochrome c oxidase; the effects of the two inhibitors being essentially additive under standard assay conditions, rather than dominated by either one. The findings afford a plausible explanation for an observed detrimental effect in mice associated with the use of the standard nitrite-thiosulfate combination therapy at sub-lethal levels of cyanide intoxication. PMID:21534623

  2. Acute, sublethal cyanide poisoning in mice is ameliorated by nitrite alone: complications arising from concomitant administration of nitrite and thiosulfate as an antidotal combination.

    Science.gov (United States)

    Cambal, Leah K; Swanson, Megan R; Yuan, Quan; Weitz, Andrew C; Li, Hui-Hua; Pitt, Bruce R; Pearce, Linda L; Peterson, Jim

    2011-07-18

    Sodium nitrite alone is shown to ameliorate sublethal cyanide toxicity in mice when given from ∼1 h before until 20 min after the toxic dose as demonstrated by the recovery of righting ability. An optimum dose (12 mg/kg) was determined to significantly relieve cyanide toxicity (5.0 mg/kg) when administered to mice intraperitoneally. Nitrite so administered was shown to rapidly produce NO in the bloodsteam as judged by the dose-dependent appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. It is argued that antagonism of cyanide inhibition of cytochrome c oxidase by NO is the crucial antidotal activity rather than the methemoglobin-forming action of nitrite. Concomitant addition of sodium thiosulfate to nitrite-treated blood resulted in the detection of sulfidomethemoblobin by EPR spectroscopy. Sulfide is a product of thiosulfate hydrolysis and, like cyanide, is known to be a potent inhibitor of cytochrome c oxidase, the effects of the two inhibitors being essentially additive under standard assay conditions rather than dominated by either one. The findings afford a plausible explanation for an observed detrimental effect in mice associated with the use of the standard nitrite-thiosulfate combination therapy at sublethal levels of cyanide intoxication. © 2011 American Chemical Society

  3. Histochemical localization of glutathione dependent NBT-reductase in mouse skin.

    Science.gov (United States)

    Shukla, Y

    2001-09-01

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

  4. Characterization of mitochondrial thioredoxin reductase from C. elegans

    International Nuclear Information System (INIS)

    Lacey, Brian M.; Hondal, Robert J.

    2006-01-01

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

  5. NADH-Ferricyanide Reductase of Leaf Plasma Membranes 1

    Science.gov (United States)

    Askerlund, Per; Laurent, Pascal; Nakagawa, Hiroki; Kader, Jean-Claude

    1991-01-01

    Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b5 reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber

  6. Hypoxia tolerance, nitric oxide, and nitrite

    DEFF Research Database (Denmark)

    Fago, Angela; Jensen, Frank Bo

    2015-01-01

    survival resides in concerted physiological responses, including strong metabolic depression, protection against oxidative damage and – in air breathing animals - redistribution of blood flow. Each of these responses is known to be tightly regulated by nitric oxide (NO) and during hypoxia by its metabolite...... nitrite. The aim of this review is to highlight recent work illustrating the widespread roles of NO and nitrite in the tolerance to extreme oxygen deprivation, in particular in the red-eared slider turtle and crucian carp, but also in diving marine mammals. The emerging picture underscores the importance...

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

    International Nuclear Information System (INIS)

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

    2001-01-01

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

  8. Two-domain laccase from Streptomyces coelicolor: a link between laccases and nitrite reductases

    Czech Academy of Sciences Publication Activity Database

    Skálová, Tereza; Dohnálek, Jan; Ostergaard, L. H.; Ostergaard, P. R.; Kolenko, Petr; Dušková, Jarmila; Štěpánková, Andrea; Hašek, Jindřich

    2009-01-01

    Roč. 16, 3a - Special Issue (2009), s. 3-4 ISSN 1211-5894. [Heart of European Crystallographic Meeting /12./. 24.09.2009-26.09.2009, Třešt´] R&D Projects: GA AV ČR IAA500500701; GA ČR GA305/07/1073 Institutional research plan: CEZ:AV0Z40500505 Keywords : laccase * oxidoreductase * multicopper blue protein Subject RIV: CD - Macromolecular Chemistry

  9. Voltammetry and In Situ Scanning Tunneling Microscopy of Cytochrome c Nitrite Reductase on Au(111)-Electrodes

    DEFF Research Database (Denmark)

    Gwyer, James; Zhang, Jingdong; Butt, Julea

    2006-01-01

    of the density and orientational distribution of NrfA molecules are disclosed. The submonolayer coverage resolved by in situ STM is readily reconciled with the failure to detect nonturnover signals in cyclic voltammetry of the NrfA films. The molecular structures show a range of lateral dimensions...... a direct approach to correlate electrocatalytic and molecular properties of the protein layer, a long-standing issue in protein film voltammetry....

  10. In cellulo monitoring of quinone reductase activity and reactive oxygen species production during the redox cycling of 1,2 and 1,4 quinones.

    Science.gov (United States)

    Cassagnes, Laure-Estelle; Perio, Pierre; Ferry, Gilles; Moulharat, Natacha; Antoine, Mathias; Gayon, Régis; Boutin, Jean A; Nepveu, Françoise; Reybier, Karine

    2015-12-01

    Quinones are highly reactive molecules that readily undergo either one- or two-electron reduction. One-electron reduction of quinones or their derivatives by enzymes such as cytochrome P450 reductase or other flavoproteins generates unstable semiquinones, which undergo redox cycling in the presence of molecular oxygen leading to the formation of highly reactive oxygen species. Quinone reductases 1 and 2 (QR1 and QR2) catalyze the two-electron reduction of quinones to form hydroquinones, which can be removed from the cell by conjugation of the hydroxyl with glucuronide or sulfate thus avoiding its autoxidation and the formation of free radicals and highly reactive oxygen species. This characteristic confers a detoxifying enzyme role to QR1 and QR2, even if this character is strongly linked to the excretion capacity of the cell. Using EPR spectroscopy and confocal microscopy we demonstrated that the amount of reactive oxygen species (ROS) produced by Chinese hamster ovary (CHO) cells overexpressing QR1 or QR2 compared to naive CHO cells was determined by the quinone structural type. Indeed, whereas the amount of ROS produced in the cell was strongly decreased with para-quinones such as menadione in the presence of quinone reductase 1 or 2, a strong increase in ROS was recorded with ortho-quinones such as adrenochrome, aminochrome, dopachrome, or 3,5-di-tert-butyl-o-benzoquinone in cells overexpressing QR, especially QR2. These differences could originate from the excretion process, which is different for para- and ortho-quinones. These results are of particular interest in the case of dopamine considering the association of QR2 with various neurological disorders such as Parkinson disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Nitrite maxima in the Northern Arabian Sea

    Digital Repository Service at National Institute of Oceanography (India)

    Sankaranarayanan, V.N.; DeSousa, S.N.; Fondekar, S.P.

    There are 2 nitrite maxima in the Northern Arabian Sea, one at the thermocline depth and the other at depths between 300 and 500 m. The 2nd maximum is more prominent in the northeastern part of the Arabian Sea. The 1st maximum is associated...

  12. Neutralization of wastewater from nitrite passivation

    International Nuclear Information System (INIS)

    Pawlowski, L.; Mientki, B.; Wasag, H.

    1982-01-01

    A method for neutralization of wastewater formed in nitrite passivation has been presented. The method consists of introducing urea into wastewater and acidifying it with sulphuric acid. Wastewater is neutralized with lime. After clarification, wastewater can be drained outside the plant

  13. Human carbonyl reductase 4 is a mitochondrial NADPH-dependent quinone reductase.

    Science.gov (United States)

    Endo, Satoshi; Matsunaga, Toshiyuki; Kitade, Yukio; Ohno, Satoshi; Tajima, Kazuo; El-Kabbani, Ossama; Hara, Akira

    2008-12-26

    A protein encoded in the gene Cbr4 on human chromosome 4q32.3 belongs to the short-chain dehydrogenase/reductase family. Contrary to the functional annotation as carbonyl reductase 4 (CBR4), we show that the recombinant tetrameric protein, composed of 25-kDa subunits, exhibits NADPH-dependent reductase activity for o- and p-quinones, but not for other aldehydes and ketones. The enzyme was insensitive to dicumarol and quercetin, potent inhibitors of cytosolic quinone reductases. The 25-kDa CBR4 was detected in human liver, kidney and cell lines on Western blotting using anti-CBR4 antibodies. The overexpression of CBR4 in bovine endothelial cells reveals that the enzyme has a non-cleavable mitochondrial targeting signal. We further demonstrate that the in vitro quinone reduction by CBR4 generates superoxide through the redox cycling, and suggest that the enzyme may be involved in the induction of apoptosis by cytotoxic 9,10-phenanthrenequinone.

  14. 9 CFR 319.2 - Products and nitrates and nitrites.

    Science.gov (United States)

    2010-01-01

    ... and nitrates and nitrites. Any product, such as frankfurters and corned beef, for which there is a... without nitrate or nitrite and labeled with such standard name when immediately preceded with the term... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Products and nitrates and nitrites...

  15. 40 CFR 721.4740 - Alkali metal nitrites.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Alkali metal nitrites. 721.4740... Substances § 721.4740 Alkali metal nitrites. (a) Chemical substances and significant new use subject to reporting. (1) The category of chemical substances which are nitrites of the alkali metals (Group IA in the...

  16. Inhibitors of sterol synthesis. Effects of fluorine substitution at carbon atom 25 of cholesterol on its spectral and chromatographic properties and on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

    Science.gov (United States)

    Wilson, W K; Swaminathan, S; Pinkerton, F D; Gerst, N; Schroepfer, G J

    1994-05-01

    25-Fluorocholesterol (III) was prepared by treatment of 25-hydroxycholesterol (IV) with hydrogen fluoride-pyridine. Compounds III, IV, and cholesterol (I) were fully characterized by 1H and 13C NMR, and stereochemical assignments were established for the C-22 and C-23 protons. The side-chain proton assignments, which apply to most other sterols with a saturated eight-carbon side chain, were based on conformational analysis and comparisons with NMR data for 25,26,26,26,27,27,27-heptafluorocholesterol (II). The chromatographic behavior of I, II, and III were compared on thin-layer chromatography, high performance liquid chromatography, and gas chromatography. Major fragment ions in electron-impact mass spectra of III were analogous to ions of either cholesterol or desmosterol, and a similar analogy was observed for the trimethylsilyl ethers. The 25-hydroxysterol IV and the 25-fluorosterol III differed markedly in their effects on the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells. Whereas 25-hydroxycholesterol caused a approximately 66% lowering of reductase activity in cells at 0.1 microM, the 25-fluorosterol III had no effect at this concentration.

  17. Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins.

    Science.gov (United States)

    Heine, Thomas; Tucker, Kathryn; Okonkwo, Nonye; Assefa, Berhanegebriel; Conrad, Catleen; Scholtissek, Anika; Schlömann, Michael; Gassner, George; Tischler, Dirk

    2017-04-01

    The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation. We find that the reductase domain proceeds through a sequential ternary-complex mechanism at low FAD concentration and a double-displacement mechanism at higher concentrations of FAD. Single-turnover studies indicate an observed rate constant for FAD-to-FAD hydride transfer of ~8 s -1 . This step is rate limiting in the styrene epoxidation reaction and helps to ensure that flavin reduction and styrene epoxidation reactions proceed without wasteful side reactions. Comparison of the reductase activity of the fusion proteins with the naturally occurring reductase, SMOB, and N-terminally histidine-tagged reductase, NSMOB, suggests that the observed changes in catalytic mechanism are due in part to an increase in flavin-binding affinity associated with the N-terminal extension of the reductase.

  18. TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation.

    Science.gov (United States)

    Amaral, Jefferson H; Montenegro, Marcelo F; Pinheiro, Lucas C; Ferreira, Graziele C; Barroso, Rafael P; Costa-Filho, Antonio J; Tanus-Santos, Jose E

    2013-12-01

    Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (N(ω)-nitro-L-arginine methyl ester; 100mg/kg; po)-induced hypertension treated with TEMPOL (18mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl

  19. Nitroxide 4-hydroxy-2,2',6,6'-tetramethylpiperidine 1-oxyl (Tempol) inhibits the reductase activity of protein disulfide isomerase via covalent binding to the Cys400residue on CXXC redox motif at the a'active site.

    Science.gov (United States)

    Santos, Gérsika Bitencourt; Gonzalez-Perilli, Lucia; Mastrogiovanni, Mauricio; Aicardo, Adrián; Cerdeira, Cláudio Daniel; Trostchansky, Andrés; Brigagão, Maísa Ribeiro Pereira Lima

    2017-06-25

    Oxidative stress arising from inflammatory processes is a serious cause of cell and tissue damage. Tempol is an efficient antioxidant with superoxide dismutase-like activity. The purpose of this paper is to address the inhibition of protein disulfide isomerase (PDI), an essential redox chaperone whose active sites contain the Cys-Gly-His-Cys (CXXC) motif, by the nitroxide Tempol. In the presence of Tempol (5-120 μM), the reductase activity of PDI was reversibly affected both in vitro and in activated mice neutrophils, with an IC 50 of 22.9 ± 10.8 μM. Inhibitory activity was confirmed by using both the insulin method and fluorescent formation of eosin-glutathione (E-GSH). The capacity of Tempol to bind the enzyme was determined by EPR and mass spectrometry. EPR Tempol signal decreased in the presence of PDI while remained unaffected when PDI thiols were previously blocked with NEM. When total protein was analyzed, 1 and 4 molecules of Tempol were bound to the protein. However, only one was found to be covalently bound to PDI at the a'active site. More specifically, Cys 400 was modified by Tempol. We have shown that the nitroxide Tempol acts as an inhibitor of PDI through covalent binding to the Cys400 of the protein structure. Since PDI is coupled with the assembly of the NADPH oxidase complex of phagocytes, these findings reveal a novel action of Tempol that presents potential clinical applications for therapeutic intervention to target PDI knockdown in pathological processes in which this protein is engaged. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Investigation of nitrite adulteration on the immunoassay and GC-MS analysis of cannabinoids in urine specimens.

    Science.gov (United States)

    Tsai, L S; ElSohly, M A; Tsai, S F; Murphy, T P; Twarowska, B; Salamone, S J

    2000-01-01

    Nitrite ion has been identified as the active ingredient of two commercial adulterants that could cause discrepant results between the immunoassay screening and gas chromatographic-mass spectrometric (GC-MS) confirmation of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in urine. Procedures to chemically convert the nitrite ion at the beginning of sample preparation for GC-MS analysis may not overcome all nitrite adulteration cases because portions of the THCCOOH might have been lost between the time of sample collection and the time of analysis. This study was conducted to further investigate the influence of both urine sample matrix and the duration of nitrite exposure on nitrite interference of THCCOOH detection. Forty clinical "THC-positive samples" that had been screened and confirmed positive for the presence of THCCOOH were spiked with 0.15M or 0.3M of nitrite. The levels of THCCOOH at various time intervals after nitrite spiking were monitored by instrument-based cannabinoids immunoassays (Syva EMIT d.a.u. and/or Roche Abuscreen ONLINE assays) and by an onsite THC immunoassay (Roche ONTRAK TESTSTIK). Results from this report demonstrate that the two outstanding "urine specimen factors" that dictated the effectiveness of the nitrite adulteration were urinary pH and the original drug concentration before nitrite spiking. Significant decreases in the immunoassay results could be observed within 4 h of nitrite treatment in the majority of samples with acidic urinary pH values. Regardless of their original concentration of THCCOOH (GC-MS ranging from 33 to 488 ng/mL), all of the 20 samples that had acidic pH values gave negative immunoassay results 1 day after nitrite adulteration. In contrast, the immunoassay results of samples with neutral or basic pH values were less affected by nitrite exposure in the same studies. Approximately two-thirds of the samples with pH values greater than 7.0 remained immunoassay-positive 3 days after nitrite

  1. Concurrent Activity of Anammox and Denitrifying Bacteria in the Black Sea

    OpenAIRE

    John B. Kirkpatrick; John B. Kirkpatrick; Clara A. Fuchsman; Evgeniy eYakushev; James T. Staley; James W. Murray

    2012-01-01

    After the discovery of ANaerobic AMMonium OXidation (anammox) in the Black Sea in 2003, the role of heterotrophic denitrification as the main marine pathway for fixed N loss was questioned. A 3 part, 15 month time series investigating Black Sea nitrite reductase (nirS) mRNA transcripts at a single location was conducted in order to better understand the activity of anammox and denitrifying bacteria. Here we show that both of these groups were active, as well as being concurrent in the lower s...

  2. Concurrent activity of anammox and denitrifying bacteria in the Black Sea

    OpenAIRE

    Kirkpatrick, John B.; Fuchsman, Clara A.; Yakushev, Evgeniy; Staley, James T.; Murray, James W.

    2012-01-01

    After the discovery of ANaerobic AMMonium OXidation (anammox) in the environment, the role of heterotrophic denitrification as the main marine pathway for fixed N loss has been questioned. A 3 part, 15 month time series investigating nitrite reductase (nirS) mRNA transcripts at a single location in the Black Sea was conducted in order to better understand the activity of anammox and denitrifying bacteria. Here we show that both of these groups were active, as well as being concurrent in the l...

  3. Molecular effects of bioactive fraction of Curcuma mangga (DLBS4847 as a downregulator of 5α-reductase activity pathways in prostatic epithelial cells

    Directory of Open Access Journals (Sweden)

    Karsono AH

    2014-06-01

    Full Text Available Agung Heru Karsono, Olivia Mayasari Tandrasasmita, Raymond R TjandrawinataSection of Molecular Pharmacology, Research Innovation and Invention, Dexa Laboratories of Biomolecular Sciences, Dexa Medica, Cikarang, IndonesiaAbstract: DLBS4847 is a standardized bioactive fraction of Curcuma mangga. In this study, we used prostate cancer (PC-3 as the cell line to study the effects of DLBS4847 on prostatic cell viability, as well as related molecular changes associated with the decreased cell number. The observation revealed that DLBS4847 inhibited the growth of PC3 cells through downregulation of the 5α-reductase (5AR pathway. At the transcription level, 5AR1 and androgen-receptor gene expressions were downregulated in a dose-dependent manner. Furthermore, 5AR-1 and dihydrotestosterone expression were also downregulated at the protein level. A microarray study was also performed to see the effects of DLBS4847 on differential gene expressions in prostate cancer 3 cells. Among others, DLBS4847 downregulated genes related to prostate growth and hypertrophy. Our results suggested that DLBS4847 could potentially become an alternative treatment for prostate disorders, such as benign prostatic hyperplasia. In this regard, DLBS4847 exerts its growth inhibition partially through downregulation of the 5AR pathway.Keywords: DLBS4847, Curcuma mangga, 5α-reductase inhibitor, benign prostatic hyperplasia (BPH, prostate cancer

  4. The Role of Human Aldo-Keto Reductases (AKRs in the Metabolic Activation and Detoxication of Polycyclic Aromatic Hydrocarbons: Interconversion of PAH-catechols and PAH o-Quinones

    Directory of Open Access Journals (Sweden)

    Li eZhang

    2012-11-01

    Full Text Available Polycyclic aromatic hydrocarbons (PAH are ubiquitous environmental pollutants. They are procarcinogens requiring metabolic activation to elicit their deleterious effects. Aldo-keto reductases (AKR catalyze the oxidation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active PAH o-quiniones. AKRs are also found to be capable of reducing PAH o-quinones to form PAH catechols. The interconversion of o-quinones and catechols results in the redox cycling of PAH o-quinones to give rise to the generation of reactive oxygen species and subsequent oxidative DNA damage. On the other hand, PAH catechols can be intercepted through phase II metabolism by which PAH o-quinones could be detoxified and eliminated. The aim of the present review is to summarize the role of human AKRs in the metabolic activation/detoxication of PAH and the relevance of phase II conjugation reactions to human lung carcinogenesis.

  5. Effect of alkalinity on nitrite accumulation in treatment of coal chemical industry wastewater using moving bed biofilm reactor.

    Science.gov (United States)

    Hou, Baolin; Han, Hongjun; Jia, Shengyong; Zhuang, Haifeng; Zhao, Qian; Xu, Peng

    2014-05-01

    Nitrogen removal via nitrite (the nitrite pathway) is more suitable for carbon-limited industrial wastewater. Partial nitrification to nitrite is the primary step to achieve nitrogen removal via nitrite. The effect of alkalinity on nitrite accumulation in a continuous process was investigated by progressively increasing the alkalinity dosage ratio (amount of alkalinity to ammonia ratio, mol/mol). There is a close relationship among alkalinity, pH and the state of matter present in aqueous solution. When alkalinity was insufficient (compared to the theoretical alkalinity amount), ammonia removal efficiency increased first and then decreased at each alkalinity dosage ratio, with an abrupt removal efficiency peak. Generally, ammonia removal efficiency rose with increasing alkalinity dosage ratio. Ammonia removal efficiency reached to 88% from 23% when alkalinity addition was sufficient. Nitrite accumulation could be achieved by inhibiting nitrite oxidizing bacteria (NOB) by free ammonia (FA) in the early period and free nitrous acid in the later period of nitrification when alkalinity was not adequate. Only FA worked to inhibit the activity of NOB when alkalinity addition was sufficient. Copyright © 2014 The Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved.

  6. Antimicrobial properties of milk: dependence on presence of xanthine oxidase and nitrite.

    Science.gov (United States)

    Hancock, John T; Salisbury, Vyv; Ovejero-Boglione, Maria Cristina; Cherry, Robert; Hoare, Catherine; Eisenthal, Robert; Harrison, Roger

    2002-10-01

    Human and bovine milk inhibited the metabolic activity of Escherichia coli, as shown by luminescence monitoring of constructs expressing the luxCDABE genes. Inhibition was dependent on both xanthine oxidase (XO) activity and on the presence of nitrite, implying that XO-generated nitric oxide functions as an antibacterial agent.

  7. Antimicrobial Properties of Milk: Dependence on Presence of Xanthine Oxidase and Nitrite

    OpenAIRE

    Hancock, John T.; Salisbury, Vyv; Ovejero-Boglione, Maria Cristina; Cherry, Robert; Hoare, Catherine; Eisenthal, Robert; Harrison, Roger

    2002-01-01

    Human and bovine milk inhibited the metabolic activity of Escherichia coli, as shown by luminescence monitoring of constructs expressing the luxCDABE genes. Inhibition was dependent on both xanthine oxidase (XO) activity and on the presence of nitrite, implying that XO-generated nitric oxide functions as an antibacterial agent.

  8. Synthesis and 2D-QSAR studies of neolignan-based diaryl-tetrahydrofuran and -furan analogues with remarkable activity against Trypanosoma cruzi and assessment of the trypanothione reductase activity.

    Science.gov (United States)

    Hartmann, Ana Paula; de Carvalho, Marcelo Rodrigues; Bernardes, Lilian Sibelle Campos; Moraes, Milena Hoehr de; de Melo, Eduardo Borges; Lopes, Carla Duque; Steindel, Mario; da Silva, João Santana; Carvalho, Ivone

    2017-11-10

    Two series of diaryl-tetrahydrofuran and -furan were synthesised and screened for anti-trypanosomal activity against trypomastigote and amastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. Based on evidence that modification of a natural product may result in a more effective drug than the natural product itself, and using known neolignan inhibitors veraguensin 1 and grandisin 2 as templates to synthesise simpler analogues, remarkable anti-trypanosomal activity and selectivity were found for 3,5-dimethoxylated diaryl-furan 5c and 2,4-dimethoxylated diaryl-tetrahydrofuran 4e analogues with EC 50 0.01 μM and EC 50 0.75 μM, respectively, the former being 260-fold more potent than veraguensin 1 and 150-fold better than benznidazole, the current available drugs for Chagas disease treatment. The ability of the most potent anti-trypanosomal compounds to penetrate LLC-MK2 cells infected with T. cruzi amastigotes parasite was tested, which revealed 4e and 5e analogues as the most effective, causing no damage to mammalian cells. In particular, the majority of the derivatives were non-toxic against mice spleen cells. 2D-QSAR studies show the rigid central core and the position of dimethoxy-aryl substituents dramatically affect the anti-trypanosomal activity. The mode of action of the most active anti-trypanosomal derivatives was investigated by exploring the anti-oxidant functions of Trypanothione reductase (TR). As a result, diarylfuran series displayed the strongest inhibition, highlighting compounds 5d-e (IC 50 19.2 and 17.7 μM) and 5f-g (IC 50 8.9 and 7.4 μM), respectively, with similar or 2-fold higher than the reference inhibitor clomipramine (IC 50 15.2 μM). Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  9. Mutagenesis breeding research of Lactobacillus brevis of nitrite reduction

    Directory of Open Access Journals (Sweden)

    LI Zeli

    2015-10-01

    Full Text Available The pollution of nitrite in food became one of the focus of food safety issues,the use of biotechnology methods degrading nitrite became hotspot.The primitive strain was Lactobacillus brevis C2,preserved in our laboratory,had the ability to degrade nitrite,through composite mutagenesis of 15 W,254 nm,20 cm ultraviolet mutagenesis (UV for 120 s and 0.8% diethyl sulfate(DES in 37℃ mutation for 40 min,after screening,we successfully obtained high efficient strain of nitrite degradation,named UV6-DS2,relative to the starting strain,under the condition of 400 mg/L nitrite,after 12 h degradation,nitrite degradation rate increased from 92.8% to 97.8%,to explore its application in food was able to effectively reduce concentration of nitrite in food.

  10. Extreme differences between hemoglobins I and II of the clam Lucina pectinalis in their reactions with nitrite.

    Science.gov (United States)

    Bonaventura, Celia; Henkens, Robert; De Jesus-Bonilla, Walleska; Lopez-Garriga, Juan; Jia, Yiping; Alayash, Abdu I; Siburt, Claire J Parker; Crumbliss, Alvin L

    2010-10-01

    The clam Lucina pectinalis supports its symbiotic bacteria by H₂S transport in the open and accessible heme pocket of Lucina Hb I and by O₂ transport in the narrow and crowded heme pocket of Lucina Hb II. Remarkably, air-equilibrated samples of Lucina Hb I were found to be more rapidly oxidized by nitrite than any previously studied Hb, while those of Lucina Hb II showed an unprecedented resistance to oxidation induced by nitrite. Nitrite-induced oxidation of Lucina Hb II was enabled only when O₂ was removed from its active site. Structural analysis revealed that O₂ "clams up" the active site by hydrogen bond formation to B10Tyr and other distal-side residues. Quaternary effects further restrict nitrite entry into the active site and stabilize the hydrogen-bonding network in oxygenated Lucina Hb II dimers. The dramatic differences in nitrite reactivities of the Lucina Hbs are not related to their O₂ affinities or anaerobic redox potentials, which were found to be similar, but are instead a result of differences in accessibility of nitrite to their active sites; i.e. these differences are due to a kinetic rather than thermodynamic effect. Comparative studies revealed heme accessibility to be a factor in human Hb oxidation by nitrite as well, as evidenced by variations of rates of nitrite-induced oxidation that do not correlate with R and T state differences and inhibition of oxidation rate in the presence of O₂. These results provide a dramatic illustration of how evolution of active sites with varied heme accessibility can moderate the rates of inner-sphere oxidative reactions of Hb and other heme proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

  11. Investigation of reduction and tolerance capability of lactic acid bacteria isolated from kimchi against nitrate and nitrite in fermented sausage condition.

    Science.gov (United States)

    Paik, Hyun-Dong; Lee, Joo-Yeon

    2014-08-01

    Lactobacillus brevis KGR3111, Lactobacillus curvatus KGR 2103, Lactobacillus plantarum KGR 5105, and Lactobacillus sakei KGR 4108 isolated from kimchi were investigated for their potential to be used as starter culture for fermented sausages with the capability to reduce and tolerate nitrate/nitrite. The reduction capability of tested strains for nitrate was not dramatic. All tested strains, however, showed the capability to produce nitrite reductase with the reduction amount of 58.46-75.80 mg/l of NO(2)(-). L. brevis and L. plantarum showed nitrate tolerance with the highest number of 8.71 log cfu/ml and 8.81 log cfu/ml, and L. brevis and L. sakei exhibited nitrite tolerance with the highest number of 8.24 log cfu/ml and 8.25 log cfu/ml, respectively. As a result, L. brevis, L. plantarum, and L. sakei isolated from kimchi showed a tolerance against nitrate or nitrite with a good nitrite reduction capability, indicating the satisfaction of one of the selection criteria to be used as starter culture for fermented sausages. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Origin of nitrite and nitrate in nasal and exhaled breath condensate and relation to nitric oxide formation.

    Science.gov (United States)

    Marteus, H; Törnberg, D C; Weitzberg, E; Schedin, U; Alving, K

    2005-03-01

    Raised concentrations of nitrate and nitrite have been found in exhaled breath condensate (EBC) in airway disease, and it has been postulated that this reflects increased nitric oxide (NO) metabolism. However, the chemical and anatomical origin of nitrate and nitrite in the airways has not yet been sufficiently studied. The fraction of exhaled NO at an exhalation flow rate of 50 ml/s (FE(NO)) and nitrite and nitrate in EBC, nasal condensate, and saliva were measured in 17 tracheostomised and 15 non-tracheostomised subjects, all of whom were non-smokers without respiratory disease. Tracheal and oral samples were taken from the tracheostomised subjects and nasal (during velum closure) and oral samples from the non-tracheostomised subjects. Measurements were performed before and after sodium nitrate ingestion (10 mg/kg) and use of antibacterial mouthwash (chlorhexidine 0.2%). In tracheostomised subjects oral FE(NO) increased by 90% (pnitrate ingestion. Oral EBC nitrite levels were increased 23-fold at 60 minutes (pnitrite levels in tracheal EBC showed only a minor increase (fourfold, pNitrate was increased the same amount in oral and tracheal EBC at 60 minutes (2.5-fold, pnitrite increased after nitrate ingestion and after chlorhexidine mouthwash they approached baseline levels again (pnitrate, and nitrite were not affected by nitrate intake or mouthwash. At baseline, mouthwash with deionised water did not affect nitrite in oral EBC or saliva, whereas significant reductions were seen after antibacterial mouthwash (pnitrate is taken up by the lower airways but not the nasal airways. Nitrate levels in EBC are thus influenced by dietary intake. Nitrate is reduced to nitrite by bacterial activity which takes place primarily in the oropharyngeal tract of healthy subjects. Only oropharyngeal nitrite seems to contribute to exhaled NO in non-inflamed airways, and there is also a substantial contribution of nitrite from the oropharyngeal tract during standard collection of EBC.

  13. Role of nitrite in regulation of fetal cephalic circulation in sheep.

    Science.gov (United States)

    Truong, Giang T; Schröder, Hobe J; Liu, Taiming; Zhang, Meijuan; Kanda, Eriko; Bragg, Shannon; Power, Gordon G; Blood, Arlin B

    2014-04-15

    Nitrite has been postulated to provide a reservoir for conversion to nitric oxide (NO), especially in tissues with reduced oxygen levels as in the fetus. Nitrite would thus provide local vasodilatation and restore a balance between oxygen supply and need, a putative mechanism of importance especially in the brain. The current experiments test the hypothesis that exogenous nitrite acts as a vasodilator in the cephalic vasculature of the intact, near term fetal sheep. Fetuses were first instrumented to measure arterial blood pressure and carotid artery blood flow and then studied 4-5 days later while in utero without anaesthesia. Initially l-nitro-arginine (LNNA) was given to block endogenous NO production. Carotid resistance to flow increased 2-fold from 0.54 ± 0.01 (SEM) to 1.20 ± 0.08 mmHg min ml(-1) (in 13 fetuses, P pressure, blood flow and vascular resistance did not change compared to fetuses receiving saline, even at plasma nitrite concentrations two orders of magnitude above the physiological range. The results indicate that while cephalic vascular tone is controlled by endogenous nitric oxide synthase activity, exogenously administered nitrite is not a vasodilator at physiological concentrations in the vasculature served by the carotid artery of fetal sheep.

  14. Effects of low concentrations of bisulfite-sulfite and nitrite on microorganisms.

    Science.gov (United States)

    Wodzinski, R S; Labeda, D P; Alexander, M

    1978-01-01

    A wide range of microorganisms was tested to determine their sensitivity to low concentrations of bisulfite-sulfite and nitrite, solubility products of SO2 and NO2, respectively. Photosynthesis by blue-green algae (cyanobacteria) was more strongly inhibited by 0.1 mM bisulfite-sulfite and 1 mM nitrite at pH 6.0 than photosynthesis by eucaryotic algae and respiration of bacteria, fungi, and protozoa. At pH 7.7, blue-green algae were still more sensitive to bisulfite-sulfite and nitrite than eucaryotic algae, but the toxicity of bisulfite-sulfite and nitrite decreased as the pH increased. Photosynthesis by Anabaena flos-aquae at pH 6.0 was inhibited 25% by a bisulfite-sulfite concentration of 10 micrometer and 15% by a nitrite concentration of 50 micrometer. Photosynthesis by the blue-green alga, Lyngbya sp., was not exceptionally sensitive to chlorate and thiosulfate. Acetylene-reducing activity of Beijerinckia indica was completely inhibited by 0.1 mM bisulfite-sulfite at pH 4.0, the suppression being decreased with increasing pH. PMID:646357

  15. Optimization of nitrogen removal from piggery waste by nitrite nitrification.

    Science.gov (United States)

    Eum, Y; Choi, E

    2002-01-01

    The piggery waste characteristics greatly vary with types of manure collections and the amount of water used. If solids are separated well, the waste strength will be greatly reduced resulting in lower TCOD/TKN ratio of 4 (average). If solids are separated by a mechanical scraper, some solids will remain and the waste strength will be increased with a TCOD/TKN ratio of 7. This study was conducted to find an optimum operating condition for nitrogen removal with these two ratios. Nitrite nitrification was targeted because it could be a short cut process for savings in oxygen for nitrification and carbon requirements for denitrification. The study results indicated that nitrogen loading rate and pH were the most important factors to be considered for stable nitrite nitrification. The applicable nitrogen loads were estimated to be 0.3 to 2.0 kgTKN/oxic m3/d for high TCOD/TKN ratio without pH control. With higher pH > 8, NO2N/NOxN ratios in oxic stages even with lower nitrogen loads were increased. The SBR with low TCOD/TKN ratio less than 4 required additional alkalinity. For a complete denitrification, the influent TCOD/TKN ratio must exceed 6 with oxic/total reactor volume ratio of 0.5. Nitrite nitrification and denitrification could save about 35% in tank volume and 50% in carbon requirement, respectively. However, 9.5% oxygen saving could be expected during the operation with low TCOD/TKN ratio. The elevated temperature due to the heat released from COD removal also enhanced microbial activities for nitrification and denitrification as well as ammonia stripping. However, careful attention must be provided for the reactor temperature not to inhibit the nitrification process.

  16. Cultivation, growth physiology, and chemotaxonomy of nitrite-oxidizing bacteria.

    Science.gov (United States)

    Spieck, Eva; Lipski, André

    2011-01-01

    Lithoautotrophic nitrite-oxidizing bacteria (NOB) are known as fastidious microorganisms, which are hard to maintain and not many groups are trained to keep them in culture. They convert nitrite stoichiometrically to nitrate and growth is slow due to the poor energy balance. NOB are comprised of five genera, which are scattered among the phylogenetic tree. Because NOB proliferate in a broad range of environmental conditions (terrestrial, marine, acidic) and have diverse lifestyles (lithoautotrophic, mixotrophic, and heterotrophic), variation in media composition is necessary to match their individual growth requirements in the laboratory. From Nitrobacter and Nitrococcus relatively high cell amounts can be achieved by consumption of high nitrite concentrations, whereas accumulation of cells belonging to Nitrospira, Nitrospina, or the new candidate genus Nitrotoga needs prolonged feeding procedures. Isolation is possible for planktonic cells by dilution series or plating techniques, but gets complicated for strains with a tendency to develop microcolonies like Nitrospira. Physiological experiments including determination of the temperature or pH-optimum can be conducted with active laboratory cultures of NOB, but the attainment of reference values like cell protein content or cell numbers might be hard to realize due to the formation of flocs and the low cell density. Monitoring of laboratory enrichments is necessary especially if several species or genera coexist within the same culture and due to population shifts over time. Chemotaxonomy is a valuable method to identify and quantify NOB in biofilms and pure cultures alike, since fatty acid profiles reflect their phylogenetic heterogeneity. This chapter focusses on methods to enrich, isolate, and characterize NOB by various cultivation-based techniques. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor.

    Science.gov (United States)

    Li, Yun; Wang, Zhaozhao; Li, Jun; Wei, Jia; Zhang, Yanzhuo; Zhao, Baihang

    2017-04-01

    Nitritation can be used as a pretreatment for anaerobic ammonia oxidation (anammox). Various control strategies for nitritation and half-nitritation of old landfill leachate in a membrane bioreactor were investigated in this study and the inhibition kinetics of substrate, product and old landfill leachate on nitritation were analyzed via batch tests. The results demonstrated that old landfill leachate nitritation in the membrane bioreactor can be achieved by adjusting the influent loading and dissolved oxygen (DO). From days 105-126 of the observation period, the average effluent concentration was 871.3 mg/L and the accumulation rate of [Formula: see text] was 97.2%. Half-nitritation was realized quickly by adjusting hydraulic retention time and DO. A low-DO control strategy appeared to best facilitate long-term and stable operation. Nitritation inhibition kinetic experiments showed that the inhibition of old landfill leachate was stronger than that of the substrate [Formula: see text] or product [Formula: see text] . The ammonia oxidation rate dropped by 22.2% when the concentration of old landfill leachate (calculated in chemical oxygen demand) was 1600.2 mg/L; further, when only free ammonia or free nitrous acid were used as a single inhibition factor, the ammonia oxidation rate dropped by 4.7-6.5% or 14.5-15.9%, respectively. Haldane, Aiba, and a revised inhibition kinetic model were adopted to separately fit the experimental data. The R 2 correlation coefficient values for these three models were 0.982, 0.996, and 0.992, respectively. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  18. A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi

    DEFF Research Database (Denmark)

    Pena Diaz, Javier; Montalvetti, A; Camacho, A

    1997-01-01

    cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly...

  19. Isolated menthone reductase and nucleic acid molecules encoding same

    Science.gov (United States)

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

    2013-04-23

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

  20. Nitric oxide formation from nitrite in zebrafish

    DEFF Research Database (Denmark)

    Jensen, Frank Bo

    2007-01-01

    nitrite levels for variable time periods, and changes in blood nitrosylhemoglobin (HbNO), methemoglobin (metHb), oxygenated hemoglobin (oxyHb) and deoxygenated hemoglobin (deoxyHb) were evaluated by spectral deconvolution. Blood HbNO (a biomarker of internal NO production) was low in controls, increased......, and the possibility that excess NO may inhibit mitochondrial respiration, whole animal routine oxygen consumption was not depressed....

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin...... diacetyl (acetoin) reductase activity with NADH as coenzyme, but not with NADPH as coenzyme, suggesting the presence of another diacetyl (acetoin)-reducing activity in L. pseudomesenteroides. Plasmid-curing experiments demonstrated that the butA gene is carried on a 20-kb plasmid in L. pseudomesenteroides....

  2. The role of red blood cell S-nitrosation in nitrite bioactivation and its modulation by leucine and glucose

    Directory of Open Access Journals (Sweden)

    Nadeem Wajih

    2016-08-01

    Full Text Available Previous work has shown that red blood cells (RBCs reduce nitrite to NO under conditions of low oxygen. Strong support for the ability of red blood cells to promote nitrite bioactivation comes from using platelet activation as a NO-sensitive process. Whereas addition of nitrite to platelet rich plasma in the absence of RBCs has no effect on inhibition of platelet activation, when RBCs are present platelet activation is inhibited by an NO-dependent mechanism that is potentiated under hypoxia. In this paper, we demonstrate that nitrite bioactivation by RBCs is blunted by physiologically-relevant concentrations of nutrients including glucose and the important signaling amino acid leucine. Our mechanistic investigations demonstrate that RBC mediated nitrite bioactivation is largely dependent on nitrosation of RBC surface proteins. These data suggest a new expanded paradigm where RBC mediated nitrite bioactivation not only directs blood flow to areas of low oxygen but also to areas of low nutrients. Our findings could have profound implications for normal physiology as well as pathophysiology in a variety of diseases including diabetes, sickle cell disease, and arteriosclerosis.

  3. Photooxidative Destruction of Chloroplasts Leads to Reduced Expression of Peroxisomal NADH-Dependent Hydroxypyruvate Reductase in Developing Cucumber Cotyledons 1

    Science.gov (United States)

    Schwartz, Brain W.; Daniel, Steven G.; Becker, Wayne M.

    1992-01-01

    Photooxidative destruction of chloroplasts by exposure of norflurazon-treated cucumber (Cucumis sativus L.) seedlings to white light leads to reduced levels of the nuclear-encoded, peroxisomal enzyme hydroxypyruvate reductase. The partial reduction in hydroxypyruvate reductase activity under photooxidative conditions is accompanied by reductions in levels of hydroxypyruvate reductase protein and transcript. The low level of hydroxypyruvate reductase gene expression in the dark is not affected by norflurazon, and nonphotooxidizing far-red light is able to induce significant increases in hydroxypyruvate reductase expression even in the presence of norflurazon. We conclude that intact plastids are required for maximal expression of hydroxypyruvate reductase in the light and that the plastids affect hydroxypyruvate reductase gene expression at a pretranslational level. ImagesFigure 2Figure 3Figure 4 PMID:16668940

  4. Optimum conditions for cotton nitrate reductase extraction and ...

    African Journals Online (AJOL)

    GREGO

    mM of glutamine in the extraction buffer stimulates significantly, in vitro, the reduction of nitrate. Enzyme activity is moreover optimal when 1 M of exogenous nitrate, as substrate, is added to the reaction medium. At these optimum conditions of nitrate reductase activity determination, the substrate was completely reduced ...

  5. Safer and healthier reduced nitrites turkey meat sausages using lyophilized Cystoseira barbata seaweed extract.

    Science.gov (United States)

    Sellimi, Sabrine; Benslima, Abdelkarim; Ksouda, Ghada; Montero, Veronique Barragan; Hajji, Mohamed; Nasri, Moncef

    2017-10-21

    Background Nitrite salts are still common additives in the meat industry. The present study provides a first approach on the employment of the lyophilized aqueous extract (WE) of the Tunisian seaweed Cystoseira barbata for the quality enhancement of turkey meat sausage. Methods WE was supplemented as a natural antioxidant agent to investigate its effectiveness in delaying lipid oxidation turkey meat sausages containing reduced amounts of sodium nitrites. Results On storage day 5, all concentrations of WE (0.01-0.4 %) reduced the meat lipid oxidation by approximately 36 %, as compared to the negative control containing only 80 mg/kg of meat of sodium nitrites as antioxidant. It was noted that within 15 days of refrigerated storage, a meat system containing 80 mg/kg of meat of sodium nitrites and 0.02 % and 0.04 % of WE had similar Thiobarbituric Acid Reactive Substances (TBARS) levels (19±1.32 and 17±1.12 µmol/kg of meat, respectively), which were comparable to the positive control containing sodium nitrites (150 mg/kg of meat) and 0.045 % vitamin C (18.46±1.27 µmol/kg of meat). In-depth, the metabolomic profiling using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-quadripole-time-of-flight-mass spectrometry (LC-QTOF-MS) analyses of the Tunisian seaweed C. barbata solvent extracts showed that the main active compounds were phenolic compounds, fatty acids and sterols. Conclusions Overall, the cold medium containing C. barbata lyophilized aqueous extrac, with strong antioxidant activity and antihypertensive properties, may open the way to the development of a natural quality enhancement strategy for new functional and ever healthier reduced nitrites meat sausages based on algae.

  6. PENURUNAN KADAR AMONIA, NITRIT, DAN NITRAT LIMBAH CAIR INDUSTRI TAHU MENGGUNAKAN ARANG AKTIF DARI AMPAS KOPI

    Directory of Open Access Journals (Sweden)

    Irmanto

    2009-11-01

    Full Text Available The tofu industry is one of food industry which the product of organic waste to environment pollution. One of alternative methode which used to overcome tofu industrial waste water pollution is adsorption methode using activated carbon from coffee waste. The aim of this researched is to know about the activated carbon from coffee waste quality which observe of rendemen, water content, ash content, and iodium adsorption, to know optimum contact of time and pH of coffee waste to decrease ammonia, nitrite and nitrate contents in tofu industry waste water and to know decrease percentage of ammonia, nitrite and nitrate contents in tofu industrial waste water using activated carbon from coffee waste. The activated carbon made by soaking of coffee waste in HCl 0.1 M solution for 2 days. The activated carbon coaled in muffle furnace at temperature 350°C. The activated carbon analyzed consist of rendemen, water content, ash content, and iodium adsorption. Optimum contact of time and pH of coffee waste determined in order to get optimum adsorption ammonia, nitrite and nitrate in tofu industrial waste water. Contact time variation are 1, 10, 30, 45, 60, 90, 120 minutes and pH variation are 4, 5, 6, 7, 8, 9, 10. The result showed that the activated carbon from coffee waste fulfill the criteria SNI number 06-3730-1995. The activated carbon from coffee waste could be used to decrease the ammonia, nitrite, and nitrate contents in tofu industrial waste water at the optimum contact of time of 30 minutes and pH 7. Decreasing percentage of ammonia, nitrite and nitrate contents in tofu industrial waste water are 64,69% , 52,35% and 86,40% respectively.

  7. Determination of Nitrite in Meat Products using a Metalloporphyrin Based Nitrite-Selective Membrane Electrode

    Directory of Open Access Journals (Sweden)

    Dana VLASCICI

    2006-07-01

    Full Text Available The potentiometric response characteristics of a nitrite-selective electrode based on Co (III tetraphenylporphyrins (TPP in o-nitrophenyloctylether plasticized polyvinyl chloride membranes are compared. To establish the optimum composition of the membrane, different molar percents of cationic derivative (0-100 mol% relative to ionophore were used. The influence of different plasticizers: o-nitrophenyloctylether, dioctylphtalate and tricresilphosphate on potentiometric answer were studied. Electrodes formulated with membranes containing 1 wt% ClCoTPP, 66 wt% o-NPOE, 33 wt% PVC (plasticizer:PVC = 2:1 and the lipophilic cationic derivative (10 mol% are shown to exhibit high selectivity for nitrite over many anions, except the lipophilic anions perchlorate and thiocyanate. The electrodes based on Co (III porphyrins were used for the potentiometric determination of nitrites in meat products. The results were compared with a colorimetric method used as the reference method. There was a good agreement between the potentiometric and colorimetric procedures.

  8. Alkene hydrogenation activity of enoate reductases for an environmentally benign biosynthesis of adipic acid† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02842j Click here for additional data file.

    Science.gov (United States)

    Khusnutdinova, Anna N.; Flick, Robert; Kim, Taeho; Bornscheuer, Uwe T.

    2017-01-01

    Adipic acid, a precursor for Nylon-6,6 polymer, is one of the most important commodity chemicals, which is currently produced from petroleum. The biosynthesis of adipic acid from glucose still remains challenging due to the absence of biocatalysts required for the hydrogenation of unsaturated six-carbon dicarboxylic acids to adipic acid. Here, we demonstrate the first enzymatic hydrogenation of 2-hexenedioic acid and muconic acid to adipic acid using enoate reductases (ERs). ERs can hydrogenate 2-hexenedioic acid and muconic acid producing adipic acid with a high conversion rate and yield in vivo and in vitro. Purified ERs exhibit a broad substrate spectrum including aromatic and aliphatic 2-enoates and a significant oxygen tolerance. The discovery of the hydrogenation activity of ERs contributes to an understanding of the catalytic mechanism of these poorly characterized enzymes and enables the environmentally benign biosynthesis of adipic acid and other chemicals from renewable resources. PMID:28616142

  9. Nitrate, nitrite, and nitrous oxide transformations in sediments along a salinity gradient in the Weser Estuary

    DEFF Research Database (Denmark)

    Nielsen, Michael; Gieseke, Armin; de Beer, Dirk

    2009-01-01

    by activity of nitrite oxidisers in oxic surface layers. In contrast, high rates of NO2- release occurred in marine sediment, where NO2- production was predominantly associated with incomplete nitrification in oxic layers. Similarly, stimulated partial nitrification due to NH4+ addition led to NO2- liberation...

  10. Effect of Electrolytes on the Adsorption of Nitrite and Nitrate from ...

    African Journals Online (AJOL)

    Michael Horsfall

    derived activated carbon in aqueous system and the effects of electrolytes investigated in this study using batch sorption process. The data showed that nitrate adsorbed nearly 1.5 times higher than that of nitrite. The adsorption is adequately ...

  11. The investigation of electrochemical properties for Fe3O4@Pt nanocomposites and an enhancement sensing for nitrite

    International Nuclear Information System (INIS)

    Liu, Yang; Zhou, Jie; Gong, Jin; Wu, Wei-Ping; Bao, Ning; Pan, Zhong-Qin; Gu, Hai-Ying

    2013-01-01

    Highlights: •Electrochemical characteristics on nanoparticles and nanocomposites were compared. •The reasons for superior electrochemical activity of Fe 3 O 4 @Pt were discussed. •We report an excellent nitrite biosensor based on Fe 3 O 4 @Pt. •Electro-analytical parameters of nitrite at Fe 3 O 4 @Pt were evaluated in detail. -- Abstract: The electrochemical differences (such as charge transfer resistivity, electroactive surface, standard electron transfer rate constant, adsorption amount and analytical performance of nitrite sensor) between Fe 3 O 4 @Pt nanocomposites with two elements and core–shell structure and NPs (Fe 3 O 4 nanoparticles and Pt nanoparticles) with single component and simple structure were investigated in detail. Above those investigations, it is believed that for the Fe 3 O 4 @Pt core–shell nanocomposites, Pt shell could provide more electro-catalytic activity while magnetic Fe 3 O 4 core could provide larger surface area and facilitate the purification of nanocomposites. After that, Fe 3 O 4 @Pt nanocomposites modified GCE served as a nitrite sensor. Electrochemical parameters of nitrite at Fe 3 O 4 @Pt nanocomposites such as electron transfer number, electron transfer coefficient, standard heterogeneous rate constant and electron diffusion coefficient were evaluated. With the proposed electrochemical sensors, nitrite in tap water and orange juice could be detected. This investigation suggested that core–shell nanocomposites were superior for the fabrication of electrochemical sensors

  12. Bioactivation of lapachol responsible for DNA scission by NADPH-cytochrome P450 reductase.

    Science.gov (United States)

    Kumagai, Y; Tsurutani, Y; Shinyashiki, M; Homma-Takeda, S; Nakai, Y; Yoshikawa, T; Shimojo, N

    1997-09-01

    The reduction of the naphthoquinone derivative, lapachol, which is responsible for its bioactivation was examined using microsomal preparations and NADPH-cytochrome P450 reductase (P450 reductase). Phenobarbital (PB) pretreatment resulted in an induction of enzyme activities for cytochrome c reduction (1.54 times) and lapachol reduction (1.20 times) by hepatic microsomal preparation of rats. The specific activity of lapachol reduction by purified P450 reductase showed 56-fold higher than that by untreated liver microsomes. Addition of antibody against P450 reductase (2 mg of IgG/mg of protein) to the microsomal incubation mixture caused an immunoinhibition of cytochrome c (32%) and lapachol (19%) reduction activities, suggesting that P450 reductase catalyzes lapachol reduction. Generation of superoxide anion radical (1321 nmol/mg per min) in approximately equivalent amounts of with NADPH consumption (941 nmol/mg per min) was detected during metabolism of lapachol by P450 reductase. Electron spin resonance (ESR) experiments confirmed generation of superoxide anion radical and hydroxyl radical as these 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) adducts. Incubation of lapachol with P450 reductase caused a cleavage of DNA which was reduced in the presence of Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase(1), and hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO) and thiourea. Taken together, these results indicate that lapachol is bioactivated by P450 reductase to reactive species, which promote DNA scission through the redox cycling based generation of superoxide anion radical.

  13. Nitrite-free Asian hot dog sausages reformulated with nitrite replacers.

    Science.gov (United States)

    Ruiz-Capillas, C; Tahmouzi, S; Triki, M; Rodríguez-Salas, L; Jiménez-Colmenero, F; Herrero, A M

    2015-07-01

    This research deals with the application of a global strategy designed to produce a nitrite-free Asian hot dog. Different ingredients such as annatto, cochineal, orange dietary fibre, vitamins E and C, lactate and celery were combined in order to study the appearance (colour), lipid oxidation stability and microbial stability of the nitrite-free formulations. The control sample contained much more (P annatto (RA) had the lowest a* values. Lipid oxidation levels were similar irrespective of formulation. The hot dog reformulated with cochineal (RC) scored higher for overall acceptability than RA, mainly due to its colour.

  14. Methylenetetrahydrofolate reductase (MTHFR) gene polymorphism ...

    African Journals Online (AJOL)

    Administrator

    2011-10-19

    Oct 19, 2011 ... Polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene are associated with abortion, early embryo loss and recurrent spontaneous abortion in human. However, information on the association between MTHFR polymorphism and cow abortion is scarce. In the present study, the effects.

  15. Association between methylenetetrahydrofolate reductase (MTHFR ...

    African Journals Online (AJOL)

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

  16. Methylenetetrahydrofolate reductase (MTHFR) gene polymorphism ...

    African Journals Online (AJOL)

    Polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene are associated with abortion, early embryo loss and recurrent spontaneous abortion in human. However, information on the association between MTHFR polymorphism and cow abortion is scarce. In the present study, the effects of MTHFR ...

  17. Trametes versicolor carboxylate reductase uncovered.

    Science.gov (United States)

    Winkler, Margit; Winkler, Christoph K

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

  18. The role of biliverdin reductase in colorectal cancer

    International Nuclear Information System (INIS)

    Bauer, M.

    2010-01-01

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

  19. Nitrite Regulates Hypoxic Vasodilation via Myoglobin–Dependent Nitric Oxide Generation

    Science.gov (United States)

    Totzeck, Matthias; Hendgen-Cotta, Ulrike B.; Luedike, Peter; Berenbrink, Michael; Klare, Johann P.; Steinhoff, Heinz-Juergen; Semmler, Dominik; Shiva, Sruti; Williams, Daryl; Kipar, Anja; Gladwin, Mark T.; Schrader, Juergen; Kelm, Malte; Cossins, Andrew R.; Rassaf, Tienush

    2012-01-01

    Background Hypoxic vasodilation is a physiological response to low oxygen (O2) tension that increases blood supply to match metabolic demands. While this response has been characterized for more than 100 years, the underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin (deoxyMb) in the heart can reduce nitrite to nitric oxide (NO˙) and thereby contribute to cardiomyocyte NO˙ signaling during ischemia. Based on recent observations that Mb is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular Mb to form NO˙. Methods and Results We here show that Mb is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO˙ from nitrite reduction by deoxyMb activates canonical soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) signaling pathways. In vivo and ex vivo vasodilation responses, the reduction of nitrite to NO˙ and the subsequent signal transduction mechanisms were all significantly impaired in mice without myoglobin (Mb−/−). Hypoxic vasodilation studies in Mb, endothelial and inducible NO synthase knockout models (eNOS−/−, iNOS−/−) suggest that only Mb contributes to systemic hypoxic vasodilatory responses in mice. Conclusions Endogenous nitrite is a physiological effector of hypoxic vasodilation. Its reduction to NO˙ via the heme globin Mb enhances blood flow and matches O2 supply to increased metabolic demands under hypoxic conditions. PMID:22685116

  20. The stress caused by nitrite with titanium dioxide nanoparticles under UVA irradiation in human keratinocyte cell

    International Nuclear Information System (INIS)

    Tu, Min; Huang, Yi; Li, Hai-Ling; Gao, Zhong-Hong

    2012-01-01

    Highlights: ► Nitrite increased photo-toxicity of nano-TiO 2 on human keratinocyte cells in a dose-dependant manner. ► Morphological study suggested the cell death may be mediated by apoptosis inducing factor. ► Protein nitration was generated in the cells, and the most abundant nitrated protein was identified as cystatin-A. ► Tyr35 was the most likely site to be nitrated in cystatin-A. -- Abstract: Our previous work found that in the presence of nitrite, titanium dioxide nanoparticles can cause protein tyrosine nitration under UVA irradiation in vivo. In this paper, the human keratinocyte cells was used as a skin cell model to further study the photo-toxicity of titanium dioxide nanoparticles when nitrite was present. The results showed that nitrite increased the photo-toxicity of titanium dioxide in a dose-dependant manner, and generated protein tyrosine nitration in keratinocyte cells. Morphological study of keratinocyte cells suggested a specific apoptosis mediated by apoptosis inducing factor. It was also found the main target nitrated in cells was cystatin-A, which expressed abundantly in cytoplasm and functioned as a cysteine protease inhibitor. The stress induced by titanium dioxide with nitrite under UVA irradiation in human keratinocyte cells appeared to trigger the apoptosis inducing factor mediated cell death and lose the inhibition of active caspase by cystatin-A. We conclude that nitrite can bring new damage and stress to human keratinocyte cells with titanium dioxide nanoparticles under UVA irradiation.

  1. Role of nitrite, urate and pepsin in the gastroprotective effects of saliva

    Directory of Open Access Journals (Sweden)

    Bárbara S. Rocha

    2016-08-01

    Full Text Available Dietary nitrate is now recognized as an alternative substrate for nitric oxide (•NO production in the gut. This novel pathway implies the sequential reduction of nitrate to nitrite, •NO and other bioactive nitrogen oxides but the physiological relevance of these oxidants has remained elusive. We have previously shown that dietary nitrite fuels an hitherto unrecognized nitrating pathway at acidic gastric pH, through which pepsinogen is nitrated in the gastric mucosa, yielding a less active form of pepsin in vitro. Here, we demonstrate that pepsin is nitrated in vivo and explore the functional impact of protein nitration by means of peptic ulcer development. Upon administration of pentagastrin and human nitrite-rich saliva or sodium nitrite to rats, nitrated pepsin was detected in the animal's stomach by immunoprecipitation. •NO was measured in the gastric headspace before and after nitrite instillation by chemiluminescence. At the end of each procedure, the stomach's lesions, ranging from gastric erosions to haemorrhagic ulcers, were scored. Nitrite increased gastric •NO by 200-fold (p<0.05 and nitrated pepsin was detected both in the gastric juice and the mucosa (p<0.05. Exogenous urate, a scavenger of nitrogen dioxide radical, blunted •NO detection and inhibited pepsin nitration, suggesting an underlining free radical-dependent mechanism for nitration. Functionally, pepsin nitration prevented the development of gastric ulcers, as the lesions were only apparent when pepsin nitration was inhibited by urate. In sum, this work unravels a novel dietary-dependent nitrating pathway in which pepsin is nitrated and inactivated in the stomach, preventing the progression of gastric ulcers.

  2. Expression and site-directed mutagenesis of human dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

    1988-05-17

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 ..-->.. Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by ..cap alpha..-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

  3. Induction of aldose reductase and sorbitol in renal inner medullary cells by elevated extracellular NaCl.

    OpenAIRE

    Bagnasco, S M; Uchida, S; Balaban, R S; Kador, P F; Burg, M B

    1987-01-01

    Aldose reductase [aldehyde reductase 2; alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21] catalyzes conversion of glucose to sorbitol. Although its activity is implicated in the progression of ocular and neurological complications of diabetes, the normal function of the enzyme in most cells is unknown. Both aldose reductase activity and substantial levels of sorbitol were previously reported in renal inner medullary cells. In this tissue, the extracellular NaCl concentration normally is high and...

  4. Mannose-6-Phosphate Reductase, a Key Enzyme in Photoassimilate Partitioning, Is Abundant and Located in the Cytosol of Photosynthetically Active Cells of Celery (Apium graveolens L.) Source Leaves.

    Science.gov (United States)

    Everard, J. D.; Franceschi, V. R.; Loescher, W. H.

    1993-06-01

    Mannitol, a major photosynthetic product and transport carbohydrate in many plants, accounts for approximately 50% of the carbon fixed by celery (Apium graveolens L.) leaves. Previous subfractionation studies of celery leaves indicated that the enzymes for mannitol synthesis were located in the cytosol, but these data are inconsistent with that published for the sites of sugar alcohol synthesis in other families and taxa, including apple (Malus) and a brown alga (Fucus). Using antibodies to a key synthetic enzyme, NADPH-dependent mannose-6-phosphate reductase (M6PR), and immunocytochemical techniques, we have resolved both the inter-cellular and intracellular sites of mannitol synthesis. In leaves, M6PR was found only in cells containing ribulose-1,5-bisphosphate carboxylase/oxygenase. M6PR was almost exclusively cytosolic in these cells, with the nucleus being the only organelle to show labeling. The key step in transport carbohydrate biosynthesis that is catalyzed by M6PR displays no apparent preferential association with vascular tissues or with the bundle sheath. These results show that M6PR and, thus, mannitol synthesis are closely associated with the distribution of photosynthetic carbon metabolism in celery leaves. The principal role of M6PR is, therefore, in the assimilation of carbon being exported from the chloroplast, and it seems unlikely that this enzyme plays even an indirect role in phloem loading of mannitol.

  5. Molecular effects of bioactive fraction of Curcuma mangga (DLBS4847) as a downregulator of 5α-reductase activity pathways in prostatic epithelial cells

    International Nuclear Information System (INIS)

    Karsono, Agung Heru; Tandrasasmita, Olivia Mayasari; Tjandrawinata, Raymond R

    2014-01-01

    DLBS4847 is a standardized bioactive fraction of Curcuma mangga. In this study, we used prostate cancer (PC)-3 as the cell line to study the effects of DLBS4847 on prostatic cell viability, as well as related molecular changes associated with the decreased cell number. The observation revealed that DLBS4847 inhibited the growth of PC3 cells through downregulation of the 5α-reductase (5AR) pathway. At the transcription level, 5AR1 and androgen-receptor gene expressions were downregulated in a dose-dependent manner. Furthermore, 5AR-1 and dihydrotestosterone expression were also downregulated at the protein level. A microarray study was also performed to see the effects of DLBS4847 on differential gene expressions in prostate cancer 3 cells. Among others, DLBS4847 downregulated genes related to prostate growth and hypertrophy. Our results suggested that DLBS4847 could potentially become an alternative treatment for prostate disorders, such as benign prostatic hyperplasia. In this regard, DLBS4847 exerts its growth inhibition partially through downregulation of the 5AR pathway

  6. Nitroreductase reactions of Arabidopsis thaliana thioredoxin reductase.

    Science.gov (United States)

    Miskiniene, V; Sarlauskas, J; Jacquot, J P; Cenas, N

    1998-09-07

    Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) catalyzed redox cycling of aromatic nitrocompounds, including the explosives 2,4,6-trinitrotoluene and tetryl, and the herbicide 3,5-dinitro-o-cresol. The yield of nitro anion radicals was equal to 70-90%. Redox cycling of tetryl was accompanied by formation of N-methylpicramide. Bimolecular rate constants of nitroaromatic reduction (kcat/Km) and reaction catalytic constants (kcat) increased upon an increase in oxidant single-electron reduction potential (E(1)7). Using compounds with an unknown E(1)7 value, the reactivity of TR increased parallelly to the increase in reactivity of ferredoxin:NADP+ reductase of Anabaena PCC 7119 (EC 1.18.1.2). This indicated that the main factor determining reactivity of nitroaromatics towards TR was their energetics of single-electron reduction. Incubation of reduced TR in the presence of tetryl or 2,4-dinitrochlorobenzene resulted in a loss of thioredoxin reductase activity, most probably due to modification of reduced catalytic disulfide, whereas nitroreductase reaction rates were unchanged. This means that on the analogy of quinone reduction by TR (D. Bironaite, Z. Anusevicius, J.-P. Jacquot, N. Cenas, Biochim. Biophys. Acta 1383 (1998) 82-92), FAD and not catalytic disulfide of TR was responsible for the reduction of nitroaromatics. Tetryl, 2,4,6-trinitrotoluene and thioredoxin increased the FAD fluorescence intensity of TR. This finding suggests that nitroaromatics may bind close to the thioredoxin-binding site at the catalytic disulfide domain of TR, and induce a conformational change of enzymes (S.B. Mulrooney, C.H. Williams Jr., Protein Sci. 6 (1997) 2188-2195). Our data indicate that certain nitroaromatic herbicides, explosives and other classes of xenobiotics may interfere with the reduction of thioredoxin by plant TR, and confer prooxidant properties to this antioxidant enzyme.

  7. Comparative analysis of nitrite uptake and hemoglobin-nitrite reactions in erythrocytes: sorting out uptake mechanisms and oxygenation dependencies

    DEFF Research Database (Denmark)

    Jensen, Frank Bo; Rohde, Sabina

    2010-01-01

    (hagfish and lamprey) anion exchanger-1 (AE1) in the membrane, with the aim to unravel the mechanisms and oxygenation dependencies of nitrite transport. Added nitrite rapidly diffused into the RBCs until equilibrium. The distribution ratio of nitrite across the membrane agreed with that expected from HNO2...... diffusion and AE1-mediated facilitated NO2- diffusion. Participation of HNO2 diffusion was emphasized by rapid transmembrane nitrite equilibration also in the natural AE1 knockouts. Following the equilibration, nitrite was consumed by reacting with Hb, which created a continued inward diffusion controlled....... We propose a model for RBC nitrite uptake that involves both HNO2 diffusion and AE1-mediated transport and which explains both the present and previous (sometimes puzzling) results....

  8. Sustained release of growth hormone and sodium nitrite from biomimetic collagen coating immobilized on silicone tubes improves endothelialization.

    Science.gov (United States)

    Salehi-Nik, Nasim; Malaie-Balasi, Zahra; Amoabediny, Ghassem; Banikarimi, Seyedeh Parnian; Zandieh-Doulabi, Behrouz; Klein-Nulend, Jenneke

    2017-08-01

    Biocompatibility of biomedical devices can be improved by endothelialization of blood-contacting parts mimicking the vascular endothelium's function. Improved endothelialization might be obtained by using biomimetic coatings that allow local sustained release of biologically active molecules, e.g. anti-thrombotic and growth-inducing agents, from nanoliposomes. We aimed to test whether incorporation of growth-inducing nanoliposomal growth hormone (nGH) and anti-thrombotic nanoliposomal sodium nitrite (nNitrite) into collagen coating of silicone tubes enhances endothelialization by stimulating endothelial cell proliferation and inhibiting platelet adhesion. Collagen coating stably immobilized on acrylic acid-grafted silicone tubes decreased the water contact angle from 102° to 56°. Incorporation of 50 or 500nmol/ml nNitrite and 100 or 1000ng/ml nGH into collagen coating decreased the water contact angle further to 48°. After 120h incubation, 58% nitrite and 22% GH of the initial amount of sodium nitrite and GH in nanoliposomes were gradually released from the nNitrite-nGH-collagen coating. Endothelial cell number was increased after surface coating of silicone tubes with collagen by 1.6-fold, and with nNitrite-nGH-collagen conjugate by 1.8-3.9-fold after 2days. After 6days, endothelial cell confluency in the absence of surface coating was 22%, with collagen coating 74%, and with nNitrite-nGH-collagen conjugate coating 83-119%. In the absence of endothelial cells, platelet adhesion was stimulated after collagen coating by 1.3-fold, but inhibited after nNitrite-nGH-collagen conjugate coating by 1.6-3.7-fold. The release of anti-thrombotic prostaglandin I 2 from endothelial cells was stimulated after nNitrite-nGH-collagen conjugate coating by 1.7-2.2-fold compared with collagen coating. Our data shows improved endothelialization and blood compatibility using nNitrite-nGH-collagen conjugate coating on silicone tubes suggesting that these coatings are highly suitable

  9. Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus1

    Science.gov (United States)

    Lesot, Agnès; Benveniste, Irène; Hasenfratz, Marie-Paule; Durst, Francis

    1992-01-01

    Monoclonal antibodies (mAbs) against a plant NADPH-cytochrome P-450 (Cyt P-450) reductase from Jerusalem artichoke (Helianthus tuberosus) tuber were prepared. These antibodies were produced by hybridoma resulting from the fusion of spleen cells from a rat immunized with a purified preparation of the reductase and mouse myeloma cells. The mAbs thus obtained were screened for their interaction with the reductases, first in western dots and then in blots, and for their ability to inhibit the NADPH-cytochrome c (Cyt c) reductase activity from Jerusalem artichoke microsomes. Among the 11 clones giving a positive response on western blots, only 6 were also able to inhibit microsomal NADPH-Cyt c reductase activity, and the microsomal Cyt P-450 monooxygenase activities dependent upon electrons transferred by the reductase. Thus, two families of mAbs were characterized: a family of mAbs that interact with epitopes of the reductase implicated in the reduction of Cyt P-450 by NADPH (binding sites for NADPH, flavin mononucleotide, flavin adenine dinucleotide, and Cyt P-450), and a structural family, whose members recognize epitopes outside the active site of the reductases. These mAbs specifically recognize the reductase, and all of them interact with all of the isoforms, indicating that important primary or secondary structural analogies exist between the isoforms, not only at the active site, but also at the level of epitopes not directly associated with catalytic activity. Images Figure 1 Figure 2 Figure 3 PMID:16653138

  10. The Nitrite-Scavenging Properties of Catechol, Resorcinol, and Hydroquinone: A Comparative Study on Their Nitration and Nitrosation Reactions.

    Science.gov (United States)

    Lu, Yunhao; Dong, Yanzuo; Li, Xueli; He, Qiang

    2016-10-14

    The nitration and nitrosation reactions of catechol, resorcinol, and hydroquinone (0.05 mmol/L) with sodium nitrite (0.05 mmol/L) at pH 3 and 37 °C were studied by using liquid chromatography and mass spectrometry (LC-MS) and atom charge analysis, which was aimed to provide chemical insight into the nitrite-scavenging behavior of polyphenols. The 3 benzenediols showed different mechanisms to scavenge nitrite due to their differences in hydroxyl position. Catechol was nitrated with 1 NO 2 group at the hydroxyl oxygen, and resorcinol was nitrosated with 2 NO groups at the C 2 and C 4 (or C 6 ) positions of the benzene ring. Hydroquinone could scavenge nitrite through both nitration and nitrosation mechanisms. The nitrated hydroquinone had 1 NO 2 group at the hydroxyl oxygen in the molecule, while the nitrosated 1 containing 2 NO groups at the benzene ring might have 3 structure probabilities. The results may provide a structure-activity understanding on the nitrite-scavenging property of polyphenols, so as to promote their application in the food industry for the removal of possibly toxic nitrites found in many vegetables and often in processed meat products. © 2016 Institute of Food Technologists®.

  11. Fluorimetry of nitrite and its applications

    International Nuclear Information System (INIS)

    Qian Hongjuan; Wu Jizong; Zhang Lihua; Liu Junling

    2007-01-01

    A new fluorimetric method for determination of nitrite was studied, based on the reaction of 5-aminofluorescein in acidic medium to form a new compound which was no fluorescence and reduced to highly fluorescence in alkaline medium. Effects of metal ions impurity were discussed. The acidity and measuring time were given. The precision of this method are 4% and 5% respectively. The recoveries are between 96% and 106% in nuclear fuel reprocessing solution and between 96% and 103% in high-level radioactive liquid waste. The method is applied to the determination of NO 2 - in nuclear fuel reprocessing solution and high- level radioactive liquid waste and nitrocompound in organic liquid waste. (authors)

  12. Optimum conditions for cotton nitrate reductase extraction and ...

    African Journals Online (AJOL)

    Conditions of nitrate reductase extraction and activity measurement should be adapted to plant species, and to the organs of the same plant, because of extreme weaknesses and instabilities of the enzyme. Different extraction and reaction media have been compared in order to define the best conditions for cotton callus ...

  13. Transcriptional modulation of genes encoding nitrate reductase in ...

    African Journals Online (AJOL)

    2016-10-26

    Oct 26, 2016 ... The free aluminum (Al) content in soil can reach levels that are toxic to plants, and this has frequently limited increased productivity of cultures. Four genes encoding nitrate reductase (NR) were identified, named ZmNR1–4. With the aim of evaluating NR activity and the transcriptional modulation of the.

  14. Transcriptional modulation of genes encoding nitrate reductase in ...

    African Journals Online (AJOL)

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

  15. Dihydrofolate reductase: A potential drug target in trypanosomes and leishmania

    Science.gov (United States)

    Zuccotto, Fabio; Martin, Andrew C. R.; Laskowski, Roman A.; Thornton, Janet M.; Gilbert, Ian H.

    1998-05-01

    Dihydrofolate reductase has successfully been used as a drug target in the area of anti-cancer, anti-bacterial and anti-malarial chemotherapy. Little has been done to evaluate it as a drug target for treatment of the trypanosomiases and leishmaniasis. A crystal structure of Leishmania major dihydrofolate reductase has been published. In this paper, we describe the modelling of Trypanosoma cruzi and Trypanosoma brucei dihydrofolate reductases based on this crystal structure. These structures and models have been used in the comparison of protozoan, bacterial and human enzymes in order to highlight the different features that can be used in the design of selective anti-protozoan agents. Comparison has been made between residues present in the active site, the accessibility of these residues, charge distribution in the active site, and the shape and size of the active sites. Whilst there is a high degree of similarity between protozoan, human and bacterial dihydrofolate reductase active sites, there are differences that provide potential for selective drug design. In particular, we have identified a set of residues which may be important for selective drug design and identified a larger binding pocket in the protozoan than the human and bacterial enzymes.

  16. Impacts of some divalent cations on periplasmic nitrate reductase and dehydrogenase enzymes of Escherichia, Pseudomonas and Acinetobacter species

    Directory of Open Access Journals (Sweden)

    Christian E. Nwanyanwu

    2008-08-01

    Full Text Available The impacts of Hg2+, Cd2+ and Zn2+ on the activities of periplasmic nitrate reductase (NAP and dehydrogenase (DHA enzymes of three organisms isolated from soil and sediment-water interface were analysed in liquid culture studies. NAP and DHA activities were estimated from nitrite and triphenyl formazan were produced respectively after 4h incubation at 28 ± 2oC. Hg2+ completely inhibited NAP activity in Escherichia and Pseudomonas spp at all the concentrations (0.2 – 1mM while progressive inhibitions of NAP activity were observed in Escherichia and Pseudomonas spp with increasing concentrations of Zn2+ and Cd2+. Both metals were stimulatory to NAP of Acinetobacter sp at 0.2 – 1mM. Apart from stimulation of DHA activity by Zn2+ (0.2 – 1mM in Escherichia sp, Cd2+ (0.4 -1.0mM in Acinetobacter sp and (1.0mM in Pseudomonas sp, all the metals progressively inhibited DHA activities in the three organisms. In Escherichia sp, the activities of the two enzymes were negatively correlated on exposure to Zn2+ (r = -0.91 and positively correlated (r = >0.90 on exposure to Cd2+ and Hg2+. Based on IC50 values of the metals for the DHA and NAP enzymes, the most resistant of the three organisms were Escherichia sp and Acinetobacter sp respectively. Quantitatively, NAP with its lower IC50 values than DHA was a more sensitive toxicity measure for Hg2+ in all the organisms. The sensitivity of microbial metabolic enzymes to the toxic effects of metals varies with the type of enzyme, metal and the microorganism involved.

  17. Bimodal effect of hydrogen peroxide and oxidative events in nitrite-induced rapid root abscission by the water fern Azolla pinnata

    Directory of Open Access Journals (Sweden)

    Michael F Cohen

    2015-07-01

    Full Text Available In the genus Azolla rapid abscission of roots from floating fronds occurs within minutes in response to a variety of stresses, including exposure to nitrite. We found that hydrogen peroxide, though itself not an inducer of root abscission, modulates nitrite-induced root abscission by Azolla pinnata in a dose-dependent manner, with 2 mM H2O2 significantly diminishing the responsiveness to 2 mM NaNO2, and 10 mM H2O2 slightly enhancing it. Hypoxia, which has been found in other plants to result in autogenic production of H2O2, dramatically stimulated root abscission of A. pinnata in response to nitrite, especially for plants previously cultivated in medium containing 5 mM KNO3 compared to plants cultivated under N2-fixing conditions without combined nitrogen. Plants, including Azolla, produce the small signaling molecule nitric oxide (NO from nitrite using nitrate reductase. We found Azolla plants to display dose-dependent root abscission in response to the NO donor spermine NONOate. Treatment of plants with the thiol-modifying agents S-methyl methanethiosulfonate or glutathione inhibited the nitrite-induced root abscission response. Synchrotron radiation-based Fourier transform infrared (SR-FTIR spectromicroscopy revealed higher levels of carbonylation in the abscission zone of dropped roots, indicative of reaction products of polysaccharides with potent free radical oxidants. We hypothesize that metabolic products of nitrite and NO react with H2O2 in the apoplast leading to free-radical-mediated cleavage of structural polysaccharides and consequent rapid root abscission.

  18. Electrocatalytic reduction of nitrite on tetraruthenated metalloporphyrins/Nafion glassy carbon modified electrode

    Energy Technology Data Exchange (ETDEWEB)

    Calfuman, Karla [Facultad de Ciencias, Departamento de Quimica, Universidad de Chile, Las Palmeras 3425, Casilla 653, Nunoa, Santiago (Chile); Aguirre, Maria Jesus [Facultad de Quimica y Biologia, Departamento de Quimica de los Materiales, Universidad de Santiago de Chile, Santiago (Chile); Canete-Rosales, Paulina; Bollo, Soledad [Facultad de Ciencias Quimicas y Farmaceuticas, Departamento de Quimica Farmacologica y Toxicologica, Universidad de Chile, Santiago (Chile); Llusar, Rosa [Departamento de Quimica Fisica y Analitica, Universidad de Jaume I, Castellon (Spain); Isaacs, Mauricio, E-mail: misaacs@uchile.cl [Facultad de Ciencias, Departamento de Quimica, Universidad de Chile, Las Palmeras 3425, Casilla 653, Nunoa, Santiago (Chile)

    2011-10-01

    Highlights: > Preparation and characterization of modified electrodes with M(II) Tetraruthenated porphyrins onto a Nafion film. > The electrodes were characterized by SEM, TEM, AFM and SECM techniques. > The modified electrodes are active in the electrochemical reduction of nitrite at -660 mV vs Ag/AgCl. > GC/Nf/CoTRP modified electrode is more electrochemically active than their Ni and Zn analogues. - Abstract: This paper describes the electrochemical reduction of nitrite ion in neutral aqueous solution mediated by tetraruthenated metalloporphyrins (Co(II), Ni(II) and Zn(II)) electrostatically assembled onto a Nafion film previously adsorbed on glassy carbon or ITO electrodes. Scanning electron microscope (SEM-EDX) and transmission electron microscopy (TEM) results have shown that on ITO electrodes the macrocycles forms multiple layers with a disordered stacking orientation over the Nafion film occupying hydrophobic and hydrophilic sites in the polyelectrolyte. Atomic force microscopy (AFM) results demonstrated that the Nafion film is 35 nm thick and tetraruthenated metalloporphyrins layers 190 nm thick presenting a thin but compacted morphology. Scanning electrochemical microscopy (SECM) images shows that the Co(II) tetraruthenated porphyrins/Nf/GC modified electrode is more electrochemically active than their Ni and Zn analogues. These modified electrodes are able to reduce nitrite at -660 mV showing enhanced reduction current and a decrease in the required overpotential compared to bare glassy carbon electrode. Controlled potential electrolysis experiments verify the production of ammonia, hydrazine and hydroxylamine at potentials where reduction of solvent is plausible demonstrating some selectivity toward the nitrite ion. Rotating disc electrode voltammetry shows that the factor that governs the kinetics of nitrite reduction is the charge propagation in the film.

  19. Noble metal catalyzed hydrogen generation from formic acid in nitrite-containing simulated nuclear waste media

    International Nuclear Information System (INIS)

    King, R.B.; Bhattacharyya, N.K.; Wiemers, K.D.

    1994-08-01

    Simulants for the Hanford Waste Vitrification Plant (HWVP) feed containing the major non-radioactive components Al, Cd, Fe, Mn, Nd, Ni, Si, Zr, Na, CO 3 2- , NO 3 -, and NO 2 - were used as media to evaluate the stability of formic acid towards hydrogen evolution by the reaction HCO 2 H → H 2 + CO 2 catalyzed by the noble metals Ru, Rh, and/or Pd found in significant quantities in uranium fission products. Small scale experiments using 40-50 mL of feed simulant in closed glass reactors (250-550 mL total volume) at 80-100 degree C were used to study the effect of nitrite and nitrate ion on the catalytic activities of the noble metals for formic acid decomposition. Reactions were monitored using gas chromatography to analyze the CO 2 , H 2 , NO, and N 2 O in the gas phase as a function of time. Rhodium, which was introduced as soluble RhCl 3 ·3H 2 O, was found to be the most active catalyst for hydrogen generation from formic acid above ∼80 degree C in the presence of nitrite ion in accord with earlier observations. The inherent homogeneous nature of the nitrite-promoted Rh-catalyzed formic acid decomposition is suggested by the approximate pseudo first-order dependence of the hydrogen production rate on Rh concentration. Titration of the typical feed simulants containing carbonate and nitrite with formic acid in the presence of rhodium at the reaction temperature (∼90 degree C) indicates that the nitrite-promoted Rh-catalyzed decomposition of formic acid occurs only after formic acid has reacted with all of the carbonate and nitrite present to form CO 2 and NO/N 2 O, respectively. The catalytic activities of Ru and Pd towards hydrogen generation from formic acid are quite different than those of Rh in that they are inhibited rather than promoted by the presence of nitrite ion

  20. Evidence of the chemical reaction of (18)O-labelled nitrite with CO2 in aqueous buffer of neutral pH and the formation of (18)OCO by isotope ratio mass spectrometry.

    Science.gov (United States)

    Tsikas, Dimitrios; Böhmer, Anke; Gros, Gerolf; Endeward, Volker

    2016-05-01

    Inorganic nitrite (NO2(-), ON-O(-) ←→ (-)O-NO) is the autoxidation product of nitric oxide (NO). Nitrite can also be formed from inorganic nitrate (ONO2(-)), the major oxidation product of NO in erythrocytes, by the catalytic action of bacterial nitrate reductase in gut and oral microflora. Nitrite can be reduced to NO by certain cellular proteins and enzymes, as well as in the gastric juice under acidic conditions. Hemoglobin, xanthine oxidoreductase and carbonic anhydrase (CA) have been reported to convert nitrite to NO. Renal CA isoforms are involved in the reabsorption of nitrite and may, therefore, play an important role in NO homeostasis. Yet, the mechanisms underlying the action of CA on nitrite are incompletely understood. The nitrate/nitrite system is regarded as a reservoir of NO. We have recently shown that nitrite reacts chemically with carbon dioxide (CO2), the regular substrate of CA. The present communication reports a stable isotope ratio mass spectrometry (IRMS) study on the reaction of NO2(-) and CO2 performed in 50 mM HEPES buffer of pH 7.4 at 37 °C. By using (18)O-labelled nitrite ((18)ON-O(-)/(-18)O-NO) and CO2 we observed formation of (18)O-labelled CO2. This finding is an unequivocal evidence of the chemical reaction of (18)ON-O(-)/(-18)O-NO with CO2. The reaction is rapid and involves nucleophilic attack of the negatively charged nitrite via one of its oxygen atoms on the partially positively charged CO2 molecule to form the putative intermediate (18)ON-O-CO2(-)/(-)O2C-(18)O-NO. The by far largest fraction of this intermediate decomposes back to (18)ON-O(-)/(-18)O-NO and CO2. A very small fraction of the intermediate, however, rearranges and finally decomposes to form (18)OCO and nitrite. This reaction is slower in the presence of an isolated erythrocytic CA isoform II. In summary, NO2(-), CO2 and CA are ubiquitous. The chemical reaction of NO2(-) with CO2 and its modulation by CA isoforms may play important roles in the transport of

  1. Ethyl nitrite is produced in the human stomach from dietary nitrate and ethanol, releasing nitric oxide at physiological pH: potential impact on gastric motility.

    Science.gov (United States)

    Rocha, Bárbara S; Gago, Bruno; Barbosa, Rui M; Cavaleiro, Carlos; Laranjinha, João

    2015-05-01

    Nitric oxide ((∙)NO), a ubiquitous molecule involved in a plethora of signaling pathways, is produced from dietary nitrate in the gut through the so-called nitrate-nitrite-NO pathway. In the stomach, nitrite derived from dietary nitrate triggers a network of chemical reactions targeting endogenous and exogenous biomolecules, thereby producing new compounds with physiological activity. The aim of this study was to ascertain whether compounds with physiological relevance are produced in the stomach upon consumption of nitrate- and ethanol-rich foods. Human volunteers consumed a serving of lettuce (source of nitrate) and alcoholic beverages (source of ethanol). After 15 min, samples of the gastric headspace were collected and ethyl nitrite was identified by GC-MS. Wistar rats were used to study the impact of ethyl nitrite on gastric smooth muscle relaxation at physiological pH. Nitrogen oxides, produced from nitrite in the stomach, induce nitrosation of ethanol from alcoholic beverages in the human stomach yielding ethyl nitrite. Ethyl nitrite, a potent vasodilator, is produced in vivo upon the consumption of lettuce with either red wine or whisky. Moreover, at physiological pH, ethyl nitrite induces gastric smooth muscle relaxation through a cGMP-dependent pathway. Overall, these results suggest that ethyl nitrite is produced in the gastric lumen and releases (∙)NO at physiological pH, which ultimately may have an impact on gastric motility. Systemic effects may also be expected if ethyl nitrite diffuses through the gastric mucosa reaching blood vessels, therefore operating as a (∙)NO carrier throughout the body. These data pinpoint posttranslational modifications as an underappreciated mechanism for the production of novel molecules with physiological impact locally in the gut and highlight the notion that diet may fuel compounds with the potential to modulate gastrointestinal welfare. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Addition of nitrite enhances the electrochemical defluorination of 2-fluoroaniline

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Huajun [School of Environmental Science and Engineering, Zhejiang Gongshang University, Hangzhou 310012 (China); Zhejiang Provincial Key Laboratory of Solid Waste Treatment and Recycling, Hangzhou 310012 (China); Laboratory of Microbial Ecology and Technology, Ghent University, Coupure Links 653, B-9000 Ghent (Belgium); Liang, Yuxiang [School of Environmental Science and Engineering, Zhejiang Gongshang University, Hangzhou 310012 (China); Zhejiang Provincial Key Laboratory of Solid Waste Treatment and Recycling, Hangzhou 310012 (China); Guo, Kun [Laboratory of Microbial Ecology and Technology, Ghent University, Coupure Links 653, B-9000 Ghent (Belgium); Long, Yuyang [School of Environmental Science and Engineering, Zhejiang Gongshang University, Hangzhou 310012 (China); Zhejiang Provincial Key Laboratory of Solid Waste Treatment and Recycling, Hangzhou 310012 (China); Cong, Yanqing, E-mail: yqcong@hotmail.com [School of Environmental Science and Engineering, Zhejiang Gongshang University, Hangzhou 310012 (China); Shen, Dongsheng [School of Environmental Science and Engineering, Zhejiang Gongshang University, Hangzhou 310012 (China); Zhejiang Provincial Key Laboratory of Solid Waste Treatment and Recycling, Hangzhou 310012 (China)

    2015-12-30

    Highlights: • A method for improving defluorination performance by in situ self-assembly of pollutants was developed. • The mechanisms of 2-FA modification and defluorination are discussed. • Positively-charged diazonium salt is used to weaken the C–F bond. - Abstract: This study introduces a novel approach that uses the interaction of pollutants with added nitrite to produce diazonium salts, which cause in situ self-assembly of the pollutants on carbon electrodes, to improve their 2-fluoroaniline (2-FA) defluorination and removal performance. The 2-FA degradation performance, electrode properties, electrochemical properties and degradation pathway were investigated. The reactor containing NO{sub 2}{sup −} achieved a 2-FA removal efficiency of 90.1% and a defluorination efficiency of 38% within 48 h, 1.4 and 2.3 times higher than the corresponding results achieved without NO{sub 2}{sup −}, respectively. The residual NO{sub 2}{sup −} was less than 0.5 mg/L in the reactor containing added NO{sub 2}{sup −}, which would not cause serious secondary pollution. Scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) results proved that the carbon anode surface was successfully modified with benzene polymer, and electrochemical tests confirmed that the electrochemical activity of the modified anode was enhanced significantly. The C–F bond was weakened by the effect of the positive charge of the benzenediazonium groups, and the high electrochemical activity of the carbon anode enhanced the electrochemical performance of the system to accelerate defluorination. Thus, the present electrical method involving nitrite nitrogen is very promising for the treatment of wastewater containing fluoroaniline compounds.

  3. In vitro killing of Mycobacterium ulcerans by acidified nitrite

    NARCIS (Netherlands)

    Phillips, R.; Kuijper, S.; Benjamin, N.; Wansbrough-Jones, M.; Wilks, M.; Kolk, A. H. J.

    2004-01-01

    Mycobacterium ulcerans, which causes Buruli ulcer, was exposed to acidified nitrite or to acid alone for 10 or 20 min. Killing was rapid, and viable counts were reduced below detectable limits within 10 min of exposure to 40 mM acidified nitrite. M. ulcerans is highly susceptible to acidified

  4. determination of nitrite, nitrate and total nitrogen in vegetable samples

    African Journals Online (AJOL)

    The above colour reaction system has been applied successfully for the determination of nitrite, nitrate and total nitrogen in vegetable samples. Unreduced samples give direct measure for nitrite whilst reduction of samples by copperized-cadmium column gives total nitrogen content and their difference shows nitrate content ...

  5. Occurrence and Toxicological Significance of Nitrate and Nitrite in ...

    African Journals Online (AJOL)

    Aim: To determine and evaluate the levels of nitrate and nitrite in some commercial infant formula in view of the health implications of these factors. Method: Nitrate and nitrite, which may create significant health problems in infants, were determined in four commercial infant formula. The public health and toxicological ...

  6. Nitrite as regulator of hypoxic signaling in mammalian physiology

    NARCIS (Netherlands)

    van Faassen, E.E.H.|info:eu-repo/dai/nl/071100938; Bahrami, S.; Feelisch, M.; Hogg, N.; Kelm, M.

    2009-01-01

    In this review we consider the effects of endogenous and pharmacological levels of nitrite under conditions of hypoxia. In humans, the nitrite anion has long been considered as metastable intermediate in the oxidation of nitric oxide radicals to the stable metabolite nitrate. This oxidation cascade

  7. Nitrate and nitrite in biology, nutrition and therapeutics

    NARCIS (Netherlands)

    Lundberg, J.O.; van Faassen, E.E.H.; Gladwin, M.T.; Ahluwalia, A.; Benjamin, N.

    2009-01-01

    Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia.

  8. Electrochemical removal of nitrite in simulated aquaculture wastewater

    African Journals Online (AJOL)

    Electrochemical removal of nitrite at a concentration of 10 mg l-1 from synthetic aquaculture wastewater was investigated in this study using a batch reactor. The effects of important operating parameters such as electrode material and applied current density were studied. The highest nitrite removal is achieved with nickel ...

  9. Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite.

    Science.gov (United States)

    Webb, Andrew J; Patel, Nakul; Loukogeorgakis, Stavros; Okorie, Mike; Aboud, Zainab; Misra, Shivani; Rashid, Rahim; Miall, Philip; Deanfield, John; Benjamin, Nigel; MacAllister, Raymond; Hobbs, Adrian J; Ahluwalia, Amrita

    2008-03-01

    Diets rich in fruits and vegetables reduce blood pressure (BP) and the risk of adverse cardiovascular events. However, the mechanisms of this effect have not been elucidated. Certain vegetables possess a high nitrate content, and we hypothesized that this might represent a source of vasoprotective nitric oxide via bioactivation. In healthy volunteers, approximately 3 hours after ingestion of a dietary nitrate load (beetroot juice 500 mL), BP was substantially reduced (Delta(max) -10.4/8 mm Hg); an effect that correlated with peak increases in plasma nitrite concentration. The dietary nitrate load also prevented endothelial dysfunction induced by an acute ischemic insult in the human forearm and significantly attenuated ex vivo platelet aggregation in response to collagen and ADP. Interruption of the enterosalivary conversion of nitrate to nitrite (facilitated by bacterial anaerobes situated on the surface of the tongue) prevented the rise in plasma nitrite, blocked the decrease in BP, and abolished the inhibitory effects on platelet aggregation, confirming that these vasoprotective effects were attributable to the activity of nitrite converted from the ingested nitrate. These findings suggest that dietary nitrate underlies the beneficial effects of a vegetable-rich diet and highlights the potential of a "natural" low cost approach for the treatment of cardiovascular disease.

  10. Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, Philippa J.L. [School of Chemistry, University of Sydney, New South Wales 2006 (Australia); Codd, Rachel, E-mail: rachel.codd@sydney.edu.au [School of Chemistry, University of Sydney, New South Wales 2006 (Australia); School of Medical Sciences (Pharmacology) and Bosch Institute, University of New South Wales, New South Wales 2006 (Australia)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Cold-adapted phenotype of NapA from the Antarctic bacterium Shewanella gelidimarina. Black-Right-Pointing-Pointer Protein homology model of NapA from S. gelidimarina and mesophilic homologue. Black-Right-Pointing-Pointer Six amino acid residues identified as lead candidates governing NapA cold adaptation. Black-Right-Pointing-Pointer Molecular-level understanding of designing cool-temperature in situ oxyanion sensors. -- Abstract: The reduction of nitrate to nitrite is catalysed in bacteria by periplasmic nitrate reductase (Nap) which describes a system of variable protein subunits encoded by the nap operon. Nitrate reduction occurs in the NapA subunit, which contains a bis-molybdopterin guanine dinucleotide (Mo-MGD) cofactor and one [4Fe-4S] iron-sulfur cluster. The activity of periplasmic nitrate reductase (Nap) isolated as native protein from the cold-adapted (psychrophilic) Antarctic bacterium Shewanella gelidimarina (Nap{sub Sgel}) and middle-temperature adapted (mesophilic) Shewanella putrefaciens (Nap{sub Sput}) was examined at varied temperature. Irreversible deactivation of Nap{sub Sgel} and Nap{sub Sput} occurred at 54.5 and 65 Degree-Sign C, respectively. When Nap{sub Sgel} was preincubated at 21-70 Degree-Sign C for 30 min, the room-temperature nitrate reductase activity was maximal and invariant between 21 and 54 Degree-Sign C, which suggested that Nap{sub Sgel} was poised for optimal catalysis at modest temperatures and, unlike Nap{sub Sput}, did not benefit from thermally-induced refolding. At 20 Degree-Sign C, Nap{sub Sgel} reduced selenate at 16% of the rate of nitrate reduction. Nap{sub Sput} did not reduce selenate. Sequence alignment showed 46 amino acid residue substitutions in Nap{sub Sgel} that were conserved in NapA from mesophilic Shewanella, Rhodobacter and Escherichia species and could be associated with the Nap{sub Sgel} cold-adapted phenotype. Protein homology modeling of Nap{sub Sgel} using a

  11. Formation of mutagenic N-nitroso compounds in vegetable extracts upon nitrite treatment: a comparison with the glucosinolate content.

    Science.gov (United States)

    Tiedink, H G; Davies, J A; van Broekhoven, L W; van der Kamp, H J; Jongen, W M

    1988-01-01

    More than 30 vegetables were screened for their potential to form biologically active N-nitroso compounds upon treatment with nitrite under acidic conditions. The total N-nitroso content was determined in the nitrite-treated and untreated extracts of the vegetables according to a modified method of Walters et al. (Analyst, Lond. 1978, 103, 1127). All treated extracts contained N-nitroso compounds at levels ranging from 23 to 789 nmol/25 mg dry matter. In the same samples the mutagenic activity was determined using the Salmonella typhimurium assay. About half of the vegetables were found to be mutagenic upon nitrite treatment. (Nitrite-treated extracts were considered to be mutagenic if the number of induced revertants was at least twice as high as that induced by the corresponding untreated extract). The content of different glucosinolates in the dry matter of the vegetables was also determined. Glucosinolates could be detected only in cruciferous vegetables, at levels ranging from 1.8 to 26.0 mumol/g dry matter. Although the nitrite-treated extracts of brassica species contained more N-nitroso compounds and induced more revertants than did other vegetables, there was no significant correlation between these parameters. However, the amounts of N-nitroso compounds formed upon nitrite treatment (expressed per fresh weight) did correlate significantly (P less than 0.01) with the amounts of glucosinolates (r = 0.95). When the glucosinolates were divided into aryl/alkyl- and indolyl-glucosinolates, the significant correlation was maintained for both subgroups (r = 0.93 and 0.95, respectively). From this it can be concluded that glucosinolates are probably involved in the formation of N-nitroso compounds in certain nitrite-treated vegetables.

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

    NARCIS (Netherlands)

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

    1988-01-01

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

  13. Increase in nitrite content and functionality of ethanolic extracts of Perilla frutescens following treatment with atmospheric pressure plasma.

    Science.gov (United States)

    Jung, Samooel; Lee, Chul Woo; Lee, Juri; Yong, Hae In; Yum, Su Jin; Jeong, Hee Gon; Jo, Cheorun

    2017-12-15

    This study investigated the effect of atmospheric pressure plasma (APP) treatment on nitrite content and functionality of plant extracts. Ethanolic extracts of Perilla frutescens (EEP) were prepared and treated with APP for 60min. Nitrite content increased from 0 to 45.8mg/l in EEP after APP treatment for 60min. Antimicrobial activity of EEP against Clostridium perfringens and Salmonella Typhimurium was increased by APP with no influence on antioxidative activity (p<0.05). Lyophilized EEP (LEEP) treated with APP for 60min contained 3.74mg/g nitrite. The control (LEEP without APP) contained no nitrite. The minimum inhibitory concentration (MIC) of LEEP for C. perfringens was 200µg/ml. The control did not inhibit C. perfringens growth between 25 and 1000µg/ml. MICs of LEEP and the control against S. Typhimurium were 25 and 50µg/ml, respectively. New nitrite sources with increased antimicrobial activity can be produced from natural plants by APP treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Fatty acyl-CoA reductase

    Energy Technology Data Exchange (ETDEWEB)

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

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

  15. N-partitioning, nitrate reductase and glutamine synthetase activities in two contrasting varieties of maize Partição de nitrogênio e atividade das enzimas nitrato redutase e glutamina sintetase em duas cultivares contrastantes de milho

    Directory of Open Access Journals (Sweden)

    Altair Toledo Machado

    2001-02-01

    Full Text Available In order to identify useful parameters for maize genetic breeding programs aiming at a more efficient use of N, two maize varieties of contrasting N efficiency, Sol da Manhã NF (efficient and Catetão (inefficient were compared. Experiments were carried out under field and greenhouse conditions, at low and high N levels. The parameters analysed included total and relative plant and grain N content, biomass and the activities of nitrate reductase and glutamine synthetase in different parts of the plant. It was found that the translocation efficiency of N and photoassimilates to the developing seeds and the source-sink relations were significantly different for the two varieties. N content of the whole plant and grain, cob weight and the relative ear dry weight were useful parameters for characterizing the variety Sol da Manhã NF as to its efficient use of N. Enzymes activity of glutamine synthetase (transferase reaction and nitrate reductase did not differ among the varieties.Com o objetivo de identificar parâmetros que possam ser utilizados em programas de melhoramento genético em milho para uso eficiente de N, duas cultivares de milho contrastantes quanto ao uso deste nutriente, Sol da Manhã NF (eficiente e Catetão (não-eficiente, foram avaliadas em dois experimentos conduzidos no campo e em casa de vegetação, respectivamente, sob nível baixo e alto de N. Os caracteres avaliados foram: teor e conteúdo de N em diferentes partes da planta; massa seca; peso dos grãos e de diferentes partes da planta; biomassa, e atividade das enzimas nitrato redutase e glutamina sintetase. O mecanismo de translocação de N e de fotoassimilados para os grãos e a relação fonte/dreno foram importantes para diferenciar a cultivar eficiente da não-eficiente. Conteúdo de N nos grãos e total das plantas, peso do sabugo e relação peso de espiga/matéria seca foram importantes para caracterizar a cultivar Sol da Manhã NF eficiente no uso do N. A

  16. Two enzymes catalyze vitamin K 2,3-epoxide reductase activity in mouse: VKORC1 is highly expressed in exocrine tissues while VKORC1L1 is highly expressed in brain.

    Science.gov (United States)

    Caspers, Michael; Czogalla, Katrin J; Liphardt, Kerstin; Müller, Jens; Westhofen, Philipp; Watzka, Matthias; Oldenburg, Johannes

    2015-05-01

    VKORC1 and VKORC1L1 are enzymes that both catalyze the reduction of vitamin K2,3-epoxide via vitamin K quinone to vitamin K hydroquinone. VKORC1 is the key enzyme of the classical vitamin K cycle by which vitamin K-dependent (VKD) proteins are γ-carboxylated by the hepatic γ-glutamyl carboxylase (GGCX). In contrast, the VKORC1 paralog enzyme, VKORC1L1, is chiefly responsible for antioxidative function by reduction of vitamin K to prevent damage by intracellular reactive oxygen species. To investigate tissue-specific vitamin K 2,3-epoxide reductase (VKOR) function of both enzymes, we quantified mRNA levels for VKORC1, VKORC1L1, GGCX, and NQO1 and measured VKOR enzymatic activities in 29 different mouse tissues. VKORC1 and GGCX are highly expressed in liver, lung and exocrine tissues including mammary gland, salivary gland and prostate suggesting important extrahepatic roles for the vitamin K cycle. Interestingly, VKORC1L1 showed highest transcription levels in brain. Due to the absence of detectable NQO1 transcription in liver, we assume this enzyme has no bypass function with respect to activation of VKD coagulation proteins. Our data strongly suggest diverse functions for the vitamin K cycle in extrahepatic biological pathways. Copyright © 2015. Published by Elsevier Ltd.

  17. Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself.

    Science.gov (United States)

    Gachhui, R; Presta, A; Bentley, D F; Abu-Soud, H M; McArthur, R; Brudvig, G; Ghosh, D K; Stuehr, D J

    1996-08-23

    Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2',5'-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.

  18. NITRATE AND NITRITE CONCENTRATION IN VEGETABLES GRAWING AROUND ISFAHAN CITY

    Directory of Open Access Journals (Sweden)

    R.A JAFARI

    2001-06-01

    Full Text Available Introduction: The improper and excess use of chemical fertilizers by the farmers special in "urea" form may cause the commulation of nitrates and nitrites in vegetables and fruits. This investigation has been done to determine the concentration of nitrates and nitrites in some vegetables growing around Isfahan. Methods: One hundred and thirteen random samples from 15 different kinds of green lest tuber vegetables were measured by Cadmium Column Procedure for nitrates and nitrites. Results: The mean of the nitrates level in green leaf and tuber-vegetables was 287.9 and 76.3 mg/kg, respectively. These amounts were more higher than standard value (67 mg/kg in the basis of wet material, significantly. The mean of the nitrite level in green leaf and tuber vegetables were 1.7 and 1 mg/kg, repectively, which were more higher than standard value (zero mg/kg in the basis of wet matrial. The most high concentration of nitrites and nitrates was seen in commondill and leek, repectively. Discussion: Amounts of nitrites and nitrates in our analysed samples exceeded the perimissible limits. The comulation of nitrates in some of the vegetables depend on the application of chemical fertilizers, temprature and wether, but the nitrites in vegetables is unusual in normal condition and the unsuitable storage of them causes it"s expansion.

  19. Structures of mammalian cytosolic quinone reductases.

    Science.gov (United States)

    Foster, C E; Bianchet, M A; Talalay, P; Faig, M; Amzel, L M

    2000-08-01

    The metabolism of quinone compounds presents one source of oxidative stress in mammals, as many pathways proceed by mechanisms that generate reactive oxygen species as by-products. One defense against quinone toxicity is the enzyme NAD(P)H:quinone oxidoreductase type 1 (QR1), which metabolizes quinones by a two-electron reduction mechanism, thus averting production of radicals. QR1 is expressed in the cytoplasm of many tissues, and is highly inducible. A closely related homologue, quinone reductase type 2 (QR2), has been identified in several mammalian species. QR2 is also capable of reducing quinones to hydroquinones, but unlike QR1, cannot use NAD(P)H. X-ray crystallographic studies of QR1 and QR2 illustrate that despite their different biochemical properties, these enzymes have very similar three-dimensional structures. In particular, conserved features of the active sites point to the close relationship between these two enzymes.

  20. Comparison of the Stereospecificity and Immunoreactivity of NADH-Ferricyanide Reductases in Plant Membranes.

    Science.gov (United States)

    Fredlund, K. M.; Struglics, A.; Widell, S.; Askerlund, P.; Kader, J. C.; Moller, I. M.

    1994-11-01

    The substrate stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane and peroxisomal membrane of potato (Solanum tuberosum L.) tubers, spinach (Spinacea oleracea L.) leaf plasma membrane, and red beetroot (Beta vulgaris L.) tonoplast were all specific for the [beta]-hydrogen of NADH, whereas the reductases in wheat root (Triticum aestivum L.) endoplasmic reticulum and potato tuber outer mitochondrial membrane were both [alpha]-hydrogen specific. In all isolated membrane fractions one or several polypeptides with an apparent size of 45 to 55 kD cross-reacted with antibodies raised against a microsomal NADH-ferricyanide reductase on western blots.

  1. Intensification of Doxorubicin-Related Oxidative Stress in the Heart by Hypothyroidism Is Not Related to the Expression of Cytochrome P450 NADPH-Reductase and Inducible Nitric Oxide Synthase, As Well As Activity of Xanthine Oxidase

    Directory of Open Access Journals (Sweden)

    Jaroslaw Dudka

    2012-01-01

    Full Text Available Cytochrome P450 NADPH-reductase (P450R, inducible synthase (iNOS and xanthine oxidase play an important role in the antracycline-related cardiotoxicity. The expression of P450R and iNOS is regulated by triiodothyronine. The aim of this study was to evaluate the effect of methimazole-induced hypothyreosis on oxidative stress secondary to doxorubicin administration. 48 hours after methimazole giving cessation, rats were exposed to doxorubicin (2.0, 5.0 and 15 mg/kg. Blood and heart were collected 4, 48 and 96 h after the drug administration. Animals exposed exclusively to doxorubicin or untreated ones were also assessed. The hypothyreosis (0.025% of methimazole significantly increased the doxorubicin effect on the cardiac carbonyl group and they may increase the glutathione level. An insignificant effect of methimazole was noticed in case of the cardiac lipid peroxidation product, the amount of DNA oxidative damages, iNOS and xanthine oxidase-enzymes responsible for red-ox activation of doxorubicin. However, the concentration of P450R was affected by a lower dose of methimazole in rats administered with doxorubicin. Since in rats receiving doxorubicin changes in oxidative stress caused by methimazole were not accompanied by elevation of bioreductive enzymes, it may be concluded that these changes in the oxidative stress were not related to the tested enzymes.

  2. Determination of Nitrate Reductase Assay Depending on the Microbial Growth

    International Nuclear Information System (INIS)

    El-Kabbany, H.M.

    2012-01-01

    A rapid micro-dilution assay for determination of the antimicrobial susceptibility of different bacterial isolates was developed. This assay is based on the ability of the most of viable organisms to reduce nitrates. The MIC or MBC could be determined by nitrate reductase (NR) only after 30 to 90 min of incubation depending on the behaviour of microbial growth. Bacterial viability is detected by a positive nitrite reduction rather than visible turbidity. The nitrate reduction assay was compared with standard micro-assay using 250 isolates of different taxa against 10 antibiotics belonging to different classes. An excellent agreement of 82.5 % was found between the two methods and only 17.5 % of 1794 trials showed difference in the determined MIC by tow-dilution interval above or below the MIC determined by the turbidimetric method under the same test conditions. However, the nitrate reduction assay was more rapid and sensitive in detecting viable bacteria and so, established an accurate estimate of the minimal inhibitory concentration (MIC) or the minimal bacterial concentration (MBC). The nitrate reduction assay offers the additional advantage that it could be used to determine the MBC without having to subculture the broth. 232 cases of resistance were detected by NR and 4 different media were tested for susceptibility test. The bacterial isolates were exposed to ultra violet (UV) light for different period

  3. Hypoxia tolerance, nitric oxide, and nitrite: Lessons from extreme animals

    DEFF Research Database (Denmark)

    Fago, Angela; B. Jensen, Frank

    2015-01-01

    survival resides in concerted physiological responses, including strong metabolic depression, protection against oxidative damage and – in air breathing animals - redistribution of blood flow. Each of these responses is known to be tightly regulated by nitric oxide (NO) and during hypoxia by its metabolite...... nitrite. The aim of this review is to highlight recent work illustrating the widespread roles of NO and nitrite in the tolerance to extreme oxygen deprivation, in particular in the red-eared slider turtle and crucian carp, but also in diving marine mammals. The emerging picture underscores the importance...... of NO and nitrite signaling in the adaptive response to hypoxia in vertebrate animals....

  4. Electrochemical oxidation of nitrite on nanodiamond powder electrode

    Energy Technology Data Exchange (ETDEWEB)

    Chen, L.H.; Zang, J.B.; Wang, Y.H.; Bian, L.Y. [State Key Laboratory of Metastable Materials Science and Technology, Yanshan University, Qinhuangdao 066004 (China); College of Materials Science and Engineering, Yanshan University, Qinhuangdao 066004 (China)

    2008-03-10

    Nanodiamond (ND) powder electrodes were fabricated and the electrochemical properties were investigated in the solution containing nitrite in this article. This electrode exhibits substantial catalytic ability toward the oxidation of nitrite anions. The electrochemical oxidation mechanism of nitrite on the ND powder electrode is discussed. The oxidation of NaNO{sub 2} is a two-electron transfer process. The electrode reaction rate constant k is estimated to be 2.013 x 10{sup -4} cm/s and (1 - {alpha})n{sub {alpha}} is 0.1643. The peak current increases linearly with the rising of the concentration of NaNO{sub 2}. (author)

  5. Role of cytochromes P450 1A1/2 in detoxication and activation of carcinogenic aristolochic acid I: studies with the hepatic NADPH:cytochrome P450 reductase null (HRN) mouse model.

    Science.gov (United States)

    Levová, Katerina; Moserová, Michaela; Kotrbová, Vera; Sulc, Miroslav; Henderson, Colin J; Wolf, C Roland; Phillips, David H; Frei, Eva; Schmeiser, Heinz H; Mares, Jaroslav; Arlt, Volker M; Stiborová, Marie

    2011-05-01

    Aristolochic acid (AA) causes aristolochic acid nephropathy, Balkan endemic nephropathy, and their urothelial malignancies. To identify enzymes involved in the metabolism of aristolochic acid I (AAI), the major toxic component of AA we used HRN (hepatic cytochrome P450 [Cyp] reductase null) mice, in which NADPH:Cyp oxidoreductase (Por) is deleted in hepatocytes. AAI was demethylated by hepatic Cyps in vitro to 8-hydroxy-aristolochic acid I (AAIa), indicating that less AAI is distributed to extrahepatic organs in wild-type (WT) mice. Indeed, AAI-DNA-adduct levels were significantly higher in organs of HRN mice, having low hepatic AAI demethylation capacity, than in WT mice. Absence of AAI demethylation in HRN mouse liver was confirmed in vitro; hepatic microsomes from WT, but not from HRN mice, oxidized AAI to AAIa. To define the role of hepatic Cyps in AAI demethylation, modulation of AAIa formation by CYP inducers was investigated. We conclude that AAI demethylation is attributable mainly to Cyp1a1/2. The higher AAI-DNA adduct levels in HRN than WT mice were the result of the lack of hepatic AAI demethylation concomitant with a higher activity of cytosolic NAD(P)H:quinone oxidoreductase (Nqo1), which activates AAI. Mouse hepatic Cyp1a1/2 also activated AAI to DNA adducts under hypoxic conditions in vitro, but in renal microsomes, Por and Cyp3a are more important than Cyp1a for AAI-DNA adduct formation. We propose that AAI activation and detoxication in mice are dictated mainly by AAI binding affinity to Cyp1a1/2 or Nqo1, by their turnover, and by the balance between oxidation and reduction of AAI by Cyp1a.

  6. Correlation between total nitrite/nitrate concentrations and monoamine oxidase (types A and B) and semicarbazide-sensitive amine oxidase enzymatic activities in human mesenteric arteries from non-diabetic and type 2 diabetic patients

    Energy Technology Data Exchange (ETDEWEB)

    Nunes, S.F.; Figueiredo, I.V. [Laboratório de Farmacologia, Faculdade de Farmácia, Universidade de Coimbra, Coimbra (Portugal); Pereira, J.S. [Instituto Português de Oncologia de Coimbra, Coimbra (Portugal); Lopes, M.C.; Caramona, M.M. [Laboratório de Farmacologia, Faculdade de Farmácia, Universidade de Coimbra, Coimbra (Portugal)

    2011-11-25

    The aim of this study was to determine the correlation between total nitrite/nitrate concentrations (NOx) and the kinetic parameters of monoamine oxidase enzymes (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) in human mesenteric arteries. Arteries were from non-diabetic and type 2 diabetic patients with sigmoid or rectum carcinoma for whom surgery was the first option and who were not exposed to neo-adjuvant therapy. Segments of human inferior mesenteric arteries from non-diabetic (61.1 ± 8.9 years old, 7 males and 5 females, N = 12) and type 2 diabetic patients (65.8 ± 6.2 years old, 8 males and 4 females, N = 12) were used to determine NOx concentrations and the kinetic parameters of MAO-A, MAO-B and SSAO by the Griess reaction and by radiochemical assay, respectively. The NOx concentrations in arteries from diabetic patients did not differ significantly from those of the non-diabetic group (10.28 ± 4.61 vs 10.71 ± 4.32 nmol/mg protein, respectively). In the non-diabetic group, there was a positive correlation between NOx concentrations and MAO-B parameters: K{sub m} (r = 0.612, P = 0.034) and V{sub max} (r = 0.593, P = 0.042), and a negative correlation with the SSAO parameters: K{sub m} (r = -0.625, P = 0.029) and V{sub max} (r = -0.754, P = 0.005). However, in the diabetic group no correlation was found between NOx concentrations and the three kinetic parameters of the enzymes. These results suggest an important function of sympathetic nerves and vascular NOx concentrations in arteries of non-diabetic patients. Thus, these results confirm the importance of a balance between oxidants and antioxidants in the maintenance of vascular homeostasis to prevent oxidative stress.

  7. Characterization of xylose reductase from Candida tropicalis ...

    African Journals Online (AJOL)

    USER

    2010-08-02

    Aug 2, 2010 ... Xylose reductase gene, enzyme cofactors and plasmids. E.coli BL21(DE3) was used as host strains for ... C. tropicalis xylose reductase gene was isolated from plasmid. pMD18-T (TaKaRa, Japan). Enzyme ..... the gels is instable, soft and even dissolve in the solution containing multivalent anions or high ...

  8. Interactive Effect of Salicylic Acid on Some Physiological Features and Antioxidant Enzymes Activity in Ginger (Zingiber officinale Roscoe

    Directory of Open Access Journals (Sweden)

    Hawa Z. E. Jaafar

    2013-05-01

    Full Text Available The effect of foliar salicylic acid (SA applications (10−3 and 10−5 M on activities of nitrate reductase, guaiacol peroxidase (POD, superoxide dismutases (SOD, catalase (CAT and proline enzymes and physiological parameters was evaluated in two ginger varieties (Halia Bentong and Halia Bara under greenhouse conditions. In both varieties, tested treatments generally enhanced photosynthetic rate and total dry weight. Photosynthetic rate increases were generally accompanied by increased or unchanged stomatal conductance levels, although intercellular CO2 concentrations of treated plants were typically lower than in controls. Lower SA concentrations were generally more effective in enhancing photosynthetic rate and plant growth. Exogenous application of SA increased antioxidant enzyme activities and proline content; the greatest responses were obtained in plants sprayed with 10–5 M SA, with significant increases observed in CAT (20.1%, POD (45.2%, SOD (44.1% and proline (43.1% activities. Increased CAT activity in leaves is naturally expected to increase photosynthetic efficiency and thus net photosynthesis by maintaining a constant CO2 supply. Our results support the idea that low SA concentrations (10–5 M may induce nitrite reductase synthesis by mobilizing intracellular NO3− and can provide protection to nitrite reductase degradation in vivo in the absence of NO3–. Observed positive correlations among proline, SOD, CAT and POD activities in the studied varieties suggest that increased SOD activity was accompanied by increases in CAT and POD activities because of the high demands of H2O2 quenching.

  9. Purification and Properties of an NADPH-Aldose Reductase (Aldehyde Reductase) from Euonymus japonica Leaves

    Science.gov (United States)

    Negm, Fayek B.

    1986-01-01

    The enzyme aldose (aldehyde) reductase was partially purified (142-fold) and characterized from Euonymus japonica leaves. The reductase, a dimer, had an average molecular weight of 67,000 as determined by gel filtration on Sephadex G-100. The enzyme was NADPH specific and reduced a broad range of substrates including aldoses, aliphatic aldehydes, and aromatic aldehydes. Maximum activity was observed at pH 8 in phosphate and Tris-HCl buffers and at pH 8.6 to 9.0 in glycine-NaOH buffer using dl-glyceraldehyde or 3-pyridinecarboxaldehyde as substrate. NADP was a competitive inhibitor with respect to NADPH with a Ki of 60 micromolar. Glycerol was an uncompetitive inhibitor to dl-glyceraldehyde (K′i = 460 millimolar). The Euonymus enzyme was inhibited by sulfhydryl inhibitor, phenobarbital, and high concentrations of Li2SO4. Pyrazol and metal chelating agents inhibited the enzyme slightly. Enzyme activity was detected in the leaves and berries of Celastrus orbiculatus and several species of Euonymus. Probable function of this enzyme is to reduce d-galactose to galactitol, a characteristic metabolite in phloem sap of members of the Celastraceae family. Images Fig. 1 PMID:16664750

  10. DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target.

    Directory of Open Access Journals (Sweden)

    Rubén Soto-Acosta

    2017-04-01

    Full Text Available Dengue is the most common mosquito-borne viral disease in humans. Changes of lipid-related metabolites in endoplasmic reticulum of dengue virus (DENV infected cells have been associated with replicative complexes formation. Previously, we reported that DENV infection inhibits HMGCR phosphorylation generating a cholesterol-enriched cellular environment in order to favor viral replication. In this work, using enzymatic assays, ELISA, and WB we found a significant higher activity of HMGCR in DENV infected cells, associated with the inactivation of AMPK. AMPK activation by metformin declined the HMGCR activity suggesting that AMPK inactivation mediates the enhanced activity of HMGCR. A reduction on AMPK phosphorylation activity was observed in DENV infected cells at 12 and 24 hpi. HMGCR and cholesterol co-localized with viral proteins NS3, NS4A and E, suggesting a role for HMGCR and AMPK activity in the formation of DENV replicative complexes. Furthermore, metformin and lovastatin (HMGCR inhibitor altered this co-localization as well as replicative complexes formation supporting that active HMGCR is required for replicative complexes formation. In agreement, metformin prompted a significant dose-dependent antiviral effect in DENV infected cells, while compound C (AMPK inhibitor augmented the viral genome copies and the percentage of infected cells. The PP2A activity, the main modulating phosphatase of HMGCR, was not affected by DENV infection. These data demonstrate that the elevated activity of HMGCR observed in DENV infected cells is mediated through AMPK inhibition and not by increase in PP2A activity. Interestingly, the inhibition of this phosphatase showed an antiviral effect in an HMGCR-independent manner. These results suggest that DENV infection increases HMGCR activity through AMPK inactivation leading to higher cholesterol levels in endoplasmic reticulum necessary for replicative complexes formation. This work provides new information

  11. Supporting palladium metal on gold nanoparticles improves its catalysis for nitrite reduction.

    Science.gov (United States)

    Qian, Huifeng; Zhao, Zhun; Velazquez, Juan C; Pretzer, Lori A; Heck, Kimberly N; Wong, Michael S

    2014-01-07

    Nitrate (NO3(-)) and nitrite (NO2(-)) anions are often found in groundwater and surface water as contaminants globally, especially in agricultural areas due to nitrate-rich fertilizer use. One popular approach to studying the removal of nitrite/nitrate from water has been their degradation to dinitrogen via Pd-based reduction catalysis. However, little progress has been made towards understanding how the catalyst structure can improve activity. Focusing on the catalytic reduction of nitrite in this study, we report that Au NPs supporting Pd metal ("Pd-on-Au NPs") show catalytic activity that varies with volcano-shape dependence on Pd surface coverage. At room temperature, in CO2-buffered water, and under H2 headspace, the NPs were maximally active at a Pd surface coverage of 80%, with a first-order rate constant (k(cat) = 576 L g(Pd)(-1) min(-1)) that was 15x and 7.5x higher than monometallic Pd NPs (~4 nm; 40 L g(Pd)(-1) min(-1)) and Pd/Al2O3 (1 wt% Pd; 76 L g(Pd)(-1) min(-1)), respectively. Accounting only for surface Pd atoms, these NPs (576 L g(surface-Pd)(-1) min(-1)) were 3.6x and 1.6x higher than monometallic Pd NPs (160 L g(surface-Pd)(-1) min(-1)) and Pd/Al2O3 (361 L g(surface-Pd)(-1) min(-1)). These NPs retained ~98% of catalytic activity at a chloride concentration of 1 mM, whereas Pd/Al2O3 lost ~50%. The Pd-on-Au nanostructure is a promising approach to improve the catalytic reduction process for nitrite and, with further development, also for nitrate anions.

  12. Increased formation of carcinogenic PAH metabolites in fish promoted by nitrite

    Digital Repository Service at National Institute of Oceanography (India)

    Shailaja, M.S.; Rajamanickam, R.; Wahidullah, S.

    -. The concentrations of the two test toxicants (PAH and nitrite) used were well within the range of field-measured levels (COMAPS, 2002). Biotransformation of PAH in the exposed fish was followed with the help of biomarkers namely, 7-ethoxyresorufin O...-deethylase (EROD) activity and biliary PAH metabolites. The liver somatic index (LSI) was used to determine the physiological status of the fish. 2. Materials and methods 2.1. Exposure protocol Juvenile Oreochromis mossambicus (8.6 to 11g) obtained...

  13. Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases.

    Science.gov (United States)

    Moura, José J G; Brondino, Carlos D; Trincão, José; Romão, Maria João

    2004-10-01

    Molybdenum and tungsten are second- and third-row transition elements, respectively, which are found in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen atom transfer reactions. Mononuclear Mo-containing enzymes have been classified into three families: xanthine oxidase, DMSO reductase, and sulfite oxidase. The proteins of the DMSO reductase family present the widest diversity of properties among its members and our knowledge about this family was greatly broadened by the study of the enzymes nitrate reductase and formate dehydrogenase, obtained from different sources. We discuss in this review the information of the better characterized examples of these two types of Mo enzymes and W enzymes closely related to the members of the DMSO reductase family. We briefly summarize, also, the few cases reported so far for enzymes that can function either with Mo or W at their active site.

  14. The cytochrome bd respiratory oxygen reductases.

    Science.gov (United States)

    Borisov, Vitaliy B; Gennis, Robert B; Hemp, James; Verkhovsky, Michael I

    2011-11-01

    Cytochrome bd is a respiratory quinol: O₂ oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O₂ and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O₂-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O₂, evidence for a proton channel connecting this