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Sample records for aldose reductase ec

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

    PubMed Central

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

    1985-01-01

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

  2. Aldose reductase, oxidative stress, and diabetic mellitus.

    PubMed

    Tang, Wai Ho; Martin, Kathleen A; Hwa, John

    2012-01-01

    Diabetes mellitus (DM) is a complex metabolic disorder arising from lack of insulin production or insulin resistance (Diagnosis and classification of diabetes mellitus, 2007). DM is a leading cause of morbidity and mortality in the developed world, particularly from vascular complications such as atherothrombosis in the coronary vessels. Aldose reductase (AR; ALR2; EC 1.1.1.21), a key enzyme in the polyol pathway, catalyzes nicotinamide adenosine dinucleotide phosphate-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS) in various tissues of DM including the heart, vasculature, neurons, eyes, and kidneys. As an example, hyperglycemia through such polyol pathway induced oxidative stress, may have dual heart actions, on coronary blood vessel (atherothrombosis) and myocardium (heart failure) leading to severe morbidity and mortality (reviewed in Heather and Clarke, 2011). In cells cultured under high glucose conditions, many studies have demonstrated similar AR-dependent increases in ROS production, confirming AR as an important factor for the pathogenesis of many diabetic complications. Moreover, recent studies have shown that AR inhibitors may be able to prevent or delay the onset of cardiovascular complications such as ischemia/reperfusion injury, atherosclerosis, and atherothrombosis. In this review, we will focus on describing pivotal roles of AR in the pathogenesis of cardiovascular diseases as well as other diabetic complications, and the potential use of AR inhibitors as an emerging therapeutic strategy in preventing DM complications. PMID:22582044

  3. Structure of aldose reductase from Giardia lamblia

    PubMed Central

    Ferrell, M.; Abendroth, J.; Zhang, Y.; Sankaran, B.; Edwards, T. E.; Staker, B. L.; Van Voorhis, W. C.; Stewart, L. J.; Myler, P. J.

    2011-01-01

    Giardia lamblia is an anaerobic aerotolerant eukaryotic parasite of the intestines. It is believed to have diverged early from eukarya during evolution and is thus lacking in many of the typical eukaryotic organelles and biochemical pathways. Most conspicuously, mitochondria and the associated machinery of oxidative phosphorylation are absent; instead, energy is derived from substrate-level phosphorylation. Here, the 1.75 Å resolution crystal structure of G. lamblia aldose reductase heterologously expressed in Escherichia coli is reported. As in other oxidoreductases, G. lamblia aldose reductase adopts a TIM-barrel conformation with the NADP+-binding site located within the eight β-strands of the interior. PMID:21904059

  4. In vitro expression of rat lens aldose reductase in Escherichia coli.

    PubMed Central

    Old, S E; Sato, S; Kador, P F; Carper, D A

    1990-01-01

    Aldose reductase (alditol:NADP+ oxidoreductase, EC 1.1.1.21), an enzyme that converts glucose to sorbitol, the first step of the polyol pathway, has been implicated in secondary complications of diabetes, such as cataracts, retinopathy, neuropathy, and nephropathy. Aldose reductase inhibitors have been observed to prevent or delay the onset of these complications; however, more potent and specific inhibitors are needed. Development of new inhibitors necessitates a better understanding of the molecular structure of this protein. To elucidate the structure-function relationships of aldose reductase and to develop methods of regulating this enzyme, large and homogeneous quantities of rat lens aldose reductase have been expressed in bacterial cells. A construction of the complete coding sequence and 3' untranslated region for rat lens aldose reductase was assembled in the expression vector pKK233-2 (Pharmacia). This construction expresses an active enzyme that has been purified and demonstrates kinetic, immunological, and inhibitory properties similar to rat lens aldose reductase. Images PMID:2114645

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

    PubMed Central

    Wolff, S P; Crabbe, M J

    1985-01-01

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

  6. Aldose reductase mediates retinal microglia activation.

    PubMed

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

    2016-04-29

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

  7. Aldose reductase inhibitory compounds from Xanthium strumarium.

    PubMed

    Yoon, Ha Na; Lee, Min Young; Kim, Jin-Kyu; Suh, Hong-Won; Lim, Soon Sung

    2013-09-01

    As part of our ongoing search for natural sources of therapeutic and preventive agents for diabetic complications, we evaluated the inhibitory effects of components of the fruit of Xanthium strumarium (X. strumarium) on aldose reductase (AR) and galactitol formation in rat lenses with high levels of glucose. To identify the bioactive components of X. strumarium, 7 caffeoylquinic acids and 3 phenolic compounds were isolated and their chemical structures were elucidated on the basis of spectroscopic evidence and comparison with published data. The abilities of 10 X. strumarium-derived components to counteract diabetic complications were investigated by means of inhibitory assays with rat lens AR (rAR) and recombinant human AR (rhAR). From the 10 isolated compounds, methyl-3,5-di-O-caffeoylquinate showed the most potent inhibition, with IC₅₀ values of 0.30 and 0.67 μM for rAR and rhAR, respectively. In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate, methyl-3,5-di-O-caffeoylquinate showed competitive inhibition of rhAR. Furthermore, methyl-3,5-di-O-caffeoylquinate inhibited galactitol formation in the rat lens and in erythrocytes incubated with a high concentration of glucose, indicating that this compound may be effective in preventing diabetic complications. PMID:23604720

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

    PubMed

    Fatmawati, Sri; Ersam, Taslim; Shimizu, Kuniyoshi

    2015-01-15

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

  9. Mapping of aldose reductase gene sequences to human chromosomes 1, 3, 7, 9, 11, and 13

    SciTech Connect

    Bateman, J.B.; Kojis, T. UCLA School of Medicine, Los Angeles, CA ); Heinzmann, C.; Sparkes, R.S.; Klisak, I.; Diep, A. ); Carper, D. ); Nishimura, Chihiro ); Mohandas, T. )

    1993-09-01

    Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase; EC 1.1.1.21) (AR) catalyzes the reduction of several aldehydes, including that of glucose, to the corresponding sugar alcohol. Using a complementary DNA clone encoding human AR, the authors mapped the gene sequences to human chromosomes 1, 3, 7, 9, 11, 13, 14, and 18 by somatic cell hybridization. By in situ hybridization analysis, sequences were localized to human chromosomes 1q32-q43, 3p12, 7q31-q35, 9q22, 11p14-p15, and 13q14-q21. As a putative functional AR gene has been mapped to chromosome 7 and a putative pseudogene to chromosome 3, the sequences on the other seven chromosomes may represent other active genes, non-aldose reductase homologous sequences, or pseudogenes. 24 refs., 3 figs., 2 tabs.

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

    PubMed

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

    2013-01-01

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

  11. Human brain aldehyde reductases: relationship to succinic semialdehyde reductase and aldose reductase.

    PubMed

    Hoffman, P L; Wermuth, B; von Wartburg, J P

    1980-08-01

    Human brain contains multiple forms of aldehyde-reducing enzymes. One major form (AR3), as previously shown, has properties that indicate its identity with NADPH-dependent aldehyde reductase isolated from brain and other organs of various species; i.e., low molecular weight, use of NADPH as the preferred cofactor, and sensitivity to inhibition by barbiturates. A second form of aldehyde reductase ("SSA reductase") specifically reduces succinic semialdehyde (SSA) to produce gamma-hydroxybutyrate. This enzyme form has a higher molecular weight than AR3, and uses NADH as well as NADPH as cofactor. SSA reductase was not inhibited by pyrazole, oxalate, or barbiturates, and the only effective inhibitor found was the flavonoid quercetine. Although AR3 can also reduce SSA, the relative specificity of SSA reductase may enhance its in vivo role. A third form of human brain aldehyde reductase, AR2, appears to be comparable to aldose reductases characterized in several species, on the basis of its activity pattern with various sugar aldehydes and its response to characteristic inhibitors and activators, as well as kinetic parameters. This enzyme is also the most active in reducing the aldehyde derivatives of biogenic amines. These studies suggest that the various forms of human brain aldehyde reductases may have specific physiological functions. PMID:6778961

  12. A flavone from Manilkara indica as a specific inhibitor against aldose reductase in vitro.

    PubMed

    Haraguchi, Hiroyuki; Hayashi, Ryosuke; Ishizu, Takashi; Yagi, Akira

    2003-09-01

    Isoaffinetin (5,7,3',4',5'-pentahydroxyflavone-6-C-glucoside) was isolated from Manilkara indica as a potent inhibitor of lens aldose reductase by bioassay-directed fractionation. This C-glucosyl flavone showed specific inhibition against aldose reductases (rat lens, porcine lens and recombinant human) with no inhibition against aldehyde reductase and NADH oxidase. Kinetic analysis showed that isoaffinetin exhibited uncompetitive inhibition against both dl-glyceraldehyde and NADPH. A structure-activity relationship study revealed that the increasing number of hydroxy groups in the B-ring contributes to the increase in aldose reductase inhibition by C-glucosyl flavones. PMID:14598214

  13. Aldose and aldehyde reductases : structure-function studies on the coenzyme and inhibitor-binding sites.

    SciTech Connect

    El-Kabbani, O.; Old, S. E.; Ginell, S. L.; Carper, D. A.; Biosciences Division; Monash Univ.; NIH

    1999-09-03

    PURPOSE: To identify the structural features responsible for the differences in coenzyme and inhibitor specificities of aldose and aldehyde reductases. METHODS: The crystal structure of porcine aldehyde reductase in complex with NADPH and the aldose reductase inhibitor sorbinil was determined. The contribution of each amino acid lining the coenzyme-binding site to the binding of NADPH was calculated using the Discover package. In human aldose reductase, the role of the non-conserved Pro 216 (Ser in aldehyde reductase) in the binding of coenzyme was examined by site-directed mutagenesis. RESULTS: Sorbinil binds to the active site of aldehyde reductase and is hydrogen-bonded to Trp 22, Tyr 50, His 113, and the non-conserved Arg 312. Unlike tolrestat, the binding of sorbinil does not induce a change in the side chain conformation of Arg 312. Mutation of Pro 216 to Ser in aldose reductase makes the binding of coenzyme more similar to that of aldehyde reductase. CONCLUSIONS: The participation of non-conserved active site residues in the binding of inhibitors and the differences in the structural changes required for the binding to occur are responsible for the differences in the potency of inhibition of aldose and aldehyde reductases. We report that the non-conserved Pro 216 in aldose reductase contributes to the tight binding of NADPH.

  14. Molecular cloning and characterization of Schistosoma japonicum aldose reductase.

    PubMed

    Liu, Jian; Wang, Jipeng; Wang, Shuqi; Xu, Bin; Liu, Xiufeng; Wang, Xiaoning; Hu, Wei

    2013-02-01

    Antioxidant defense is an essential mechanism for schistosomes to cope with damage from host immune-generated reactive oxygen species. The evaluation of the effects of aldose reductase, an important enzyme that may be involved in this system, has long been neglected. In the present study, aldose reductase of Schistosoma japonicum (SjAR) was cloned and characterized. The activity of SjAR was assessed in vitro and was suppressed by the reported inhibitor, epalrestat. RT-PCR analysis revealed that SjAR was expressed at each of the development stages analyzed with increased levels in cercariae. The results also showed that SjAR was expressed at higher levels in adult male worms than in adult female worms. Indirect enzyme-linked immunosorbent assay and western blot analysis indicated that the purified recombinant SjAR (rSjAR) protein displayed a significant level of antigenicity. Immunolocalization analysis revealed that SjAR was mainly distributed in the gynecophoral canal of adult male worms. BALB/c mice immunized with rSjAR induced a 32.91 % worm reduction compared to the adjuvant group (P < 0.01). Moreover, a 28.27 % reduction in egg development in the liver (P > 0.05) and a 42.75 % reduction in egg development in the fecal samples (P < 0.05) were also observed. These results suggested that SjAR may be a potential new drug target or vaccine candidate for schistosomes. PMID:23160889

  15. Virtual screening of plant derived compounds for aldose reductase inhibition using molecular docking.

    PubMed

    Muppalaneni, Naresh Babu; Rao, Allam Appa

    2012-01-01

    The role of the aldose reductase in type 2 diabetes is widely described. Therefore, it is of interest to identify plant derived compounds to inhibit its activity. We studied the protein-ligand interaction of 267 compounds from different parts of seven plants (Allium sativum, Coriandrum sativum, Dacus carota, Murrayyakoneigii, Eucalyptus, Calendula officinalis and Lycopersicon esculentum) with aldose reductase as the target protein. Molecular docking and re-scoring of top ten compounds (using GOLD, AutoDock Vina, eHiTS, PatchDock and MEDock) followed by rank-sum technique identified compound allium38 with high binding affinity for aldose reductase. PMID:23275691

  16. Studies on WF-3681, a novel aldose reductase inhibitor. I. Taxonomy, fermentation, isolation and characterization.

    PubMed

    Nishikawa, M; Tsurumi, Y; Namiki, T; Yoshida, K; Okuhara, M

    1987-10-01

    WF-3681 was isolated from a cultured filtrate of Chaetomella raphigera as a novel inhibitor of aldose reductase. It was extracted with ethyl acetate and then purified with silica gel chromatography. Its molecular formula was determined to be C13H12O5 by elemental analysis and high resolution electron impact mass spectrometry. IC50 of WF-3681 was 2.5 X 10(-7) M for partially purified aldose reductase of rabbit lens. PMID:3119547

  17. Isoquinoline alkaloids from Tinospora cordifolia inhibit rat lens aldose reductase.

    PubMed

    Patel, Mayurkumar B; Mishra, Shrihari

    2012-09-01

    The inhibitory activity of Tinospora cordifolia stem-derived alkaloids was evaluated against lens aldose reductase (AR) isolated from male Wistar rats. Anticataract potential of the alkaloids of T. cordifolia was evaluated in vitro in rat lenses, considering the activity of normal rat lenses as 100%. The biologically active constituents of T. cordifolia extract were characterized as the isoquinoline alkaloids, jatrorrhizine, palmatine and magnoflorine, by spectral analysis. The inhibitory effects varied with all chemicals and concentrations used. The inhibitory concentration (IC₅₀) values of jatrorrhizine, palmatine and magnoflorine are 3.23, 3.45 and 1.25 µg/mL respectively. The concentration of maximum activity was selected for its effect on galactose-induced polyol accumulation in vitro. The percentage inhibition of galactose-induced polyol accumulation was 62.6, 58.8 and 27.7% in the presence of jatrorrhizine, palmatine and magnoflorine, respectively. Magnoflorine may be useful as lead compounds and new agents for AR inhibition. PMID:22294283

  18. Autoimmunity in Membranous Nephropathy Targets Aldose Reductase and SOD2

    PubMed Central

    Prunotto, Marco; Carnevali, Maria Luisa; Candiano, Giovanni; Murtas, Corrado; Bruschi, Maurizio; Corradini, Emilia; Trivelli, Antonella; Magnasco, Alberto; Petretto, Andrea; Santucci, Laura; Mattei, Silvia; Gatti, Rita; Scolari, Francesco; Kador, Peter; Allegri, Landino

    2010-01-01

    Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti–aldose reductase (AR) and anti–manganese superoxide dismutase (SOD2) IgG4 in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG4 from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG4 and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression. PMID:20150532

  19. Analysis of enzyme-catalyzed nucleotide modification by aldose reductase

    SciTech Connect

    Grimshaw, C.E.

    1987-05-01

    Homogeneous bovine kidney aldose reductase catalyzes two reactions in addition to the normal aldehyde-dependent oxidation of NADPH. First, adduct formation between the oxidized nucleotide and the oxidized substrate is observed during turnover due to initial formation of a reversible E:NADP/sup +/:R-CHO ternary complex, which subsequently reacts to give the covalent complex (E:NADP/sup +/-R-CHO). The reaction is enzyme-catalyzed with substantial enhancement of both the pseudo-first order rate constant and the overall K/sub eq/ relative to the reaction with free NADP/sup +/ in aqueous buffer. Analysis of the concentration dependence and time-course for reversible dead-end and covalent complex formation are described for several aldehyde and nucleotide substrates. Non-linear time courses for aldehyde reduction and substrate inhibition by the aldehyde substrate in initial velocity studies are completely accounted for by this mechanism, thereby eliminating a simple Dalziel-type explanation for the substrate activation by aldehyde which is also observed. Second, enzyme-catalyzed oxidation of NADPH occurs in the absence of aldehyde substrate with a rate equal to .03% of V/sub max/ for the normal reduction of glyceraldehyde. By 500 MHz /sup 1/H-NMR, the enzyme-catalyzed oxidation of (4-/sup 2/H)NADPH appears to be greater than 95% stereospecific. Spectroscopic evidence for a similar oxidation reaction is observed for the covalent E:NADP/sup +/-R-CHO adduct with glyceraldehyde, but not with glycolaldehyde.

  20. Aldose reductase inhibition improves nerve conduction velocity in diabetic patients.

    PubMed

    Judzewitsch, R G; Jaspan, J B; Polonsky, K S; Weinberg, C R; Halter, J B; Halar, E; Pfeifer, M A; Vukadinovic, C; Bernstein, L; Schneider, M; Liang, K Y; Gabbay, K H; Rubenstein, A H; Porte, D

    1983-01-20

    To assess the potential role of polyol-pathway activity in diabetic neuropathy, we measured the effects of sorbinil--a potent inhibitor of the key polyol-pathway enzyme aldose reductase--on nerve conduction velocity in 39 stable diabetics in a randomized, double-blind, cross-over trial. During nine weeks of treatment with sorbinil (250 mg per day), nerve conduction velocity was greater than during a nine-week placebo period for all three nerves tested: the peroneal motor nerve (mean increase [+/- S.E.M.], 0.70 +/- 0.24 m per second, P less than 0.008), the median motor nerve (mean increase, 0.66 +/- 0.27, P less than 0.005), and the median sensory nerve (mean increase, 1.16 +/- 0.50, P less than 0.035). Conduction velocity for all three nerves declined significantly within three weeks after cessation of the drug. These effects of sorbinil were not related to glycemic control, which was constant during the study. Although the effect of sorbinil in improving nerve conduction velocity in diabetics was small, the findings suggest that polyol-pathway activity contributes to slowed nerve conduction in diabetics. The clinical applicability of these observations remains to be determined, but they encourage further exploration of this approach to the treatment or prevention of diabetic neuropathy. PMID:6401351

  1. Overexpression of Aldose Reductase Render Mouse Hepatocytes More Sensitive to Acetaminophen Induced Oxidative Stress and Cell Death.

    PubMed

    Ahmed, Munzir M E; Al-Obosi, J A S; Osman, H M; Shayoub, M E

    2016-04-01

    Acetaminophen (APAP) a commonly used drug for decrease the fever and pain but is capable to induced hepatotoxicity at over dose. This study was carried out to investigate the effect of APAP on the expression of anti-apoptotic and antioxidative defense genes, and whether aldose reductase over-expressing plasmid capable to protect against APAP-induced oxidative stress and cell death. APAP treatment induced oxidative stress and hepatotoxicity, and significantly increased aldose reductase mRNA and protein expression in mouse hepatocyte (AML-12). Unexpectedly, AML-12 cells over-expressing aldose reductase augmented APAP-induced reduction in cell viability, reactive oxygen species (ROS) production, glutathione (GSH) depletion and glutathione S-transferase A2 expression. Moreover, over-expression of aldose reductase potentiated APAP induced reduction on proliferating cell nuclear antigen, B cell lymphoma-extra large (bcl-xL), catalase, glutathione peroxidase-1 (GPx-1) and abolished APAP-induced B-cell lymphoma 2 (bcl-2) inductions. Further, over-expression of aldose reductase significantly abolished AMP activated protein kinase (AMPK) activity in APAP-treated cells and induced p53 expression. This results demonstrate that APAP induced toxicity in AML-12, increased aldose reductase expression, and over-expression of aldose reductase render this cell more susceptible to APAP induced oxidative stress and cell death, this probably due to inhibition AMPK or bcl-2 activity, or may due to competition between aldose reductase and glutathione reductase for NADPH. PMID:27069324

  2. Highly selective aldose reductase inhibitors. 3. Structural diversity of 3-(arylmethyl)-2,4,5-trioxoimidazolidine-1-acetic acids.

    PubMed

    Kotani, T; Nagaki, Y; Ishii, A; Konishi, Y; Yago, H; Suehiro, S; Okukado, N; Okamoto, K

    1997-02-28

    Accumulation of intracellular sorbitol, the reduced product of glucose, catalyzed by aldose reductase (AR) (EC 1.1.1.21), is thought to be the cause of the development of diabetic complications. Our attention is focused on finding compounds which inhibit AR without significantly inhibiting aldehyde reductase (ALR) (EC 1.1.1.2). The uracil or 2,4-dioxoimidazolidine skeleton having the benzothiazolyl or 4-chloro-3-nitrophenyl group as an aryl part indicated not only extremely high AR inhibitory activity but also AR selectivity. The ratio of IC50(ALR)/IC50(AR) of 3-[(5-chlorobenzothiazol-2-yl)methyl]-1,2,3,4-tetrahydro-2,4- dioxopyrimidine-1-acetic acid (47d) was more than 17 500. The uracil skeleton with the benzothiazolyl moiety seemed to be the best combination for selective AR inhibition. PMID:9057855

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

    PubMed

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

    2016-07-01

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

  4. Affinity purifications of aldose reductase and xylitol dehydrogenase from the xylose-fermenting yeast Pachysolen tannophilus

    SciTech Connect

    Bolen, P.L.; Roth, K.A.; Freer, S.N.

    1986-10-01

    Although xylose is a major product of hydrolysis of lignocellulosic materials, few yeasts are able to convert it to ethanol. In Pachysolen tannophilus, one of the few xylose-fermenting yeasts found, aldose reductase and xylitol dehydrogenase were found to be key enzymes in the metabolic pathway for xylose fermentation. This paper presents a method for the rapid and simultaneous purification of both aldose reductase and xylitol dehydrogenase from P. tannophilus. Preliminary studies indicate that this method may be easily adapted to purify similar enzymes from other xylose-fermenting yeasts.

  5. Aldose reductase expression as a risk factor for cataract

    PubMed Central

    Snow, Anson; Shieh, Biehuoy; Chang, Kun-Che; Pal, Arttatrana; Lenhart, Patricia; Ammar, David; Ruzycki, Philip; Palla, Suryanarayana; Reddy, G. Bhanuprakesh; Petrash, J. Mark

    2015-01-01

    Aldose reductase (AR) is thought to play a role in the pathogenesis of diabetic eye diseases, including cataract and retinopathy. However, not all diabetics develop ocular complications. Paradoxically, some diabetics with poor metabolic control appear to be protected against retinopathy, while others with a history of excellent metabolic control develop severe complications. These observations indicate that one or more risk factors may influence the likelihood that an individual with diabetes will develop cataracts and/or retinopathy. We hypothesize that an elevated level of AR gene expression could confer higher risk for development of diabetic eye disease. To investigate this hypothesis, we examined the onset and severity of diabetes-induced cataract in transgenic mice, designated AR-TG, that were either heterozygous or homozygous for the human AR (AKR1B1) transgene construct. AR-TG mice homozygous for the transgene demonstrated a conditional cataract phenotype, whereby they developed lens vacuoles and cataract-associated structural changes only after induction of experimental diabetes; no such changes were observed in AR-TG heterozygotes or nontransgenic mice with or without experimental diabetes induction. We observed that nondiabetic AR-TG mice did not show lens structural changes even though they had lenticular sorbitol levels almost as high as the diabetic AR-TG lenses that showed early signs of cataract. Over-expression of AR led to increases in the ratio of activated to total levels of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal (JNK1/2), which are known to be involved in cell growth and apoptosis respectively. After diabetes induction, AR-TG but not WT controls had decreased levels of phosphorylated as well as total ERK1/2 and JNK1/2 compared to their nondiabetic counterparts. These results indicate that high AR expression in the context of hyperglycemia and insulin deficiency may constitute a risk factor that could predispose the

  6. Aldose reductase expression as a risk factor for cataract.

    PubMed

    Snow, Anson; Shieh, Biehuoy; Chang, Kun-Che; Pal, Arttatrana; Lenhart, Patricia; Ammar, David; Ruzycki, Philip; Palla, Suryanarayana; Reddy, G Bhanuprakesh; Petrash, J Mark

    2015-06-01

    Aldose reductase (AR) is thought to play a role in the pathogenesis of diabetic eye diseases, including cataract and retinopathy. However, not all diabetics develop ocular complications. Paradoxically, some diabetics with poor metabolic control appear to be protected against retinopathy, while others with a history of excellent metabolic control develop severe complications. These observations indicate that one or more risk factors may influence the likelihood that an individual with diabetes will develop cataracts and/or retinopathy. We hypothesize that an elevated level of AR gene expression could confer higher risk for development of diabetic eye disease. To investigate this hypothesis, we examined the onset and severity of diabetes-induced cataract in transgenic mice, designated AR-TG, that were either heterozygous or homozygous for the human AR (AKR1B1) transgene construct. AR-TG mice homozygous for the transgene demonstrated a conditional cataract phenotype, whereby they developed lens vacuoles and cataract-associated structural changes only after induction of experimental diabetes; no such changes were observed in AR-TG heterozygotes or nontransgenic mice with or without experimental diabetes induction. We observed that nondiabetic AR-TG mice did not show lens structural changes even though they had lenticular sorbitol levels almost as high as the diabetic AR-TG lenses that showed early signs of cataract. Over-expression of AR led to increases in the ratio of activated to total levels of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal (JNK1/2), which are known to be involved in cell growth and apoptosis, respectively. After diabetes induction, AR-TG but not WT controls had decreased levels of phosphorylated as well as total ERK1/2 and JNK1/2 compared to their nondiabetic counterparts. These results indicate that high AR expression in the context of hyperglycemia and insulin deficiency may constitute a risk factor that could predispose the

  7. In vitro antidiabetic effects of selected fruits and vegetables against glycosidase and aldose reductase.

    PubMed

    Wu, Tong; Luo, Jiaqiang; Xu, Baojun

    2015-11-01

    In vitro antidiabetic effect of fruits and vegetables with reports as folk remedies were investigated. The antidiabetic effects were evaluated by comparing the inhibitory properties of α-glycosidase, aldose reductase, and antioxidant activity. The results indicated that lychee extract exhibited the best dose-dependent inhibitory activity against α-glycosidase with IC 50 of 10.4 mg/mL, and lemon peel extract exhibited aldose reductase inhibitory potential with IC 50 value at 3.63 mg/mL. Besides, the result also showed that the inhibitory effects of blueberry and plum against α-glycosidase were strong among the fruits samples. Bitter gourd and eggplant demonstrated significant inhibitory potential against aldose reductase, with IC 50 values at 8.55 mg/mL and 8.06 mg/mL, respectively. The result from correlation analysis part showed that the antioxidant activities of selected fruits and vegetables were found related to their health beneficial effects, as there was positive correlations between total flavonoids content (TFC) and aldose reductase inhibitory activity (r (2) = 0.556). PMID:26788291

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

    SciTech Connect

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

    2007-11-01

    Preliminary X-ray diffraction studies of apo maize aldose reductase at 2.0 Å resolution are reported. Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 Å and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 Å was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR.

  9. Endotoxin causes pulmonary hypertension by upregulating smooth muscle endothelin type-B receptors: role of aldose reductase.

    PubMed

    Dschietzig, Thomas; Alexiou, Kosta; Richter, Christoph; Bobzin, Martin; Baumann, Gert; Stangl, Karl; Brunner, Friedrich

    2008-08-01

    Endothelin-1 (ET-1), a potent vasoconstrictor and mitogen, is upregulated in pulmonary tissue during endotoxemia and contributes markedly to endotoxin-induced pulmonary hypertension. It is, however, unknown whether the ET receptors, ET(A) and ET(B), are differentially regulated in endotoxemic pulmonary vasculature and how this may impact on pulmonary vascular tone. To investigate this topic, we used isolated perfused lungs, pulmonary endothelial cells (ECs), and pulmonary vascular smooth muscle cells (SMCs) of the rat. During a 6-h endotoxin exposure, isolated perfused lungs developed significant pulmonary hypertension that was markedly attenuated by antagonizing ET(A) or ET(B) receptors using subtype-selective or a mixed ET(A/B) receptor antagonist. Peptide levels of big ET-1 and ET-1 and gene expression of prepro-ET-1 were increased after endotoxin challenge in all tissues. In endotoxemic isolated perfused lungs and ECs, the significant rise of mature ET-1 seen in controls after ET(B) receptor or mixed antagonism disappeared completely. However, this effect was preserved in endotoxemic SMCs. In ECs, endotoxin markedly downregulated maximum ET(B) receptor sites and ET(B) mRNA levels, whereas in SMCs, it generated substantial ET(B) receptor upregulation and moderate ET(A) receptor downregulation. The aldose reductase inhibitors sorbinil and zopolrestat mitigated endotoxin-induced pulmonary hypertension, ET-1 stimulation, and differential ET(B) receptor regulation. We conclude that endotoxin-induced pulmonary hypertension in the rat results from a loss of endothelial and concomitant gain of vascular smooth muscle ET(B) receptors. These changes are at least partly mediated by aldose reductase. PMID:18091567

  10. Effects of aldose reductase inhibitor treatment in diabetic polyneuropathy - a clinical and neurophysiological study.

    PubMed Central

    Fagius, J; Jameson, S

    1981-01-01

    The efficacy of treatment with an aldose reductase inhibitor (1,3-dioxo-1 H-benz-de-isoquinoline-2(3H)-acetic acid, AY-22,284, Alrestatin) on peripheral nerve function in diabetic polyneuropathy was assessed. Thirty patients with long-standing diabetes and slight to moderate neuropathy participated in the double-blind placebo trial. Clinical examination, sensory threshold determinations for vibratory, tactile and thermal stimuli, conduction velocity measurements and studies of automatic function were performed to evaluate the treatment. Significant differences favouring Alrestatin over placebo were found for many of the measured variables, whereas no changes occurred on placebo. The apparent improvement of neuropathy occurred despite persisting hyperglycaemia. The results indicate that aldose reductase inhibitor treatment may be of value in diabetic polyneuropathy, and provide support for the sorbitol pathway hypothesis of diabetic polyneuropathy. PMID:6801211

  11. Inhibitory effects of Colocasia esculenta (L.) Schott constituents on aldose reductase.

    PubMed

    Li, Hong Mei; Hwang, Seung Hwan; Kang, Beom Goo; Hong, Jae Seung; Lim, Soon Sung

    2014-01-01

    The goal of this study was to determine the rat lens aldose reductase-inhibitory effects of 95% ethanol extracts from the leaves of C. esculenta and, its organic solvent soluble fractions, including the dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (BuOH) and water (H2O) layers, using dl-glyceraldehyde as a substrate. Ten compounds, namely tryptophan (1), orientin (2), isoorientin (3), vitexin (4), isovitexin (5), luteolin-7-O-glucoside (6), luteolin-7-O-rutinoside (7), rosmarinic acid (8), 1-O-feruloyl-d-glucoside (9) and 1-O-caffeoyl-d-glucoside (10) were isolated from the EtOAc and BuOH fractions of C. esculenta. The structures of compounds 1-10 were elucidated by spectroscopic methods and comparison with previous reports. All the isolates were subjected to an in vitro bioassay to evaluate their inhibitory activity against rat lens aldose reductase. Among tested compounds, compounds 2 and 3 significantly inhibited rat lens aldose reductase, with IC50 values of 1.65 and 1.92 μM, respectively. Notably, the inhibitory activity of orientin was 3.9 times greater than that of the positive control, quercetin (4.12 μM). However, the isolated compounds showed only moderate ABTS+ [2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] activity. These results suggest that flavonoid derivatives from Colocasia esculenta (L.) Schott represent potential compounds for the prevention and/or treatment of diabetic complications. PMID:25255750

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-01

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

  15. GP-1447, an inhibitor of aldose reductase, prevents the progression of diabetic cataract in rats.

    PubMed

    Kawakubo, Ken; Mori, Asami; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2012-01-01

    We examined the effects of GP-1447 (3-[(4,5,7-trifluorobenzothiazol-2-yl)methyl]-5-methylphenyl acetic acid) on existing cataracts and sorbitol content in the lens in rats with streptozotocin-induced diabetes. GP-1447 is an inhibitor of aldose reductase, which is the first enzyme in the polyol pathway. Cataracts in the central region of the lens were observed in 7 of 14 eyes (50%) by the fifth week after induction of diabetes, and development of mature cataracts was observed in most lenses by the ninth week. In diabetic rats that received GP-1447 treatment beginning in the fifth week after induction of diabetes, progression of cataracts was observed for 1 week after initiation of treatment. Thereafter, the severity of cataracts did not change substantially. Sorbitol levels in the lens peaked during the first week of diabetes, and this increase was maintained during the 9-week observation period. Elevated sorbitol levels in the lenses of diabetic rats gradually declined after GP-1447 treatment was started on the fifth week after induction of diabetes. Cataracts and sorbitol elevation were not observed in the lenses of controls or diabetic rats treated with GP-1447 immediately after induction of diabetes. These results suggest that the polyol pathway plays an important role in both the appearance and progression of cataracts in diabetic rats. Inhibition of aldose reductase could significantly prevent progression of existing cataracts. PMID:22687477

  16. Clinical and neurophysiological studies of aldose reductase inhibitor ponalrestat in chronic symptomatic diabetic peripheral neuropathy.

    PubMed

    Florkowski, C M; Rowe, B R; Nightingale, S; Harvey, T C; Barnett, A H

    1991-01-01

    Increased flux through the polyol pathway mediated by the enzyme aldose reductase may be associated with the development of diabetic neuropathy. Fifty-four diabetic patients (median age 56 yr, range 25-65 yr) with chronic neuropathic symptoms were randomly allocated to placebo or aldose reductase inhibition (300 or 600 mg ponalrestat ICI 128436) groups for 24 wk. Patients with vibration perception thresholds (VPTs) greater than 35 V at the great toe or thermal difference thresholds (TTs) greater than 10 degrees C on the dorsum of the foot were excluded from the trial. No significant changes were observed in symptoms of pain, numbness, or paresthesia between ponalrestat and placebo groups, and there were no improvements in VPT or TT at several sites. Posterior tibial nerve conduction velocity changed from 35.3 +/- 4.9 m/s at baseline to 33.4 +/- 4.0 m/s at 24 wk (NS) with placebo compared with 37.6 +/- 5.6 vs. 37.2 +/- 8.7 m/s (NS) with 300 mg ponalrestat and 34.5 +/- 6.1 vs. 36.2 +/- 6.8 m/s (NS) with 600 mg ponalrestat. Further studies are indicated with intervention at an earlier stage in the evolution of neuropathy and for longer periods. PMID:1901808

  17. Phytochemical analysis with the antioxidant and aldose reductase inhibitory capacities of Tephrosia humilis aerial parts' extracts.

    PubMed

    Plioukas, Michael; Gabrieli, Chrysi; Lazari, Diamanto; Kokkalou, Eugene

    2016-06-01

    The aerial parts of Tephrosia humilis were tested about their antioxidant potential, their ability to inhibit the aldose/aldehyde reductase enzymes and their phenolic content. The plant material was exhaustively extracted with petroleum ether, dichloromethane and methanol, consecutively. The concentrated methanol extract was re-extracted, successively, with diethyl ether, ethyl acetate and n-butanol. All extracts showed significant antioxidant capacity, but the most effective was the ethyl acetate extract. As about the aldose reductase inhibition, all fractions, except the aqueous, were strong inhibitors of the enzyme, with the n-butanolic and ethyl acetate fractions to inhibit the enzyme above 75%. These findings provide support to the ethnopharmacological usage of the plant as antioxidant and validate its potential to act against the long-term diabetic complications. The phytochemical analysis showed the presence of 1,4-dihydroxy-3,4-(epoxyethano)-5-cyclohexene(1), cleroindicin E(2), lupeol(3), methyl p-coumarate(4), methyl 4-hydroxybenzoate(5), prunin(6), 5,7,2',5'-tetrahydroxyflavanone 7-rutinoside(7), protocatechuic acid(8), luteolin 7-glucoside(9), apigenin(10), naringin(11), rhoifolin(12) and luteolin 7-glucuronate(13). PMID:26209262

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

    PubMed

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

    2006-09-01

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

  19. Model of the catalytic mechanism of human aldose reductase based on quantum chemical calculations.

    SciTech Connect

    Cachau, R. C.; Howard, E. H.; Barth, P. B.; Mitschler, A. M.; Chevrier, B. C.; Lamour, V.; Joachimiak, A.; Sanishvili, R.; Van Zandt, M.; Sibley, E.; Moras, D.; Podjarny, A.; UPR de Biologie Structurale; National Cancer Inst.; Univ. Louis Pasteur; Inst. for Diabetes Discovery, Inc.

    2000-01-01

    Aldose Reductase is an enzyme involved in diabetic complications, thoroughly studied for the purpose of inhibitor development. The structure of an enzyme-inhibitor complex solved at sub-atomic resolution has been used to develop a model for the catalytic mechanism. This model has been refined using a combination of Molecular Dynamics and Quantum calculations. It shows that the proton donation, the subject of previous controversies, is the combined effect of three residues: Lys 77, Tyr 48 and His 110. Lys 77 polarises the Tyr 48 OH group, which donates the proton to His 110, which becomes doubly protonated. His 110 then moves and donates the proton to the substrate. The key information from the sub-atomic resolution structure is the orientation of the ring and the single protonafion of the His 110 in the enzyme-inhibitor complex. This model is in full agreement with all available experimental data.

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2016-06-15

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

  2. Aldose reductase, ocular diabetic complications and the development of topical Kinostat(®).

    PubMed

    Kador, Peter F; Wyman, Milton; Oates, Peter J

    2016-09-01

    Diabetes mellitus (DM) is a major health problem with devastating effects on ocular health in both industrialized and developing countries. The control of hyperglycemia is critical to minimizing the impact of DM on ocular tissues because inadequate glycemic control leads to ocular tissue changes that range from a temporary blurring of vision to permanent vision loss. The biochemical mechanisms that promote the development of diabetic complications have been extensively studied. As a result, a number of prominent biochemical pathways have been identified. Among these, the two-step sorbitol pathway has been the most extensively investigated; nevertheless, it remains controversial. To date, long-term pharmacological studies in animal models of diabetes have demonstrated that the onset and development of ocular complications that include keratopathy, retinopathy and cataract can be ameliorated by the control of excess metabolic flux through aldose reductase (AR). Clinically the alleles of AR have been linked to the rapidity of onset and severity of diabetic ocular complications in diabetic patient populations around the globe. In spite of these promising preclinical and human genetic rationales, several clinical trials of varying durations with structurally diverse aldose reductase inhibitors (ARIs) have shown limited success or failure in preventing or arresting diabetic retinopathy. Despite these clinical setbacks, topical ARI Kinostat(®) promises to find a home in clinical veterinary ophthalmology where its anticipated approval by the FDA will present an alternative treatment paradigm to cataract surgery in diabetic dogs. Here, we critically review the role of AR in diabetes mellitus-linked ocular disease and highlight the development of Kinostat(®) for cataract prevention in diabetic dogs. In addition to the veterinary market, we speculate that with further safety and efficacy studies in humans, Kinostat(®) or a closely related product could have a future role

  3. Development of novel pyrazolone derivatives as inhibitors of aldose reductase: an eco-friendly one-pot synthesis, experimental screening and in silico analysis.

    PubMed

    Kadam, Aparna; Dawane, Bhaskar; Pawar, Manisha; Shegokar, Harshala; Patil, Kapil; Meshram, Rohan; Gacche, Rajesh

    2014-04-01

    Aldose reductase is the key enzyme of polypol pathway leading to accumulation of sorbitol. Sorbitol does not diffuse across the cell membranes easily and therefore accumulates within the cell, causing osmotic damage which leads to retinopathy (cataractogenesis), neuropathy and other diabetic complications. Currently, aldose reductase inhibitors like epalrestat, ranirestat and fidarestat are used for the amelioration of diabetic complications. However, such drugs are effective in patients having good glycemic control and less severe diabetic complications. In present study we have designed novel pyrazolone derivative and performed eco-friendly synthesis approach and tested the synthesized compounds as potential inhibitors of aldose reductase activity. Additional in silico analysis in current study indicates presence of highly conserved chemical environment in active site of goat lens aldose reductase. The reported data is expected to be useful for developing novel pyrazolone derivatives as lead compounds in the management of diabetic complications. PMID:24607578

  4. Aldose reductase C-106T polymorphism is associated with the risk of essential hypertension.

    PubMed

    Wang, Yaqin; Yu, Min; Mo, Long; Li, Zhenyu; Wang, Junjie; Zhou, Hong-Hao; Ouyang, Dong-Sheng

    2016-10-10

    Aldose Reductase (AR), encoded by AKR1B1, is a member of NADPH-dependent aldo-keto reductase superfamily. The C-106T polymorphism of AKR1B1 is closely related to the diabetic complications. Our previous studies have indicated that the expression of AR was increased in spontaneously hypertensive rats, suggesting the effect of AR in hypertension. Here we investigated whether AKR1B1 C-106T polymorphism was associated with essential hypertension (EH). AKR1B1 C-106T polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the direct sequencing methods. 383 healthy subjects and 383 essential hypertensive patients were recruited in this study. The polymorphism of AKR1B1 C-106T in EH and normal tensive (NT) groups was in agreement with Hardy-Weinberg equilibrium. -106T allele of AKR1B1 C-106T variants was more frequent in EH patients compared with normal tensive subjects, indicating that -106T allele was a risk factor of EH (OR=1.841, 95%CI=1.366-2.481). In male patients, C-106T polymorphism was associated significantly with decreased serum high density lipoprotein cholesterol and higher systolic blood pressure levels. Our results suggest that -106T allele of AKR1B1 C-106T polymorphism may be associated with increased risk for EH in Chinese Han population. PMID:27343777

  5. Aldose reductase participates in the downregulation of T cell functions due to suppressor macrophages

    PubMed Central

    Shimizu, Toshiaki; Tatano, Yutaka; Tomioka, Haruaki

    2016-01-01

    The cell-to-cell contact of T lymphocytes with immunosuppressive macrophages causes marked changes in the tyrosine phosphorylation of some cytosolic proteins of T cells. By phosphoproteome analysis, we identified a 36-kDa protein as aldose reductase (AR). The AR expression in T cells was not changed by TCR stimulation or due to cell-to-cell transmission of suppressor signals from immunosuppressive macrophages. Therefore, AR phosphorylation/dephosphorylation is essential for the transduction of TCR-mediated T-cell stimulatory signals, and moreover plays important roles for the cross-talk of immunosuppressive macrophage-derived suppressor signals with the signaling pathways for T-cell activation. Moreover, AR played important roles in the upregulation of ERK1/2-mediated signaling pathways in T lymphocytes. Notably, the enzymatic activity of AR was not required for its signaling action. Taken together, it is concluded that AR mediates intracellular transmission of the suppressor signal of immunosuppressive macrophages toward downstream ERK1/2 pathways, possibly through its direct interaction with acceptor proteins. PMID:26868163

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

    PubMed Central

    Somani, Gauresh; Sathaye, Sadhana

    2015-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-11-01

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

  9. Design and synthesis of potent and multifunctional aldose reductase inhibitors based on quinoxalinones.

    PubMed

    Qin, Xiangyu; Hao, Xin; Han, Hui; Zhu, Shaojuan; Yang, Yanchun; Wu, Bobin; Hussain, Saghir; Parveen, Shagufta; Jing, Chaojun; Ma, Bing; Zhu, Changjin

    2015-02-12

    Quinoxalin-2(1H)-one based design and synthesis produced several series of aldose reductase (ALR2) inhibitor candidates. In particular, phenolic structure was installed in the compounds for the combination of antioxidant activity and strengthening the ability to fight against diabetic complications. Most of the series 6 showed potent and selective effects on ALR2 inhibition with IC50 values in the range of 0.032-0.468 μM, and 2-(3-(2,4-dihydroxyphenyl)-7-fluoro-2-oxoquinoxalin-1(2H)-yl)acetic acid (6e) was the most active. More significantly, most of the series 8 revealed not only good activity in the ALR2 inhibition but also potent antioxidant activity, and 2-(3-(3-methoxy-4-hydroxystyryl)-2-oxoquinoxalin-1(2H)-yl)acetic acid (8d) was even as strong as the well-known antioxidant Trolox at a concentration of 100 μM, verifying the C3 p-hydroxystyryl side chain as the key structure for alleviating oxidative stress. These results therefore suggest an achievement of multifunctional ALR2 inhibitors having both potency for ALR2 inhibition and as antioxidants. PMID:25602762

  10. Aldose Reductase acts as a Selective Derepressor of PPARγ and Retinoic Acid Receptor

    PubMed Central

    Thiagarajan, Devi; Ananthakrishnan, Radha; Zhang, Jinghua; O’Shea, Karen M.; Quadri, Nosirudeen; Li, Qing; Sas, Kelli; Jing, Xiao; Rosario, Rosa; Pennathur, Subramaniam; Schmidt, Ann Marie; Ramasamy, Ravichandran

    2016-01-01

    Summary Histone deacetylase 3 (HDAC3), a chromatin modifying enzyme, requires association with the deacetylase containing domain (DAD) of the nuclear receptor co-repressors NCOR1 and SMRT for its stability and activity. Here we show that aldose reductase (AR), the rate-limiting enzyme of the polyol pathway, competes with HDAC3 to bind the NCOR1/SMRT DAD. Increased AR expression leads to HDAC3 degradation followed by increased PPARγ signaling resulting in lipid accumulation in the heart. AR also downregulates expression of nuclear corepressor complex cofactors including Gps2 and Tblr1, thus affecting activity of the nuclear corepressor complex itself. Though AR reduces HDAC3-corepressor complex formation, it specifically de-represses the retinoic acid receptor (RAR), but not other nuclear receptors such as the thyroid receptor (TR) and liver X receptor (LXR). In summary, this work defines a distinct role for AR in lipid and retinoid metabolism through HDAC3 regulation and consequent de-repression of PPARγ and RAR. PMID:27052179

  11. Stress tolerance of transgenic barley accumulating the alfalfa aldose reductase in the cytoplasm and the chloroplast.

    PubMed

    Nagy, Bettina; Majer, Petra; Mihály, Róbert; Pauk, János; Horváth, Gábor V

    2016-09-01

    Barley represents one of the major crops grown worldwide; its genetic transformation provides an important tool for the improvement of crop quality and tolerance to environmental stress factors. Biotic and abiotic stresses produce reactive oxygen species in the plant cells that can directly oxidize the cellular components including lipid membranes; resulting in lipid peroxidation and subsequently the accumulation of reactive carbonyl compounds. In order to protect barley plants from the effects of stress-produced reactive carbonyls, an Agrobacterium-mediated transformation was carried out using the Medicago sativa aldose reductase (MsALR) gene. In certain transgenic lines the produced MsALR enzyme was targeted to the chloroplasts to evaluate its protective effect in these organelles. The dual fluorescent protein-based method was used for the evaluation of tolerance of young seedlings to diverse stresses; our results demonstrated that this technique could be reliably applied for the detection of cellular stress in a variety of conditions. The chlorophyll and carotenoid content measurements also supported the results of the fluorescent protein-based method and the stress-protective effect of the MsALR enzyme. Targeting of MsALR into the chloroplast has also resulted in increased stress tolerance, similarly to the observed effect of the cytosolic MsALR accumulation. The results of the DsRed/GFP fluorescent protein-based method indicated that both the cytosol and chloroplast accumulation of MsALR can increase the abiotic stress tolerance of transgenic barley lines. PMID:27469099

  12. Diallyl sulfide protects against N-nitrosodiethylamine-induced liver tumorigenesis: Role of aldose reductase

    PubMed Central

    Ibrahim, Safinaz S; Nassar, Noha N

    2008-01-01

    AIM: To evaluate the protective effect of diallyl sulfide (DAS) against N-nitrosodiethylamine (NDEA)-induced liver carcinogenesis. METHODS: Male Wistar rats received either NDEA or NDEA together with DAS as protection. Liver energy metabolism was assessed in terms of lactate, pyruvate, lactate/pyruvate, ATP levels, lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PD) activities. In addition, membrane disintegration of the liver cells was evaluated by measuring lipid-peroxidation products, measured as malondialdehyde (MDA); nitric oxide (NO) levels; glucose-6-phosphatase (G6Pase), catalase (CAT) and superoxide dismutase (SOD) activities. Liver DNA level, glutathione-S-transferase (GST) and cytochrome c oxidase activities were used as DNA fragmentation indices. Aldose reductase (AR) activity was measured as an index for cancer cells resistant to chemotherapy and histopathological examination was performed on liver sections from different groups. RESULTS: NDEA significantly disturbed liver functions and most of the aforementioned indices. Treatment with DAS significantly restored liver functions and hepatocellular integrity; improved parameters of energy metabolism and suppressed free-radical generation. CONCLUSION: We provide evidence that DAS exerts a protective role on liver functions and tissue integrity in face of enhanced tumorigenesis caused by NDEA, as well as improving cancer-cell sensitivity to chemotherapy. This is mediated through combating oxidative stress of free radicals, improving the energy metabolic state of the cell, and enhancing the activity of G6Pase, GST and AR enzymes. PMID:18985804

  13. Altered aldose reductase gene regulation in cultured human retinal pigment epithelial cells.

    PubMed Central

    Henry, D N; Del Monte, M; Greene, D A; Killen, P D

    1993-01-01

    Aldose reductase (AR2), a putative "hypertonicity stress protein" whose gene is induced by hyperosmolarity, protects renal medullary cells against the interstitial hyperosmolarity of antidiuresis by catalyzing the synthesis of millimolar concentrations of intracellular sorbitol from glucose. Although AR2 gene induction has been noted in a variety of renal and nonrenal cells subjected to hypertonic stress in vitro, the functional significance of AR2 gene expression in cells not normally exposed to a hyperosmolar milieu is not fully understood. The physiological impact of basal AR2 expression in such cells may be limited to hyperglycemic states in which AR2 promotes pathological polyol accumulation, a mechanism invoked in the pathogenesis of diabetic complications. Since AR2 overexpression in the retinal pigment epithelium has been associated with diabetic retinopathy, the regulation of AR2 gene expression and associated changes in sorbitol and myo-inositol were studied in human retinal pigment epithelial cells in culture. The relative abundance of aldehyde reductase (AR1) and AR2 mRNA was quantitated by filter hybridization of RNA from several human retinal pigment epithelial cell lines exposed to hyperglycemic and hyperosmolar conditions in vitro. AR2 but not AR1 mRNA was significantly increased some 11- to 18-fold by hyperosmolarity in several retinal pigment epithelial cell lines. A single cell line with a 15-fold higher basal level of AR2 mRNA than other cell lines tested demonstrated no significant increase in AR2 mRNA in response to hypertonic stress. This cell line demonstrated accelerated and exaggerated production of sorbitol and depletion of myo-inositol upon exposure to 20 mM glucose. Therefore, abnormal AR2 expression may enhance the sensitivity of cells to the biochemical consequences of hyperglycemia potentiating the development of diabetic complications. Images PMID:8349800

  14. Melatonin Reduces Cataract Formation and Aldose Reductase Activity in Lenses of Streptozotocin-induced Diabetic Rat

    PubMed Central

    Khorsand, Marjan; Akmali, Masoumeh; Sharzad, Sahab; Beheshtitabar, Mojtaba

    2016-01-01

    Background: The relationship between the high activity of aldose reductase (AR) and diabetic cataract formation has been previously investigated. The purpose of the present study was to determine the preventing effect of melatonin on streptozotocin (STZ)-induced diabetic cataract in rats. Methods: 34 adult healthy male Sprague-Dawely rats were divided into four groups. Diabetic control and diabetic+melatonin received a single dose of STZ (50 mg/kg, intraperitoneally), whereas the normal control and normal+melatonin received vehicle. The melatonin groups were gavaged with melatonin (5 mg/kg) daily for a period of 8 weeks, whereas the rats in the normal control and diabetic control groups received only the vehicle. The rats’ eyes were examined every week and cataract formation scores (0-4) were determined by slit-lamp microscope. At the end of the eighth week, the rats were sacrificed and markers of the polyol pathway and antioxidative (Glutathione, GSH) in their lens were determined. The levels of blood glucose, HbA1c and plasma malondialdhyde (MDA), as a marker of lipid peroxidation, were also measured. Results: Melatonin prevented STZ-induced hyperglycemia by decreased blood glucose and HbA1c levels. Slit lamp examination indicated that melatonin delayed cataract progression in diabetic rats. The results revealed that melatonin feeding increased the GSH levels, decreased the activities of AR and sorbitol dehydrogenase (SDH) and sorbitol formation in catractous lenses as well as plasma MDA content. Conclusion: In summary, for the first time we demonstrated that melatonin delayed the formation and progression of cataract in diabetic rat lenses. PMID:27365552

  15. Aldose Reductase Is Involved in the Development of Murine Diet-Induced Nonalcoholic Steatohepatitis

    PubMed Central

    Qiu, Longxin; Lin, Jianhui; Ying, Miao; Chen, Weiqiang; Yang, Jinmei; Deng, Tiantian; Chen, Jinfeng; Shi, Duanyu; Yang, James Y.

    2013-01-01

    Hepatic aldose reductase (AR) expression is known to be induced in liver diseases, including hepatitis and hepatocellular carcinoma. However, the role of AR in the development of these diseases remains unclear. We performed this current study to determine whether and how AR might be involved in the development of diet-induced nonalcoholic steatohepatitis. Our results showed that the level of AR protein expression was significantly higher in db/db mice fed the methionine-choline-deficient (MCD) diet than in mice fed the control diet. In parallel with the elevation in AR, steatohepatitis was observed in MCD diet-fed mice, and this diet-induced steatohepatitis was significantly attenuated by lentiviral-mediated knock-down of the AR gene. This suppressive effect of AR knock-down was associated with repressed levels of serum alanine aminotransferase and hepatic lipoperoxides, reduced mRNA and protein expression of hepatic cytochrome P450 2E1 (CYP2E1), and decreased mRNA expression of pro-inflammatory tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Moreover, AR-induced elevations on the level of CYP2E1 expression, reactive oxygen species, mRNA expression of TNF-α and IL-6 were confirmed in AML12 hepatocytes. Further, lentiviral-mediated knock-down of AR ameliorated MCD diet-induced collagen deposition in the livers of db/db mice. With the improvement in liver fibrosis, the mRNA levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-2 (MMP-2), two genes involved in hepatic fibrogenesis, were found to be significantly suppressed, while TIMP-2 and MMP-13 were unaffected. Together these data indicate that inhibition of AR alleviates the MCD diet-induced liver inflammation and fibrosis in db/db mice, probably through dampening CYP2E1 mediated-oxidative stress and ameliorating the expression of pro-inflammatory cytokines. PMID:24066058

  16. Structural and kinetic modifications of aldose reductase by S-nitrosothiols.

    PubMed Central

    Srivastava, S; Dixit, B L; Ramana, K V; Chandra, A; Chandra, D; Zacarias, A; Petrash, J M; Bhatnagar, A; Srivastava, S K

    2001-01-01

    Modification of aldose reductase (AR) by the nitrosothiols S-nitroso-N-acetyl penicillamine (SNAP) and N-(beta-glucopyranosyl)-N(2)-acetyl-S-nitrosopenicillamide (glyco-SNAP) resulted in a 3-7-fold increase in its k(cat) and a 25-40-fold increase in its K(m) for glyceraldehyde. In comparison with the native protein, the modified enzyme was less sensitive to inhibition by sorbinil and was not activated by SO(2-)(4) anions. The active-site residue, Cys-298, was identified as the main site of modification, because the site-directed mutant in which Cys-298 was replaced by serine was insensitive to glyco-SNAP. The extent of modification was not affected by P(i) or O(2), indicating that it was not due to spontaneous release of nitric oxide (NO) by the nitrosothiols. Electrospray ionization MS revealed that the modification reaction proceeds via the formation of an N-hydroxysulphenamide-like adduct between glyco-SNAP and AR. In time, the adduct dissociates into either nitrosated AR (AR-NO) or a mixed disulphide between AR and glyco-N-acetylpenicillamine (AR-S-S-X). Removal of the mixed-disulphide form of the protein by lectin-column chromatography enriched the preparation in the high-K(m)-high-k(cat) form of the enzyme, suggesting that the kinetic changes are due to the formation of AR-NO, and that the AR-S-S-X form of the enzyme is catalytically inactive. Modification of AR by the non-thiol NO donor diethylamine NONOate (DEANO) increased enzyme activity and resulted in the formation of AR-NO. However, no adducts between AR and DEANO were formed. These results show that nitrosothiols cause multiple structural and functional changes in AR. Our observations also suggest the general possibility that transnitrosation reactions can generate both nitrosated and thiolated products, leading to non-unique changes in protein structure and function. PMID:11485558

  17. Structural and thermodynamic study on aldose reductase: nitro-substituted inhibitors with strong enthalpic binding contribution.

    PubMed

    Steuber, Holger; Heine, Andreas; Klebe, Gerhard

    2007-05-01

    To prevent diabetic complications derived from enhanced glucose flux via the polyol pathway the development of aldose reductase inhibitors (ARIs) has been established as a promising therapeutic concept. In order to identify novel lead compounds, a virtual screening (VS) was performed successfully suggesting carboxylate-type inhibitors of sub-micromolar to micromolar affinity. Here, we combine a structural characterization of the binding modes observed by X-ray crystallography with isothermal titration calorimetry (ITC) measurements providing insights into the driving forces of inhibitor binding, particularly of the first leads from VS. Characteristic features of this novel inhibitor type include a carboxylate head group connected via an alkyl spacer to a heteroaromatic moiety, which is linked to a further nitro-substituted aromatic portion. The crystal structures of two enzyme-inhibitor complexes have been determined at resolutions of 1.43 A and 1.55 A. Surprisingly, the carboxylic group of the most potent VS lead occupies the catalytic pocket differently compared to the interaction geometry observed in almost all other crystal structures with structurally related ligands and obtained under similar conditions, as an interstitial water molecule is picked up upon ligand binding. The nitro-aromatic moiety of both leads occupies the specificity pocket of the enzyme, however, adopting a different geometry compared to the docking prediction: unexpectedly, the nitro group binds to the bottom of the specificity pocket and provokes remarkable induced-fit adaptations. A peptide group located at the active site orients in such a way that H-bond formation to one nitro group oxygen atom is enabled, whereas a neighbouring tyrosine side-chain performs a slight rotation off from the binding cavity to accommodate the nitro group. Identically constituted ligands, lacking this nitro group, exhibit an affinity drop of one order of magnitude. In addition, thermodynamic data suggest a

  18. Aldose reductase modulates cardiac glycogen synthase kinase-3β phosphorylation during ischemia-reperfusion

    PubMed Central

    Abdillahi, Mariane; Ananthakrishnan, Radha; Vedantham, Srinivasan; Shang, Linshan; Zhu, Zhengbin; Rosario, Rosa; Zirpoli, Hylde; Bohren, Kurt M.; Gabbay, Kenneth H.

    2012-01-01

    Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3β (p-GSK3β). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/without GSK3β inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O2) and reoxygenation (20.9% O2) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3β was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P < 0.05). p-GSK3β was decreased in ARTg hearts compared with WT and ARKO (P < 0.05). In ARKO, p-GSK3β and apoptotic markers were decreased compared with WT (P < 0.05). WT and ARTg hearts perfused with GSK3β inhibitors improved p-GSK3β expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P < 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O2) and reoxygenation (20.9% O2), had greater LDH release compared with control HL-1 cells (P < 0.05). p-GSK3β was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P < 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC) α/β inhibitor

  19. Aldose reductase modulates cardiac glycogen synthase kinase-3β phosphorylation during ischemia-reperfusion.

    PubMed

    Abdillahi, Mariane; Ananthakrishnan, Radha; Vedantham, Srinivasan; Shang, Linshan; Zhu, Zhengbin; Rosario, Rosa; Zirpoli, Hylde; Bohren, Kurt M; Gabbay, Kenneth H; Ramasamy, Ravichandran

    2012-08-01

    Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3β (p-GSK3β). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/without GSK3β inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3β was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P < 0.05). p-GSK3β was decreased in ARTg hearts compared with WT and ARKO (P < 0.05). In ARKO, p-GSK3β and apoptotic markers were decreased compared with WT (P < 0.05). WT and ARTg hearts perfused with GSK3β inhibitors improved p-GSK3β expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P < 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)), had greater LDH release compared with control HL-1 cells (P < 0.05). p-GSK3β was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P < 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC)

  20. High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase.

    PubMed

    Hazemann, I; Dauvergne, M T; Blakeley, M P; Meilleur, F; Haertlein, M; Van Dorsselaer, A; Mitschler, A; Myles, D A A; Podjarny, A

    2005-10-01

    Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples. PMID:16204895

  1. High-resolution neutron protein crystallography with radically small crystal volumes: Application of perdeuteration to human aldose reductase

    SciTech Connect

    Hazemann, I.; Dauvergne, M. T.; Blakeley, M. P.; Meilleur, Flora; Haertlein, M.; Van Dorsselaer, A.; Mitschler, A.; Myles, Dean A A; Podjarny, A.

    2005-08-01

    Neutron diffraction data have been collected to 2.2 {angstrom} resolution from a small (0.15 mm{sup 3}) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm{sup 3} are reported. Neutron data were recorded to 2 {angstrom} resolution, with subsequent data analysis using data to 2.2 {angstrom}. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

  2. Osmotic Stress, not Aldose Reductase Activity, Directly induces Growth Factors and MAPK Signaling changes during Sugar Cataract Formation

    PubMed Central

    Zhang, Peng; Xing, Kuiyi; Randazzo, James; Blessing, Karen; Lou, Marjorie F.; Kador, Peter F.

    2012-01-01

    In sugar cataract formation in rats, aldose reductase (AR) actitvity is not only linked to lenticular sorbitol (diabetic) or galactitol (galactosemic) formation but also to signal transduction changes, cytotoxic signals and activation of apoptosis. Using both in vitro and in vivo techniques, the interrelationship between AR activity, polyol (sorbitol and galactitol) formation, osmotic stress, growth factor induction, and cell signaling changes have been investigated. For in vitro studies, lenses from Sprague Dawley rats were cultured for up to 48 hrs in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galctose with/without the aldose reductase inhibitors AL1576 or tolrestat, the sorbitol dehydrogenase inhibitor (SDI) CP-470,711, or 15 mM mannitol (osmotic-compensated media). For in vivo studies, lenses were obtained from streptozotocin-induced diabetic Sprague Dawley rats fed diet with/without the ARIs AL1576 or tolrestat for 10 weeks. As expected, lenses cultured in high glucose / galactose media or from untreated diabetic rats all showed a decrease in the GSH pool that was lessened by ARI treatment. Lenses either from diabetic rats or from glucose/galactose culture conditions showed increased expression of basic-FGF, TGF-β, and increased signaling through P-Akt, P-ERK1/2 and P-SAPK/JNK which were also normalized by ARIs to the expression levels observed in non-diabetic controls. Culturing rat lenses in osomotically compensated media containing 30 mM glucose or galactose did not lead to increased growth factor expression or altered signaling. These studies indicate that it is the biophysical response of the lens to osmotic stress that results in an increased intralenticular production of basic-FGF and TGF-β and the altered cytotoxic signaling that is observed during sugar cataract formation. PMID:22710095

  3. Bioactivity Focus of α-Cyano-4-hydroxycinnamic acid (CHCA) Leads to Effective Multifunctional Aldose Reductase Inhibitors.

    PubMed

    Zhang, Laitao; Li, Yi-Fang; Yuan, Sheng; Zhang, Shijie; Zheng, Huanhuan; Liu, Jie; Sun, Pinghua; Gu, Yijun; Kurihara, Hiroshi; He, Rong-Rong; Chen, Heru

    2016-01-01

    Bioactivity focus on α-cyano-4-hydroxycinnamic acid (CHCA) scaffold results in a small library of novel multifunctional aldose reductase (ALR2) inhibitors. All the entities displayed good to excellent inhibition with IC50 72-405 nM. (R,E)-N-(3-(2-acetamido-3-(benzyloxy)propanamido)propyl)-2-cyano-3-(4-hydroxy phenyl)acrylamide (5f) was confirmed as the most active inhibitor (IC50 72.7 ± 1.6 nM), and the best antioxidant. 5f bound to ALR2 with new mode without affecting the aldehyde reductase (ALR1) activity, implicating high selectivity to ALR2. 5f was demonstrated as both an effective ALR2 inhibitor (ARI) and antioxidant in a chick embryo model of hyperglycemia. It attenuated hyperglycemia-induced incidence of neural tube defects (NTD) and death rate, and significantly improved the body weight and morphology of the embryos. 5f restored the expression of paired box type 3 transcription factor (Pax3), and reduced the hyperglycemia-induced increase of ALR2 activity, sorbitol accumulation, and the generation of ROS and MDA to normal levels. All the evidences support that 5f may be a potential agent to treat diabetic complications. PMID:27109517

  4. Bioactivity Focus of α-Cyano-4-hydroxycinnamic acid (CHCA) Leads to Effective Multifunctional Aldose Reductase Inhibitors

    PubMed Central

    Zhang, Laitao; Li, Yi-Fang; Yuan, Sheng; Zhang, Shijie; Zheng, Huanhuan; Liu, Jie; Sun, Pinghua; Gu, Yijun; Kurihara, Hiroshi; He, Rong-Rong; Chen, Heru

    2016-01-01

    Bioactivity focus on α-cyano-4-hydroxycinnamic acid (CHCA) scaffold results in a small library of novel multifunctional aldose reductase (ALR2) inhibitors. All the entities displayed good to excellent inhibition with IC50 72–405 nM. (R,E)-N-(3-(2-acetamido-3-(benzyloxy)propanamido)propyl)-2-cyano-3-(4-hydroxy phenyl)acrylamide (5f) was confirmed as the most active inhibitor (IC50 72.7 ± 1.6 nM), and the best antioxidant. 5f bound to ALR2 with new mode without affecting the aldehyde reductase (ALR1) activity, implicating high selectivity to ALR2. 5f was demonstrated as both an effective ALR2 inhibitor (ARI) and antioxidant in a chick embryo model of hyperglycemia. It attenuated hyperglycemia-induced incidence of neural tube defects (NTD) and death rate, and significantly improved the body weight and morphology of the embryos. 5f restored the expression of paired box type 3 transcription factor (Pax3), and reduced the hyperglycemia-induced increase of ALR2 activity, sorbitol accumulation, and the generation of ROS and MDA to normal levels. All the evidences support that 5f may be a potential agent to treat diabetic complications. PMID:27109517

  5. Synthesis and biological evaluation of some new pyrazoline substituted benzenesulfonylurea/thiourea derivatives as anti-hyperglycaemic agents and aldose reductase inhibitors.

    PubMed

    Ovais, Syed; Pushpalatha, H; Reddy, G Bhanuprakash; Rathore, Pooja; Bashir, Rafia; Yaseen, Shafiya; Dheyaa, Alhamza; Yaseen, Raed; Tanwar, Omprakash; Akthar, Mymoona; Samim, Mohammed; Javed, Kalim

    2014-06-10

    Seventeen new pyrazoline substituted benzenesulfonylurea/thiourea derivatives (2a-q) were synthesized and characterized by elemental analysis and various spectroscopic techniques viz; IR, (1)H NMR, (13)C NMR, and MS data. Thirteen compounds showed moderate to good anti-hyperglycaemic activity in glucose fed hyperglycaemic normal rats at the dose of 0.05 mM/kg b.w. On the basis of docking results nine compounds (2a, 2c, 2e, 2h, 2k, 2l, 2n, 2o and 2q) were evaluated for their ability to inhibit rat lens aldose reductase. Out of these six compounds (2h, 2k, 2l, 2n, 2o and 2q) were found more effective than the known ARI sorbinil. Five compounds (2h, 2k, 2l, 2n and 2o) showed significant dual action (anti-hyperglycaemic and aldose reductase inhibition). PMID:24780598

  6. Anti-neuroinflammatory efficacy of the aldose reductase inhibitor FMHM via phospholipase C/protein kinase C-dependent NF-κB and MAPK pathways

    SciTech Connect

    Zeng, Ke-Wu; Li, Jun; Dong, Xin; Wang, Ying-Hong; Ma, Zhi-Zhong; Jiang, Yong; Jin, Hong-Wei; Tu, Peng-Fei

    2013-11-15

    Aldose reductase (AR) has a key role in several inflammatory diseases: diabetes, cancer and cardiovascular diseases. Therefore, AR inhibition seems to be a useful strategy for anti-inflammation therapy. In the central nervous system (CNS), microglial over-activation is considered to be a central event in neuroinflammation. However, the effects of AR inhibition in CNS inflammation and its underlying mechanism of action remain unknown. In the present study, we found that FMHM (a naturally derived AR inhibitor from the roots of Polygala tricornis Gagnep.) showed potent anti-neuroinflammatory effects in vivo and in vitro by inhibiting microglial activation and expression of inflammatory mediators. Mechanistic studies showed that FMHM suppressed the activity of AR-dependent phospholipase C/protein kinase C signaling, which further resulted in downstream inactivation of the IκB kinase/IκB/nuclear factor-kappa B (NF-κB) inflammatory pathway. Therefore, AR inhibition-dependent NF-κB inactivation negatively regulated the transcription and expression of various inflammatory genes. AR inhibition by FMHM exerted neuroprotective effects in lipopolysaccharide-induced neuron–microglia co-cultures. These findings suggested that AR is a potential target for neuroinflammation inhibition and that FMHM could be an effective agent for treating or preventing neuroinflammatory diseases. - Highlights: • FMHM is a natural-derived aldose reductase (AR) inhibitor. • FMHM inhibits various neuroinflammatory mediator productions in vitro and in vivo. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent NF-κB pathway. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent MAPK pathway. • FMHM protects neurons against inflammatory injury in microglia-neuron co-cultures.

  7. Quantum mechanical calculation of electric fields and vibrational Stark shifts at active site of human aldose reductase.

    PubMed

    Wang, Xianwei; Zhang, John Z H; He, Xiao

    2015-11-14

    Recent advance in biophysics has made it possible to directly measure site-specific electric field at internal sites of proteins using molecular probes with C = O or C≡N groups in the context of vibrational Stark effect. These measurements directly probe changes of electric field at specific protein sites due to, e.g., mutation and are very useful in protein design. Computational simulation of the Stark effect based on force fields such as AMBER and OPLS, while providing good insight, shows large errors in comparison to experimental measurement due to inherent difficulties associated with point charge based representation of force fields. In this study, quantum mechanical calculation of protein's internal electrostatic properties and vibrational Stark shifts was carried out by using electrostatically embedded generalized molecular fractionation with conjugate caps method. Quantum calculated change of mutation-induced electric field and vibrational Stark shift is reported at the internal probing site of enzyme human aldose reductase. The quantum result is in much better agreement with experimental data than those predicted by force fields, underscoring the deficiency of traditional point charge models describing intra-protein electrostatic properties. PMID:26567650

  8. Quantum mechanical calculation of electric fields and vibrational Stark shifts at active site of human aldose reductase

    NASA Astrophysics Data System (ADS)

    Wang, Xianwei; Zhang, John Z. H.; He, Xiao

    2015-11-01

    Recent advance in biophysics has made it possible to directly measure site-specific electric field at internal sites of proteins using molecular probes with C = O or C≡N groups in the context of vibrational Stark effect. These measurements directly probe changes of electric field at specific protein sites due to, e.g., mutation and are very useful in protein design. Computational simulation of the Stark effect based on force fields such as AMBER and OPLS, while providing good insight, shows large errors in comparison to experimental measurement due to inherent difficulties associated with point charge based representation of force fields. In this study, quantum mechanical calculation of protein's internal electrostatic properties and vibrational Stark shifts was carried out by using electrostatically embedded generalized molecular fractionation with conjugate caps method. Quantum calculated change of mutation-induced electric field and vibrational Stark shift is reported at the internal probing site of enzyme human aldose reductase. The quantum result is in much better agreement with experimental data than those predicted by force fields, underscoring the deficiency of traditional point charge models describing intra-protein electrostatic properties.

  9. Aldose Reductase-Mediated Phosphorylation of p53 Leads to Mitochondrial Dysfunction, and Damage in Diabetic Platelets

    PubMed Central

    Tang, Wai Ho; Stitham, Jeremiah; Jin, Yu; Liu, Renjing; Lee, Seung Hee; Du, Jing; Atteya, Gourg; Gleim, Scott; Spollett, Geralyn; Martin, Kathleen; Hwa, John

    2014-01-01

    Background Platelet abnormalities are well-recognized complications of diabetes mellitus (DM). Mitochondria play a central role in platelet metabolism and activation. Mitochondrial dysfunction is evident in DM. The molecular pathway for hyperglycemia-induced mitochondrial dysfunction in DM platelets is unknown. Methods and Results Using both human and humanized mouse models, we report that hyperglycemia-induced aldose reductase (AR) activation, and subsequent reactive oxygen species (ROS) production, leads to increased p53 phosphorylation (Ser15), which promotes mitochondrial dysfunction, damage and rupture by sequestration of the anti-apoptotic protein Bcl-xL. In a glucose dose dependent manner, severe mitochondrial damage leads to loss of mitochondrial membrane potential and platelet apoptosis (cytochrome c release, caspase 3 activation and phosphatidylserine exposure). Although platelet hyperactivation, mitochondrial dysfunction, AR activation, ROS production and p53 phosphorylation are all induced by hyperglycemia, we demonstrate that platelet apoptosis and hyperactivation are two distinct states, dependent upon the severity of the hyperglycemia and mitochondrial damage. Combined, both lead to increased thrombus formation in a mouse blood stasis model. Conclusions AR contributes to diabetes-mediated mitochondrial dysfunction and damage through the activation of p53. The degree of mitochondrial dysfunction and damage determines whether hyperactivity (mild damage) or apoptosis (severe damage) will ensue. These signaling components provide novel therapeutic targets for DM thrombotic complications. PMID:24474649

  10. Quantum mechanical calculation of electric fields and vibrational Stark shifts at active site of human aldose reductase

    SciTech Connect

    Wang, Xianwei; Zhang, John Z. H.; He, Xiao

    2015-11-14

    Recent advance in biophysics has made it possible to directly measure site-specific electric field at internal sites of proteins using molecular probes with C = O or C≡N groups in the context of vibrational Stark effect. These measurements directly probe changes of electric field at specific protein sites due to, e.g., mutation and are very useful in protein design. Computational simulation of the Stark effect based on force fields such as AMBER and OPLS, while providing good insight, shows large errors in comparison to experimental measurement due to inherent difficulties associated with point charge based representation of force fields. In this study, quantum mechanical calculation of protein’s internal electrostatic properties and vibrational Stark shifts was carried out by using electrostatically embedded generalized molecular fractionation with conjugate caps method. Quantum calculated change of mutation-induced electric field and vibrational Stark shift is reported at the internal probing site of enzyme human aldose reductase. The quantum result is in much better agreement with experimental data than those predicted by force fields, underscoring the deficiency of traditional point charge models describing intra-protein electrostatic properties.

  11. Exclusion of aldose reductase as a mediator of ERG deficits in a mouse model of diabetic eye disease

    PubMed Central

    SAMUELS, IVY S.; LEE, CHIEH-ALLEN; PETRASH, J. MARK; PEACHEY, NEAL S.; KERN, TIMOTHY S.

    2013-01-01

    Streptozotocin (STZ)-induced diabetes is associated with reductions in the electrical response of the outer retina and retinal pigment epithelium (RPE) to light. Aldose reductase (AR) is the first enzyme required in the polyol-mediated metabolism of glucose, and AR inhibitors have been shown to improve diabetes-induced electroretinogram (ERG) defects. Here, we used control and AR−/− mice to determine if genetic inactivation of this enzyme likewise inhibits retinal electrophysiological defects observed in a mouse model of type 1 diabetes. STZ was used to induce hyperglycemia and type 1 diabetes. Diabetic and age-matched nondiabetic controls of each genotype were maintained for 22 weeks, after which ERGs were used to measure the light-evoked components of the RPE (dc-ERG) and the neural retina (a-wave, b-wave). In comparison to their nondiabetic controls, wildtype (WT) and AR−/− diabetic mice displayed significant decreases in the c-wave, fast oscillation, and off response components of the dc-ERG but not in the light peak response. Nondiabetic AR−/− mice displayed larger ERG component amplitudes than did nondiabetic WT mice; however, the amplitude of dc-ERG components in diabetic AR−/− animals were similar to WT diabetics. ERG a-wave amplitudes were not reduced in either diabetic group, but b-wave amplitudes were lower in WT and AR−/− diabetic mice. These findings demonstrate that the light-induced responses of the RPE and outer retina are disrupted in diabetic mice, but these defects are not due to photoreceptor dysfunction, nor are they ameliorated by deletion of AR. This latter finding suggests that benefits observed in other studies utilizing pharmacological inhibitors of AR might have been secondary to off-target effects of the drugs. PMID:23101909

  12. SIRT1 contributes to aldose reductase expression through modulating NFAT5 under osmotic stress: In vitro and in silico insights.

    PubMed

    Timucin, Ahmet Can; Bodur, Cagri; Basaga, Huveyda

    2015-11-01

    So far, a myriad of molecules were characterized to modulate NFAT5 and its downstream targets. Among these NFAT5 modifiers, SIRT1 was proposed to have a promising role in NFAT5 dependent events, yet the exact underlying mechanism still remains obscure. Hence, the link between SIRT1 and NFAT5-aldose reductase (AR) axis under osmotic stress, was aimed to be delineated in this study. A unique osmotic stress model was generated and its mechanistic components were deciphered in U937 monocytes. In this model, AR expression and nuclear NFAT5 stabilization were revealed to be positively regulated by SIRT1 through utilization of pharmacological modulators. Overexpression and co-transfection studies of NFAT5 and SIRT1 further validated the contribution of SIRT1 to AR and NFAT5. The involvement of SIRT1 activity in these events was mediated via modification of DNA binding of NFAT5 to AR ORE region. Besides, NFAT5 and SIRT1 were also shown to co-immunoprecipitate under isosmotic conditions and this interaction was disrupted by osmotic stress. Further in silico experiments were conducted to investigate if SIRT1 directly targets NFAT5. In this regard, certain lysine residues of NFAT5, when kept deacetylated, were found to contribute to its DNA binding and SIRT1 was shown to directly bind K282 of NFAT5. Based on these in vitro and in silico findings, SIRT1 was identified, for the first time, as a novel positive regulator of NFAT5 dependent AR expression under osmotic stress in U937 monocytes. PMID:26297866

  13. Amelioration of Acute Kidney Injury in Lipopolysaccharide-Induced Systemic Inflammatory Response Syndrome by an Aldose Reductase Inhibitor, Fidarestat

    PubMed Central

    Takahashi, Kazunori; Mizukami, Hiroki; Kamata, Kosuke; Inaba, Wataru; Kato, Noriaki; Hibi, Chihiro; Yagihashi, Soroku

    2012-01-01

    Background Systemic inflammatory response syndrome is a fatal disease because of multiple organ failure. Acute kidney injury is a serious complication of systemic inflammatory response syndrome and its genesis is still unclear posing a difficulty for an effective treatment. Aldose reductase (AR) inhibitor is recently found to suppress lipopolysaccharide (LPS)-induced cardiac failure and its lethality. We studied the effects of AR inhibitor on LPS-induced acute kidney injury and its mechanism. Methods Mice were injected with LPS and the effects of AR inhibitor (Fidarestat 32 mg/kg) before or after LPS injection were examined for the mortality, severity of renal failure and kidney pathology. Serum concentrations of cytokines (interleukin-1β, interleukin-6, monocyte chemotactic protein-1 and tumor necrosis factor-α) and their mRNA expressions in the lung, liver, spleen and kidney were measured. We also evaluated polyol metabolites in the kidney. Results Mortality rate within 72 hours was significantly less in LPS-injected mice treated with AR inhibitor both before (29%) and after LPS injection (40%) than untreated mice (90%). LPS-injected mice showed marked increases in blood urea nitrogen, creatinine and cytokines, and AR inhibitor treatment suppressed the changes. LPS-induced acute kidney injury was associated with vacuolar degeneration and apoptosis of renal tubular cells as well as infiltration of neutrophils and macrophages. With improvement of such pathological findings, AR inhibitor treatment suppressed the elevation of cytokine mRNA levels in multiple organs and renal sorbitol accumulation. Conclusion AR inhibitor treatment ameliorated LPS-induced acute kidney injury, resulting in the lowered mortality. PMID:22253906

  14. Effect of SG-210, a novel aldose reductase inhibitor, on impaired polyol pathway in rats received diabetic manipulations.

    PubMed

    Horie, S; Nagai, H; Yuuki, T; Narita, Y; Tsuda, Y; Nakajima, T; Nakamura, N

    1998-01-01

    To investigate the effect of SG-210, a potent inhibitor selective to aldose reductase (ARI), on the impaired polyol pathway, we examined biochemically and histologically the potencies of this compound in streptozotocin-induced diabetic or galactosemic rats. The study with diabetic rats showed that SG-210 (1-10 mg x kg(-1)) dose-dependently inhibited sorbitol accumulations in erythrocytes, sciatic nerves, lens, and retina with ED50 values of 1.4, 1.3, 3.5, and 4.6 mg x kg(-1), respectively. Zenarestat, currently under clinical trials both in Japan and the United States, was about two or over five times less potent than SG-210 in suppressing sorbitol contents of erythrocytes or other tissues, respectively. Epalrestat, commercially available, was much less potent in reducing the contents with ED50 values of more than 30 mg x kg(-1) in all of the cells and the tissues examined. An extensive study using galactosemic rats indicated that SG-210 (3-30 mg x kg(-1)) inhibited galactitol accumulations in lens and retina as well as in erythrocytes, preventing the progression of histological abnormalities in lens accompanied by the reduction in galactitol contents. Epalrestat (3-30 mg x kg(-1)) failed to show any significant effects. Pharmacokinetic studies suggested that SG-210 has a high bioavailability and possesses a long half-life in rats (ca. 10 h). Taken together with its excellent pharmacokinetic profiles, the potent suppressive effects of SG-210 observed in this study may be available as a new treatment of diabetic complications. PMID:9618072

  15. Deficiency of aldose reductase attenuates inner retinal neuronal changes in a mouse model of retinopathy of prematurity.

    PubMed

    Fu, Zhongjie; Nian, Shen; Li, Suk-Yee; Wong, David; Chung, Sookja K; Lo, Amy C Y

    2015-09-01

    Retinopathy of prematurity (ROP) is a leading cause of childhood blindness where vascular abnormality and retinal dysfunction are reported. We showed earlier that genetic deletion of aldose reductase (AR), the rate-limiting enzyme in the polyol pathway, reduced the neovascularization through attenuating oxidative stress induction in the mouse oxygen-induced retinopathy (OIR) modeling ROP. In this study, we further investigated the effects of AR deficiency on retinal neurons in the mouse OIR. Seven-day-old wild-type and AR-deficient mice were exposed to 75 % oxygen for 5 days and then returned to room air. Electroretinography was used to assess the neuronal function at postnatal day (P) 30. On P17 and P30, retinal cytoarchitecture was examined by morphometric analysis and immunohistochemistry for calbindin, protein kinase C alpha, calretinin, Tuj1, and glial fibrillary acidic protein. In OIR, attenuated amplitudes and delayed implicit time of a-wave, b-wave, and oscillatory potentials were observed in wild-type mice, but they were not significantly changed in AR-deficient mice. The morphological changes of horizontal, rod bipolar, and amacrine cells were shown in wild-type mice and these changes were partly preserved with AR deficiency. AR deficiency attenuated the Müller cell gliosis induced in OIR. Our observations demonstrated AR deficiency preserved retinal functions in OIR and AR deficiency could partly reduce the extent of retinal neuronal histopathology. These findings suggested a therapeutic potential of AR inhibition in ROP treatment with beneficial effects on the retinal neurons. PMID:25921391

  16. Design of an Amide N-glycoside Derivative of β-Glucogallin: A Stable, Potent, and Specific Inhibitor of Aldose Reductase

    PubMed Central

    Li, Linfeng; Chang, Kun-Che; Zhou, Yaming; Shieh, Biehuoy; Ponder, Jessica; Abraham, Adedoyin D.; Ali, Hadi; Snow, Anson; Petrash, J. Mark; LaBarbera, Daniel V.

    2014-01-01

    β-glucogallin (BGG), a major component of the Emblica officinalis medicinal plant, is a potent and selective inhibitor of aldose-reductase (AKR1B1). New linkages (ether/triazole/amide) were introduced via high yielding, efficient syntheses to replace the labile ester, and an original 2-step (90%) preparation of BGG was developed. Inhibition of AKR1B1was assessed in vitro and using transgenic lens organ cultures, which identified the amide linked glucoside (BGA) as a stable, potent and selective lead therapeutic toward the treatment of diabetic eye disease. PMID:24341381

  17. Quantum Model of Catalysis Based on a Mobile Proton Revealed by Subatomic X-ray and Neutron Diffraction Studies of h-aldose Reductase

    SciTech Connect

    Blakeley, M. P.; Ruiz, Fredrico; Cachau, Raul; Hazemann, I.; Meilleur, Flora; Mitschler, A.; Ginell, Stephan; Afonine, Pavel; Ventura, Oscar; Cousido-Siah, Alexandra; Haertlein, M.; Joachimiak, Andrzej; Myles, Dean A A; Podjarny, A.

    2008-01-01

    We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 Angstroms, 100K; 0.80 Angstroms, 15K; 1.75 Angstroms, 293K), neutron Laue data (2.2 Angstroms, 293K), and quantum mechanical modeling. These complementary techniques unveil the internal organization and mobility of the hydrogen bond network that defines the properties of the catalytic engine, explaining how this promiscuous enzyme overcomes the simultaneous requirements of efficiency and promiscuity offering a general mechanistic view for this class of enzymes.

  18. [Role of heat shock proteins, aldose reductase, Bcl-2 protein and microRNA in the mechanism of delayed preconditioning of heart].

    PubMed

    Lishmanov, Iu B; Maslov, L N; Khaliulin, I G; Zhang, Y; Pei, J -M

    2010-05-01

    Analysis of published data allows affirming that heat shock proteins (HSP) play an important role in the mechanism of cardioprotective effect of delayed preconditioning. However, HSP in all probability are non-end effectors but mediators of preconditioning because a peak of their levels in myocardium does not concur with maximal elevation of cardiac tolerance to impact of ischemia and reperfusion. There are bases to think that aldose reductase and Bcl-2 protein are claimants to the role of end-effectors of delayed preconditioning but microRNAs serve as mediators of forming increased cardiac tolerance to ischemia-reperfusion. PMID:20583571

  19. A defect in sodium-dependent amino acid uptake in diabetic rabbit peripheral nerve. Correction by an aldose reductase inhibitor or myo-inositol administration.

    PubMed Central

    Greene, D A; Lattimer, S A; Carroll, P B; Fernstrom, J D; Finegold, D N

    1990-01-01

    A myo-inositol-related defect in nerve sodium-potassium ATPase activity in experimental diabetes has been suggested as a possible pathogenetic factor in diabetic neuropathy. Because the sodium-potassium ATPase is essential for other sodium-cotransport systems, and because myo-inositol-derived phosphoinositide metabolites regulate multiple membrane transport processes, sodium gradient-dependent amino acid uptake was examined in vitro in endoneurial preparations derived from nondiabetic and 14-d alloxan diabetic rabbits. Untreated alloxan diabetes reduced endoneurial sodium-gradient dependent uptake of the nonmetabolized amino acid 2-aminoisobutyric acid by greater than 50%. Administration of an aldose reductase inhibitor prevented reductions in both nerve myo-inositol content and endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Myo-inositol supplementation that produced a transient pharmacological elevation in plasma myo-inositol concentration, but did not raise nerve myo-inositol content, reproduced the effect of the aldose reductase inhibitor on endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Phorbol myristate acetate, which acutely normalizes sodium-potassium ATPase activity in diabetic nerve, did not acutely correct 2-aminoisobutyric uptake when added in vitro. These data suggest that depletion of a small myo-inositol pool may be implicated in the pathogenesis of defects in amino acid uptake in diabetic nerve and that rapid correction of sodium-potassium ATPase activity with protein kinase C agonists in vitro does not acutely normalize sodium-dependent 2-aminoisobutyric acid uptake. PMID:2185278

  20. Preventive effect of long-term aldose reductase inhibition (ponalrestat) on nerve conduction and sural nerve structure in the spontaneously diabetic Bio-Breeding rat.

    PubMed Central

    Sima, A A; Prashar, A; Zhang, W X; Chakrabarti, S; Greene, D A

    1990-01-01

    To test the hypothesis that aldose reductase inhibition may prevent or delay the development of functional and structural neuropathy in the insulin-deficient diabetic Bio-Breeding rat (BB-rat), hyperglycemic rats were begun on the aldose reductase inhibitor (ARI) ponalrestat 25 mg/kg body wt soon after the onset of diabetes and followed for 4 or 6 mo. Ponalrestat treatment completely prevented the characteristic nerve conduction slowing and structural abnormalities of the node of Ranvier for 4 mo despite only partial preservation of axonal integrity. Ponalrestat treatment for 6 mo achieved a partial but significant prevention of nerve conduction slowing, axoglial dysjunction, and axonal degenerative changes. This incomplete but significant prevention of neuropathy by ponalrestat suggests that additional mechanisms besides polyol-pathway activation may be of importance in the pathogenesis of diabetic neuropathy. Alternatively, the dosage used in the present study may not have been sufficient to achieve a complete prevention. Despite the only partial protective effect of ARI treatment on degenerative peripheral nerve changes in hyperglycemic BB-rats, 6 mo of treatment resulted in a more than threefold increase in regenerating nerve fibers. These data suggest that prophylactic ARI treatment may be efficacious in delaying the development of diabetic neuropathy. Images PMID:2110189

  1. A series of pyrido[2,3-b]pyrazin-3(4H)-one derivatives as aldose reductase inhibitors with antioxidant activity.

    PubMed

    Han, Zhongfei; Hao, Xin; Ma, Bing; Zhu, Changjin

    2016-10-01

    A series of pyrido[2,3-b]pyrazin-3(4H)-one based derivatives were designed as inhibitors of aldose reductase (ALR2), the enzyme which plays a key role in the development of diabetes complications as well as in the oxidative stress processes associated with diabetes and other pathologies. Most of the derivatives, having a substituted C2 aromatic group and a N4 acetic acid group on the core structure, were found to be potent and selective aldose reductase inhibitors with submicromolar IC50 values, and 9c was the most active with IC50 value 0.009 μM. Particularly, a number of the designed compounds bearing phenolic hydroxyl substituted C2-styryl side chain showed excellent not only in ALR2 inhibition but also in antioxidant, and among these 11i was proved to be the top one with an antioxidant ability even comparable to that of the well-known antioxidant Trolox. Structure-activity relationship and molecular docking studies highlighted the importance of phenolic hydroxyl substituents and vinyl spacer in C2 side chain of the scaffold for the construction of efficient and multifunctional ALR2 inhibitors. PMID:27267001

  2. Phenolic Compounds from the Leaves and Twigs of Osteomeles schwerinae That Inhibit Rat Lens Aldose Reductase and Vessel Dilation in Zebrafish Larvae.

    PubMed

    Lee, Ik-Soo; Jung, Seung-Hyun; Lee, Yun Mi; Choi, So-Jin; Sun, Hang; Kim, Jin Sook

    2015-09-25

    Three new phenolic biphenyl derivatives (1-3) and one new lignan glycoside (4) were isolated from the leaves and twigs of Osteomeles schwerinae. The structures of the new compounds were established by spectroscopic data interpretation. The inhibitory effects of 1-4 on rat lens aldose reductase in vitro were examined, and compounds 1-3 markedly inhibited the enzyme with IC50 values of 3.8 to 13.8 μM. In addition, the effects of these isolates on the dilation of hyaloid-retinal vessels induced by high glucose (HG) in zebrafish larvae were investigated. Compound 1 was the most effective in reducing HG-induced dilation of hyaloid-retinal vessels. PMID:26331986

  3. Detoxifying Enzymes at the Cross-Roads of Inflammation, Oxidative Stress, and Drug Hypersensitivity: Role of Glutathione Transferase P1-1 and Aldose Reductase

    PubMed Central

    Sánchez-Gómez, Francisco J.; Díez-Dacal, Beatriz; García-Martín, Elena; Agúndez, José A. G.; Pajares, María A.; Pérez-Sala, Dolores

    2016-01-01

    Phase I and II enzymes are involved in the metabolism of endogenous reactive compounds as well as xenobiotics, including toxicants and drugs. Genotyping studies have established several drug metabolizing enzymes as markers for risk of drug hypersensitivity. However, other candidates are emerging that are involved in drug metabolism but also in the generation of danger or costimulatory signals. Enzymes such as aldo-keto reductases (AKR) and glutathione transferases (GST) metabolize prostaglandins and reactive aldehydes with proinflammatory activity, as well as drugs and/or their reactive metabolites. In addition, their metabolic activity can have important consequences for the cellular redox status, and impacts the inflammatory response as well as the balance of inflammatory mediators, which can modulate epigenetic factors and cooperate or interfere with drug-adduct formation. These enzymes are, in turn, targets for covalent modification and regulation by oxidative stress, inflammatory mediators, and drugs. Therefore, they constitute a platform for a complex set of interactions involving drug metabolism, protein haptenation, modulation of the inflammatory response, and/or generation of danger signals with implications in drug hypersensitivity reactions. Moreover, increasing evidence supports their involvement in allergic processes. Here, we will focus on GSTP1-1 and aldose reductase (AKR1B1) and provide a perspective for their involvement in drug hypersensitivity. PMID:27540362

  4. Detoxifying Enzymes at the Cross-Roads of Inflammation, Oxidative Stress, and Drug Hypersensitivity: Role of Glutathione Transferase P1-1 and Aldose Reductase.

    PubMed

    Sánchez-Gómez, Francisco J; Díez-Dacal, Beatriz; García-Martín, Elena; Agúndez, José A G; Pajares, María A; Pérez-Sala, Dolores

    2016-01-01

    Phase I and II enzymes are involved in the metabolism of endogenous reactive compounds as well as xenobiotics, including toxicants and drugs. Genotyping studies have established several drug metabolizing enzymes as markers for risk of drug hypersensitivity. However, other candidates are emerging that are involved in drug metabolism but also in the generation of danger or costimulatory signals. Enzymes such as aldo-keto reductases (AKR) and glutathione transferases (GST) metabolize prostaglandins and reactive aldehydes with proinflammatory activity, as well as drugs and/or their reactive metabolites. In addition, their metabolic activity can have important consequences for the cellular redox status, and impacts the inflammatory response as well as the balance of inflammatory mediators, which can modulate epigenetic factors and cooperate or interfere with drug-adduct formation. These enzymes are, in turn, targets for covalent modification and regulation by oxidative stress, inflammatory mediators, and drugs. Therefore, they constitute a platform for a complex set of interactions involving drug metabolism, protein haptenation, modulation of the inflammatory response, and/or generation of danger signals with implications in drug hypersensitivity reactions. Moreover, increasing evidence supports their involvement in allergic processes. Here, we will focus on GSTP1-1 and aldose reductase (AKR1B1) and provide a perspective for their involvement in drug hypersensitivity. PMID:27540362

  5. Part 1: synthesis of irreversible inhibitors of aldose reductase with subsequent development of a carbon-13 NMR protein probe. Part 2: synthesis of selenium analogs of dopamine as potential dopamine receptor agonists

    SciTech Connect

    Ares, J.J.

    1986-01-01

    Aldose reductase converts glucose into sorbitol using NADPH as a cofactor. Sorbitol accumulation in various tissues is believed to play a major role in the development of debilitating complications of diabetes; thus, much effort has been directed toward the preparation of aldose reductase inhibitors. Of the compounds prepared, the most active are the isothiocyanate and azide analogs of the reversible aldose reductase inhibitor alrestatin. The potency of the alrestatin isothiocyanate prompted the authors to examine the possibility that isothiocyanates enriched with carbon-13 could be used as carbon-13 NMR protein probes. Toward this end, a synthesis of carbon-13 enriched phenylisothiocyanate has been developed. This reagent has been successfully utilized to study peptides via carbon-13 NMR spectroscopy. Research in their laboratory over the years has focused on answering two fundamental questions regarding the interaction of dopamine with its receptor. First, can the concept of bioisosterism be applied to dopamine agonists. Secondly, what is the actual molecular species of dopamine which interacts with the dopamine receptor. In an effort to answer these questions, methyl selenide and dimethyl selenonium analogs of dopamine have been synthesized.

  6. The Prostaglandin F Synthase Activity of the Human Aldose Reductase AKR1B1 Brings New Lenses to Look at Pathologic Conditions

    PubMed Central

    Bresson, Eva; Lacroix-Pépin, Nicolas; Boucher-Kovalik, Sofia; Chapdelaine, Pierre; Fortier, Michel A.

    2012-01-01

    Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2α are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2/EP2 and PGF2α/FP may constitute a functional dyad with physiological relevance comparable to the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2α production in response to IL-1β in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231) also known as ALDR1 or ALR2 is a functional PGF2α synthase in different models of living cells and tissues. Using human endometrial cells, prostate, and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1β is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2α production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1β particularly around the multiple stress response region containing two putative antioxidant response elements adjacent to TonE and AP1. We also show that AKR1B1 is able to regulate PGE2 production through PGF2α acting on its FP receptor and that aldose reductase inhibitors like alrestatin, Statil (ponalrestat), and EBPC exhibit distinct and characteristic inhibition of PGF2α production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies. PMID:22654757

  7. Excretion of tectoridin metabolites in rat urine and bile orally administrated at different dosages and their inhibitory activity against aldose reductase.

    PubMed

    Qu, Jialin; Wu, Zhizhen; Gao, Jie; Wen, Hao; Wang, Tao; Yuan, Dan

    2014-12-01

    This study investigated the urinary and biliary excretion of tectoridin, a major active isoflavonoid found in the flowers of Pueraria thomsonii Benth. and the rhizomes of Belamcanda chinensis (L.) DC. Using UHPLC/Q-TOFMS, seven glucuronides and/or sulfated metabolites and four Phase I metabolites were simultaneously quantified in rat urine after oral administration of tectoridin at 100 and 200 mg/kg. Over a 72-h period, 14.2% and 14.7% of the tectoridin were excreted as eleven metabolites in urine, among which, two major metabolites tectorigenin-7-O-β-D-glucuronide (Te-7G) and tectorigenin accounted for 5.5-5.5% and 4.3-4.4%. Furthermore, the cumulative excretion of four glucuronides and sulfated metabolites in bile accounted for 7.3% and 3.9% of the dose within 60 h, among which, Te-7G and tectorigenin-7-O-glucuronide-4'-O-sulfate (Te-7G-4'S) accounted for 2.3-3.0% and 1.4-3.9%, respectively. The results indicate that the urine was the primary elimination route, and glucuronidation after deglycosylation at C-7 position was the major metabolic pathway of tectoridin in vivo. Moreover, the inhibitory activities of tectoridin and its five metabolites on rat lens aldose reductase were confirmed (IC₅₀: 1.4-15.5 μM), whereas irisolidone-7-O-glucuronide (Ir-7G) and irisolidone showed little activity. PMID:25256063

  8. SIRT6 Is a Positive Regulator of Aldose Reductase Expression in U937 and HeLa cells under Osmotic Stress: In Vitro and In Silico Insights.

    PubMed

    Timucin, Ahmet Can; Basaga, Huveyda

    2016-01-01

    SIRT6 is a protein deacetylase, involved in various intracellular processes including suppression of glycolysis and DNA repair. Aldose Reductase (AR), first enzyme of polyol pathway, was proposed to be indirectly associated to these SIRT6 linked processes. Despite these associations, presence of SIRT6 based regulation of AR still remains ambiguous. Thus, regulation of AR expression by SIRT6 was investigated under hyperosmotic stress. A unique model of osmotic stress in U937 cells was used to demonstrate the presence of a potential link between SIRT6 and AR expression. By overexpressing SIRT6 in HeLa cells under hyperosmotic stress, its role on upregulation of AR was revealed. In parallel, increased SIRT6 activity was shown to upregulate AR in U937 cells under hyperosmotic milieu by using pharmacological modulators. Since these modulators also target SIRT1, binding of the inhibitor, Ex-527, specifically to SIRT6 was analyzed in silico. Computational observations indicated that Ex-527 may also target SIRT6 active site residues under high salt concentration, thus, validating in vitro findings. Based on these evidences, a novel regulatory step by SIRT6, modifying AR expression under hyperosmotic stress was presented and its possible interactions with intracellular machinery was discussed. PMID:27536992

  9. 3D-QSAR (CoMFA and CoMSIA) and pharmacophore (GALAHAD) studies on the differential inhibition of aldose reductase by flavonoid compounds.

    PubMed

    Caballero, Julio

    2010-11-01

    Inhibitory activities of flavonoid derivatives against aldose reductase (AR) enzyme were modelled by using CoMFA, CoMSIA and GALAHAD methods. CoMFA and CoMSIA methods were used for deriving quantitative structure-activity relationship (QSAR) models. All QSAR models were trained with 55 compounds, after which they were evaluated for predictive ability with additional 14 compounds. The best CoMFA model included both steric and electrostatic fields, meanwhile, the best CoMSIA model included steric, hydrophobic and H-bond acceptor fields. These models had a good predictive quality according to both internal and external validation criteria. On the other hand, GALAHAD was used for deriving a 3D pharmacophore model. Twelve active compounds were used for deriving this model. The obtained model included hydrophobe, hydrogen bond acceptor and hydrogen bond donor features; it was able to identify the active AR inhibitors from the remaining compounds. These in silico tools might be useful in the rational design of new AR inhibitors. PMID:20863730

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

    PubMed Central

    Chandra De, Utpal; Debnath, Tanusree; Sen, Debanjan; Debnath, Sudhan

    2015-01-01

    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 pIC50 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, R2 = 0.9502 and variance ratio of regression 122 has been found as the best statistical model. PMID:25709964

  11. SIRT6 Is a Positive Regulator of Aldose Reductase Expression in U937 and HeLa cells under Osmotic Stress: In Vitro and In Silico Insights

    PubMed Central

    Timucin, Ahmet Can; Basaga, Huveyda

    2016-01-01

    SIRT6 is a protein deacetylase, involved in various intracellular processes including suppression of glycolysis and DNA repair. Aldose Reductase (AR), first enzyme of polyol pathway, was proposed to be indirectly associated to these SIRT6 linked processes. Despite these associations, presence of SIRT6 based regulation of AR still remains ambiguous. Thus, regulation of AR expression by SIRT6 was investigated under hyperosmotic stress. A unique model of osmotic stress in U937 cells was used to demonstrate the presence of a potential link between SIRT6 and AR expression. By overexpressing SIRT6 in HeLa cells under hyperosmotic stress, its role on upregulation of AR was revealed. In parallel, increased SIRT6 activity was shown to upregulate AR in U937 cells under hyperosmotic milieu by using pharmacological modulators. Since these modulators also target SIRT1, binding of the inhibitor, Ex-527, specifically to SIRT6 was analyzed in silico. Computational observations indicated that Ex-527 may also target SIRT6 active site residues under high salt concentration, thus, validating in vitro findings. Based on these evidences, a novel regulatory step by SIRT6, modifying AR expression under hyperosmotic stress was presented and its possible interactions with intracellular machinery was discussed. PMID:27536992

  12. Tonicity-responsive enhancer binding protein regulates the expression of aldose reductase and protein kinase C δ in a mouse model of diabetic retinopathy.

    PubMed

    Park, Jeongsook; Kim, Hwajin; Park, So Yun; Lim, Sun Woo; Kim, Yoon Sook; Lee, Dong Hoon; Roh, Gu Seob; Kim, Hyun Joon; Kang, Sang Soo; Cho, Gyeong Jae; Jeong, Bo-Young; Kwon, H Moo; Choi, Wan Sung

    2014-05-01

    Recent studies revealed that Tonicity-responsive enhancer binding protein (TonEBP) directly regulates the transcription of aldose reductase (AR), which catalyzes the first step of the polyol pathway of glucose metabolism. Activation of protein kinase C δ (PKCδ) is dependent on AR and it has been linked to diabetic complications. However, whether TonEBP affects expressions of AR and PKCδ in diabetic retinopathy was not clearly shown. In this study, we used TonEBP heterozygote mice to study the role of TonEBP in streptozotocin (STZ)-induced diabetic retinopathy. We performed immunofluorescence staining and found that retinal expressions of AR and PKCδ were significantly reduced in the heterozygotes compared to wild type littermates, particularly in ganglion cell layer. To examine further the effect of TonEBP reduction in retinal tissues, we performed intravitreal injection of TonEBP siRNA and confirmed the decrease in AR and PKCδ levels. In addition, we found that a proapoptotic factor, Bax level was reduced and a survival factor, Bcl2 level was increased after injection of TonEBP siRNA, indicating that TonEBP mediates apoptotic cell death. In parallel, TonEBP siRNA was applied to the in vitro human retinal pigment epithelial (ARPE-19) cells cultured in high glucose media. We have consistently found the decrease in AR and PKCδ levels and changes in apoptotic factors for survival. Together, these results clearly demonstrated that hyperglycemia-induced TonEBP plays a crucial role in increasing AR and PKCδ levels and leading to apoptotic death. Our findings suggest that TonEBP reduction is an effective therapeutic strategy for diabetic retinopathy. PMID:24631337

  13. Kinetic and molecular docking studies of loganin and 7-O-galloyl-D-sedoheptulose from Corni Fructus as therapeutic agents for diabetic complications through inhibition of aldose reductase.

    PubMed

    Lee, Chan Mee; Jung, Hyun Ah; Oh, Sang Ho; Park, Chan Hum; Tanaka, Takashi; Yokozawa, Takako; Choi, Jae Sue

    2015-06-01

    Aldose reductase (AR) is a key enzyme in the polyol pathway that is strongly implicated in the pathogenesis of diabetic complications. AR inhibitors have been proposed as therapeutic agents for diabetic complications through suppression of sorbitol formation and accumulation. In this study, we evaluated whether two major compounds of Corni Fructus, loganin and 7-O-galloyl-D-sedoheptulose, had an inhibitory effect on diabetic complications through AR inhibition. Because the iridoid glycoside loganin and the low-molecular-weight polyphenol 7-O-galloyl-D-sedoheptulose showed marginal inhibitory activities against rat lens AR (RLAR) and human recombinant AR (HRAR) in inhibition assays, we performed enzyme kinetic analyses and molecular simulation of the interaction of these two compounds with AR to further investigate their potential as inhibitors of diabetic complications. In kinetic analysis using Lineweaver-Burk plots and Dixon plots, loganin and 7-O-galloyl-D-sedoheptulose were both mixed inhibitors of RLAR with inhibition constants (K i) of 27.99 and 128.68 μΜ, respectively. Moreover, molecular docking simulation of both compounds demonstrated negative binding energies (Autodock 4.0 = -6.7; -7.5 kcal/mol; Fred 2.0 = -59.4; -63.2 kcal/mol) indicating a high affinity and tight binding capacity for the active site of the enzyme. Iridoid nucleus and aromatic ring systems and glycoside and sedoheptulose moieties were found to bind tightly to the specificity pocket and the anion binding pocket in RLAR through Phe123, His111, Trp21, Tyr49, His111, and Trp112 residues. Our results clearly indicate that loganin and 7-O-galloyl-D-sedoheptulose have great promise for the treatment of diabetic complications through inhibition of AR. PMID:25315636

  14. Aldose Reductase Regulates Microglia/Macrophages Polarization Through the cAMP Response Element-Binding Protein After Spinal Cord Injury in Mice.

    PubMed

    Zhang, Qian; Bian, Ganlan; Chen, Peng; Liu, Ling; Yu, Caiyong; Liu, Fangfang; Xue, Qian; Chung, Sookja K; Song, Bing; Ju, Gong; Wang, Jian

    2016-01-01

    Inflammatory reactions are the most critical pathological processes occurring after spinal cord injury (SCI). Activated microglia/macrophages have either detrimental or beneficial effects on neural regeneration based on their functional polarized M1/M2 subsets. However, the mechanism of microglia/macrophage polarization to M1/M2 at the injured spinal cord environment remains unknown. In this study, wild-type (WT) or aldose reductase (AR)-knockout (KO) mice were subjected to SCI by a spinal crush injury model. The expression pattern of AR, behavior tests for locomotor activity, and lesion size were assessed at between 4 h and 28 days after SCI. We found that the expression of AR is upregulated in microglia/macrophages after SCI in WT mice. In AR KO mice, SCI led to smaller injury lesion areas compared to WT. AR deficiency-induced microglia/macrophages induce the M2 rather than the M1 response and promote locomotion recovery after SCI in mice. In the in vitro experiments, microglia cell lines (N9 or BV2) were treated with the AR inhibitor (ARI) fidarestat. AR inhibition caused 4-hydroxynonenal (HNE) accumulation, which induced the phosphorylation of the cAMP response element-binding protein (CREB) to promote Arg1 expression. KG501, the specific inhibitor of phosphorylated CREB, could cancel the upregulation of Arg1 by ARI or HNE stimulation. Our results suggest that AR works as a switch which can regulate microglia by polarizing cells to either the M1 or the M2 phenotype under M1 stimulation based on its states of activity. We suggest that inhibiting AR may be a promising therapeutic method for SCI in the future. PMID:25520004

  15. Rabbit corneal and conjunctival permeability of the novel aldose reductase inhibitors: N-[[4-(benzoylamino)phenyl] sulphonyl]glycines and N-benzoyl-N-phenylglycines.

    PubMed

    Kompella, U B; Sunkara, G; Thomas, E; Clark, C R; Deruiter, J

    1999-08-01

    Corneal and conjunctival permeability has been investigated for novel aldose reductase inhibitors (ARIs) of the N{[4-(benzoylamino)phenyl]sulphonyl}glycine (benzoylaminophenylsulphonylglycine) and N-benzoyl-N-phenylglycine (benzoylphenylglycine) series, compounds developed for prevention of cataract formation in diabetic subjects. Six benzoylaminophenylsulphonylglycines were synthesized with modifications either of the phenyl group or of the glycine structure and three benzoylphenylglycines were synthesized with modification in the phenyl group of the benzoyl moiety. Transport of ARIs in the mucosal to serosal direction was evaluated across rabbit cornea and conjunctiva bathed in glutathione-bicarbonate Ringer's solution maintained at pH 7.4 and 37 degrees C. The permeability coefficients of the novel ARIs across cornea and conjunctiva ranged from 1.87 to 8.95 x 10(-6) cm s(-1) and from 4.6 to 19.15 x 10(-6) cm s(-1), respectively. The ratio of corneal to conjunctival permeability ranged from 0.12 to 0.79. The calculated log partition coefficient (log P) values for the ARIs were in the range 0.84 to 2.78. The log distribution coefficients (log D) were in the range -2.87 to -0.89. There was no apparent relationship between log P or log D and the permeability coefficients of the ARIs for either tissue. Cornea was more resistant to ARI transport than was conjunctiva. Substitution of a phenyl group for hydrogen in the glycine methylene group reduced the permeability coefficient. Permeability coefficients were different for different stereoisomers. Compared with the permeability coefficient of benzoylaminophenylsulphonylglycine, that of 4-fluorobenzoylaminophenylsulphonylglycine was lower in the cornea but similar in the conjunctiva. In both tissues, the permeability coefficient of 2-nitrobenzoylaminophenylsulphonylglycine was less than that of 4-nitrobenzoylaminophenylsulphonylglycine. There was no significant difference between the permeability coefficients of 3-nitro

  16. Gedunin abrogates aldose reductase, PI3K/Akt/mToR, and NF-κB signaling pathways to inhibit angiogenesis in a hamster model of oral carcinogenesis.

    PubMed

    Kishore T, Kranthi Kiran; Ganugula, Raghu; Gade, Deepak Reddy; Reddy, Geereddy Bhanuprakash; Nagini, Siddavaram

    2016-02-01

    Aberrant activation of oncogenic signaling pathways plays a central role in tumor development and progression. The aim of this present study was to investigate the chemopreventive effects of the neem limonoid gedunin in the hamster model of oral cancer based on its ability to modulate aldose reductase (AR), phosphatidyl inositol-3-kinase (PI3K)/Akt, and nuclear factor kappa B (NF-κB) pathways to block angiogenesis. Administration of gedunin suppressed the development of HBP carcinomas by inhibiting PI3K/Akt and NF-κB pathways through the inactivation of Akt and inhibitory kappa B kinase (IKK), respectively. Immunoblot and molecular docking interactions revealed that inhibition of these signaling pathways may be mediated via inactivation of AR by gedunin. Gedunin blocked angiogenesis by downregulating the expression of miR-21 and the pro-angiogenic factors vascular endothelial growth factor and hypoxia inducible factor-1 alpha (HIF-1α). In conclusion, the results of the present study provide compelling evidence that gedunin prevents progression of hamster buccal pouch (HBP) carcinomas via inhibition of the kinases Akt, IKK, and AR, and the oncogenic transcription factors NF-κB and HIF-1α to block angiogenesis. PMID:26342697

  17. Inhibition of aldose reductase prevents growth factor-induced G1-S phase transition through the AKT/phosphoinositide 3-kinase/E2F-1 pathway in human colon cancer cells.

    PubMed

    Ramana, Kota V; Tammali, Ravinder; Srivastava, Satish K

    2010-04-01

    Colon cancer is the leading cause of cancer death in both men and women worldwide. The deregulated cell cycle control or decreased apoptosis of normal epithelial cells leading to uncontrolled proliferation is one of the major features of tumor progression. We have previously shown that aldose reductase (AR), a NADPH-dependent aldo-keto reductase, has been shown to be involved in growth factor-induced proliferation of colon cancer cells. Herein, we report that inhibition of AR prevents epidermal growth factor (EGF)- and basic fibroblast growth factor (bFGF)-induced HT29 cell proliferation by accumulating cells at G(1) phase of cell cycle. Similar results were observed in SW480 and HCT-116 colon cancer cells. Treatment of HT29 cells with AR inhibitor, sorbinil or zopolrestat, prevented the EGF- and bFGF-induced DNA binding activity of E2F-1 and phosphorylation of retinoblastoma protein. Inhibition of AR also prevented EGF- and bFGF-induced phosphorylation of cyclin-dependent kinase (cdk)-2 and expression of G(1)-S transition regulatory proteins such as cyclin D1, cdk4, proliferating cell nuclear antigen, cyclin E, and c-myc. More importantly, inhibition of AR prevented the EGF- and bFGF-induced activation of phosphoinositide 3-kinase/AKT and reactive oxygen species generation in colon cancer cells. Further, inhibition of AR also prevented the tumor growth of human colon cancer cells in nude mouse xenografts. Collectively, these results show that AR mediates EGF- and bFGF-induced colon cancer cell proliferation by activating or expressing G(1)-S phase proteins such as E2F-1, cdks, and cyclins through the reactive oxygen species/phosphoinositide 3-kinase/AKT pathway, indicating the use of AR inhibitors in the prevention of colon carcinogenesis. Mol Cancer Ther; 9(4); 813-24. (c)2010 AACR. PMID:20354121

  18. Multiple aldehyde reductases of human brain.

    PubMed

    Hoffman, P L; Wermuth, B; von Wartburg, J P

    1980-01-01

    Human brain contains four forms of aldehyde reducing enzymes. One major activity, designated AR3, has properties indicating its identity with the NADPH-dependent aldehyde reductase, EC 1.1.1.2. The other major form of human brain enzyme, AR1, which is also NADPH-dependent, reduces both aldehyde and ketone-containing substrates, including vitamin K3 (menadione) and daunorubicin, a cancer chemotherapeutic agent. This enzyme is very sensitive to inhibition by the flavonoids quercitrin and quercetine, and may be analogous to a daunorubicin reductase previously described in liver of other species. One minor form of human brain aldehyde reductase, AR2, demonstrates substrate specificity and inhibitor sensitivity which suggest its similarity to aldose reductases found in lens and other tissues of many species. This enzyme, which can also use NADH as cofactor to some extent, is the most active in reducing the aldehyde derivatives of the biogenic amines. The fourth human brain enzyme ("SSA reductase") differs from the other forms in its ability to use NADH as well as or better than NADPH as cofactor, and in its molecular weight, which is nearly twice that of the other forms. It is quite specific for succinic semialdehyde (SSA) as substrate, and was found to be significantly inhibited only by quercetine and quercitrin. AR3 can also reduce SSA, and both enzymes may contribute to the production of gamma-hydroxybutyric acid in vivo. These results indicate that the human brain aldehyde reductases can play relatively specific physiologic roles. PMID:7424738

  19. Characterization of a unique Caulobacter crescentus aldose-aldose oxidoreductase having dual activities.

    PubMed

    Andberg, Martina; Maaheimo, Hannu; Kumpula, Esa-Pekka; Boer, Harry; Toivari, Mervi; Penttilä, Merja; Koivula, Anu

    2016-01-01

    We describe here the characterization of a novel enzyme called aldose-aldose oxidoreductase (Cc AAOR; EC 1.1.99) from Caulobacter crescentus. The Cc AAOR exists in solution as a dimer, belongs to the Gfo/Idh/MocA family and shows homology with the glucose-fructose oxidoreductase from Zymomonas mobilis. However, unlike other known members of this protein family, Cc AAOR is specific for aldose sugars and can be in the same catalytic cycle both oxidise and reduce a panel of monosaccharides at the C1 position, producing in each case the corresponding aldonolactone and alditol, respectively. Cc AAOR contains a tightly-bound nicotinamide cofactor, which is regenerated in this oxidation-reduction cycle. The highest oxidation activity was detected on D-glucose but significant activity was also observed on D-xylose, L-arabinose and D-galactose, revealing that both hexose and pentose sugars are accepted as substrates by Cc AAOR. The configuration at the C2 and C3 positions of the saccharides was shown to be especially important for the substrate binding. Interestingly, besides monosaccharides, Cc AAOR can also oxidise a range of 1,4-linked oligosaccharides having aldose unit at the reducing end, such as lactose, malto- and cello-oligosaccharides as well as xylotetraose. (1)H NMR used to monitor the oxidation and reduction reaction simultaneously, demonstrated that although D-glucose has the highest affinity and is also oxidised most efficiently by Cc AAOR, the reduction of D-glucose is clearly not as efficient. For the overall reaction catalysed by Cc AAOR, the L-arabinose, D-xylose and D-galactose were the most potent substrates. PMID:26428243

  20. Structure and function of Caulobacter crescentus aldose-aldose oxidoreductase.

    PubMed

    Taberman, Helena; Andberg, Martina; Koivula, Anu; Hakulinen, Nina; Penttilä, Merja; Rouvinen, Juha; Parkkinen, Tarja

    2015-12-15

    Aldose-aldose oxidoreductase (Cc AAOR) is a recently characterized enzyme from the bacterial strain Caulobacter crescentus CB15 belonging to the glucose-fructose oxidoreductase/inositol dehydrogenase/rhizopine catabolism protein (Gfo/Idh/MocA) family. Cc AAOR catalyses the oxidation and reduction of a panel of aldose monosaccharides using a tightly bound NADP(H) cofactor that is regenerated in the catalytic cycle. Furthermore, Cc AAOR can also oxidize 1,4-linked oligosaccharides. In the present study, we present novel crystal structures of the dimeric Cc AAOR in complex with the cofactor and glycerol, D-xylose, D-glucose, maltotriose and D-sorbitol determined to resolutions of 2.0, 1.8, 1.7, 1.9 and 1.8 Å (1 Å=0.1 nm), respectively. These complex structures allowed for a detailed analysis of the ligand-binding interactions. The structures showed that the C1 carbon of a substrate, which is either reduced or oxidized, is close to the reactive C4 carbon of the nicotinamide ring of NADP(H). In addition, the O1 hydroxy group of the substrate, which is either protonated or deprotonated, is unexpectedly close to both Lys(104) and Tyr(189), which may both act as a proton donor or acceptor. This led us to hypothesize that this intriguing feature could be beneficial for Cc AAOR to catalyse the reduction of a linear form of a monosaccharide substrate and the oxidation of a pyranose form of the same substrate in a reaction cycle, during which the bound cofactor is regenerated. PMID:26438878

  1. Purification and characterization of glutathione reductase (E.C. 1.8.1.7) from bovine filarial worms Setaria cervi.

    PubMed

    Arora, Kavita; Ahmad, Rumana; Srivastava, Arvind K

    2013-04-01

    Antioxidant enzymes are the parasite's premier resource to defend themselves against reactive oxygen species generated by macrophages, neutrophils and eosinophils of the host. These enzymes may be particularly important for parasites involved in chronic infections, such as parasitic helminths. Glutathione (GSH) and glutathione reductase (GR) are parts of the GSH redox cycle, which protects cells against damage by oxidants. Both GSH and GR are present in significant amounts in Setaria cervi female worms. GR has a central role in glutathione metabolism and as such is a potential target for chemotherapy. The aim of the work was to purify and characterize GR from S. cervi and to compare the properties of the helminth enzyme with its mammalian counterpart. GR was purified from filarial parasites S. cervi and preliminary steady state kinetics was performed. The purified protein was observed to be a dimer of 55 kDa subunit as evident from SDS-PAGE analysis. Kinetic studies revealed significant differences in the properties of S. cervi GR from its mammalian counterpart which may be exploited in chemotherapy of filariasis. Filarial GR is thus proposed as a potential drug target. PMID:24431548

  2. Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae.

    PubMed Central

    Kuhn, A; van Zyl, C; van Tonder, A; Prior, B A

    1995-01-01

    A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (Km = 46 microM; kcat/Km = 52,100 s-1 M-1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (Km = 1.44 mM; kcat/Km = 1,790 s-1 M-1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5'-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases. PMID:7747971

  3. A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-L-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-D-gluconate.

    PubMed

    Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

    2015-12-25

    Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼ 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. PMID:26555267

  4. Functional studies of aldo-keto reductases in Saccharomyces cerevisiae*

    PubMed Central

    Chang, Qing; Griest, Terry A.; Harter, Theresa M.; Petrash, J. Mark

    2007-01-01

    SUMMARY We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast. PMID:17140678

  5. Extreme ultraviolet photoionization of aldoses and ketoses

    NASA Astrophysics Data System (ADS)

    Shin, Joong-Won; Dong, Feng; Grisham, Michael E.; Rocca, Jorge J.; Bernstein, Elliot R.

    2011-04-01

    Gas phase monosaccharides (2-deoxyribose, ribose, arabinose, xylose, lyxose, glucose galactose, fructose, and tagatose), generated by laser desorption of solid sample pellets, are ionized with extreme ultraviolet photons (EUV, 46.9 nm, 26.44 eV). The resulting fragment ions are analyzed using a time of flight mass spectrometer. All aldoses yield identical fragment ions regardless of size, and ketoses, while also generating same ions as aldoses, yields additional features. Extensive fragmentation of the monosaccharides is the result the EUV photons ionizing various inner valence orbitals. The observed fragmentation patterns are not dependent upon hydrogen bonding structure or OH group orientation.

  6. Molecular cloning of mannose-6-phosphate reductase and its developmental expression in celery.

    PubMed Central

    Everard, J D; Cantini, C; Grumet, R; Plummer, J; Loescher, W H

    1997-01-01

    Compared with other primary photosynthetic products (e.g. sucrose and starch), little is known about sugar alcohol metabolism, its regulation, and the manner in which it is integrated with other pathways. Mannose-6-phosphate reductase (M6PR) is a key enzyme that is involved in mannitol biosynthesis in celery (Apium graveolens L.). The M6PR gene was cloned from a leaf cDNA library, and clonal authenticity was established by assays of M6PR activity, western blots, and comparisons of the deduced amino acid sequence with a celery M6PR tryptic digestion product. Recombinant M6PR, purified from Escherichia coli, had specific activity, molecular mass, and kinetic characteristics indistinguishable from those of authentic celery M6PR. Sequence analyses showed M6PR to be a member of the aldo-keto reductase superfamily, which includes both animal and plant enzymes. The greatest sequence similarity was with aldose-6-phosphate reductase (EC 1.1.1.200), a key enzyme in sorbitol synthesis in Rosaceae. Developmental studies showed M6PR to be limited to green tissues and to be under tight transcriptional regulation during leaf initiation, expansion, and maturation. These data confirmed a close relationship between the development of photosynthetic capacity, mannitol synthesis, and M6PR activity. PMID:9112783

  7. High-level expression of human dihydropteridine reductase (EC 1.6.99.7), without N-terminal amino acid protection, in Escherichia coli.

    PubMed Central

    Armarego, W L; Cotton, R G; Dahl, H H; Dixon, N E

    1989-01-01

    The cDNA coding for human dihydropteridine reductase [Dahl, Hutchinson, McAdam, Wake, Morgan & Cotton (1987) Nucleic Acids Res. 15, 1921-1936] was inserted downstream of tandem bacteriophage lambda PR and PL promoters in Escherichia coli vector pCE30. Since pCE30 also expresses the lambda c1857ts gene, transcription may be controlled by variation of temperature. The recombinant plasmid in an E. coli K12 strain grown at 30 degrees C, then at 45 degrees C, directed the synthesis of dihydropteridine reductase to very high levels. The protein was soluble, at least as active as the authentic human enzyme, and lacked the N-terminal amino acid protection. Images Fig. 1. Fig. 2. PMID:2673215

  8. Catalytic anomeric aminoalkynylation of unprotected aldoses.

    PubMed

    Kimura, Yasuaki; Ito, Soichi; Shimizu, Yohei; Kanai, Motomu

    2013-08-16

    A copper(I)-catalyzed anomeric aminoalkynylation reaction of unprotected aldoses was realized. Use of an electron-deficient phosphine ligand, boric acid to stabilize the iminium intermediate, and a protic additive (IPA) to presumably enhance reversible carbohydrate-boron complexation were all essential for efficient conversion. The reaction proceeded well even with a natural disaccharide substrate, suggesting that the developed catalytic reaction could be useful for the synthesis of glycoconjugates with minimum use of protecting groups. PMID:23901780

  9. Sulphur flux through the sulphate assimilation pathway is differently controlled by adenosine 5′-phosphosulphate reductase under stress and in transgenic poplar plants overexpressing γ-ECS, SO, or APR

    PubMed Central

    Scheerer, Ursula; Haensch, Robert; Mendel, Ralf R.; Kopriva, Stanislav; Rennenberg, Heinz; Herschbach, Cornelia

    2010-01-01

    Sulphate assimilation provides reduced sulphur for the synthesis of cysteine, methionine, and numerous other essential metabolites and secondary compounds. The key step in the pathway is the reduction of activated sulphate, adenosine 5′-phosphosulphate (APS), to sulphite catalysed by APS reductase (APR). In the present study, [35S]sulphur flux from external sulphate into glutathione (GSH) and proteins was analysed to check whether APR controls the flux through the sulphate assimilation pathway in poplar roots under some stress conditions and in transgenic poplars. (i) O-Acetylserine (OAS) induced APR activity and the sulphur flux into GSH. (ii) The herbicide Acetochlor induced APR activity and results in a decline of GSH. Thereby the sulphur flux into GSH or protein remained unaffected. (iii) Cd treatment increased APR activity without any changes in sulphur flux but lowered sulphate uptake. Several transgenic poplar plants that were manipulated in sulphur metabolism were also analysed. (i) Transgenic poplar plants that overexpressed the γ-glutamylcysteine synthetase (γ-ECS) gene, the enzyme catalysing the key step in GSH formation, showed an increase in sulphur flux into GSH and sulphate uptake when γ-ECS was targeted to the cytosol, while no changes in sulphur flux were observed when γ-ECS was targeted to plastids. (ii) No effect on sulphur flux was observed when the sulphite oxidase (SO) gene from Arabidopsis thaliana, which catalyses the back reaction of APR, that is the reaction from sulphite to sulphate, was overexpressed. (iii) When Lemna minor APR was overexpressed in poplar, APR activity increased as expected, but no changes in sulphur flux were observed. For all of these experiments the flux control coefficient for APR was calculated. APR as a controlling step in sulphate assimilation seems obvious under OAS treatment, in γ-ECS and SO overexpressing poplars. A possible loss of control under certain conditions, that is Cd treatment, Acetochlor

  10. Sulphur flux through the sulphate assimilation pathway is differently controlled by adenosine 5'-phosphosulphate reductase under stress and in transgenic poplar plants overexpressing gamma-ECS, SO, or APR.

    PubMed

    Scheerer, Ursula; Haensch, Robert; Mendel, Ralf R; Kopriva, Stanislav; Rennenberg, Heinz; Herschbach, Cornelia

    2010-01-01

    Sulphate assimilation provides reduced sulphur for the synthesis of cysteine, methionine, and numerous other essential metabolites and secondary compounds. The key step in the pathway is the reduction of activated sulphate, adenosine 5'-phosphosulphate (APS), to sulphite catalysed by APS reductase (APR). In the present study, [(35)S]sulphur flux from external sulphate into glutathione (GSH) and proteins was analysed to check whether APR controls the flux through the sulphate assimilation pathway in poplar roots under some stress conditions and in transgenic poplars. (i) O-Acetylserine (OAS) induced APR activity and the sulphur flux into GSH. (ii) The herbicide Acetochlor induced APR activity and results in a decline of GSH. Thereby the sulphur flux into GSH or protein remained unaffected. (iii) Cd treatment increased APR activity without any changes in sulphur flux but lowered sulphate uptake. Several transgenic poplar plants that were manipulated in sulphur metabolism were also analysed. (i) Transgenic poplar plants that overexpressed the gamma-glutamylcysteine synthetase (gamma-ECS) gene, the enzyme catalysing the key step in GSH formation, showed an increase in sulphur flux into GSH and sulphate uptake when gamma-ECS was targeted to the cytosol, while no changes in sulphur flux were observed when gamma-ECS was targeted to plastids. (ii) No effect on sulphur flux was observed when the sulphite oxidase (SO) gene from Arabidopsis thaliana, which catalyses the back reaction of APR, that is the reaction from sulphite to sulphate, was overexpressed. (iii) When Lemna minor APR was overexpressed in poplar, APR activity increased as expected, but no changes in sulphur flux were observed. For all of these experiments the flux control coefficient for APR was calculated. APR as a controlling step in sulphate assimilation seems obvious under OAS treatment, in gamma-ECS and SO overexpressing poplars. A possible loss of control under certain conditions, that is Cd treatment

  11. Medicinal flowers. XXXX . Structures of dihydroisocoumarin glycosides and inhibitory effects on aldose reducatase from the flowers of Hydrangea macrophylla var.thunbergii.

    PubMed

    Liu, Jiang; Nakamura, Seikou; Zhuang, Yan; Yoshikawa, Masayuki; Hussein, Ghazi Mohamed Eisa; Matsuo, Kyohei; Matsuda, Hisashi

    2013-01-01

    Six dihydroisocoumarin glycosides, florahydrosides I and II, thunberginol G 8-O-β-d-glucopyranoside, thunberginol C 8-O-β-d-glucopyranoside, 4-hydroxythunberginol G 3'-O-β-d-glucopyranoside, and thunberginol D 3'-O-β-d-glucopyranoside, have been isolated from the flowers of Hydrangea macrophylla Seringe var. thunbergii Makino (Saxifragaceae) together with 20 known compounds. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. Among the constituents, acylated quinic acid analog, neochlorogenic acid, was shown to substantially inhibit aldose reductase [IC50=5.6 µm]. In addition, the inhibitory effects on aldose reductase of several caffeoylquinic acid analogs were examined for structure-activity relationship study. As the results, 4,5-O-trans-p-dicaffeoyl-d-quinic acid was found to exhibit a potent inhibitory effect [IC50=0.29 µm]. PMID:23727779

  12. Catalytic Isomerization of Biomass-Derived Aldoses: A Review.

    PubMed

    Delidovich, Irina; Palkovits, Regina

    2016-03-21

    Selected aldohexoses (d-glucose, d-mannose, and d-galactose) and aldopentoses (d-xylose, l-arabinose, and d-ribose) are readily available components of biopolymers. Isomerization reactions of these substances are very attractive as carbon-efficient processes to broaden the portfolio of abundant monosaccharides. This review focuses on the chemocatalytic isomerization of aldoses into the corresponding ketoses as well as epimerization of aldoses at C2. Recent advances in the fields of catalysis by bases and Lewis acids are considered. The emphasis is laid on newly uncovered catalytic systems and mechanisms of carbohydrate transformations. PMID:26948404

  13. Dehydration of different ketoses and aldoses to 5-hydroxymethylfurfural.

    PubMed

    van Putten, Robert-Jan; Soetedjo, Jenny N M; Pidko, Evgeny A; van der Waal, Jan C; Hensen, Emiel J M; de Jong, Ed; Heeres, Hero J

    2013-09-01

    5-Hydroxymethylfurfural (HMF) is considered an important building block for future bio-based chemicals. Here, we present an experimental study using different ketoses (fructose, sorbose, tagatose) and aldoses (glucose, mannose, galactose) under aqueous acidic conditions (65 g L(-1) substrate, 100-160 °C, 33-300 mM H2 SO4 ) to gain insights into reaction pathways for hexose dehydration to HMF. Both reaction rates and HMF selectivities were significantly higher for ketoses than for aldoses, which is in line with literature. Screening and kinetic experiments showed that the reactivity of the different ketoses is a function of the hydroxyl group orientation at the C3 and C4 positions. These results, in combination with DFT calculations, point to a dehydration mechanism involving cyclic intermediates. For aldoses, no influence of the hydroxyl group orientation was observed, indicating a different rate-determining step. The combination of the knowledge from the literature and the findings in this work indicates that aldoses require an isomerization to ketose prior to dehydration to obtain high HMF yields. PMID:24039165

  14. A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.

    PubMed

    Wiebe, Marilyn G; Nygård, Yvonne; Oja, Merja; Andberg, Martina; Ruohonen, Laura; Koivula, Anu; Penttilä, Merja; Toivari, Mervi

    2015-11-01

    An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis (Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor) showed some Zm GFOR type of activity, producing D-gluconate and D-sorbitol when a mixture of D-glucose and D-fructose was used as substrate. However, the activity in Cc aaor expressing strain was >100-fold lower compared to strains expressing Zm gfor. Interestingly, C. crescentus AAOR was clearly more efficient than the Zm GFOR in converting in vitro a single sugar substrate D-xylose (10 mM) to xylitol without an added cofactor, whereas this type of activity was very low with Zm GFOR. Furthermore, when cultured in the presence of D-xylose, the S. cerevisiae strain expressing Cc aaor produced nearly equal concentrations of D-xylonate and xylitol (12.5 g D-xylonate l(-1) and 11.5 g D-xylitol l(-1) from 26 g D-xylose l(-1)), whereas the control strain and strain expressing Zm gfor produced only D-xylitol (5 g l(-1)). Deletion of the gene encoding the major aldose reductase, Gre3p, did not affect xylitol production in the strain expressing Cc aaor, but decreased xylitol production in the strain expressing Zm gfor. In addition, expression of Cc aaor together with the D-xylonolactone lactonase encoding the gene xylC from C. crescentus slightly increased the final concentration and initial volumetric production rate of both D-xylonate and D-xylitol. These results suggest that C. crescentus AAOR is a novel type of oxidoreductase able to convert the single aldose substrate D-xylose to both its oxidized and reduced product. PMID:26264136

  15. Aldose-ketose interconversion in pyridine in the presence of aluminium oxide.

    PubMed

    Ekeberg, Dag; Morgenlie, Svein; Stenstrøm, Yngve

    2007-10-15

    The reaction rate of the Lobry de Bruyn-Alberda van Ekenstein transformation of aldoses to ketoses in boiling pyridine was strongly increased by the addition of aluminium oxide. In addition to aldose-ketose transformation, 2-epimers of the starting aldoses and 3-epimers of the primarily produced ketoses were formed to some extent, as reported also when these reactions are carried out without aluminium oxide. The relative amounts of the primary ketose and the starting aldose in the reaction mixtures may be explained on the basis of their stability, predicted from reported free energy calculations. Isomerisation of ketoses to aldoses was much slower than the reverse reaction. The relative free energies are also in these cases important, the very stable xylo-2-hexulose gave only 7% and 6% of the aldoses gulose and idose, respectively, after boiling for 7h in pyridine in the presence of aluminium oxide. PMID:17606255

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

    PubMed Central

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

    2016-01-01

    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions. PMID:27499746

  17. A kinetic estimate of the free aldehyde content of aldoses

    NASA Technical Reports Server (NTRS)

    Dworkin, J. P.; Miller, S. L.; Bada, J. L. (Principal Investigator)

    2000-01-01

    The relative free aldehyde content of eight hexoses and four pentoses has been estimated within about 10% from the rate constants for their reaction with urazole (1,2,4-triazole-3,5-dione). These values of the percent free aldehyde are in agreement with those estimated from CD measurements, but are more accurate. The relative free aldehyde contents for the aldoses were then correlated to various literature NMR measurements to obtain the absolute values. This procedure was also done for three deoxyaldoses, which react much more rapidly than can be accounted for by the free aldehyde content. This difference in reactivity between aldoses and deoxyaldoses is due to the inductive effect of the H versus the OH on C-2'. This may help explain why deoxyribonucleosides hydrolyze much more rapidly than ribonucleosides.

  18. Color polymorphs of aldose reductase inhibitor epalrestat: configurational, conformational and synthon differences.

    PubMed

    Swapna, Battini; Suresh, Kuthuru; Nangia, Ashwini

    2016-03-01

    We report five crystalline polymorphs and an amorphous phase of epalrestat together with configurational isomerism and color behavior: form I (deep red), form II (deep orange), form III (bright yellow), form IV (yellow), and form V (orange) are in the E,Z configuration of the drug, and a Z,Z isomer (bright yellow). Two pathways are identified for polymorph conversion: direct transformation of the E,Z isomer and another pathway via the Z,Z isomer to the E,Z polymorphs. From a pharmaceutical perspective, the stability of polymorphs was established under grinding, solvent slurry and thermal conditions: form I (thermodynamic) > form II > form V > form III > form IV (least stable). PMID:26889760

  19. Comparative anatomy of the aldo-keto reductase superfamily.

    PubMed Central

    Jez, J M; Bennett, M J; Schlegel, B P; Lewis, M; Penning, T M

    1997-01-01

    The aldo-keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (alpha/beta)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo-keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo-keto reductase active site is highly conserved. However, our alignments indicate that variation ofa single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon-carbon double-bond reduction, as in the 3-oxo-5beta-steroid 4-dehydrogenases (Delta4-3-ketosteroid 5beta-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17beta-hydroxysteroid dehydrogenase and rat ovary 20alpha-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo-keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short

  20. Thioredoxin reductase.

    PubMed

    Mustacich, D; Powis, G

    2000-02-15

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

  1. Aldo–Keto Reductase 1B10 and Its Role in Proliferation Capacity of Drug-Resistant Cancers

    PubMed Central

    Matsunaga, Toshiyuki; Wada, Yasuhiro; Endo, Satoshi; Soda, Midori; El-Kabbani, Ossama; Hara, Akira

    2011-01-01

    The human aldo–keto reductase AKR1B10, originally identified as an aldose reductase-like protein and human small intestine aldose reductase, is a cytosolic NADPH-dependent reductase that metabolizes a variety of endogenous compounds, such as aromatic and aliphatic aldehydes and dicarbonyl compounds, and some drug ketones. The enzyme is highly expressed in solid tumors of several tissues including lung and liver, and as such has received considerable interest as a relevant biomarker for the development of those tumors. In addition, AKR1B10 has been recently reported to be significantly up-regulated in some cancer cell lines (medulloblastoma D341 and colon cancer HT29) acquiring resistance toward chemotherapeutic agents (cyclophosphamide and mitomycin c), suggesting the validity of the enzyme as a chemoresistance marker. Although the detailed information on the AKR1B10-mediated mechanisms leading to the drug resistance process is not well understood so far, the enzyme has been proposed to be involved in functional regulations of cell proliferation and metabolism of drugs and endogenous lipids during the development of chemoresistance. This article reviews the current literature focusing mainly on expression profile and roles of AKR1B10 in the drug resistance of cancer cells. Recent developments of AKR1B10 inhibitors and their usefulness in restoring sensitivity to anticancer drugs are also reviewed. PMID:22319498

  2. Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

    PubMed Central

    Kwak, Young Hak; Lee, Dong Seok; Kim, Han Bok

    2003-01-01

    The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5α and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione S-transferase (GST) and E. coli DH5α NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5α NfsB (GST-EcNfsB), and a fusion between GST and V. harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification. GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates. The Km values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 μM, respectively. The Vmax values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively. GST-VhNfsA was the most effective of the three chromate reductases, as determined by each Vmax/Km value. The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30°C, respectively. Thus, it is confirmed that nitroreductase can also act as a chromate reductase. Nitroreductases may be used in chromate remediation. GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution. Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor. GST-VhNfsA reduced Cr(VI) to Cr(III). Cr(III) was much less toxic to E. coli than Cr(VI). PMID:12902220

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

    PubMed Central

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

    1996-01-01

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

  4. The C-terminal loop of aldehyde reductase determines the substrate and inhibitor specificity.

    PubMed

    Barski, O A; Gabbay, K H; Bohren, K M

    1996-11-12

    Human aldehyde reductase has a preference for carboxyl group-containing negatively charged substrates. It belongs to the NADPH-dependent aldo-keto reductase superfamily whose members are in part distinguished by unique C-terminal loops. To probe the role of the C-terminal loops in determining substrate specificities in these enzymes, two arginine residues, Arg308 and Arg311, located in the C-terminal loop of aldehyde reductase, and not found in any other C-terminal loop, were replaced with alanine residues. The catalytic efficiency of the R311A mutant for aldehydes containing a carboxyl group is reduced 150-250-fold in comparison to that of the wild-type enzyme, while substrates not containing a negative charge are unaffected. The R311A mutant is also significantly less sensitive to inhibition by dicarboxylic acids, indicating that Arg311 interacts with one of the carboxyl groups. The inhibition pattern indicates that the other carboxyl group binds to the anion binding site formed by Tyr49, His112, and the nicotinamide moiety of NADP+. The correlation between inhibitor potency and the length of the dicarboxylic acid molecules suggests a distance of approximately 10 A between the amino group of Arg311 and the anion binding site in the aldehyde reductase molecule. The sensitivity of inhibition of the R311A mutant by several commercially available aldose reductase inhibitors (ARIs) was variable, with tolrestat and zopolrestat becoming more potent inhibitors (30- and 5-fold, respectively), while others remained the same or became less potent. The catalytic properties, substrate specificity, and susceptibility to inhibition of the R308A mutant remained similar to that of the wild-type enzyme. The data provide direct evidence for C-terminal loop participation in determining substrate and inhibitor specificity of aldo-keto reductases and specifically identifies Arg311 as the basis for the carboxyl-containing substrate preference of aldehyde reductase. PMID:8916913

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

    NASA Technical Reports Server (NTRS)

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

    1987-01-01

    Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system--NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.

  6. Quinone Reductase 2 Is a Catechol Quinone Reductase

    SciTech Connect

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2008-09-05

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

  7. ECS communications success

    NASA Astrophysics Data System (ADS)

    Dinwiddy, S. E.

    1985-09-01

    The European Communications Satellite (ECS) which supplies satellite links for national telecommunication, long-distance international telephone traffic, and the distribution of television programs is described. The ECS concept was tested by the Orbital Test Satellite and proved the applicability of the ECS for television transmission and high-speed data links provided by small earth stations. The industrial development, operation, and cost of the project, which was shared by the European Space Agency members, are discussed. Extra repeater chains for small-dish services employed by ECS operate in the 14.0-14.25 GHz uplink and 12.5-12.75 GHz downlink frequency band and are utilized by small earth stations. The advantages and disadvantages of transmission services provided by the small earth stations are studied. The utilization of the point-to-multipoint service of the small earth station for the transmission of data is analyzed. The television distribution services available with the ECS system are examined; the ECS provides ten 20-W channels for a lifetime of seven years.

  8. An Expeditious Synthesis of Sialic Acid Derivatives by Copper(I)-Catalyzed Stereodivergent Propargylation of Unprotected Aldoses

    PubMed Central

    2016-01-01

    We developed a copper(I)-catalyzed stereodivergent anomeric propargylation of unprotected aldoses as a facile synthetic pathway to a broad variety of sialic acid derivatives. The soft allenylcopper(I) species, catalytically generated from stable allenylboronic acid pinacolate (2), is unusually inert to protonolysis by the multiple hydroxy groups of the substrates and thereby functions as a carbon nucleophile. The key additive B(OMe)3 facilitated ring-opening of the nonelectrophilic cyclic hemiacetal forms of aldoses to the reactive aldehyde forms. The chirality of the catalyst, and not the internal stereogenic centers of substrates, predominantly controlled the stereochemistry of the propargylation step; i.e., the diastereoselectivity was switched simply by changing the catalyst chirality. This is the first nonenzyme catalyst-controlled stereodivergent C–C bond elongation at the anomeric center of unprotected aldoses, which contain multiple protic functional groups and stereogenic centers. The propargylation products can be expeditiously transformed into naturally occurring and synthetic sialic acid derivatives in a simple three-step sequence. This synthetic method, which requires no protecting groups, can be performed on a gram-scale and thus offers general and practical access to various sialic acid derivatives from unprotected aldoses. PMID:27163022

  9. An Expeditious Synthesis of Sialic Acid Derivatives by Copper(I)-Catalyzed Stereodivergent Propargylation of Unprotected Aldoses.

    PubMed

    Wei, Xiao-Feng; Shimizu, Yohei; Kanai, Motomu

    2016-01-27

    We developed a copper(I)-catalyzed stereodivergent anomeric propargylation of unprotected aldoses as a facile synthetic pathway to a broad variety of sialic acid derivatives. The soft allenylcopper(I) species, catalytically generated from stable allenylboronic acid pinacolate (2), is unusually inert to protonolysis by the multiple hydroxy groups of the substrates and thereby functions as a carbon nucleophile. The key additive B(OMe)3 facilitated ring-opening of the nonelectrophilic cyclic hemiacetal forms of aldoses to the reactive aldehyde forms. The chirality of the catalyst, and not the internal stereogenic centers of substrates, predominantly controlled the stereochemistry of the propargylation step; i.e., the diastereoselectivity was switched simply by changing the catalyst chirality. This is the first nonenzyme catalyst-controlled stereodivergent C-C bond elongation at the anomeric center of unprotected aldoses, which contain multiple protic functional groups and stereogenic centers. The propargylation products can be expeditiously transformed into naturally occurring and synthetic sialic acid derivatives in a simple three-step sequence. This synthetic method, which requires no protecting groups, can be performed on a gram-scale and thus offers general and practical access to various sialic acid derivatives from unprotected aldoses. PMID:27163022

  10. Cotton Benzoquinone Reductase: Up-regulation During Early Cotton Fiber Developement

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme that catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2-D PAGE comparisons, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage but ...

  11. CONTROL OF NITRATE REDUCTASE BY CIRCADIAN AND DURRAL RHYTHM IN TOMATO

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrate reductase (NR) (E.C. 1.6.6.1) is a key, regulatory step in the assimilation of nitrate into amino acids in plant leaves. NR activity is intricately controlled by multifarious regulatory mechanisms acting at different levels ranging from transcription to protein degradation. It is one of a ...

  12. Asymmetric assembly of aldose carbohydrates from formaldehyde and glycolaldehyde by tandem biocatalytic aldol reactions.

    PubMed

    Szekrenyi, Anna; Garrabou, Xavier; Parella, Teodor; Joglar, Jesús; Bujons, Jordi; Clapés, Pere

    2015-09-01

    The preparation of multifunctional chiral molecules can be greatly simplified by adopting a route via the sequential catalytic assembly of achiral building blocks. The catalytic aldol assembly of prebiotic compounds into stereodefined pentoses and hexoses is an as yet unmet challenge. Such a process would be of remarkable synthetic utility and highly significant with regard to the origin of life. Pursuing an expedient enzymatic approach, here we use engineered D-fructose-6-phosphate aldolase from Escherichia coli to prepare a series of three- to six-carbon aldoses by sequential one-pot additions of glycolaldehyde. Notably, the pertinent selection of the aldolase variant provides control of the sugar size. The stereochemical outcome of the addition was also altered to allow the synthesis of L-glucose and related derivatives. Such engineered biocatalysts may offer new routes for the straightforward synthesis of natural molecules and their analogues that circumvent the intricate enzymatic pathways forged by evolution. PMID:26291944

  13. Asymmetric assembly of aldose carbohydrates from formaldehyde and glycolaldehyde by tandem biocatalytic aldol reactions

    NASA Astrophysics Data System (ADS)

    Szekrenyi, Anna; Garrabou, Xavier; Parella, Teodor; Joglar, Jesús; Bujons, Jordi; Clapés, Pere

    2015-09-01

    The preparation of multifunctional chiral molecules can be greatly simplified by adopting a route via the sequential catalytic assembly of achiral building blocks. The catalytic aldol assembly of prebiotic compounds into stereodefined pentoses and hexoses is an as yet unmet challenge. Such a process would be of remarkable synthetic utility and highly significant with regard to the origin of life. Pursuing an expedient enzymatic approach, here we use engineered D-fructose-6-phosphate aldolase from Escherichia coli to prepare a series of three- to six-carbon aldoses by sequential one-pot additions of glycolaldehyde. Notably, the pertinent selection of the aldolase variant provides control of the sugar size. The stereochemical outcome of the addition was also altered to allow the synthesis of L-glucose and related derivatives. Such engineered biocatalysts may offer new routes for the straightforward synthesis of natural molecules and their analogues that circumvent the intricate enzymatic pathways forged by evolution.

  14. Evolution Alters the Enzymatic Reaction Coordinate of Dihydrofolate Reductase

    PubMed Central

    2015-01-01

    How evolution has affected enzyme function is a topic of great interest in the field of biophysical chemistry. Evolutionary changes from Escherichia coli dihydrofolate reductase (ecDHFR) to human dihydrofolate reductase (hsDHFR) have resulted in increased catalytic efficiency and an altered dynamic landscape in the human enzyme. Here, we show that a subpicosecond protein motion is dynamically coupled to hydride transfer catalyzed by hsDHFR but not ecDHFR. This motion propagates through residues that correspond to mutational events along the evolutionary path from ecDHFR to hsDHFR. We observe an increase in the variability of the transition states, reactive conformations, and times of barrier crossing in the human system. In the hsDHFR active site, we detect structural changes that have enabled the coupling of fast protein dynamics to the reaction coordinate. These results indicate a shift in the DHFR family to a form of catalysis that incorporates rapid protein dynamics and a concomitant shift to a more flexible path through reactive phase space. PMID:25369552

  15. EC Tube Fits

    SciTech Connect

    Kurita, C.H.; /Fermilab

    1987-03-03

    In the design of the EC, the beam tube, through which the beam line travels, can be found in the IH tube which is centrally located in the IH module. However, also between the beam tube and the IH tube lie both the vacuum and inner tubes of the vacuum and inner vessels. It is the vacuum between these vessels which provides insulation between the ambient beam tube and liquid argon in the cryostat. while the vacuum tube is supported along its length with the inner tube as best as possible, the inner tube will only be supported at the ends. The beam tube will also be end-supported, but it will be allowed to rest directly on the inner surface of the vacuum tube. It is required that the beam tube be able to slide in and out of the vacuum tube with relative ease in order that the EC's can be moved away from the CC when necessary (repair work, etc.). Although the frequency of such a move is not known, it is hoped to be low, and it would therefore be desirable, for cost reasons, to be able to use stock tubing for the vacuum and beam tubes instead of using specially machined tubing.

  16. Ascorbate free radical reductase mRNA levels are induced by wounding.

    PubMed Central

    Grantz, A A; Brummell, D A; Bennett, A B

    1995-01-01

    A cDNA clone encoding ascorbate free radical (AFR) reductase (EC 1.6.5.4) was isolated from tomato (Lycopersicon esculentum Mill.) and its mRNA levels were analyzed. The cDNA encoded a deduced protein of 433 amino acids and possessed amino acid domains characteristic of flavin adenine dinucleotide- and NAD(P)H-binding proteins but did not possess typical eukaryotic targeting sequences, suggesting that it encodes a cytosolic form of AFR reductase. Low-stringency genomic DNA gel blot analysis indicated that a single nuclear gene encoded this enzyme. Total ascorbate contents were greatest in leaves, with decreasing amounts in stems and roots and relatively constant levels in all stages of fruit. AFR reductase activity was inversely correlated with total ascorbate content, whereas the relative abundance of AFR reductase mRNA was directly correlated with enzyme activity in tissues examined. AFR reductase mRNA abundance increased dramatically in response to wounding, a treatment that is known to also induce ascorbate-dependent prolyl hydroxylation required for the accumulation of hydroxyproline-rich glycoproteins. In addition, AFR reductase may contribute to maintaining levels of ascorbic acid for protection against wound-induced free radical-mediated damage. Collectively, the results suggest that AFR reductase activity is regulated at the level of mRNA abundance by low ascorbate contents or by factors that promote ascorbate utilization. PMID:7784511

  17. Nitrate and periplasmic nitrate reductases

    PubMed Central

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

    2014-01-01

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

  18. Aldo-Keto Reductases 1B in Endocrinology and Metabolism

    PubMed Central

    Pastel, Emilie; Pointud, Jean-Christophe; Volat, Fanny; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie

    2012-01-01

    The aldose reductase (AR; human AKR1B1/mouse Akr1b3) has been the focus of many research because of its role in diabetic complications. The starting point of these alterations is the massive entry of glucose in polyol pathway where it is converted into sorbitol by this enzyme. However, the issue of AR function in non-diabetic condition remains unresolved. AR-like enzymes (AKR1B10, Akr1b7, and Akr1b8) are highly related isoforms often co-expressed with bona fide AR, making functional analysis of one or the other isoform a challenging task. AKR1B/Akr1b members share at least 65% protein identity and the general ability to reduce many redundant substrates such as aldehydes provided from lipid peroxidation, steroids and their by-products, and xenobiotics in vitro. Based on these properties, AKR1B/Akr1b are generally considered as detoxifying enzymes. Considering that divergences should be more informative than similarities to help understanding their physiological functions, we chose to review specific hallmarks of each human/mouse isoforms by focusing on tissue distribution and specific mechanisms of gene regulation. Indeed, although the AR shows ubiquitous expression, AR-like proteins exhibit tissue-specific patterns of expression. We focused on three organs where certain isoforms are enriched, the adrenal gland, enterohepatic, and adipose tissues and tried to connect recent enzymatic and regulation data with endocrine and metabolic functions of these organs. We presented recent mouse models showing unsuspected physiological functions in the regulation of glucido-lipidic metabolism and adipose tissue homeostasis. Beyond the widely accepted idea that AKR1B/Akr1b are detoxification enzymes, these recent reports provide growing evidences that they are able to modify or generate signal molecules. This conceptually shifts this class of enzymes from unenviable status of scavenger to upper class of messengers. PMID:22876234

  19. Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation 1

    PubMed Central

    Laliberté, Gilles; Hellebust, Johan A.

    1989-01-01

    Pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2), which catalyzes the reduction of P5C to proline, was partially purified from two Chlorella species; Chlorella autotrophica, a euryhaline marine alga that responds to increases in salinity by accumulating proline and ions, and Chlorella saccharophila, which does not accumulate proline for osmoregulation. From the elution profile of this enzyme from an anion exchange column in Tris-HCl buffer (pH 7.6), containing sorbitol and glycine betaine, it was shown that P5C reductase from C. autotrophica was a neutral protein whereas the enzyme from C. saccharophila was negatively charged. The kinetic mechanisms of the reductase was characteristic of a ping-pong mechanism with double competitive substrate inhibition. Both enzymes showed high specificity for NADH as cofactor. The affinities of the reductases for their substrates did not change when the cells were grown at different salinities. In both algae, the apparent Km values of the reductase for P5C and NADH were 0.17 and 0.10 millimolar, respectively. A fourfold increase in maximal velocity of the reductase was observed when C. autotrophica was transferred from 50 to 150% artificial sea water. Even though the reductase was inhibited by NaCl, KCl, and proline, it still showed appreciable activity in the presence of these compounds at molar concentrations. A possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase is discussed in relation to the specificity of P5C reductase for NADH and its responses to salt treatments. PMID:16667157

  20. Molecular distribution and degradation status of combined aldoses in sinking particulate organic matter

    NASA Astrophysics Data System (ADS)

    Panagiotopoulos, C.; Sempéré, R.

    2003-04-01

    Particulate samples were collected by using floating sediment traps (50--300 m) and in situ pumps (30 and 200 m) in the Southern Indian Ocean (Polar Front Zone (PFZ) and Sub-Tropical Zone (STZ)), Mediterranean Sea (Ligurian and Ionian Seas) and Atlantic Ocean (Upwelling (UPW) of Agadir-Morocco). They were studied for monosaccharide composition after acid hydrolysis (HCl 0.09 M, 20 h, 100^oC) by using High Performance Anion Exchange Chromatography followed by Pulsed Amperometric Detection (HPAEC-PAD). Our results indicated that higher PCHO yields (calculated as PCHO-C/POC ratios) were associated to higher C:N ratios (Med. Sea sample, PCHO yields = 12.7 ± 7.7%; C:N ratios = 8.3 ± 1.6; n = 12) whether the opposite trend was found for Southern Ocean samples (PCHO yields = 3.3 ± 0.75%; C:N ratios = 5.7 ± 0.59, n = 5) indicating significant variability in the sugar content of particles which might be due to the degradation degree of the particles as well as to the initial chemical composition of plankton. Alternatively, other processes such as high production of extracellular polysaccharides (type transparent exopolymer polysaccharides (TEP)) due to phosphorus limitation of some phytoplanktonic species may increase the sugar content in Mediterranean particles and the C/N ratio. In any case, glucose appeared to be the most abundant monosaccharide in Mediterranean Sea or UPW samples (range 23--59 wt% of the total aldoses) whereas ribose (17--39 wt%) and galactose (range 10--28 wt%) were the predominant aldoses in Southern Indian Ocean. These sugars (glucose + ribose) exhibited a strong negative relationship with C:N (r = -0.53, p >0.01; n = 30) in sediment traps (data from this study) and sediment (data from literature) particulate material which further indicates that these two monosaccharides are selectively extracted from the carbohydrate pool in sediment. In vitro biodegradation experiments performed with large particles (>60 μm) sampled using in situ pumps in

  1. Imaging the activity of nitrate reductase by means of a scanning electrochemical microscope.

    PubMed

    Zaumseil, J; Wittstock, G; Bahrs, S; Steinrücke, P

    2000-06-01

    Scanning electrochemical microscopy (SECM) was used to characterize immobilized nitrate reductase (NaR) from Pseudonomonas stutzeri (E.C. 1.7.99.4). Nitrate reductase with membrane fragment was embedded in a polyurethane hydrogel in a capillary and solubilized NaR without membrane fragment was covalently coupled to a diaminoethyl-cellulose-carbamitate film on glass. After systematic studies of possible mediators, SECM feedback imaging of both forms of immobilized NaR was accomplished with methylviologen as redox mediator. PMID:11225859

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

    PubMed Central

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

    1995-01-01

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

  3. Bioactive constituents of Clausena lansium and a method for discrimination of aldose enantiomers.

    PubMed

    Shen, De-Yang; Chao, Chih-Hua; Chan, Hsiu-Hui; Huang, Guan-Jhong; Hwang, Tsong-Long; Lai, Chin-Yu; Lee, Kuo-Hsiung; Thang, Tran Dinh; Wu, Tian-Shung

    2012-10-01

    Glycosides, clausenosides A and B, and carbazole alkaloids, clausenaline A, claulamine A, and claulamine B, together with 50 known compounds, were isolated from the stems of Clausena lansium. Their structures were determined by means of spectroscopic methods, including that of CD and 1D/2D NMR analysis. Claulamine A has a 1-oxygenated carbazole skeleton with a rare 2,3-lactone ring, and claulamine B represents an hitherto unknown acetal carbazole alkaloid. Thirty-one of the isolated known compounds were evaluated in various assays for anti-inflammatory activity. Among them, imperatorin, isoheraclenin, and osthol exhibited selective and potent inhibition of formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation, and lansiumarin C also decreased nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production in lipopolysaccharide (LPS)-induced macrophages. In addition, a modified HPLC method of pre-column derivatization was developed that is more practical for simultaneous analysis of aldose enantiomers as compared to the literature method. The absolute configurations of the sugar moieties in clausenosides A and B were determined with this modified method. PMID:22818357

  4. Purification and partial characterization of NADPH-cytochrome c reductase from Petunia hybrida flowers.

    PubMed Central

    Menting, J G; Cornish, E; Scopes, R K

    1994-01-01

    NADPH-cytochrome c reductase was solubilized from the microsomal fraction of Petunia hybrida flowers by 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate detergent and purified by adenosine 2',5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. Two proteins with molecular sizes of 75 and 81 kD were detected in the purified preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis showed that both purified proteins cross-reacted with two different monoclonal antibodies raised against P. hybrida NADPH-cytochrome c reductase and rabbit anti-Jerusalem artichoke NADPH-cytochrome P450 reductase antibodies. Only one 84-kD protein was detected by western blot analysis of fresh microsomal extracts. Amino acid sequence analysis of tryptic peptides revealed significant similarity to the NADPH binding region of plant and animal NADPH-cytochrome P450 reductases and Bacillus megaterium cytochrome P450:NADPH-cytochrome P450 reductase. The pH optimum for reduction of ferricytochrome c was 7.4 and the Km values for the binding of NADPH and ferricytochrome c were 9.2 and 2.8 microM, respectively. We believe that the purified enzyme is a P. hybrida NADPH-cytochrome P450 reductase (EC 1.6.2.4). PMID:7991686

  5. Photoaffinity labelling of nuclear steroid 5 alpha-reductase of rat ventral prostate.

    PubMed

    Enderle-Schmitt, U; Seitz, J; Aumüller, G

    1989-09-01

    In order to get more information on the molecular structure of the rat prostatic 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3:1.22) a systematic photoaffinity labelling study has been performed. To irreversibly freeze the status quo of interaction, either testosterone, the physiological ligand, or diazo-MAPD (21-diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione), a specific 5 alpha-reductase inhibitor, was irradiated with isolated nuclei or with purified nuclear membranes or with solubilized nuclear membrane proteins and checked for optimal labelling conditions. The principal substances covalently labelled were phospholipids and at a minor ratio proteins. Analysis by SDS-PAGE and autoradiofluorography revealed two labelled polypeptides with molecular weights of 20 kDa and 26 kDa. The following evidence indicates that these polypeptides might be derived from the enzyme 5 alpha-reductase: both proteins are labelled only when specific ligands for 5 alpha-reductase are used; binding can be reduced by the addition of an excess of unlabelled ligand; enzyme activity is irreversibly suppressed when irradiated in the presence of these ligands; only subcellular fractions containing 5 alpha-reductase reveal the labelled proteins; in all 5 alpha-reductase containing preparations with increasing specific activity, independent of the polypeptide pattern, the same proteins are labelled. PMID:2779229

  6. Ascorbate synthesis pathway, dual role of ascorbate in bone homeostasis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using mouse gene knock-out models, we identify aldehyde reductase (EC 1.1.1.2, Akr1a4 (GR)) and aldose reductase (EC 1.1.1.21, Akr1b3 (AR)) as the enzymes responsible for conversion of D-glucuronate to L-gulonate, a key step in the ascorbate (ASC) synthesis pathway in mice. The gene knock-out (KO) m...

  7. Isolated menthone reductase and nucleic acid molecules encoding same

    DOEpatents

    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.

  8. Zeatin reductase in Phaseolus embryos

    SciTech Connect

    Martin, R.C.; Mok, David, W.S.; Mok, M.C. )

    1989-04-01

    Zeatin was converted to O-xylosylzeatin in embryos of Phaseolus vulgaris . O-xylosyldihydrozeatin was also identified as a zeatin metabolite. Incubation of embryo extracts with {sup 14}C-zeatin and {sup 14}C-O-xylosylzeatin revealed that reduction preceeds the O-xylosylation of zeatin. An enzyme responsible for reducing the N{sup 6}-side chain was isolated and partially purified using ammonium sulfate fractionation and affinity, gel filtration and anion exchange chromatography. The NADPH dependent reductase was zeatin specific and did not recognize cis-zeatin, ribosylzeatin, i{sup 6}Ade or i{sup 6}Ado. Two forms of the reductase could be separated by either gel filtration or anion exchange HPLC. The HMW isozyme (Mr. 55,000) eluted from the anion exchange column later than the LMW isozyme (Mr. 25,000). Interspecific differences in zeatin reductase activity were also detected.

  9. Genetics Home Reference: 5-alpha reductase deficiency

    MedlinePlus

    ... gene provides instructions for making an enzyme called steroid 5-alpha reductase 2. This enzyme is involved ... external genitalia. Mutations in the SRD5A2 gene prevent steroid 5-alpha reductase 2 from effectively converting testosterone ...

  10. Identification, cloning and regulation of cDNA encoding aldo-keto reductase 1B7 in the adrenal gland of two Saharan rodents Meriones libycus (Libyan jird) and Gerbillus gerbillus (gerbil).

    PubMed

    Mataoui-Mazari, Houria; Amirat, Zaïna; Khammar, Farida; Martinez, Antoine

    2011-12-01

    Aldo-Keto Reductase 1B7 (AKR1B7) is a mouse aldose reductase-like protein with two major sites of expression, the vas deferens and the adrenal cortex. In the adrenal cortex, Akr1b7 is an adrenocorticotropin (ACTH)-responsive-gene whose product scavenges harmful byproducts of steroidogenesis and limits stress response through the biosynthesis of prostaglandin F2α. The purpose of the present study was to explore the possible expression of AKR1B7 in the adrenal glands of two saharan rodents, Libyan jird and Lesser Egyptian gerbil. Western blot analyses demonstrated that a protein related to murine/rat AKR1B7 was highly expressed in adrenals and absent from vas deferens of both saharan species. Based on conserved sequences between mouse and rat, full length cDNA were cloned and sequenced in both species while hormonal regulation and tissue localization were explored in Libyan jird. Both cDNA encoded the expected 316 amino acids protein typical of AKR1B subfamily and contained the highly conserved catalytic tetrad consisting in Asp-44, Tyr-49, Lys-78 and His-111 residues. The deduced proteins shared higher identities with aldose reductase-like, i.e. AKR1B7 (86-94%), AKR1B8 and AKR1B10 (83-86%) than with aldose reductase group, i.e. AKR1B1 and AKR1B3 (70%). Phylogenetic analysis showed that the Libyan jird and gerbil enzymes were more closely related to murine and rat AKR1B7 than to the other AKR1B members. Northern blot analyses of total RNA from Libyan jird adrenals showed a single mRNA transcript of 1.4 kb whose expression was dependent on circulating ACTH levels. In conclusion, we demonstrate here that adrenal glands of Libyan jird and gerbil express both an ortholog of the murine/rat Akr1b7 gene and that ACTH-responsiveness is at least conserved in Libyan jird. PMID:21963864

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

    PubMed Central

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

    1973-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Lang, F.

    1991-01-01

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

  13. Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae

    PubMed Central

    Karhumaa, Kaisa; Sanchez, Rosa Garcia; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie-F

    2007-01-01

    Background Two heterologous pathways have been used to construct recombinant xylose-fermenting Saccharomyces cerevisiae strains: i) the xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway and ii) the xylose isomerase (XI) pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose reductase gene GRE3). The two isogenic strains and the industrial xylose-fermenting strain TMB 3400 were studied regarding their xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate. Results In defined mineral medium, the xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected. Conclusion Despite by-product formation, the XR-XDH xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed. PMID:17280608

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

    PubMed

    Balotf, Sadegh; Kavoosi, Gholamreza; Kholdebarin, Bahman

    2016-03-01

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

  15. Dihydrofolate reductase as a model for studies of enzyme dynamics and catalysis

    PubMed Central

    Kohen, Amnon

    2015-01-01

    Dihydrofolate reductase from Escherichia coli (ecDHFR) serves as a model system for investigating the role of protein dynamics in enzyme catalysis. We discuss calculations predicting a network of dynamic motions that is coupled to the chemical step catalyzed by this enzyme. Kinetic studies testing these predictions are presented, and their potential use in better understanding the role of these dynamics in enzyme catalysis is considered. The cumulative results implicate motions across the entire protein in catalysis. PMID:26918149

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

    PubMed Central

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

    2013-01-01

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

  17. Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes

    PubMed Central

    Spite, Matthew; Baba, Shahid P.; Ahmed, Yonis; Barski, Oleg A.; Nijhawan, Kanchan; Petrash, J. Mark; Bhatnagar, Aruni; Srivastava, Sanjay

    2007-01-01

    Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone (‘core’ aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte–endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C16:0-20:4 phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C16:0-20:4 phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are

  18. Prostaglandin (PG) F2 Alpha Synthesis in Human Subcutaneous and Omental Adipose Tissue: Modulation by Inflammatory Cytokines and Role of the Human Aldose Reductase AKR1B1

    PubMed Central

    Michaud, Andréanne; Lacroix-Pépin, Nicolas; Pelletier, Mélissa; Veilleux, Alain; Noël, Suzanne; Bouchard, Céline; Marceau, Picard; Fortier, Michel A.; Tchernof, André

    2014-01-01

    Introduction PGF2α may be involved in the regulation of adipose tissue function. Objectives 1) To examine PGF2α release by primary preadipocytes, mature adipocytes and whole tissue explants from the subcutaneous and omental fat compartments; 2) To assess which PGF synthase is the most relevant in human adipose tissue. Methods Fat samples were obtained by surgery in women. PGF2α release by preadipocytes, adipocytes and explants under stimulation by TNF-α, IL-1β or both was measured. Messenger RNA expression levels of AKR1B1 and AKR1C3 were measured by RT-PCR in whole adipose tissue and cytokine-treated preadipocytes. The effect of AKR1B1 inhibitor ponalrestat on PGF2α synthesis was investigated. Results PGF2α release was significantly induced in response to cytokines compared to control in omental (p = 0.01) and to a lesser extent in subcutaneous preadipocytes (p = 0.02). Messenger RNA of COX-2 was significantly higher in omental compared to subcutaneous preadipocytes in response to combined TNF-α and IL-1β (p = 0.01). Inflammatory cytokines increased AKR1B1 mRNA expression and protein levels (p≤0.05), but failed to increase expression levels of AKR1C3 in cultured preadipocytes. Accordingly, ponalrestat blunted PGF2α synthesis by preadipocytes in basal and stimulated conditions (p≤0.05). Women with the highest PGF2α release by omental adipocytes had a higher BMI (p = 0.05), waist circumference (p≤0.05) and HOMAir index (p≤0.005) as well as higher mRNA expression of AKR1B1 in omental (p<0.10) and subcutaneous (p≤0.05) adipose tissue compared to women with low omental adipocytes PGF2α release. Positive correlations were observed between mRNA expression of AKR1B1 in both compartments and BMI, waist circumference as well as HOMAir index (p≤0.05 for all). Conclusion PGF2α release by omental mature adipocytes is increased in abdominally obese women. Moreover, COX-2 expression and PGF2α release is particularly responsive to inflammatory stimulation in omental preadipocytes. Yet, blockade of PGF synthase AKR1B1 inhibits most of the PGF2α release. PMID:24663124

  19. LC-MS-MS Characterization of Forced Degradation Products of Fidarestat, a Novel Aldose Reductase Inhibitor: Development and Validation of a Stability-Indicating RP-HPLC Method.

    PubMed

    Talluri, M V N Kumar; Khatoon, Lubna; Kalariya, Pradipbhai D; Chavan, Balasaheb B; Ragampeta, Srinivas

    2015-10-01

    An accurate, precise, robust and selective stability-indicating liquid chromatographic (LC) method has been developed for the monitoring of fidarestat in the presence of its forced degradants. The drug was subjected to hydrolysis (acid, alkali and neutral degradation), oxidation, photolysis and thermal stress conditions. The drug degraded significantly under hydrolytic (basic, acidic and neutral) and oxidative stress conditions, whereas it was found to be stable in photolytic and thermal conditions. The chromatographic separation was achieved on a Grace C18, (250 mm × 4.6 mm × 5 μm) column using gradient mobile phase system consisting of 10 mM of ammonium acetate buffer at pH 4 and acetonitrile at a flow rate of 1 mL/min with UV detection at 283 nm. The developed method was extended to liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS-MS) for characterization of all the degradation products. A total of five new degradation products were identified and characterized by LC-QTOF-MS-MS. The developed LC method was validated as per ICH guideline Q2 (R1). The proposed method was found to be successively applied for the quality control of fidarestat in bulk drug analysis. PMID:26014964

  20. Automatic Assignment of EC Numbers

    PubMed Central

    Egelhofer, Volker; Schomburg, Ida; Schomburg, Dietmar

    2010-01-01

    A wide range of research areas in molecular biology and medical biochemistry require a reliable enzyme classification system, e.g., drug design, metabolic network reconstruction and system biology. When research scientists in the above mentioned areas wish to unambiguously refer to an enzyme and its function, the EC number introduced by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) is used. However, each and every one of these applications is critically dependent upon the consistency and reliability of the underlying data for success. We have developed tools for the validation of the EC number classification scheme. In this paper, we present validated data of 3788 enzymatic reactions including 229 sub-subclasses of the EC classification system. Over 80% agreement was found between our assignment and the EC classification. For 61 (i.e., only 2.5%) reactions we found that their assignment was inconsistent with the rules of the nomenclature committee; they have to be transferred to other sub-subclasses. We demonstrate that our validation results can be used to initiate corrections and improvements to the EC number classification scheme. PMID:20126531

  1. Loop interactions during catalysis by dihydrofolate reductase from Moritella profunda.

    PubMed

    Behiry, Enas M; Evans, Rhiannon M; Guo, Jiannan; Loveridge, E Joel; Allemann, Rudolf K

    2014-07-29

    Dihydrofolate reductase (DHFR) is often used as a model system to study the relation between protein dynamics and catalysis. We have studied a number of variants of the cold-adapted DHFR from Moritella profunda (MpDHFR), in which the catalytically important M20 and FG loops have been altered, and present a comparison with the corresponding variants of the well-studied DHFR from Escherichia coli (EcDHFR). Mutations in the M20 loop do not affect the actual chemical step of transfer of hydride from reduced nicotinamide adenine dinucleotide phosphate to the substrate 7,8-dihydrofolate in the catalytic cycle in either enzyme; they affect the steady state turnover rate in EcDHFR but not in MpDHFR. Mutations in the FG loop also have different effects on catalysis by the two DHFRs. Despite the two enzymes most likely sharing a common catalytic cycle at pH 7, motions of these loops, known to be important for progression through the catalytic cycle in EcDHFR, appear not to play a significant role in MpDHFR. PMID:25014120

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

    PubMed

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases. PMID:12626517

  3. Purification and thermal dependence of glutathione reductase from two forage legume species.

    PubMed

    Kidambi, S P; Mahan, J R; Matches, A G

    1990-02-01

    Alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.) are forage legumes that differ in their responses to high and low temperature stresses. Thermal limitations on the function of glutathione reductase (EC 1.6.4.2) could adversely affect the ability of the plant to cope with adverse temperatures. Our objectives were to (a) purify glutathione reductase from ;Cimarron' alfalfa and ;PI 212241' sainfoin and (b) investigate the intraspecies variation in the thermal dependency of glutathione reductase from each of three cultivars of alfalfa and two cultivars and an introduction of sainfoin. Glutathione reductase was purified 1222-and 1948-fold to a specific activity of 281 and 273 units per milligram of protein, from one species each of alfalfa and sainfoin, respectively. The relative molecular mass of the protein was approximately 140 kilodaltons with subunits of 57 and 37 kilodaltons under denaturing conditions. The activation energies were approximately 50 kilojoules per mole for both species. Over a 5 to 45 degrees C temperature gradient, large variation among species and genotypes within species was found for: (a) the minimum apparent Michaelis constant (0.6-2.1 micromoles of NADPH), (b) the temperature at which the minimum apparent Michaelis constant was observed (10-25 degrees C), and (c) the thermal kinetic windows (6-19 degrees C width). Future studies will focus on relating the thermal dependence of the Michaelis constant of the glutathione reductases and plant growth rates and forage quality of these species throughout the growing season. PMID:16667283

  4. Argonne's SpEC Module

    SciTech Connect

    Harper, Jason

    2014-05-05

    Jason Harper, an electrical engineer in Argonne National Laboratory's EV-Smart Grid Interoperability Center, discusses his SpEC Module invention that will enable fast charging of electric vehicles in under 15 minutes. The module has been licensed to BTCPower.

  5. Argonne's SpEC Module

    ScienceCinema

    Harper, Jason

    2014-06-05

    Jason Harper, an electrical engineer in Argonne National Laboratory's EV-Smart Grid Interoperability Center, discusses his SpEC Module invention that will enable fast charging of electric vehicles in under 15 minutes. The module has been licensed to BTCPower.

  6. Tungstate, a Molybdate Analog Inactivating Nitrate Reductase, Deregulates the Expression of the Nitrate Reductase Structural Gene

    PubMed Central

    Deng, Mingde; Moureaux, Thérèse; Caboche, Michel

    1989-01-01

    Nitrate reductase (NR, EC 1.6.6.1) from higher plants is a homodimeric enzyme carrying a molybdenum cofactor at the catalytic site. Tungsten can be substituted for molybdenum in the cofactor structure, resulting in an inactive enzyme. When nitratefed Nicotiana tabacum plants were grown on a nutrient solution in which tungstate was substituted for molybdate, NR activity in the leaves decreased to a very low level within 24 hours while NR protein accumulated progressively to a level severalfold higher than the control after 6 days. NR mRNA level in molybdate-grown plants exhibited a considerable day-night fluctuation. However, when plants were treated with tungstate, NR mRNA level remained very high. NR activity and protein increased over a 24-hour period when nitrate was added back to N-starved molybdate-grown plants. NR mRNA level increased markedly during the first 2 hours and then decreased. In the presence of tungstate, however, the induction of NR activity by nitrate was totally abolished while high levels of NR protein and mRNA were both induced, and the high level of NR mRNA was maintained over a 10-hour period. These results suggest that the substitution of tungsten for molybdenum in NR complex leads to an overexpression of the NR structural gene. Possible mechanisms involved in this deregulation are discussed. Images Figure 2 Figure 3 Figure 5 PMID:16667015

  7. Fatty acyl-CoA reductase

    SciTech Connect

    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.

  8. Preliminary joint X-ray and neutron protein crystallographic studies of ecDHFR complexed with folate and NADP{sup +}

    SciTech Connect

    Wan, Qun Kovalevsky, Andrey Y.; Wilson, Mark A.; Bennett, Brad C.; Langan, Paul; Dealwis, Chris

    2014-05-25

    A 2.0 Å resolution neutron data set and a 1.6 Å resolution X-ray data set were collected for joint X-ray/neutron refinement of the ecDHFR–folate–NADP{sup +} complex in order to study the reaction mechanism of dihydrofolate reductase.

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

    PubMed

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

    2015-12-01

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

  10. Dihydropteridine reductase from Escherichia coli.

    PubMed Central

    Vasudevan, S G; Shaw, D C; Armarego, W L

    1988-01-01

    A dihydropteridine reductase from Escherichia coli was purified to apparent homogeneity. It is a dimeric enzyme with identical subunits (Mr 27000) and a free N-terminal group. It can use NADH (Vmax./Km 3.36 s-1) and NADPH (Vmax./Km 1.07 s-1) when 6-methyldihydro-(6H)-pterin is the second substrate, as well as quinonoid dihydro-(6H)-biopterin (Vmax./Km 0.69 s-1), dihydro-(6H)-neopterin (Vmax./Km 0.58 s-1), dihydro-(6H)-monapterin 0.66 s-1), 6-methyldihydro-(6H)-pterin and cis-6,7-dimethyldihydro-(6H)-pterin (Vmax./Km 0.66 s-1) when NADH is the second substrate. The pure reductase has a yellow colour and contains bound FAD. The enzyme also has pterin-independent NADH and NADPH oxidoreductase activities when potassium ferricyanide is the electron acceptor. Images Fig. 2. PMID:3060113

  11. Optical observation of correlated motions in dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2015-03-01

    Enzyme function relies on its structural flexibility to make conformational changes for substrate binding and product release. An example of a metabolic enzyme where such structural changes are vital is dihydrofolate reductase (DHFR). DHFR is essential in both prokaryotes and eukaryotes for the nucleotide biosynthesis by catalyzing the reduction of dihydrofolate to tetrahydrofolate. NMR dynamical measurements found large amplitude fast dynamics that could indicate rigid-body, twisting-hinge motion for ecDHFR that may mediate flux. The role of such long-range correlated motions in function was suggested by the observed sharp decrease in enzyme activity for the single point mutation G121V, which is remote from active sites. This decrease in activity may be caused by the mutation interfering with the long-range intramolecular vibrations necessary for rapid access to functional configurations. We use our new technique of crystal anisotropy terahertz microscopy (CATM), to observe correlated motions in ecDHFR crystals with the bonding of NADPH and methotrexate. We compare the measured intramolecular vibrational spectrum with calculations using normal mode analysis.

  12. The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor.

    PubMed Central

    Eliasson, R; Fontecave, M; Jörnvall, H; Krook, M; Pontis, E; Reichard, P

    1990-01-01

    Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2+ ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E. coli. After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions. One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several defined protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein B1) of the aerobic reductase but not by monoclonal anti-B1 antibodies. Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component. Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established. The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction. One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal. We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP. Images PMID:2185465

  13. The Role of Large-Scale Motions in Catalysis by Dihydrofolate Reductase

    PubMed Central

    2011-01-01

    Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR. PMID:22060818

  14. An electrogenic nitric oxide reductase.

    PubMed

    Al-Attar, Sinan; de Vries, Simon

    2015-07-22

    Nitric oxide reductases (Nors) are members of the heme-copper oxidase superfamily that reduce nitric oxide (NO) to nitrous oxide (N₂O). In contrast to the proton-pumping cytochrome oxidases, Nors studied so far have neither been implicated in proton pumping nor have they been experimentally established as electrogenic. The copper-A-dependent Nor from Bacillus azotoformans uses cytochrome c₅₅₁ as electron donor but lacks menaquinol activity, in contrast to our earlier report (Suharti et al., 2001). Employing reduced phenazine ethosulfate (PESH) as electron donor, the main NO reduction pathway catalyzed by Cu(A)Nor reconstituted in liposomes involves transmembrane cycling of the PES radical. We show that Cu(A)Nor reconstituted in liposomes generates a proton electrochemical gradient across the membrane similar in magnitude to cytochrome aa₃, highlighting that bacilli using Cu(A)Nor can exploit NO reduction for increased cellular ATP production compared to organisms using cNor. PMID:26149211

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

    SciTech Connect

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

    1986-04-29

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

  16. Co-Expression of Monodehydroascorbate Reductase and Dehydroascorbate Reductase from Brassica rapa Effectively Confers Tolerance to Freezing-Induced Oxidative Stress

    PubMed Central

    Shin, Sun-Young; Kim, Myung-Hee; Kim, Yul-Ho; Park, Hyang-Mi; Yoon, Ho-Sung

    2013-01-01

    Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing. PMID:24170089

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

    PubMed Central

    Chen, Y P; Yoch, D C

    1989-01-01

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

  18. EC decay of 244Bk

    NASA Astrophysics Data System (ADS)

    Sodaye, Suparna; Tripathi, R.; Sudarshan, K.; Sharma, S. K.; Pujari, P. K.; Palit, R.; Mukhopadhyay, S.

    2014-12-01

    Berkelium isotopes have been produced in 11B-induced reaction on 238U. The EC decay of 244Bk → 244Cm has been studied by carrying out the single and coincidence measurements of the γ-rays emitted during the de-excitation of the 244Cm levels. Radiochemical separations have been carried out to minimize the contribution from the fission products and target. The new half-life of 244Bk is obtained as 5.02 ± 0.03 h, which is close to the theoretically calculated value. The relative intensities of the decay γ-rays have been re-evaluated. Based on the coincidence measurements, a tentative partial level scheme for 244Bk → 244Cm decay has been proposed.

  19. Genetics Home Reference: sepiapterin reductase deficiency

    MedlinePlus

    ... reductase enzyme. This enzyme is involved in the production of a molecule called tetrahydrobiopterin (also known as ... is responsible for the last step in the production of tetrahydrobiopterin. Tetrahydrobiopterin helps process several building blocks ...

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

    PubMed

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

    1994-01-01

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

  1. A dissimilatory nitrite reductase in Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

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

    1984-01-01

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

  2. Overexpression of human NADPH:cytochrome c (P450) reductase confers enhanced sensitivity to both tirapazamine (SR 4233) and RSU 1069.

    PubMed Central

    Patterson, A. V.; Saunders, M. P.; Chinje, E. C.; Talbot, D. C.; Harris, A. L.; Strafford, I. J.

    1997-01-01

    P450 reductase (NADPH: cytochrome c (P450) reductase, EC 1.6.2.4) plays an important role in the reductive activation of the bioreductive drug tirapazamine (SR4233). Thus, in a panel of human breast cancer cell lines, expression of P450 reductase correlated with both the hypoxic toxicity and the metabolism of tirapazamine [Patterson et al (1995) Br J Cancer 72: 1144-1150]. To examine this dependence in more detail, the MDA231 cell line, which has the lowest activity of P450 reductase in our breast cell line panel, was transfected with the human P450 reductase cDNA. Isolated clones expressed a 78-kDa protein, which was detected with anti-P450 reductase antibody, and were shown to have up to a 53-fold increase in activity of the enzyme. Using six stable transfected clones covering the 53-fold range of activity of P450 reductase, it was shown that the enzyme activity correlated directly with both hypoxic and aerobic toxicity of tirapazamine, and metabolism of the drug under hypoxic conditions. No metabolism was detected under aerobic conditions. For RSU1069, toxicity was also correlated with P450 reductase activity, but only under hypoxic conditions. Measurable activity of P450 reductase was found in a selection of 14 primary human breast tumours. Activity covered an 18-fold range, which was generally higher than that seen in cell lines but within the range of activity measured in the transfected clones. These results suggest that if breast tumours have significant areas of low oxygen tension, then they are likely to be highly sensitive to the cytotoxic action of tirapazamine and RSU 1069. Images Figure 1 PMID:9374381

  3. Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110

    PubMed Central

    2010-01-01

    Background Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. Candida magnoliae JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from C. magnoliae JH110. Results The gene encoding a novel ER was isolated from an osmophilic yeast C. magnoliae JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from Trichosporonoides megachiliensis SNG-42. The intact coding region of ER from C. magnoliae JH110 was cloned, functionally expressed in Escherichia coli using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42°C and 5.5, respectively. Among various aldoses, the C. magnoliae JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could account for the enzyme

  4. Thioredoxin Reductase and its Inhibitors

    PubMed Central

    Saccoccia, Fulvio; Angelucci, Francesco; Boumis, Giovanna; Carotti, Daniela; Desiato, Gianni; Miele, Adriana E; Bellelli, Andrea

    2014-01-01

    Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis. PMID:24875642

  5. Characterization of thyroidal glutathione reductase

    SciTech Connect

    Raasch, R.J.

    1989-01-01

    Glutathione levels were determined in bovine and rat thyroid tissue by enzymatic conjugation with 1-chloro-2,4-dinitrobenzene using glutathione S-transferase. Bovine thyroid tissue contained 1.31 {+-} 0.04 mM reduced glutathione (GSH) and 0.14 {+-} 0.02 mM oxidized glutathione (GSSG). In the rat, the concentration of GSH was 2.50 {+-} 0.05 mM while GSSG was 0.21 {+-} 0.03 mM. Glutathione reductase (GR) was purified from bovine thyroid to electrophoretic homogeneity by ion exchange, affinity and molecular exclusion chromatography. A molecular weight range of 102-109 kDa and subunit size of 55 kDa were determined for GR. Thyroidal GR was shown to be a favoprotein with one FAD per subunit. The Michaelis constants of bovine thyroidal GR were determined to be 21.8 {mu}M for NADPH and 58.8 {mu}M for GSSG. The effect of thyroid stimulating hormone (TSH) and thyroxine (T{sub 4}) on in vivo levels of GR and glucose 6-phosphate dehydrogenase were determined in rat thyroid homogenates. Both enzymes were stimulated by TSH treatment and markedly reduced following T{sub 4} treatment. Lysosomal hydrolysis of ({sup 125}I)-labeled and unlabeled thyroglobulin was examined using size exclusion HPLC.

  6. ECS Prepares to Set Agenda, Find President

    ERIC Educational Resources Information Center

    Hoff, David J.

    2006-01-01

    The ECS, like other nonprofit groups serving state officials, has faced financial difficulties in recent years, starting when states faced severe revenue shortfalls early in the decade. But its problems became public this spring when Kathy Christie, the group's No. 2 official and a 17-year ECS employee, resigned and said in a letter to the ECS…

  7. Alteration of organic matter during infaunal polychaete gut passage and links to sediment organic geochemistry. Part II: Fatty acids and aldoses

    NASA Astrophysics Data System (ADS)

    Woulds, Clare; Middelburg, Jack J.; Cowie, Greg L.

    2014-07-01

    The activities of sediment-dwelling fauna are known to influence the rates of and pathways through which organic matter is cycled in marine sediments, and thus to influence eventual organic carbon burial or decay. However, due to methodological constraints, the role of faunal gut passage in determining the subsequent composition and thus degradability of organic matter is relatively little studied. Previous studies of organic matter digestion by benthic fauna have been unable to detect uptake and retention of specific biochemicals in faunal tissues, and have been of durations too short to fit digestion into the context of longer-term sedimentary degradation processes. Therefore this study aimed to investigate the aldose and fatty acid compositional alterations occurring to organic matter during gut passage by the abundant and ubiquitous polychaetes Hediste diversicolor and Arenicola marina, and to link these to longer-term changes typically observed during organic matter decay. This aim was approached through microcosm experiments in which selected polychaetes were fed with 13C-labelled algal detritus, and organisms, sediments, and faecal pellets were sampled at three timepoints over ∼6 weeks. Samples were analysed for their 13C-labelled aldose and fatty acid contents using GC-MS and GC-IRMS. Compound-selective net accumulation of biochemicals in polychaete tissues was observed for both aldoses and fatty acids, and the patterns of this were taxon-specific. The dominant patterns included an overall loss of glucose and polyunsaturated fatty acids; and preferential preservation or production of arabinose, microbial compounds (rhamnose, fucose and microbial fatty acids), and animal-synthesised fatty acids. These patterns may have been driven by fatty acid essentiality, preferential metabolism of glucose, and A. marina grazing on bacteria. Fatty acid suites in sediments from faunated microcosms showed greater proportions of saturated fatty acids and bacterial markers

  8. Regulation of synthesis and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by low density lipoprotein and 25-hydroxycholesterol in UT-1 cells.

    PubMed Central

    Faust, J R; Luskey, K L; Chin, D J; Goldstein, J L; Brown, M S

    1982-01-01

    UT-1 cells are a clone of Chinese hamster ovary cells that were selected to grow in the presence of compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]. These cells have 100- to 1,000-fold more immunoprecipitable reductase than normal. The enzyme activity is rapidly decreased when low density lipoprotein (LDL) or 25-hydroxycholesterol is added to the culture medium. In this current study, a quantitative immunoprecipitation assay was used to determine whether LDL and 25-hydroxycholesterol inhibit the synthesis or stimulate the degradation of reductase in UT-1 cells. Each of these agents inhibited the incorporation of [35S]methionine into immunoprecipitable reductase by more than 98%. Pulse-chase experiments showed that reductase was degraded with a half-life of 10-13 hr in UT-1 cells and that the rate of degradation of preformed enzyme was increased 3-fold by the addition of either LDL or 25-hydroxycholesterol. We conclude that the predominant mechanism by which LDL and 25-hydroxycholesterol decrease reductase activity in UT-1 cells is a profound suppression of synthesis of the enzyme. Images PMID:6957860

  9. Magnesium Deficiency and High Light Intensity Enhance Activities of Superoxide Dismutase, Ascorbate Peroxidase, and Glutathione Reductase in Bean Leaves 1

    PubMed Central

    Cakmak, Ismail; Marschner, Horst

    1992-01-01

    The influence of varied Mg supply (10-1000 micromolar) and light intensity (100-580 microeinsteins per square meter per second) on the concentrations of ascorbate (AsA) and nonprotein SH-compounds and the activities of superoxide dismutase (SOD; EC 1.15.11) and the H2O2 scavenging enzymes, AsA peroxidase (EC 1.11.1.7), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were studied in bean (Phaseolus vulgaris L.) leaves over a 13-day period. The concentrations of AsA and SH-compounds and the activities of SOD and H2O2 scavenging enzymes increased with light intensity, in particular in Mg-deficient leaves. Over the 12-day period of growth for a given light intensity, the concentrations of AsA and SH-compounds and the activities of these enzymes remained more or less constant in Mg-sufficient leaves. In contrast, in Mg-deficient leaves, a progressive increase was recorded, particularly in concentrations of AsA and activities of AsA peroxidase and glutathione reductase, whereas the activities of guaiacol peroxidase and catalase were only slightly enhanced. Partial shading of Mg-deficient leaf blades for 4 days prevented chlorosis, and the activities of the O2.− and H2O2 scavenging enzymes remained at a low level. The results demonstrate the role of both light intensity and Mg nutritional status on the regulation of O2.− and H2O2 scavenging enzymes in chloroplasts. PMID:16668779

  10. Explosive Effectiveness Capability (ExEC)

    SciTech Connect

    Nakafuji, G.; Daily, L.; Leake, J.

    2000-07-26

    Gaining accurate predictions of damage inflicted by high explosive devices is vital in order to minimize collateral damage effects on a target. ExEC provides a means for conducting very detailed analysis of weapons effects on targets for advanced mission planning purposes. ExEC is composed of a suite of high fidelity physics codes, which have been used for decades by the nuclear weapons laboratories for assessing high explosive, thermal, and structural effects. The ExEC capability should not be confused with the fast running empirical codes MEA and MEVA, which are good for fast scoping analysis. MEA and MEVA rely on look-up tables or simple approximations to quickly obtain a rough estimate of weapon damage on a target. In contrast, the ExEC capability provides a much higher fidelity damage prediction and a limitless number of target configurations by solving the time dependent conservation equations for mass, momentum, and energy, in order to directly simulate the interaction of a weapon with a target component. In contrast, MEA/MEVA must be calibrated for every different weapon type and target. ExEC has produced accurate simulations for weapon disablement, shape charge penetration, sympathetic detonation, fragment damage effects, and blast effects. For example, ExEC was used to look at a DTRA test performed at White Sands Missile Range, Dipole Orbit 1 (DO1). In DO1, a BLU-109 bomb was statically detonated in a partially buried four-room building that was storing chemical and biological weapon simulants, as well as inactive equipment typical of a CBW (Chemical and Biological Warfare) facility. The blast wave (overpressure) histories were recorded and compared to an ExEC and MEVA simulation. A direct comparison between experimental data and calculations, for a single pressure gauge, indicated that MEVA had a 200% error compared to 5% for ExEC. In addition, ExEC tracks the time dependant fluctuations in pressure (for an accurate prediction of the impulse), while MEVA

  11. Developing Electrocaloric (EC) Materials with Giant EC Response and Chip-Scale EC Cooling Devices

    NASA Astrophysics Data System (ADS)

    Zhang, Qiming

    2015-03-01

    The direct and efficient coupling between the electric signals and the elastic, thermal, optical and magnetic signals in ferroelectric based electroactive materials makes them attractive for exploiting a broad range of cross-coupling phenomena which have great promise for new device technologies. This talk will present the recent advances at Penn State in developing electrocaloric materials which may provide alternative cooling technology to replace the century old vapor compression cycle (VCC) based cooling which employs strong greenhouse gases as the refrigerants. Electrocaloric effect (ECE), which is the temperature and entropy change of insulating dielectric materials under electric fields, is attractive to realize efficient cooling devices. However, the relatively small ECE observed in dielectrics in the last century make it unimpressive for any practical applications. Experimental results on the ECE in the relaxor ferroelectric polymers and general theoretical considerations for achieving large ECE will be presented. This talk will also discuss considerations on and present recent works in using nanocomposites to further enhancing the ECE beyond the pure relaxor polymers, on the giant ECE in a class of dielectric liquid, and in bulk ferroelectric ceramics near the invariant critical point. The works related to developing the chip-scale EC cooling devices, exploiting the newly discovered large ECE in ferroelectric materials and featuring high cooling power density and high efficiency, will also be presented. This work has been supported by DoE BES and by ARO.

  12. Structural and mechanistic insights on nitrate reductases.

    PubMed

    Coelho, Catarina; Romão, Maria João

    2015-12-01

    Nitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data. PMID:26362109

  13. Progesterone 5β-reductase genes of the Brassicaceae family as function-associated molecular markers.

    PubMed

    Munkert, J; Costa, C; Budeanu, O; Petersen, J; Bertolucci, S; Fischer, G; Müller-Uri, F; Kreis, W

    2015-11-01

    This study aimed to define progesterone 5β-reductases (P5βR, EC 1.3.99.6, enone 1,4-reductases) as function-associated molecular markers at the plant family level. Therefore cDNAs were isolated from 25 Brassicaceae species, including two species, Erysimum crepidifolium and Draba aizoides, known to produce cardiac glycosides. The sequences were used in a molecular phylogeny study. The cladogram created is congruent to the existing molecular analyses. Recombinant His-tagged forms of the P5βR cDNAs from Aethionema grandiflorum, Draba aizoides, Nasturtium officinale, Raphanus sativus and Sisymbrium officinale were expressed in E. coli. Enone 1,4-reductase activity was demonstrated in vitro using progesterone and 2-cyclohexen-1-one as substrates. Evidence is provided that functional P5βRs are ubiquitous in the Brassicaceae. The recombinant P5βR enzymes showed different substrate preferences towards progesterone and 2-cyclohexen-1-one. Sequence comparison of the catalytic pocket of the P5βR enzymes and homology modelling using Digitalis lanata P5βR (PDB ID: 2V6G) as template highlighted the importance of the hydrophobicity of the binding pocket for substrate discrimination. It is concluded that P5βR genes or P5βR proteins can be used as valuable function-associated molecular markers to infer taxonomic relationship and evolutionary diversification from a metabolic/catalytic perspective. PMID:26108256

  14. Human leukemia and normal leukocytes contain a species of immunoreactive but nonfunctional dihydrofolate reductase

    SciTech Connect

    Rothenbery, S.P.; Iqbal, M.P.

    1982-01-01

    A quantitative radioimmunoassay has been developed for human dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahdrofolate:NADP/sup +/ oxidoreductase, EC 1.5.1.3) by using antiserum raised in rabbits against the active enzyme purified from calf liver. An immunoreactive protein could be identified in the cytoplasm of chronic myelogenous leukemia cells, which contained no functional dihydrofolate reductase activity. Its concentration was stoichiometric to the volume of cytoplasm assayed and paralleled the standard curve obtained with purified enzyme, indicating that this protein in the human cells is antigenically similar to the homologous antigen. The concentration of this immunoreactive protein in the cytoplasm of human leukemia and normal leukocytes in all instances greatly exceeded the concentration of functional dihydrofolate reductase, which was measured by the binding of (/sup 3/H)methotrexate. This nonfunctional immunoreactive protein in the cytoplasm and cytosol from two different samples of chronic myelogenous leukemia cells analyzed by gel filtration had an apparent molecular weight of 41,000, which is twice the molecular weight of the functional enzyme.

  15. Human leukemia and normal leukocytes contain a species of immunoreactive but nonfunctional dihydrofolate reductase.

    PubMed Central

    Rothenberg, S P; Iqbal, M P

    1982-01-01

    A quantitative radioimmunoassay has been developed for human dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) by using antiserum raised in rabbits against the active enzyme purified from calf liver. An immunoreactive protein could be identified in the cytoplasm of chronic myelogenous leukemia cells, which contained no functional dihydrofolate reductase activity. Its concentration was stoichiometric to the volume of cytoplasm assayed and paralleled the standard curve obtained with purified enzyme, indicating that this protein in the human cells is antigenically similar to the homologous antigen. The concentration of this immunoreactive protein in the cytoplasm of human leukemia and normal leukocytes in all instances greatly exceeded the concentration of functional dihydrofolate reductase, which was measured by the binding of [3H]methotrexate. This nonfunctional immunoreactive protein in the cytoplasm and cytosol from two different samples of chronic myelogenous leukemia cells analyzed by gel filtration had an apparent molecular weight of 41,000, which is twice the molecular weight of the functional enzyme. Images PMID:6952216

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

    SciTech Connect

    Higashi, Yasuhiro; Kutchan, Toni M.; Smith, Thomas J.

    2011-11-18

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

  17. Respiratory arsenate reductase as a bidirectional enzyme

    USGS Publications Warehouse

    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.

  18. Respiratory arsenate reductase as a bidirectional enzyme

    SciTech Connect

    Richey, Christine; Chovanec, Peter; Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282 ; Hoeft, Shelley E.; Oremland, Ronald S.; Basu, Partha; Stolz, John F.

    2009-05-01

    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.

  19. Phylogenomics of Mycobacterium Nitrate Reductase Operon.

    PubMed

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

    2015-07-01

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

  20. IN VITRO INHIBITION OF GLUTATHIONE REDUCTASE BY ARSENOTRI-GLUTATHIONE

    EPA Science Inventory

    Arsenotriglutathione, a product of the reduction of arsenate and the complexation of arsenite by glutathione, is a mixed type inhibitor of the reduction of glutathione disulfide by purified yeast glutathione reductase or the glutathione reductase activity in rabbit erythrocyte ly...

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

    PubMed Central

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

    2013-01-01

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

  2. Evaluation of nitrate reductase activity in Rhizobium japonicum

    SciTech Connect

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

    1983-08-01

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

  3. Comparative preclinical activity of the folate-targeted Vinca alkaloid conjugates EC140 and EC145.

    PubMed

    Leamon, Christopher P; Reddy, Joseph A; Vlahov, Iontcho R; Westrick, Elaine; Parker, Nikki; Nicoson, Jeffrey S; Vetzel, Marilynn

    2007-10-01

    EC140 is a water soluble folate conjugate of desacetylvinblastine monohydrazide (DAVLBH), which is constructed with an endosome-cleavable acyl hydrazone bond. This agent has proven to be active and specific against well established, subcutaneous folate receptor (FR)-positive tumors in multiple animal models. Recent structure-activity and optimization studies have yielded a disulfide bond-containing counterpart to EC140, herein referred to as EC145. This new conjugate was found to retain high affinity for FR-positive cells, and it produced specific, dose-responsive activity in vitro. Comparative in vivo efficacy tests confirmed that, like EC140, EC145 displays activity against both syngeneic and xenograft tumor models. However, EC145 was found to be more active and better tolerated than EC140; hence, more durable complete responses were consistently observed in EC145-treated tumor-bearing animals. Furthermore, EC145 was not found to be active against a FR-negative tumor model. Additional preclinical studies are therefore warranted to better understand EC145's breadth of activity against FR-positive tumors. PMID:17551919

  4. Metabolism and drug interactions of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in transplant patients: are the statins mechanistically similar?

    PubMed

    Christians, U; Jacobsen, W; Floren, L C

    1998-10-01

    3-Hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.88) inhibitors are the most effective drugs to lower cholesterol in transplant patients. However, immunosuppressants and several other drugs used after organ transplantation are cytochrome P4503A (CYP3A, EC 1.14.14.1) substrates. Pharmacokinetic interaction with some of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, specifically lovastatin and simvastatin, leads to an increased incidence of muscle skeletal toxicity in transplant patients. It is our objective to review the role of drug metabolism and drug interactions of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, and cerivastatin. In the treatment of transplant patients, from a drug interaction perspective, pravastatin, which is not significantly metabolized by CYP enzymes, and fluvastatin, presumably a CYP2C9 substrate, compare favorably with the other statins for which the major metabolic pathways are catalyzed by CYP3A. PMID:9804052

  5. Post-translational Regulation of Nitrate Reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Promiscuity and diversity in 3-ketosteroid reductases.

    PubMed

    Penning, Trevor M; Chen, Mo; Jin, Yi

    2015-07-01

    Many steroid hormones contain a Δ(4)-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1-AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1-AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'. PMID:25500069

  7. Promiscuity and diversity in 3-ketosteroid reductases

    PubMed Central

    Penning, Trevor M.; Chen, Mo; Jin, Yi

    2014-01-01

    Many steroid hormones contain a Δ4-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1–AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1–AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled ‘Steroid/Sterol signaling’. PMID:25500069

  8. Ferrisiderophore reductase activity in Agrobacterium tumefaciens.

    PubMed Central

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

    1982-01-01

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

  9. Crystallization and preliminary X-ray diffraction studies of maleylacetate reductase from Rhizobium sp. strain MTP-10005

    SciTech Connect

    Fujii, Tomomi; Goda, Yuko; Yoshida, Masahiro; Oikawa, Tadao; Hata, Yasuo

    2008-08-01

    Maleylacetate reductase from Rhizobium sp. strain MTP-10005 has been crystallized using the sitting-drop vapour-diffusion method and microseeding. The crystals contained one dimeric molecule per asymmetric unit and diffracted to 1.79 Å resolution. Maleylacetate reductase (EC 1.3.1.32), which catalyzes the reduction of maleylacetate to 3-oxoadipate, plays an important role in the aerobic microbial catabolism of resorcinol. The enzyme has been crystallized at 293 K by the sitting-drop vapour-diffusion method supplemented with a microseeding technique, using ammonium sulfate as the precipitating agent. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 56.85, b = 121.13, c = 94.09 Å, β = 101.48°, and contained one dimeric molecule in the asymmetric unit. It diffracted to 1.79 Å resolution.

  10. Cloning and characterization of dihydrofolate reductases from deep-sea bacteria.

    PubMed

    Murakami, Chiho; Ohmae, Eiji; Tate, Shin-Ichi; Gekko, Kunihiko; Nakasone, Kaoru; Kato, Chiaki

    2010-04-01

    Enzymes from organisms living in deep-sea are thought to have characteristic pressure-adaptation mechanisms in structure and function. To better understand these mechanisms in dihydrofolate reductase (DHFR), an essential enzyme in living cells, we cloned, overexpressed and purified four new DHFRs from the deep-sea bacteria Shewanella violacea (svDHFR), Photobacterium profundum (ppDHFR), Moritella yayanosii (myDHFR) and Moritella japonica (mjDHFR), and compared their structure and function with those of Escherichia coli DHFR (ecDHFR). These deep-sea DHFRs showed 33-56% primary structure identity to ecDHFR while far-ultraviolet circular dichroism and fluorescence spectra suggested that their secondary and tertiary structures were not largely different. The optimal temperature and pH for deep-sea DHFRs activity were lower than those of ecDHFR and different from each other. Deep-sea DHFRs kinetic parameters K(m) and k(cat) were larger than those of ecDHFR, resulting in 1.5-2.8-fold increase of k(cat)/K(m) except for mjDHFR which had a 28-fold decrease. The enzyme activity of ppDHFR and mjDHFR (moderate piezophilic bacteria) as well as ecDHFR decreased as pressure increased, while svDHFR and myDHFR (piezophilic bacteria) showed a significant tolerance to pressure. These results suggest that DHFRs from deep-sea bacteria possess specific enzymatic properties adapted to their life under high pressure. PMID:20040594

  11. Determination of primary combustion source organic carbon-to-elemental carbon (OC / EC) ratio using ambient OC and EC measurements: secondary OC-EC correlation minimization method

    NASA Astrophysics Data System (ADS)

    Wu, Cheng; Zhen Yu, Jian

    2016-05-01

    Elemental carbon (EC) has been widely used as a tracer to track the portion of co-emitted primary organic carbon (OC) and, by extension, to estimate secondary OC (SOC) from ambient observations of EC and OC. Key to this EC tracer method is to determine an appropriate OC / EC ratio that represents primary combustion emission sources (i.e., (OC / EC)pri) at the observation site. The conventional approaches include regressing OC against EC within a fixed percentile of the lowest (OC / EC) ratio data (usually 5-20 %) or relying on a subset of sampling days with low photochemical activity and dominated by local emissions. The drawback of these approaches is rooted in its empirical nature, i.e., a lack of clear quantitative criteria in the selection of data subsets for the (OC / EC)pri determination. We examine here a method that derives (OC / EC)pri through calculating a hypothetical set of (OC / EC)pri and SOC followed by seeking the minimum of the coefficient of correlation (R2) between SOC and EC. The hypothetical (OC / EC)pri that generates the minimum R2(SOC,EC) then represents the actual (OC / EC)pri ratio if variations of EC and SOC are independent and (OC / EC)pri is relatively constant in the study period. This Minimum R Squared (MRS) method has a clear quantitative criterion for the (OC / EC)pri calculation. This work uses numerically simulated data to evaluate the accuracy of SOC estimation by the MRS method and to compare with two commonly used methods: minimum OC / EC (OC / ECmin) and OC / EC percentile (OC / EC10 %). Log-normally distributed EC and OC concentrations with known proportion of SOC are numerically produced through a pseudorandom number generator. Three scenarios are considered, including a single primary source, two independent primary sources, and two correlated primary sources. The MRS method consistently yields the most accurate SOC estimation. Unbiased SOC estimation by OC / ECmin and OC / EC10 % only occurs when the left tail of

  12. The EC4 quality manual model.

    PubMed

    Queraltó, J M

    2001-07-20

    One of the priorities of the European Confederation of Clinical Chemistry (EC4) is the harmonisation of the clinical laboratory profession in Europe. One of the first steps is to try to harmonise the quality systems, that is, the clinical laboratory organisational structure, responsibilities, procedures, processes and resources involved in quality management. The "EC4 Essential Criteria" were published by the Working Group on Harmonisation of Quality Systems in order to facilitate the development or the update of a quality system in a clinical laboratory, and to encourage international bodies to produce specific Standards for the clinical laboratory. Furthermore, the EC4 Working Group has produced a Quality Manual Model, which includes a sample of quality policy documents and some operational directions for an imaginary laboratory. This Quality Manual Model was prepared following the "EC4 Essential Criteria." Its purpose is that any quality system developed following the Manual could be accredited or certified against any Standard. The EC4 Quality Manual Model will be available, free of charge, to clinical laboratory professionals. PMID:11438291

  13. Preliminary joint X-ray and neutron protein crystallographic studies of ecDHFR complexed with folate and NADP+

    PubMed Central

    Wan, Qun; Kovalevsky, Andrey Y.; Wilson, Mark A.; Bennett, Brad C.; Langan, Paul; Dealwis, Chris

    2014-01-01

    A crystal of Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP+ of 4 × 1.3 × 0.7 mm (3.6 mm3) in size was obtained by sequential application of microseeding and macroseeding. A neutron diffraction data set was collected to 2.0 Å resolution using the IMAGINE diffractometer at the High Flux Isotope Reactor within Oak Ridge National Laboratory. A 1.6 Å resolution X-ray data set was also collected from a smaller crystal at room temperature. The neutron and X-ray data were used together for joint refinement of the ecDHFR–folate–NADP+ ternary-complex structure in order to examine the protonation state, protein dynamics and solvent structure of the complex, furthering understanding of the catalytic mechanism. PMID:24915100

  14. A Scalability Model for ECS's Data Server

    NASA Technical Reports Server (NTRS)

    Menasce, Daniel A.; Singhal, Mukesh

    1998-01-01

    This report presents in four chapters a model for the scalability analysis of the Data Server subsystem of the Earth Observing System Data and Information System (EOSDIS) Core System (ECS). The model analyzes if the planned architecture of the Data Server will support an increase in the workload with the possible upgrade and/or addition of processors, storage subsystems, and networks. The approaches in the report include a summary of the architecture of ECS's Data server as well as a high level description of the Ingest and Retrieval operations as they relate to ECS's Data Server. This description forms the basis for the development of the scalability model of the data server and the methodology used to solve it.

  15. ECS - The European Communication Satellite system

    NASA Astrophysics Data System (ADS)

    Wooster, C. B.

    1981-09-01

    The evolution of the European Communication Satellite system (ECS) is traced from feasibility studies in 1970 to the development and launch in 1978 of the Orbital Test Satellite (OTS) by the European Space Agency to prove the new satellite and radio transmission technology being used on ECS. This was followed by the establishment of 'Interim EUTELSAT' in 1979 as the organization to operate ECS. The satellite, which operates at 11/14 GHz, covers all the capitals in Europe via three spot beam antennas, supplemented by a 'Eurobeam' regional coverage antenna which extends the range to cover all of Europe and the Mediterranean basin. Telephony channels are transmitted digitally using time division multiple access (TDMA) with digital speech interpolation (DSI) to optimize satellite capacity. Television transmission is by analog FM over the Eurobeam antenna to North African as well as European capitals. System implications of TDMA operation are discussed, and the EUTELSAT policy for Special Services or satellite business systems is discussed.

  16. Public Availability to ECS Collected Datasets

    NASA Astrophysics Data System (ADS)

    Henderson, J. F.; Warnken, R.; McLean, S. J.; Lim, E.; Varner, J. D.

    2013-12-01

    Coastal nations have spent considerable resources exploring the limits of their extended continental shelf (ECS) beyond 200 nm. Although these studies are funded to fulfill requirements of the UN Convention on the Law of the Sea, the investments are producing new data sets in frontier areas of Earth's oceans that will be used to understand, explore, and manage the seafloor and sub-seafloor for decades to come. Although many of these datasets are considered proprietary until a nation's potential ECS has become 'final and binding' an increasing amount of data are being released and utilized by the public. Data sets include multibeam, seismic reflection/refraction, bottom sampling, and geophysical data. The U.S. ECS Project, a multi-agency collaboration whose mission is to establish the full extent of the continental shelf of the United States consistent with international law, relies heavily on data and accurate, standard metadata. The United States has made it a priority to make available to the public all data collected with ECS-funding as quickly as possible. The National Oceanic and Atmospheric Administration's (NOAA) National Geophysical Data Center (NGDC) supports this objective by partnering with academia and other federal government mapping agencies to archive, inventory, and deliver marine mapping data in a coordinated, consistent manner. This includes ensuring quality, standard metadata and developing and maintaining data delivery capabilities built on modern digital data archives. Other countries, such as Ireland, have submitted their ECS data for public availability and many others have made pledges to participate in the future. The data services provided by NGDC support the U.S. ECS effort as well as many developing nation's ECS effort through the U.N. Environmental Program. Modern discovery, visualization, and delivery of scientific data and derived products that span national and international sources of data ensure the greatest re-use of data and

  17. EC Transmission Line Risk Identification and Analysis

    SciTech Connect

    Bigelow, Tim S

    2012-04-01

    The purpose of this document is to assist in evaluating and planning for the cost, schedule, and technical project risks associated with the delivery and operation of the EC (Electron cyclotron) transmission line system. In general, the major risks that are anticipated to be encountered during the project delivery phase associated with the implementation of the Procurement Arrangement for the EC transmission line system are associated with: (1) Undefined or changing requirements (e.g., functional or regulatory requirements) (2) Underperformance of prototype, first unit, or production components during testing (3) Unavailability of qualified vendors for critical components Technical risks associated with the design and operation of the system are also identified.

  18. Methylenetetrahydrofolate reductase deficiency: importance of early diagnosis.

    PubMed

    Fattal-Valevski, A; Bassan, H; Korman, S H; Lerman-Sagie, T; Gutman, A; Harel, S

    2000-08-01

    Methylenetetrahydrofolate reductase deficiency is the most common inborn error of folate metabolism and should be suspected when homocystinuria is combined with hypomethioninemia. The main clinical findings are neurologic signs such as severe developmental delay, marked hypotonia, seizures, microcephaly, apnea, and coma. Most patients present in early life. The infantile form is severe, with rapid deterioration leading to death usually within 1 year. Treatment with betaine has been shown to be efficient in lowering homocysteine concentrations and returning methionine to normal, but the clinical response is variable. We report two brothers with methylenetetrahydrofolate reductase deficiency: the first was undiagnosed and died at 8 months of age from neurologic deterioration and apnea, while his brother, who was treated with betaine from the age of 4 months, is now 3 years old and has developmental delay. PMID:10961793

  19. Characterization of erythrose reductases from filamentous fungi.

    PubMed

    Jovanović, Birgit; Mach, Robert L; Mach-Aigner, Astrid R

    2013-01-01

    Proteins with putative erythrose reductase activity have been identified in the filamentous fungi Trichoderma reesei, Aspergillus niger, and Fusarium graminearum by in silico analysis. The proteins found in T. reesei and A. niger had earlier been characterized as glycerol dehydrogenase and aldehyde reductase, respectively. Corresponding genes from all three fungi were cloned, heterologously expressed in Escherichia coli, and purified. Subsequently, they were used to establish optimal enzyme assay conditions. All three enzymes strictly require NADPH as cofactor, whereas with NADH no activity could be observed. The enzymatic characterization of the three enzymes using ten substrates revealed high substrate specificity and activity with D-erythrose and D-threose. The enzymes from T. reesei and A. niger herein showed comparable activities, whereas the one from F. graminearum reached only about a tenth of it for all tested substrates. In order to proof in vivo the proposed enzyme function, we overexpressed the erythrose reductase-encoding gene in T. reesei. An increased production of erythritol by the recombinant strain compared to the parental strain could be detected. PMID:23924507

  20. EC Detector at SciBooNE

    NASA Astrophysics Data System (ADS)

    Mariani, Camillo

    2009-04-01

    SciBooNE is an experiment to measure neutrino and anti-neutrino cross-sections on the Booster Neutrino Beam at Fermilab. The EC is an extruded lead sheets and scintillating fibers "spaghetti calorimeter" to provide longitudinal containment and energy measurement for electrons and photons.

  1. Grout Analysis for EC and CC Calorimeters

    SciTech Connect

    Engstrom, L.L.; /Fermilab

    1987-01-06

    The EC and CC calorimeters roll on Two parallel hardened steel ways which reside on the top of the D0 platform's center beam. The ways will be grouted to the center beam once their correct elevation has been established. The purpose of this report is to evaluate and compare three different epoxy grouts and their properties for this application.

  2. Km and kcat. values for [6,6,7,7-2H]7,8(6H)-dihydropterin and 2,6-diamino-5-iminopyrimidin-4-one with dihydropteridine reductase.

    PubMed Central

    Armarego, W L; Randles, D; Taguchi, H

    1983-01-01

    The Km and kcat. values for [6,6,7,7-2H]7,8(6H)-dihydropterin and 2,6-diamino-5-iminopyrimidin-4-one were determined for dihydropteridine reductase (EC 1.6.99.10) from two sources. The parameters of the pterin are of the same order as those of the most effective substrates of dihydropteridine reductase. The Km values of the pterin are one order of magnitude smaller than those of the pyrimidinone, although the kcat. values are of the same order. PMID:6870836

  3. Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

    PubMed Central

    Ma, Yan; Ludden, Paul W.

    2001-01-01

    Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding. PMID:11114923

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

    PubMed

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

    1988-11-25

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

  5. Low resolution solution structure of an enzymatic active AhpC10:AhpF2 ensemble of the Escherichia coli Alkyl hydroperoxide Reductase.

    PubMed

    Kamariah, Neelagandan; Nartey, Wilson; Eisenhaber, Birgit; Eisenhaber, Frank; Grüber, Gerhard

    2016-01-01

    The ability of bacteria to combat oxidative stress is imperative for their survival. The Alkyl hydroperoxide Reductase (AhpR) system, composed of the AhpC and AhpF proteins, is one of the dominant antioxidant defense systems required for scavenging hydrogen peroxide and organic peroxide. Therefore, it is necessary to understand the mechanism of the AhpR ensemble formation. In previous studies, we were able to elucidate conformational flexibility of Escherichia coli AhpF during the catalytic cycle and its binding site, the N-terminal domain (NTD), to AhpC. We proposed the novel binding and release mechanism of EcAhpC-AhpF, which is mediated by the well defined redox-state linked conformational changes associated with the C-terminal tail and active site regions of EcAhpC. Here, we have proceeded further to elucidate the solution structure of E. coli AhpC and the stable ensemble formation with EcAhpF using size-exclusion chromatography (SEC), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) techniques. The EcAhpC-AhpF complex structure with a stoichiometry of AhpC10:AhpF2 reveals that dimeric EcAhpF in its extended conformation enables the NTD disulphide centers to come in close proximity to the redox-active disulphide centers of EcAhpC, and provides an efficient electron transfer. Furthermore, the significance of the C-terminal tail of EcAhpC in ensemble formation is elucidated. SAXS data-based modeling revealed the flexible C-terminal tail of EcAhpC in solution, and its exposed nature, making it possible to contact the NTD of EcAhpF for stable complex formation. PMID:26584540

  6. The regulation of gelation of Phloem exudate from cucurbita fruit by dilution, glutathione, and glutathione reductase.

    PubMed

    Alosi, M C; Melroy, D L; Park, R B

    1988-04-01

    The average glutathione equivalent concentration in phloem exudate collected from squash fruit (Cucurbita moschata [Duchesne] Poir. var Butternut) and pumpkin fruit (Cucurbita pepo [L.] var Jack-o-lattern) was 1.02 and 0.60 millimolar, respectively. Glutathione reductase (EC 1.6.4.2) activity in phloem exudate from squash and pumpkin fruit averaged 0.48 and 1.74 micromole NADPH oxidized per minute per milliliter, respectively. Protein concentrations in fruit phloem exudates averaged 67 milligrams per milliliter for squash and 57 milligrams per milliliter for pumpkin. The phloem-specific P-proteins account for most of the protein content of exudate. Pure exudate from fruit does not gel for hours or days, but when diluted with neutral or alkaline aqueous solutions, exudate gels rapidly. Exudate solutions undergo biphasic pH changes with dilution. We suggest that P-protein undergoes conformational change upon dilution, exposing titratable groups and sulfhydryl residues. Oxidation of the latter forms the intermolecular disulfide bridges of the gel. The gelation of diluted exudate is regulated by factors (oxygen, pH, glutathione, NADPH) which affect the maintenance of reduced sulfhydryl residues and the activity of glutathione reductase. While these factors may also act in vivo to regulate redox conditions in phloem, their relationship to hypothetical sol/gel transitions or motile and nonmotile phases in the transport conduit is unknown. PMID:16666036

  7. Complementation cloning and characterization of the pyrroline 5-carboxylate reductase gene from Drosophila melanogaster.

    PubMed

    Misener, S R; Walker, V K

    2001-02-01

    The first insect cDNA and genomic sequences encoding pyrroline 5-carboxylate reductase (EC 1.5.1.2) have been isolated from Drosophila melanogaster. The cDNA sequence was identified by interspecies complementation of an E. coli proline auxotroph and encodes a protein 280 amino acids in length with 25-41% identity to pyrroline 5-carboxylate reductases isolated from other organisms. The corresponding gene is single copy and is located at cytological position 91E-F, and in one of the P1 clones in that region. With a single 61-bp intron, and an impressively small 135- to 200-bp region that presumably acts as a bidirectional promoter, the gene itself shows remarkable economy. The calculated molecular weight of 29,700 predicts that the native enzyme is likely an octomer. Sequencing of the promoter region and expression studies, as well as the known function of the enzyme in redox regulation and the high levels of free proline in insects, suggest that this housekeeping gene encodes an enzyme with a crucial role in intermediary metabolism. PMID:11444018

  8. Spatial distribution and seasonal variation of char-EC and soot-EC in the atmosphere over China

    NASA Astrophysics Data System (ADS)

    Han, Y. M.; Lee, S. C.; Cao, J. J.; Ho, K. F.; An, Z. S.

    2009-12-01

    A previous study on PM 2.5 carbonaceous aerosols measured with the thermal optical reflectance (TOR) method in fourteen Chinese cities is extended by subdividing total EC into char-EC and soot-EC. Average char-EC concentrations show great differences between the fourteen cities and between winter and summer periods, with concentrations of 8.67 and 2.41 μg m -3 in winter and summer, respectively. Meanwhile spatial and seasonal soot-EC variations are small, with average concentrations of 1.26 and 1.21 μg m -3 in winter and summer, respectively. Spatial and temporal distributions of char-EC, similar to EC, are mainly influenced by local fuel consumption, as well as the East Asian monsoon and some meteorological factors such as the mixing height and wet precipitation. The small spatial and seasonal variation of soot-EC is consistent with its regional-to-global dispersion, which may suggest that soot carbon is not local carbon, but regional carbon. Char-EC/soot-EC ratios show summer minimum and winter maximum in all cities, which is in good agreement with the difference in source contributions between the two periods. As OC/EC ratio is affected by the formation of the secondary organic aerosol (SOA), char-EC/soot-EC ratio is a more effective indicator for source identification of carbonaceous aerosol than previously used OC/EC ratio.

  9. Crystallization and preliminary X-ray analysis of enoyl-acyl carrier protein reductase (FabK) from Streptococcus pneumoniae

    SciTech Connect

    Saito, Jun Yamada, Mototsugu; Watanabe, Takashi; Kitagawa, Hideo; Takeuchi, Yasuo

    2006-06-01

    Enoyl-acyl carrier protein (ACP) reductases are responsible for bacterial type II fatty-acid biosynthesis and are attractive targets for developing novel antibiotics. The S. pneumoniae enoyl-ACP reductase (FabK) was crystallized and selenomethionine MAD data were collected to 2 Å resolution. The enoyl-acyl carrier protein (ACP) reductase from Streptococcus pneumoniae (FabK; EC 1.3.1.9) is responsible for catalyzing the final step in each elongation cycle of fatty-acid biosynthesis. Selenomethionine-substituted FabK was purified and crystallized by the hanging-drop vapour-diffusion method at 277 K. The crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 50.26, b = 126.70, c = 53.63 Å, β = 112.46°. Diffraction data were collected to 2.00 Å resolution using synchrotron beamline BL32B2 at SPring-8. Two molecules were estimated to be present in the asymmetric unit, with a solvent content of 45.1%.

  10. Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains.

    PubMed

    Crawford, N M; Smith, M; Bellissimo, D; Davis, R W

    1988-07-01

    The sequence of nitrate reductase (EC 1.6.6.1) mRNA from the plant Arabidopsis thaliana has been determined. A 3.0-kilobase-long cDNA was isolated from a lambda gt10 cDNA library of Arabidopsis leaf poly(A)+ RNA. The cDNA hybridized to a 3.2-kilobase mRNA whose level increased 15-fold in response to treatment of the plant with nitrate. An open reading frame encoding a 917 amino acid protein was found in the sequence. This protein is very similar to tobacco nitrate reductase, being greater than 80% identical within a section of 450 amino acids. By comparing the Arabidopsis protein sequence with other protein sequences, three functional domains were deduced: (i) a molybdenum-pterin-binding domain that is similar to the molybdenum-pterin-binding domain of rat liver sulfite oxidase, (ii) a heme-binding domain that is similar to proteins in the cytochrome b5 superfamily, and (iii) an FAD-binding domain that is similar to NADH-cytochrome b5 reductase. PMID:3393528

  11. Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

    SciTech Connect

    Iyanagi, Takashi . E-mail: iyanagi@spring8.or.jp

    2005-12-09

    NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH {sup {center_dot}}/FMNH{sub 2} couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.

  12. Preliminary conceptual design of DEMO EC system

    SciTech Connect

    Garavaglia, S. Bin, W.; Bruschi, A.; Granucci, G.; Moro, A.; Rispoli, N.; Grossetti, G.; Strauss, D.; Jelonnek, J.; Tran, Q. M.; Franke, T.

    2015-12-10

    In the framework of EUROfusion Consortium the Work Package Heating and Current Drive addresses the engineering design and R&D for the electron cyclotron, ion cyclotron and neutral beam systems. This paper reports the activities performed in 2014, focusing on the work done regarding the input for the conceptual design of the EC system, particularly for the gyrotron, the transmission line and the launchers.

  13. Preliminary conceptual design of DEMO EC system

    NASA Astrophysics Data System (ADS)

    Garavaglia, S.; Bin, W.; Bruschi, A.; Granucci, G.; Grossetti, G.; Jelonnek, J.; Moro, A.; Rispoli, N.; Strauss, D.; Tran, Q. M.; Franke, T.

    2015-12-01

    In the framework of EUROfusion Consortium the Work Package Heating and Current Drive addresses the engineering design and R&D for the electron cyclotron, ion cyclotron and neutral beam systems. This paper reports the activities performed in 2014, focusing on the work done regarding the input for the conceptual design of the EC system, particularly for the gyrotron, the transmission line and the launchers.

  14. Reduction of tetrathionate by mammalian thioredoxin reductase

    PubMed Central

    Narayan, Vivek; Kudva, Avinash K.; Prabhu, K. Sandeep

    2016-01-01

    Tetrathionate, a polythionate oxidation product of microbial hydrogen sulfide and reactive oxygen species from immune cells in the gut, serves as a terminal electron acceptor to confer growth advantage for Salmonella and other enterobacteria. Here we show that the rat liver selenoen-zyme thioredoxin reductase (Txnrd1; TR1) efficiently reduces tetrathionate in vitro. Furthermore, lysates of selenium-supplemented murine macrophages also displayed activity towards tetrathionate, while cells lacking TR1 were unable to reduce tetrathionate. These studies suggest that upregulation of TR1 expression, via selenium supplementation, may modulate the gut microbiome, particularly during inflammation, by regulating the levels of tetrathionate. PMID:26252619

  15. Biliverdin reductase: a target for cancer therapy?

    PubMed Central

    Gibbs, Peter E. M.; Miralem, Tihomir; Maines, Mahin D.

    2015-01-01

    Biliverdin reductase (BVR) is a multifunctional protein that is the primary source of the potent antioxidant, bilirubin. BVR regulates activities/functions in the insulin/IGF-1/IRK/PI3K/MAPK pathways. Activation of certain kinases in these pathways is/are hallmark(s) of cancerous cells. The protein is a scaffold/bridge and intracellular transporter of kinases that regulate growth and proliferation of cells, including PKCs, ERK and Akt, and their targets including NF-κB, Elk1, HO-1, and iNOS. The scaffold and transport functions enable activated BVR to relocate from the cytosol to the nucleus or to the plasma membrane, depending on the activating stimulus. This enables the reductase to function in diverse signaling pathways. And, its expression at the transcript and protein levels are increased in human tumors and the infiltrating T-cells, monocytes and circulating lymphocytes, as well as the circulating and infiltrating macrophages. These functions suggest that the cytoprotective role of BVR may be permissive for cancer/tumor growth. In this review, we summarize the recent developments that define the pro-growth activities of BVR, particularly with respect to its input into the MAPK signaling pathway and present evidence that BVR-based peptides inhibit activation of protein kinases, including MEK, PKCδ, and ERK as well as downstream targets including Elk1 and iNOS, and thus offers a credible novel approach to reduce cancer cell proliferation. PMID:26089799

  16. Flavodiiron Oxygen Reductase from Entamoeba histolytica

    PubMed Central

    Gonçalves, Vera L.; Vicente, João B.; Pinto, Liliana; Romão, Célia V.; Frazão, Carlos; Sarti, Paolo; Giuffrè, Alessandro; Teixeira, Miguel

    2014-01-01

    Flavodiiron proteins (FDPs) are a family of enzymes endowed with bona fide oxygen- and/or nitric-oxide reductase activity, although their substrate specificity determinants remain elusive. After a comprehensive comparison of available three-dimensional structures, particularly of FDPs with a clear preference toward either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Tyr271 and Lys53 in the oxygen reducing Entamoeba histolytica EhFdp1. The biochemical and biophysical properties of these mutants were studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies coupled to potentiometry. Their reactivity with O2 and NO was analyzed by stopped-flow absorption spectroscopy and amperometric methods. These mutations, whereas keeping the overall properties of the redox cofactors, resulted in increased NO reductase activity and faster inactivation of the enzyme in the reaction with O2, pointing to a role of the mutated residues in substrate selectivity. PMID:25151360

  17. Preliminary neutron diffraction studies of Escherichia coli dihydrofolate reductase bound to the anticancer drug methotrexate

    SciTech Connect

    Bennett, Brad C.; Meilleur, Flora; Myles, Dean A A; Howell, Elizabeth E.; Dealwis, Chris G.

    2005-01-01

    The contribution of H atoms in noncovalent interactions and enzymatic reactions underlies virtually all aspects of biology at the molecular level, yet their 'visualization' is quite difficult. To better understand the catalytic mechanism of Escherichia coli dihydrofolate reductase (ecDHFR), a neutron diffraction study is under way to directly determine the accurate positions of H atoms within its active site. Despite exhaustive investigation of the catalytic mechanism of DHFR, controversy persists over the exact pathway associated with proton donation in reduction of the substrate, dihydrofolate. As the initial step in a proof-of-principle experiment which will identify ligand and residue protonation states as well as precise solvent structures, a neutron diffraction data set has been collected on a 0.3 mm{sup 3} D{sub 2}O-soaked crystal of ecDHFR bound to the anticancer drug methotrexate (MTX) using the LADI instrument at ILL. The completeness in individual resolution shells dropped to below 50% between 3.11 and 3.48 {angstrom} and the I/{sigma}(I) in individual shells dropped to below 2 at around 2.46 {angstrom}. However, reflections with I/{sigma}(I) greater than 2 were observed beyond these limits (as far out as 2.2 {angstrom}). To our knowledge, these crystals possess one of the largest primitive unit cells (P6{sub 1}, a = b = 92, c = 73 {angstrom}) and one of the smallest crystal volumes so far tested successfully with neutrons.

  18. Is EC class predictable from reaction mechanism?

    PubMed Central

    2012-01-01

    Background We investigate the relationships between the EC (Enzyme Commission) class, the associated chemical reaction, and the reaction mechanism by building predictive models using Support Vector Machine (SVM), Random Forest (RF) and k-Nearest Neighbours (kNN). We consider two ways of encoding the reaction mechanism in descriptors, and also three approaches that encode only the overall chemical reaction. Both cross-validation and also an external test set are used. Results The three descriptor sets encoding overall chemical transformation perform better than the two descriptions of mechanism. SVM and RF models perform comparably well; kNN is less successful. Oxidoreductases and hydrolases are relatively well predicted by all types of descriptor; isomerases are well predicted by overall reaction descriptors but not by mechanistic ones. Conclusions Our results suggest that pairs of similar enzyme reactions tend to proceed by different mechanisms. Oxidoreductases, hydrolases, and to some extent isomerases and ligases, have clear chemical signatures, making them easier to predict than transferases and lyases. We find evidence that isomerases as a class are notably mechanistically diverse and that their one shared property, of substrate and product being isomers, can arise in various unrelated ways. The performance of the different machine learning algorithms is in line with many cheminformatics applications, with SVM and RF being roughly equally effective. kNN is less successful, given the role that non-local information plays in successful classification. We note also that, despite a lack of clarity in the literature, EC number prediction is not a single problem; the challenge of predicting protein function from available sequence data is quite different from assigning an EC classification from a cheminformatics representation of a reaction. PMID:22530800

  19. Permeation of Telone EC through protective gloves.

    PubMed

    Zainal, Hanaa; Que Hee, Shane S

    2005-09-30

    Telone is a potent fumigant that is based on the chlorinated unsaturated hydrocarbon, 1,3-dichloropropene (1,3-DCP). It is often applied without dilution and so poses severe inhalation and air pollution threats. Urinary metabolites of 1,3-DCP have been detected after Telone skin exposure, so that preventing dermal exposure is also important. The objective of the study was to assess if nitrile and multi-layer ("laminated") gloves provide adequate protection against Telone EC formulation. To accomplish this, disposable (Safeskin) and chemically resistant (Sol-Vex) nitrile and laminated (Barrier mark and Silver Shield) glove materials were challenged by Telone EC with hexane liquid collection in an ASTM-type I-PTC-600 permeation cell. Analyses of cis- and trans-1,3-DCP in the collection fluid at specified times were performed on a moderately polar capillary column by gas chromatography-electron capture detection. Telone EC caused microholes in both nitrile materials, though the chemically protective material was degraded slower than the disposable nitrile. The laminated gloves offered limited protection. Silver Shield protected best because 1.5-2.3 mg 1,3-DCP permeated by 8 h relative to 2.5-7.6 mg for Barrier, implying about 2.5 times more protection for 8 h. Even for Silver Shield, the extent of protection was inadequate as illustrated by a risk assessment of the skin exposure situation. The normalized breakthrough times for both types of laminated gloves varied between 27 and 60 min. It is recommended that Viton gloves still be worn for protection. PMID:15982807

  20. Summary of ECE Presentations at EC-15

    SciTech Connect

    Caughman, John B

    2009-01-01

    The presentations in the area of electron cyclotron emission continued a tradition of high quality and variety for EC-15. There were a total of 20 presentations/posters in this area. The topics included a review of the history of ECE diagnostics and modeling, unresolved issues in the area of temperature measurements via ECE compared to Thomson Scattering, and many applications of ECE for understanding plasma physics in fusion experiments, including ITER. ECE is being used to study temperature fluctuations, ELMs, MHD instabilities, transport, and feedback control of tearing modes. In addition, the emission of electron Bernstein waves is also being used to understand mode conversion physics on several experiments.

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

    SciTech Connect

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

    1997-01-01

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

  2. Transcripts of anthocyanidin reductase and leucoanthocyanidin reductase and measurement of catechin and epicatechin in tartary buckwheat.

    PubMed

    Kim, Yeon Bok; Thwe, Aye Aye; Kim, Yeji; Li, Xiaohua; Cho, Jin Woong; Park, Phun Bum; Valan Arasu, Mariadhas; Abdullah Al-Dhabi, Naif; Kim, Sun-Ju; Suzuki, Tastsuro; Hyun Jho, Kwang; Park, Sang Un

    2014-01-01

    Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions. PMID:24605062

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

    PubMed Central

    Kim, Yeon Bok; Thwe, Aye Aye; Kim, YeJi; Li, Xiaohua; Cho, Jin Woong; Park, Phun Bum; Valan Arasu, Mariadhas; Abdullah Al-Dhabi, Naif; Kim, Sun-Ju; Suzuki, Tastsuro; Hyun Jho, Kwang; Park, Sang Un

    2014-01-01

    Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions. PMID:24605062

  4. Chaperone properties of Escherichia coli thioredoxin and thioredoxin reductase.

    PubMed Central

    Kern, Renée; Malki, Abderrahim; Holmgren, Arne; Richarme, Gilbert

    2003-01-01

    Thioredoxin, thioredoxin reductase and NADPH form the thioredoxin system and are the major cellular protein disulphide reductase. We report here that Escherichia coli thioredoxin and thioredoxin reductase interact with unfolded and denatured proteins, in a manner similar to that of molecular chaperones that are involved in protein folding and protein renaturation after stress. Thioredoxin and/or thioredoxin reductase promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. They also promote the functional folding of the bacterial galactose receptor, a protein without any cysteines. Furthermore, redox cycling of thioredoxin/thioredoxin reductase in the presence of NADPH and cystine stimulates the renaturation of the galactose receptor, suggesting that the thioredoxin system functions like a redox-powered chaperone machine. Thioredoxin reductase prevents the aggregation of citrate synthase under heat-shock conditions. It forms complexes that are more stable than those formed by thioredoxin with several unfolded proteins such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. These results suggest that the thioredoxin system, in addition to its protein disulphide isomerase activity possesses chaperone-like properties, and that its thioredoxin reductase component plays a major role in this function. PMID:12549977

  5. Ec-135 Fiber Optic Technology Review

    NASA Astrophysics Data System (ADS)

    Schultz, Jan R...; Hodges, Harry N.

    1984-10-01

    Fiber optic technology offers many advantages for upgrading nuclear survivability in systems such as the Airborne Command Post EC-135 aircraft, including weight and cost savings, EMI and EMC immunity, high data rates. The greatest advantage seen for nuclear survivable systems, however, is that a fiber optic system's EMP hardness can be maintained more easily with the use of fiber optics than with shielded cables or other protective methods. TRW recently completed a study to determine the feasibility of using fiber optic technology in an EC-135 aircraft environment. Since this study was conducted for a USAF Logistics Command Agency, a feasible system had to be one which could be realistically priced by an integrating contractor. Thus, any fiber optic approach would have to be well developed before it could be considered feasible. During the course of the study problem areas were encountered which are associated with the readiness of the technology for use rather than with the technology itself. These included connectors, standards, fiber radiation resistance, busing, maintenance, and logistics. Because these problems areas have not been resolved, it was concluded that fiber optic technology, despite its advantages, is not ready for directed procurement (i.e., included as a requirement in a prime mission equipment specification). However, offers by a manufacturer to use fiber optic technology in lieu of conventional technology should be considered. This paper treats these problems in more detail, addresses the areas which need further development, and discusses the hardness maintenance advantages of using fiber optic technology.

  6. Ribonucleotide reductase metallocofactor: assembly, maintenance and inhibition

    PubMed Central

    ZHANG, Caiguo; LIU, Guoqi; HUANG, Mingxia

    2014-01-01

    Ribonucleotide reductase (RNR) supplies cellular deoxyribonucleotide triphosphates (dNTP) pools by converting ribonucleotides to the corresponding deoxy forms using radical-based chemistry. Eukaryotic RNR comprises α and β subunits: α contains the catalytic and allosteric sites; β houses a diferric-tyrosyl radical cofactor (FeIII2-Y•) that is required to initiates nucleotide reduction in α. Cells have evolved multi-layered mechanisms to regulate RNR level and activity in order to maintain the adequate sizes and ratios of their dNTP pools to ensure high-fidelity DNA replication and repair. The central role of RNR in nucleotide metabolism also makes it a proven target of chemotherapeutics. In this review, we discuss recent progress in understanding the function and regulation of eukaryotic RNRs, with a focus on studies revealing the cellular machineries involved in RNR metallocofactor biosynthesis and its implication in RNR-targeting therapeutics. PMID:24899886

  7. Dynamics of trimethoprim bound to dihydrofolate reductase

    SciTech Connect

    Searle, M.S.; Forster, M.J.; Birdsall, B.; Roberts, G.C.K.; Feeney, J.; Cheung, H.T.A.; Kompis, I.; Geddes, A.J. )

    1988-06-01

    The conformation of a small molecule in its binding site on a protein is a major factor in the specificity of the interaction between them. In this paper, the authors report the use of {sup 1}H and {sup 13}C NMR spectroscopy to study the fluctuations in conformation of the anti-bacterial drug trimethoprim when it is bound to its target, dihydrofolate reductase. {sup 13}C relaxation measurements reveal dihedral angle changes of {plus minus}25{degree} to {plus minus}35{degree} on the subnanosecond time scale, while {sup 13}C line-shape analysis demonstrates dihedral angle changes of at least {plus minus}65{degree} on the millisecond time scale. {sup 1}H NMR shows that a specific hydrogen bond between the inhibitor and enzyme, which is believed to make an important contribution to binding, makes and breaks rapidly at room temperature.

  8. Nitrite Reductase Activity in Engineered Azurin Variants.

    PubMed

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

    2016-05-01

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

  9. Molecular evolution of nitrate reductase genes.

    PubMed

    Zhou, J; Kleinhofs, A

    1996-04-01

    To understand the evolutionary mechanisms and relationships of nitrate reductases (NRs), the nucleotide sequences encoding 19 nitrate reductase (NR) genes from 16 species of fungi, algae, and higher plants were analyzed. The NR genes examined show substantial sequence similarity, particularly within functional domains, and large variations in GC content at the third codon position and intron number. The intron positions were different between the fungi and plants, but conserved within these groups. The overall and nonsynonymous substitution rates among fungi, algae, and higher plants were estimated to be 4.33 x 10(-10) and 3.29 x 10(-10) substitutions per site per year. The three functional domains of NR genes evolved at about one-third of the rate of the N-terminal and the two hinge regions connecting the functional domains. Relative rate tests suggested that the nonsynonymous substitution rates were constant among different lineages, while the overall nucleotide substitution rates varied between some lineages. The phylogenetic trees based on NR genes correspond well with the phylogeny of the organisms determined from systematics and other molecular studies. Based on the nonsynonymous substitution rate, the divergence time of monocots and dicots was estimated to be about 340 Myr when the fungi-plant or algae-higher plant divergence times were used as reference points and 191 Myr when the rice-barley divergence time was used as a reference point. These two estimates are consistent with other estimates of divergence times based on these reference points. The lack of consistency between these two values appears to be due to the uncertainty of the reference times. PMID:8642612

  10. The cytochrome bd respiratory oxygen reductases

    PubMed Central

    Borisov, Vitaliy B.; Gennis, Robert B.; Hemp, James; Verkhovsky, Michael I.

    2011-01-01

    Summary 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 active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated. PMID:21756872

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

    PubMed

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

    2012-10-01

    Current generation by mediated enzyme electron transfer at electrode surfaces can be harnessed to provide biosensors and redox reactions in enzymatic fuel cells. A glucose/oxygen enzymatic fuel cell can provide power for portable and implantable electronic devices. High volume production of enzymatic fuel cell prototypes will likely require printing of electrode and catalytic materials. Here we report on preparation and performance of, completely enzymatic, printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks, enzyme and mediator. A comparison of cell performance using a range of mediators for either glucose oxidase (GOx) or aldose dehydrogenase (ALDH) oxidation of glucose at the anode and ABTS and a fungal laccase, for reduction of oxygen at the cathode, is reported. Highest power output, although of limited stability, is observed for ALDH anodes mediated by an osmium complex, providing a maximum power density of 3.5 μW cm(-2) at 0.34 V, when coupled to a laccase/ABTS cathode. The stability of cell voltage in a biobattery format, above a threshold of 200 mV under a moderate 75 kΩ load, is used to benchmark printed fuel cell performance. Highest stability is obtained for printed fuel cells using ALDH, providing cell voltages over the threshold for up to 74 h, compared to only 2 h for cells with anodes using GOx. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells. PMID:22200380

  12. Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase

    PubMed Central

    Erb, Tobias J.; Brecht, Volker; Fuchs, Georg; Müller, Michael; Alber, Birgit E.

    2009-01-01

    Chemo- and stereoselective reductions are important reactions in chemistry and biology, and reductases from biological sources are increasingly applied in organic synthesis. In contrast, carboxylases are used only sporadically. We recently described crotonyl-CoA carboxylase/reductase, which catalyzes the reduction of (E)-crotonyl-CoA to butyryl-CoA but also the reductive carboxylation of (E)-crotonyl-CoA to ethylmalonyl-CoA. In this study, the complete stereochemical course of both reactions was investigated in detail. The pro-(4R) hydrogen of NADPH is transferred in both reactions to the re face of the C3 position of crotonyl-CoA. In the course of the carboxylation reaction, carbon dioxide is incorporated in anti fashion at the C2 atom of crotonyl-CoA. For the reduction reaction that yields butyryl-CoA, a solvent proton is added in anti fashion instead of the CO2. Amino acid sequence analysis showed that crotonyl-CoA carboxylase/reductase is a member of the medium-chain dehydrogenase/reductase superfamily and shares the same phylogenetic origin. The stereospecificity of the hydride transfer from NAD(P)H within this superfamily is highly conserved, although the substrates and reduction reactions catalyzed by its individual representatives differ quite considerably. Our findings led to a reassessment of the stereospecificity of enoyl(-thioester) reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Further considerations on the reaction mechanism indicated that crotonyl-CoA carboxylase/reductase may have evolved from enoyl-CoA reductases. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis. PMID:19458256

  13. Crystallographic and solution studies of NAD(+)- and NADH-bound alkylhydroperoxide reductase subunit F (AhpF) from Escherichia coli provide insight into sequential enzymatic steps.

    PubMed

    Kamariah, Neelagandan; Manimekalai, Malathy Sony Subramanian; Nartey, Wilson; Eisenhaber, Frank; Eisenhaber, Birgit; Grüber, Gerhard

    2015-10-01

    Redox homeostasis is significant for the survival of pro- and eukaryotic cells and is crucial for defense against reactive oxygen species like superoxide and hydrogen peroxide. In Escherichia coli, the reduction of peroxides occurs via the redox active disulfide center of the alkyl hydroperoxide reductase C subunit (AhpC), whose reduced state becomes restored by AhpF. The 57kDa EcAhpF contains an N-terminal domain (NTD), which catalyzes the electron transfer from NADH via an FAD of the C-terminal domain into EcAhpC. The NTD is connected to the C-terminal domain via a linker. Here, the first crystal structure of E. coli AhpF bound with NADH and NAD(+) has been determined at 2.5Å and 2.4Å resolution, respectively. The NADH-bound form of EcAhpF reveals that the NADH-binding domain is required to alter its conformation to bring a bound NADH to the re-face of the isoalloxazine ring of the flavin, and thereby render the NADH-domain dithiol center accessible to the NTD disulfide center for electron transfer. The NAD(+)-bound form of EcAhpF shows conformational differences for the nicotinamide end moieties and its interacting residue M467, which is proposed to represent an intermediate product-release conformation. In addition, the structural alterations in EcAhpF due to NADH- and NAD(+)-binding in solution are shown by small angle X-ray scattering studies. The EcAhpF is revealed to adopt many intermediate conformations in solution to facilitate the electron transfer from the substrate NADH to the C-terminal domain, and subsequently to the NTD of EcAhpF for the final step of AhpC reduction. PMID:26092085

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

    NASA Technical Reports Server (NTRS)

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

    1984-01-01

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

  15. Summary of ECE Presentations at EC-18

    NASA Astrophysics Data System (ADS)

    Taylor, G.

    2015-03-01

    There were nine ECE and one EBE presentation at EC-18. Four of the presentations were on various aspects of ECE on ITER. The ITER ECE diagnostic has entered an important detailed preliminary design phase and faces several design challenges in the next 2-3 years. Most of the other ECE presentations at the workshop were focused on applications of ECE diagnostics to plasma measurements, rather than improvements in technology, although it was apparent that heterodyne receiver technology continues to improve. CECE, ECE imaging and EBE imaging are increasingly providing valuable insights into plasma behavior that is important to understand if future burning plasma devices, such as ITER, FNSF and DEMO, are to be successful.

  16. Decadal Prediction Experiments using EC-EARTH

    NASA Astrophysics Data System (ADS)

    Wouters, Bert; Hazeleger, Wilco; van Oldenborgh, Geert Jan

    2010-05-01

    We present the first results of decadal prediction experiments with EC-EARTH 2.1 in the framework of the EU-THOR project. This model consists of the ECMWF IFSc31 model at T159/L62 resolution, the NEMO2 ocean model at 1° resolution and the LIM2 sea-ice model. The purpose is to predict decadal variability in the Atlantic Ocean and the resulting predictability in the weather at these time scales. As expected, the model shows a bias in the first years due to the initialization shock from the full initial state (NEMOVAR), but stabilizes afterwards. The bias and first estimates of the skill are shown for a partial CMIP5 ensemble, covering ocean, surface and atmospheric components of the coupled model.

  17. Summary of ECE presentations at EC-18

    DOE PAGESBeta

    Taylor, G.

    2015-03-12

    There were nine ECE and one EBE presentation at EC-18. Four of the presentations were on various aspects of ECE on ITER. The ITER ECE diagnostic has entered an important detailed preliminary design phase and faces several design challenges in the next 2-3 years. Most of the other ECE presentations at the workshop were focused on applications of ECE diagnostics to plasma measurements, rather than improvements in technology, although it was apparent that heterodyne receiver technology continues to improve. CECE, ECE imaging and EBE imaging are increasingly providing valuable insights into plasma behavior that is important to understand if futuremore » burning plasma devices, such as ITER, FNSF and DEMO, are to be successful.« less

  18. Summary of ECE presentations at EC-18

    SciTech Connect

    Taylor, G.

    2015-03-12

    There were nine ECE and one EBE presentation at EC-18. Four of the presentations were on various aspects of ECE on ITER. The ITER ECE diagnostic has entered an important detailed preliminary design phase and faces several design challenges in the next 2-3 years. Most of the other ECE presentations at the workshop were focused on applications of ECE diagnostics to plasma measurements, rather than improvements in technology, although it was apparent that heterodyne receiver technology continues to improve. CECE, ECE imaging and EBE imaging are increasingly providing valuable insights into plasma behavior that is important to understand if future burning plasma devices, such as ITER, FNSF and DEMO, are to be successful.

  19. COMPARISON OF THE METHYL REDUCTASE GENES AND GENE PRODUCTS

    EPA Science Inventory

    The DNA sequences encoding component C of methyl coenzyme M reductase (mcr genes) in Methanothermus fervidus, Methanobacterium thermoautotrophicum, Methanococcus vannielii, and Methanosarcina barkeri have been published. omparisons of transcription initiation and termination site...

  20. Structural features of the ribonucleotide reductase of Aujeszky's disease virus.

    PubMed

    Kaliman, A V; Boldogköi, Z; Fodor, I

    1994-01-01

    A gene construct of the Aujeszky's disease virus (ADV) genome was prepared and the DNA fragment encoding the ribonucleotide reductase was structurally characterized. We determined the entire DNA sequence of two adjacent open reading frames of the ribonucleotide reductase genes with the intergenic sequence of nine base pairs. From the sequence analysis we predict that Aujeszky's disease virus encodes a ribonucleotide reductase which comprises two polypeptides--large and small subunits, with sizes of 835 and 303 amino acids, respectively. Nucleotide and amino acid sequences of the large and small subunits of the Aujeszky's disease virus ribonucleotide reductase have been compared with that of other herpesviruses, and structural features of both proteins have been characterized. PMID:7810419

  1. Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae.

    PubMed Central

    Rensing, C; Kües, U; Stahl, U; Nies, D H; Friedrich, B

    1992-01-01

    The gene merA coding for bacterial mercuric ion reductase was cloned under the control of the yeast promoter for alcohol dehydrogenase I in the yeast-Escherichia coli shuttle plasmid pADH040-2 and transformed into Saccharomyces cerevisiae AH22. The resulting transformant harbored stable copies of the merA-containing hybrid plasmid, displayed a fivefold increase in the MIC of mercuric chloride, and synthesized mercuric ion reductase activity. Images PMID:1735719

  2. Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase.

    PubMed

    Sun, Qi-An; Su, Dan; Novoselov, Sergey V; Carlson, Bradley A; Hatfield, Dolph L; Gladyshev, Vadim N

    2005-11-01

    Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes. PMID:16262253

  3. Effects of thioredoxin reductase-1 deletion on embryogenesis and transcriptome

    PubMed Central

    Bondareva, Alla A.; Capecchi, Mario R.; Iverson, Sonya V.; Li, Yan; Lopez, Nathan I.; Lucas, Olivier; Merrill, Gary F.; Prigge, Justin R.; Siders, Ashley M.; Wakamiya, Maki; Wallin, Stephanie L.; Schmidt, Edward E.

    2007-01-01

    Thioredoxin reductases (Txnrd)1 maintain intracellular redox homeostasis in most organisms. Metazoans Txnrds also participate in signal transduction. Mouse embryos homozygous for a targeted null mutation of the txnrd1 gene, encoding the cytosolic thioredoxin reductase, were viable at embryonic day 8.5 (E8.5) but not at E9.5. Histology revealed that txnrd1−/− cells were capable of proliferation and differentiation; however, mutant embryos were smaller than wild-type littermates and failed to gastrulate. In situ marker gene analyses indicated primitive streak mesoderm did not form. Microarray analyses on E7.5 txnrd−/− and txnrd+/+ littermates showed similar mRNA levels for peroxiredoxins, glutathione reductases, mitochondrial Txnrd2, and most markers of cell proliferation. Conversely, mRNAs encoding sulfiredoxin, IGF-binding protein 1, carbonyl reductase 3, glutamate cysteine ligase, glutathione S-transferases, and metallothioneins were more abundant in mutants. Many gene expression responses mirrored those in thioredoxin reductase 1-null yeast; however mice exhibited a novel response within the peroxiredoxin catalytic cycle. Thus, whereas yeast induce peroxiredoxin mRNAs in response to thioredoxin reductase disruption, mice induced sulfiredoxin mRNA. In summary, Txnrd1 was required for correct patterning of the early embryo and progression to later development. Conserved responses to Txnrd1 disruption likely allowed proliferation and limited differentiation of the mutant embryo cells. PMID:17697936

  4. An overview on 5alpha-reductase inhibitors.

    PubMed

    Aggarwal, Saurabh; Thareja, Suresh; Verma, Abhilasha; Bhardwaj, Tilak Raj; Kumar, Manoj

    2010-02-01

    Benign prostatic hyperplasia (BPH) is the noncancerous proliferation of the prostate gland associated with benign prostatic obstruction and lower urinary tract symptoms (LUTS) such as frequency, hesitancy, urgency, etc. Its prevalence increases with age affecting around 70% by the age of 70 years. High activity of 5alpha-reductase enzyme in humans results in excessive dihydrotestosterone levels in peripheral tissues and hence suppression of androgen action by 5alpha-reductase inhibitors is a logical treatment for BPH as they inhibit the conversion of testosterone to dihydrotestosterone. Finasteride (13) was the first steroidal 5alpha-reductase inhibitor approved by U.S. Food and Drug Administration (USFDA). In human it decreases the prostatic DHT level by 70-90% and reduces the prostatic size. Dutasteride (27) another related analogue has been approved in 2002. Unlike Finasteride, Dutasteride is a competitive inhibitor of both 5alpha-reductase type I and type II isozymes, reduced DHT levels >90% following 1 year of oral administration. A number of classes of non-steroidal inhibitors of 5alpha-reductase have also been synthesized generally by removing one or more rings from the azasteroidal structure or by an early non-steroidal lead (ONO-3805) (261). In this review all categories of inhibitors of 5alpha-reductase have been covered. PMID:19879888

  5. Regulation of the Neurospora crassa assimilatory nitrate reductase.

    PubMed Central

    Ketchum, P A; Zeeb, D D; Owens, M S

    1977-01-01

    Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase from Neurospora crassa was purified and found to be stimulated by certain amino acids, citrate, and ethylenediaminetetraacetic acid (EDTA). Stimulation by citrate and the amino acids was dependent upon the prior removal of EDTA from the enzyme preparations, since low quantities of EDTA resulted in maximal stimulation. Removal of EDTA from enzyme preparations by dialysis against Chelex-containing buffer resulted in a loss of nitrate reductase activity. Addition of alanine, arginine, glycine, glutamine, glutamate, histidine, tryptophan, and citrate restored and stimulated nitrate reductase activity from 29- to 46-fold. The amino acids tested altered the Km of NADPH-nitrate reductase for NADPH but did not significantly change that for nitrate. The Km of nitrate reductase for NADPH increased with increasing concentrations of histidine but decreased with increasing concentrations of glutamine. Amino acid modulation of NADPH-nitrate reductase activity is discussed in relation to the conservation of energy (NADPH) by Neurospora when nitrate is the nitrogen source. PMID:19423

  6. Novel biodegradation pathways of cyclohexane by Rhodococcus sp. EC1.

    PubMed

    Yi, Taewoo; Lee, Eun-Hee; Ahn, Yun Gyong; Hwang, Geum-Sook; Cho, Kyung-Suk

    2011-07-15

    The metabolism of cyclohexanes by Rodococcus sp. EC1 was investigated using a sequential tracking method of degradation intermediate. Evidence for the formation of cyclohexanol, cyclohexaone, 2-cyclohexen-1-one, and phenol was presented. EC1 metabolized cyclohexane to phenol by aromatization of 2-cyclohexen-1-one, and furthermore gamma-butyrolactone as an intermediate of 2-cyclohexen-1-one was formed. Aromatization by EC1 was confirmed using tetrahydrofuran. Tetrahydrofuran was metabolized through aromatization reaction, involving furan and 2,3-dihydrofuran as key intermediates. EC1 can degrade cyclohexane and tetrahydrofuran in aromatization via desaturation. PMID:21571424

  7. Maleylacetate reductase of Pseudomonas sp. strain B13: specificity of substrate conversion and halide elimination.

    PubMed Central

    Kaschabek, S R; Reineke, W

    1995-01-01

    Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channelling maleylacetate and some chlorinated derivatives into the 3-oxoadipate pathway. Several substituted maleylacetates were prepared in situ by alkaline or enzymatic hydrolysis of dienelactones as the precursor. The conversion of these methyl-, chloro-, fluoro-, and bromo-substituted maleylacetates by malelacetate reductase from 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. Two moles of NADH per mole of substrate was consumed for the conversion of maleylacetates which contain a halogen substituent in the 2 position. In contrast, only 1 mol of NADH was necessary to convert 1 mol of substrates without a halogen substituent in the 2 position. The conversion of 2-fluoro-, 2-chloro-, 2,3-dichloro-, 2,5-dichloro-, 2,3,5-trichloro-, 2-bromo-, 2,3-dibromo-, 2,5-dibromo-, 2-bromo-5-chloro-, 2-chloro-3-methyl-, and 2-chloro-5-methylmaleylacetate was accompanied by the elimination of halide from the 2 position and the temporary occurrence of the corresponding dehalogenated maleylacetate as an intermediate consuming the second mole equivalent of NADH. The properties of the halogen substituents influenced the affinity to the enzyme in the following manner. Km values increased with increasing van der Waals radii and with decreasing electronegativity of the halogen substituents (i.e., low steric hindrance and high electronegativity positively influenced the binding).The Km values obtained with 2-methyl-,3-methyl-, and 5-methylmaleylacetate showed that a methyl substituent negatively affected the affinity in the following order: 2 position >/ = 3 position >> 5 position. A reaction mechanism explaining the exclusive elimination of halogen substituents from the 2 position is proposed. PMID:7814320

  8. Malaria antifolate resistance with contrasting Plasmodium falciparum dihydrofolate reductase (DHFR) polymorphisms in humans and Anopheles mosquitoes

    PubMed Central

    Mharakurwa, Sungano; Kumwenda, Taida; Mkulama, Mtawa A. P.; Musapa, Mulenga; Chishimba, Sandra; Shiff, Clive J.; Sullivan, David J.; Thuma, Philip E.; Liu, Kun; Agre, Peter

    2011-01-01

    Surveillance for drug-resistant parasites in human blood is a major effort in malaria control. Here we report contrasting antifolate resistance polymorphisms in Plasmodium falciparum when parasites in human blood were compared with parasites in Anopheles vector mosquitoes from sleeping huts in rural Zambia. DNA encoding P. falciparum dihydrofolate reductase (EC 1.5.1.3) was amplified by PCR with allele-specific restriction enzyme digestions. Markedly prevalent pyrimethamine-resistant mutants were evident in human P. falciparum infections—S108N (>90%), with N51I, C59R, and 108N+51I+59R triple mutants (30–80%). This resistance level may be from selection pressure due to decades of sulfadoxine/pyrimethamine use in the region. In contrast, cycloguanil-resistant mutants were detected in very low frequency in parasites from human blood samples—S108T (13%), with A16V and 108T+16V double mutants (∼4%). Surprisingly, pyrimethamine-resistant mutants were of very low prevalence (2–12%) in the midguts of Anopheles arabiensis vector mosquitoes, but cycloguanil-resistant mutants were highly prevalent—S108T (90%), with A16V and the 108T+16V double mutant (49–57%). Structural analysis of the dihydrofolate reductase by in silico modeling revealed a key difference in the enzyme within the NADPH binding pocket, predicting the S108N enzyme to have reduced stability but the S108T enzyme to have increased stability. We conclude that P. falciparum can bear highly host-specific drug-resistant polymorphisms, most likely reflecting different selective pressures found in humans and mosquitoes. Thus, it may be useful to sample both human and mosquito vector infections to accurately ascertain the epidemiological status of drug-resistant alleles. PMID:22065788

  9. Malaria antifolate resistance with contrasting Plasmodium falciparum dihydrofolate reductase (DHFR) polymorphisms in humans and Anopheles mosquitoes.

    PubMed

    Mharakurwa, Sungano; Kumwenda, Taida; Mkulama, Mtawa A P; Musapa, Mulenga; Chishimba, Sandra; Shiff, Clive J; Sullivan, David J; Thuma, Philip E; Liu, Kun; Agre, Peter

    2011-11-15

    Surveillance for drug-resistant parasites in human blood is a major effort in malaria control. Here we report contrasting antifolate resistance polymorphisms in Plasmodium falciparum when parasites in human blood were compared with parasites in Anopheles vector mosquitoes from sleeping huts in rural Zambia. DNA encoding P. falciparum dihydrofolate reductase (EC 1.5.1.3) was amplified by PCR with allele-specific restriction enzyme digestions. Markedly prevalent pyrimethamine-resistant mutants were evident in human P. falciparum infections--S108N (>90%), with N51I, C59R, and 108N+51I+59R triple mutants (30-80%). This resistance level may be from selection pressure due to decades of sulfadoxine/pyrimethamine use in the region. In contrast, cycloguanil-resistant mutants were detected in very low frequency in parasites from human blood samples-S108T (13%), with A16V and 108T+16V double mutants (∼4%). Surprisingly, pyrimethamine-resistant mutants were of very low prevalence (2-12%) in the midguts of Anopheles arabiensis vector mosquitoes, but cycloguanil-resistant mutants were highly prevalent-S108T (90%), with A16V and the 108T+16V double mutant (49-57%). Structural analysis of the dihydrofolate reductase by in silico modeling revealed a key difference in the enzyme within the NADPH binding pocket, predicting the S108N enzyme to have reduced stability but the S108T enzyme to have increased stability. We conclude that P. falciparum can bear highly host-specific drug-resistant polymorphisms, most likely reflecting different selective pressures found in humans and mosquitoes. Thus, it may be useful to sample both human and mosquito vector infections to accurately ascertain the epidemiological status of drug-resistant alleles. PMID:22065788

  10. A dual role for plant quinone reductases in host-fungus interaction.

    PubMed

    Heyno, Eiri; Alkan, Noam; Fluhr, Robert

    2013-11-01

    Quinone reductases (QR, EC 1.5.6.2) are flavoproteins that protect organisms from oxidative stress. The function of plant QRs has not as yet been addressed in vivo despite biochemical evidence for their involvement in redox reactions. Here, using knock-out (KO) and overexpressing lines, we studied the protective role of two groups of Arabidopsis thaliana cytosolic QRs, Nqr (NAD(P)H:quinone oxidoreductase) and Fqr (flavodoxin-like quinone reductase), in response to infection by necrotrophic fungi. The KO lines nqr(-) and fqr1(-) displayed significantly slower development of lesions of Botrytis cinerea and Sclerotinia sclerotium in comparison to the wild type (WT). Consistent with this observation, the overexpressing line FQR1(+) was hypersensitive to the pathogens. Both the nqr(-) and fqr1(-) displayed increased fluorescence of 2',7'-dichlorofluorescein,‬ a reporter for reactive oxygen species in response to B. cinerea. Infection by B. cinerea was accompanied with increased Nqr and Fqr1 protein levels in the WT as revealed by western blotting. In addition, a marked stimulation of salicylic acid-sensitive transcripts and suppression of jasmonate-sensitive transcripts was observed in moderately wounded QR KO mutant leaves, a condition mimicking the early stage of infection. In contrast to the above observations, germination of conidia was accelerated on leaves of QR KO mutants in comparison with the WT and FQR1(+). The same effect was observed in water-soluble leaf surface extracts. It is proposed that the altered interaction between B. cinerea and the QR mutants is a consequence of subtly altered redox state of the host, which perturbs host gene expression in response to environmental stress such as fungal growth.‬‬‬‬‬‬ PMID:23464356

  11. Si pixel detectors in the detection of EC/EC decay

    SciTech Connect

    Jose, J. M.; Čermák, P.; Fajt, L.; Štekl, I.; Rukhadze, N. I.; Shitov, Yu. A.

    2015-08-17

    The SPT collaboration has been investigating the applicability of pixel detectors in the detection of two neutrino double electron capture (2νEC/EC) in{sup 106}Cd. The collaboration has proposed a Silicon Pixel Telescope (SPT) where a pair of Si pixel detectors with enriched Cd foil in the middle forms the detection unit. The Pixel detector gives spatial information along with energy of the particle, thus helps to identify and remove the background signals. Four units of SPT prototype (using 0.5 and 1 mm Si sensors) were fabricated and installed in the LSM underground laboratory, France. Recent progress in the SPT experiment and preliminary results from background measurements are presented.

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

    PubMed

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

    2014-02-15

    Sources of nitric oxide alternative to nitric oxide synthases are gaining significant traction as crucial mediators of vessel function under hypoxic inflammatory conditions. For example, capacity to catalyze the one electron reduction of nitrite (NO2-) to ·NO has been reported for hemoglobin, myoglobin and molybdopterin-containing enzymes including xanthine oxidoreductase (XOR) and aldehyde oxidase (AO). For XOR and AO, use of selective inhibition strategies is therefore crucial when attempting to assign relative contributions to nitrite-mediated ·NO formation in cells and tissue. To this end, XOR inhibition has been accomplished with application of classic pyrazolopyrimidine-based inhibitors allo/oxypurinol or the newly FDA-approved XOR-specific inhibitor, Uloric® (febuxostat). Likewise, raloxifene, an estrogen receptor antagonist, has been identified as a potent (Ki=1.0 nM) inhibitor of AO. Herein, we characterize the inhibition kinetics of raloxifene for XOR and describe the resultant effects on inhibiting XO-catalyzed ·NO formation. Exposure of purified XO to raloxifene (PBS, pH 7.4) resulted in a dose-dependent (12.5-100 μM) inhibition of xanthine oxidation to uric acid. Dixon plot analysis revealed a competitive inhibition process with a Ki=13 μM. This inhibitory process was more effective under acidic pH; similar to values encountered under hypoxic/inflammatory conditions. In addition, raloxifene also inhibited anoxic XO-catalyzed reduction of NO2- to NO (EC50=64 μM). In contrast to having no effect on XO-catalyzed uric acid production, the AO inhibitor menadione demonstrated potent inhibition of XO-catalyzed NO2- reduction (EC50=60 nM); somewhat similar to the XO-specific inhibitor, febuxostat (EC50=4 nM). Importantly, febuxostat was found to be a very poor inhibitor of human AO (EC50=613 μM) suggesting its usefulness for validating XO-dependent contributions to NO2- reduction in biological systems. Combined, these data indicate care should be taken

  13. Oncogenic Role of the Ec Peptide of the IGF-1Ec Isoform in Prostate Cancer

    PubMed Central

    Armakolas, Athanasios; Kaparelou, Maria; Dimakakos, Andreas; Papageorgiou, Efstathia; Armakolas, Nikolaos; Antonopoulos, Athanasios; Petraki, Constantina; Lekarakou, Maria; Lelovas, Pavlos; Stathaki, Martha; Psarros, Constantinos; Donta, Ismene; Galanos, Panos S; Msaouel, Paul; Gorgoulis, Vassilis G; Koutsilieris, Michael

    2015-01-01

    IGF-1 is one of the key molecules in cancer biology; however, little is known about the role of the preferential expression of the premature IGF-1 isoforms in prostate cancer. We have examined the role of the cleaved COO– terminal peptide (PEc) of the third IGF-1 isoform, IGF-1Ec, in prostate cancer. Our evidence suggests that endogenously produced PEc induces cellular proliferation in the human prostate cancer cells (PC-3) in vitro and in vivo, by activating the ERK1/2 pathway in an autocrine/paracrine manner. PEc overexpressing cells and tumors presented evidence of epithelial to mesenchymal transition, whereas the orthotopic injection of PEc-overexpressing, normal prostate epithelium cells (HPrEC) in SCID mice was associated with increased metastatic rate. In humans, the IGF-1Ec expression was detected in prostate cancer biopsies, where its expression correlates with tumor stage. Our data describes the action of PEc in prostate cancer biology and defines its potential role in tumor growth, progression and metastasis. PMID:25569803

  14. Oncogenic Role of the Ec Peptide of the IGF-1Ec Isoform in Prostate Cancer.

    PubMed

    Armakolas, Athanasios; Kaparelou, Maria; Dimakakos, Andreas; Papageorgiou, Efstathia; Armakolas, Nikolaos; Antonopoulos, Athanasios; Petraki, Constantina; Lekarakou, Maria; Lelovas, Pavlos; Stathaki, Martha; Psarros, Constantinos; Donta, Ismene; Galanos, Panos S; Msaouel, Paul; Gorgoulis, Vassilis G; Koutsilieris, Michael

    2015-01-01

    IGF-1 is one of the key molecules in cancer biology; however, little is known about the role of the preferential expression of the premature IGF-1 isoforms in prostate cancer. We have examined the role of the cleaved COO- terminal peptide (PEc) of the third IGF-1 isoform, IGF-1Ec, in prostate cancer. Our evidence suggests that endogenously produced PEc induces cellular proliferation in the human prostate cancer cells (PC-3) in vitro and in vivo, by activating the ERK1/2 pathway in an autocrine/paracrine manner. PEc overexpressing cells and tumors presented evidence of epithelial to mesenchymal transition, whereas the orthotopic injection of PEc-overexpressing, normal prostate epithelium cells (HPrEC) in SCID mice was associated with increased metastatic rate. In humans, the IGF-1Ec expression was detected in prostate cancer biopsies, where its expression correlates with tumor stage. Our data describes the action of PEc in prostate cancer biology and defines its potential role in tumor growth, progression and metastasis. PMID:25569803

  15. Inactivation of corticosteroids in intestinal mucosa by 11 beta-hydroxysteroid: NADP oxidoreductase (EC 1. 1. 1. 146)

    SciTech Connect

    Burton, A.F.; Anderson, F.H.

    1983-10-01

    Activity of the enzyme 11 beta-hydroxysteroid:NADP oxidoreductase (EC 1.1.1.146) in human intestinal mucosa was determined by incubating scraped mucosa with /sup 3/H-cortisone and /sup 14/C-cortisol; these steroids were then extracted, separated chromatographically, and the radioactivity assayed to determine simultaneously both reductase and dehydrogenase activities. This was the only significant metabolic alteration which the substrate underwent. Only two cases had slight (5 and 13%) reductase activity. In 35 patients, 16 male and 19 female, including seven cases of Crohn's disease, three ulcerative colitis, five diverticulitis, two undergoing surgery for repair of injuries and 18 for carcinoma of colon or rectum, cortisol was converted to cortisone in 15 min with a wide range of values distributed uniformly up to 85% dehydrogenation, with a mean of 42%. When tissue homogenates were fortified with coenzymes, excess NADPH lowered dehydrogenase activity 81%; excess NADP increased dehydrogenase activity 2-fold in three cases. It is possible that a value is characteristic of an individual but perhaps more likely enzyme activity varies with metabolic events involving changes in the coenzyme levels in mucosa, and a random sampling might be expected to yield such a distribution of values. In any event, where activity is high most of the cortisol is inactivated within minutes. It is suggested that synthetic corticoids which escape such metabolic alteration might, except during pregnancy, prove superior in the treatment of conditions such as inflammatory bowel disease.

  16. Insights into anticancer activity and mechanism of action of a ruthenium(II) complex in human esophageal squamous carcinoma EC109 cells.

    PubMed

    Guo, Liubin; Lv, Gaochao; Qiu, Ling; Yang, Hui; Zhang, Li; Yu, Huixin; Zou, Meifen; Lin, Jianguo

    2016-09-01

    A ruthenium(II) complex [Ru(p-cymene)(NHC)Cl2] (NHC=1,3-bis(4-(tert-butyl)benzylimidazol-2-ylidene), referred to as L-4, has been designed and synthesized recently in order to look for new anticancer drugs with high efficacy and low side effects. The anticancer activity and mechanism of action of L-4 in human esophageal squamous carcinoma EC109 cells were systematically investigated. The results revealed that L-4 exerted strong inhibitory effect on the proliferation of EC109 cells, and it arrested EC109 cells at G2/M phase, accompanied with the up-regulation of p53 and p21 and the down-regulation of cyclin D1. The results also showed that the reactive oxygen species (ROS)-dependent apoptosis of EC109 can be induced by L-4 via inhibiting the activity of glutathione reductase (GR), decreasing the ratio of glutathione to oxidized glutathione (GSH/GSSG), and leading to the generation of reactive oxygen species. The mitochondria-mediated apoptosis of EC109 induced by L-4 was also observed from the increase of Bax/Bcl-2 ratio, overload of Ca(2+), disruption of mitochondrial membrane potential (MMP), redistribution of cytochrome c, and activation of caspase-3/-9. However, the effects of L-4 on the cell viability, GR activity, GSH/GSSG ratio, reactive oxygen species level, mitochondria dysfunction and apoptosis induction were remarkably attenuated by adding the reactive oxygen species scavenger, NAC. Therefore, it was concluded that L-4 can inhibit the proliferation of EC109 cells via blocking cell cycle progression and inducing reactive oxygen species-dependent and mitochondria-mediated apoptosis. These findings suggested that the ruthenium(II) complex might be a potential effective chemotherapeutic agent for human esophageal squamous carcinoma (ESCC) and worthy of further investigation. PMID:27262377

  17. Overproduction of a Mr 92,000 protomer of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in compactin-resistant C100 cells

    PubMed Central

    Hardeman, Edna C.; Jenke, Hans-Stephan; Simoni, Robert D.

    1983-01-01

    We describe a cell line, designated C100, that displays a 100-fold increase in the major regulatory enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase [HMG-CoA; mevalonate:NADP+ oxido-reductase (CoA-acylating), EC 1.1.1.34]. Immunoprecipitation of [35S]methionine-labeled enzyme from C100 microsomal membranes prepared in the presence of the protease inhibitors phenyl-methylsulfonyl fluoride and leupeptin revealed two up regulated proteins: a major band of Mr 92,000 and a minor band of Mr 63,000. We conclude that the Mr 92,000 protein is probably the intact form of HMG-CoA reductase protomer based on the following criteria. (i) It is a highly up regulated microsomal membrane protein that coincides with the increase in HMG-CoA reductase specific activity in this cell line. (ii) It is recognized by a specific HMG-CoA reductase antiserum under a variety of stringencies. (iii) Isolation and solubilization of [35S]methionine-labeled C100 microsomal membranes in the absence of protease inhibitors resulted in the disappearance of the Mr 92,000 protein and the appearance of two proteins of Mr 52,000 and 38,000. (iv) Analysis of cells labeled for 30 min with [35S]methionine, well under the half-life of HMG-CoA reductase, revealed only the Mr 92,000 protein to be present in total cell extract. (v) The previously reported single immunoprecipitation polypeptide for HMG-CoA reductase of Mr 62,000 [Chin, D. J., Luskey, K. L., Anderson, R. G. W., Faust, J. R., Goldstein, J. L. & Brown, M. S. (1982) Proc. Natl. Acad. Sci. USA 79, 1185-1189] can be isolated and appears to be the result of both proteolysis and sample preparation for NaDodSO4 gel electrophoresis. Analysis of C100 cells labeled with [35S]methionine for 24 hr indicates that the predominant steady-state form of the enzyme is the Mr 92,000, rather than the Mr 63,000, protein, further suggesting that the two proteins do not have a classical precursor-product relationship. Images

  18. Dihydropteridine reductase activity in dried blood spots: effects of aging and senile dementia of the Alzheimer type.

    PubMed Central

    Jeeps, C M; Silcox, A; Lloyd, B; Clayton, B E

    1986-01-01

    Dihydropteridine reductase (EC 1.6.99.7) (DHPR) activity was measured in blood spots from 50 neonates, 52 healthy adults aged 30-62 years, and 21 elderly controls aged 67-97 years, as well as 32 demented patients of whom 25 had senile dementia of the Alzheimer type. Enzyme activity was stable for seven days at 4 degrees C and for at least 14 days at -20 degrees C. No important difference was found between the DHPR activity of venous and capillary blood. DHPR activity was considerably lower in the healthy adult group compared with neonates and the elderly group, and there was no sex difference at any age. The erythrocyte DHPR activity of patients with senile dementia of the Alzheimer type was similar to that of elderly controls. This result differs from that previously reported for leucocytes. PMID:3950042

  19. Pyrithione-based ruthenium complexes as inhibitors of aldo-keto reductase 1C enzymes and anticancer agents.

    PubMed

    Kljun, Jakob; Anko, Maja; Traven, Katja; Sinreih, Maša; Pavlič, Renata; Peršič, Špela; Ude, Žiga; Codina, Elisa Esteve; Stojan, Jure; Lanišnik Rižner, Tea; Turel, Iztok

    2016-08-01

    Four ruthenium complexes of clinically used zinc ionophore pyrithione and its oxygen analog 2-hydroxypyridine N-oxide were prepared and evaluated as inhibitors of enzymes of the aldo-keto reductase subfamily 1C (AKR1C). A kinetic study assisted with docking simulations showed a mixed type of inhibition consisting of a fast reversible and a slow irreversible step in the case of both organometallic compounds 1A and 1B. Both compounds also showed a remarkable selectivity towards AKR1C1 and AKR1C3 which are targets for breast cancer drug design. The organoruthenium complex of ligand pyrithione as well as pyrithione itself also displayed toxicity on the hormone-dependent MCF-7 breast cancer cell line with EC50 values in the low micromolar range. PMID:27357845

  20. Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa

    PubMed Central

    Lu, Wanxiang; Yang, Li; Karim, Abdul; Luo, Keming

    2013-01-01

    Proanthocyanidins (PAs) contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA) and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are two key enzymes of the PA biosynthesis that produce the main subunits: (+)-catechin and (−)-epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05) in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus. PMID:23741362

  1. EC Driver - 41" Stroke Hydraulic Cylinder

    SciTech Connect

    Jaques, A.; /Fermilab

    1990-05-24

    It was decided to use a hydraulic cylinder resting on the floor of the argon spill trough in the EC carriage to drive the EC's motion on the center beam. Space was limited due to the spill bellows and their required support and containment system. The 0.0. of the cylinder had to be limited to 3 to 3-1/2 inches, maximum. The weight of a wet EC and carriage is estimated to be 320 tons. The rolling coefficient of friction of the Tychoway rollers chosen to guide the EC and carriage along the hardened centerbeam ways is claimed to be less than 0.0025. The driver will also need to overcome the forces produced by moving (rotating) the numerous bayonets located at the top of the cryostats in the many piping systems. These forces were conservatively estimated at 1000 lbs. The drive force required to overcome these forces was then calculated to be: 320(2,000) x 0.0025 + 1,000 = 2.600 lbs. (min. required). Due to the uncertainty in the actual roller coefficient of friction and the various unknowns in estimating the resistive forces contained in the piping and cabling systems attached to the cryostat, a conservative design factor of 5 was chosen. This should account for any uncertainty in our estimation of the minimum required drive force and also leaves us with a reserve to fall back on in case any unforeseen problems might arise. Thus the desired capacity of the driver was set at: (2,600) x 5 = 13,000 lbs. (design capacity). Assuming a 3 inch O.D. cylinder with a 1/2 inch wall (2 inch bore), we first analyzed a 1-3/8 inch diameter piston rod. Using Shigley & Mischke's 'Mechanical Engineering Design' (5th Ed.) and it's formulas for long columns with central loading, it was determined that a 1-3/8 inch diameter rod would not suffice, given our safety factor of 2. Increasing the piston rod diameter to 1-1/2 inches proved to be sufficient. The maximum allowable load came out to be approximately 17,000 lbs., which is greater than the 13,000 lbs. design capacity. With a 1-1/2 inch

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

    PubMed

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

    2016-10-01

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

  3. ECS Special Education Handbook: 2008/2009 School Year

    ERIC Educational Resources Information Center

    Alberta Education, 2008

    2008-01-01

    This handbook explains basic funding requirements for Early Childhood Services (ECS) and how to complete application forms required for the services. It also outlines the age of eligibility for funding for all types of ECS programming. The handbook explains other funding that is provided for children identified with mild to moderate…

  4. Status of Europe's contribution to the ITER EC system

    NASA Astrophysics Data System (ADS)

    Albajar, F.; Aiello, G.; Alberti, S.; Arnold, F.; Avramidis, K.; Bader, M.; Batista, R.; Bertizzolo, R.; Bonicelli, T.; Braunmueller, F.; Brescan, C.; Bruschi, A.; von Burg, B.; Camino, K.; Carannante, G.; Casarin, V.; Castillo, A.; Cauvard, F.; Cavalieri, C.; Cavinato, M.; Chavan, R.; Chelis, J.; Cismondi, F.; Combescure, D.; Darbos, C.; Farina, D.; Fasel, D.; Figini, L.; Gagliardi, M.; Gandini, F.; Gantenbein, G.; Gassmann, T.; Gessner, R.; Goodman, T. P.; Gracia, V.; Grossetti, G.; Heemskerk, C.; Henderson, M.; Hermann, V.; Hogge, J. P.; Illy, S.; Ioannidis, Z.; Jelonnek, J.; Jin, J.; Kasparek, W.; Koning, J.; Krause, A. S.; Landis, J. D.; Latsas, G.; Li, F.; Mazzocchi, F.; Meier, A.; Moro, A.; Nousiainen, R.; Purohit, D.; Nowak, S.; Omori, T.; van Oosterhout, J.; Pacheco, J.; Pagonakis, I.; Platania, P.; Poli, E.; Preis, A. K.; Ronden, D.; Rozier, Y.; Rzesnicki, T.; Saibene, G.; Sanchez, F.; Sartori, F.; Sauter, O.; Scherer, T.; Schlatter, C.; Schreck, S.; Serikov, A.; Siravo, U.; Sozzi, C.; Spaeh, P.; Spichiger, A.; Strauss, D.; Takahashi, K.; Thumm, M.; Tigelis, I.; Vaccaro, A.; Vomvoridis, J.; Tran, M. Q.; Weinhorst, B.

    2015-03-01

    The electron cyclotron (EC) system of ITER for the initial configuration is designed to provide 20MW of RF power into the plasma during 3600s and a duty cycle of up to 25% for heating and (co and counter) non-inductive current drive, also used to control the MHD plasma instabilities. The EC system is being procured by 5 domestic agencies plus the ITER Organization (IO). F4E has the largest fraction of the EC procurements, which includes 8 high voltage power supplies (HVPS), 6 gyrotrons, the ex-vessel waveguides (includes isolation valves and diamond windows) for all launchers, 4 upper launchers and the main control system. F4E is working with IO to improve the overall design of the EC system by integrating consolidated technological advances, simplifying the interfaces, and doing global engineering analysis and assessments of EC heating and current drive physics and technology capabilities. Examples are the optimization of the HVPS and gyrotron requirements and performance relative to power modulation for MHD control, common qualification programs for diamond window procurements, assessment of the EC grounding system, and the optimization of the launcher steering angles for improved EC access. Here we provide an update on the status of Europe's contribution to the ITER EC system, and a summary of the global activities underway by F4E in collaboration with IO for the optimization of the subsystems.

  5. A Novel Metagenomic Short-Chain Dehydrogenase/Reductase Attenuates Pseudomonas aeruginosa Biofilm Formation and Virulence on Caenorhabditis elegans

    PubMed Central

    Bijtenhoorn, Patrick; Mayerhofer, Hubert; Müller-Dieckmann, Jochen; Utpatel, Christian; Schipper, Christina; Hornung, Claudia; Szesny, Matthias; Grond, Stephanie; Thürmer, Andrea; Brzuszkiewicz, Elzbieta; Daniel, Rolf; Dierking, Katja; Schulenburg, Hinrich; Streit, Wolfgang R.

    2011-01-01

    In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies. PMID:22046268

  6. In silico characterization of a nitrate reductase gene family and analysis of the predicted proteins from the moss Physcomitrella patens.

    PubMed

    Medina-Andrés, Rigoberto; Lira-Ruan, Verónica

    2012-01-01

    Assimilatory nitrate reductase (NR; EC 1.7.1.1-3) catalyzes the reduction of nitrate to nitrite. This enzyme has a conserved structure common to fungi, algae and plants. However, some differences in the amino acid sequence between plant and algal NR suggest that the activity regulation mechanisms have changed during plant evolution. Since only NRs from angiosperms have been studied, the search and analysis of NR genes and proteins from the moss Physcomitrella patens, a basal land plant, was performed to widen the knowledge of land plant NR structure. A family of three nr genes, named ppnia1;1, ppnia1;2 and ppnia2, was localized in the P. patens genome. The predicted proteins are canonical NRs with the conserved domains Molybdene-Cytochorme b -Cytochrome b reductase and possess 20 amino acid residues important for the enzymatic function conserved in plant and algal NRs. Interestingly, moss NRs lack a consensus sequence, common to angiosperm NRs, that is a target for posttranslational regulation. A phylogenetic tree with embryophyte and green algae NR sequences was constructed and P. patens NRs localized at the base of embryophyte NR evolution. The data presented here suggest that bryophytes and vascular plants have different systems to regulate NR activity. PMID:22482004

  7. In silico characterization of a nitrate reductase gene family and analysis of the predicted proteins from the moss Physcomitrella patens

    PubMed Central

    Medina-Andrés, Rigoberto

    2012-01-01

    Assimilatory nitrate reductase (NR; EC 1.7.1.1-3) catalyzes the reduction of nitrate to nitrite. This enzyme has a conserved structure common to fungi, algae and plants. However, some differences in the amino acid sequence between plant and algal NR suggest that the activity regulation mechanisms have changed during plant evolution. Since only NRs from angiosperms have been studied, the search and analysis of NR genes and proteins from the moss Physcomitrella patens, a basal land plant, was performed to widen the knowledge of land plant NR structure. A family of three nr genes, named ppnia1;1, ppnia1;2 and ppnia2, was localized in the P. patens genome. The predicted proteins are canonical NRs with the conserved domains Molybdene-Cytochorme b –Cytochrome b reductase and possess 20 amino acid residues important for the enzymatic function conserved in plant and algal NRs. Interestingly, moss NRs lack a consensus sequence, common to angiosperm NRs, that is a target for posttranslational regulation. A phylogenetic tree with embryophyte and green algae NR sequences was constructed and P. patens NRs localized at the base of embryophyte NR evolution. The data presented here suggest that bryophytes and vascular plants have different systems to regulate NR activity. PMID:22482004

  8. Purification, characterization and preliminary X-ray crystallographic studies of monodehydroascorbate reductase from Oryza sativa L. japonica

    PubMed Central

    Do, Hackwon; Kim, Il-Sup; Kim, Young-Saeng; Shin, Sun-Young; Kim, Jin-Ju; Mok, Ji-Eun; Park, Seong-Im; Wi, Ah Ram; Park, Hyun; Lee, Jun Hyuck; Yoon, Ho-Sung; Kim, Han-Woo

    2014-01-01

    Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is a key enzyme in the reactive oxygen species (ROS) detoxification system of plants. The participation of MDHAR in ascorbate (AsA) recycling in the ascorbate–glutathione cycle is important in the acquired tolerance of crop plants to abiotic environmental stresses. Thus, MDHAR represents a strategic target protein for the improvement of crop yields. Although physiological studies have intensively characterized MDHAR, a structure-based functional analysis is not available. Here, a cytosolic MDHAR (OsMDHAR) derived from Oryza sativa L. japonica was expressed using Escherichia coli strain NiCo21 (DE3) and purified. The purified OsMDHAR showed specific enzyme activity (approximately 380 U per milligram of protein) and was crystallized using the hanging-drop vapour-diffusion method at pH 8.0 and 298 K. The crystal diffracted to 1.9 Å resolution and contained one molecule in the asymmetric unit (the Matthews coefficient V M is 1.98 Å3 Da−1, corresponding to a solvent content of 38.06%) in space group P41212 with unit-cell parameters a = b = 81.89, c = 120.4 Å. The phase of the OsMDHAR structure was resolved by the molecular-replacement method using a ferredoxin reductase from Acidovorax sp. strain KKS102 (PDB entry 4h4q) as a model. PMID:25195901

  9. Probing the Electrostatics of Active Site Microenvironments along the Catalytic Cycle for Escherichia coli Dihydrofolate Reductase

    PubMed Central

    2015-01-01

    Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and 13C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor–acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for

  10. DNA damage induction of ribonucleotide reductase.

    PubMed Central

    Elledge, S J; Davis, R W

    1989-01-01

    RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidase activity in yeast strains containing the RNR2-lacZ fusion was inducible in response to DNA-damaging agents (UV light, 4-nitroquinoline-1-oxide [4-NQO], and methyl methanesulfonate [MMS]) and agents that block DNA replication (hydroxyurea [HU] and methotrexate) but not heat shock. When MATa cells were arrested in G1 by alpha-factor, RNR2 mRNA was still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. In addition, RNR2 induction was not blocked by the presence of cycloheximide and is therefore likely to be independent of protein synthesis. A mutation, rnr2-314, was found to confer hypersensitivity to HU and increased sensitivity to MMS. In rnr2-314 mutant strains, the DNA damage stress response was found to be partially constitutive as well as hypersensitive to induction by HU but not MMS. The induction properties of RNR2 were examined in a rad4-2 mutant background; in this genetic background, RNR2 was hypersensitive to induction by 4-NQO but not MMS. Induction of the RNR2-lacZ fusion in a RAD(+) strain in response to 4-NQO was not enhanced by the presence of an equal number of rad4-2 cells that lacked the fusion, implying that the DNA damage stress response in cell autonomous. Images PMID:2513480

  11. Induction of NAD(P)H:quinone reductase in murine hepatoma cells by phenolic antioxidants, azo dyes, and other chemoprotectors: a model system for the study of anticarcinogens

    SciTech Connect

    De Long, M.J.; Prochaska, H.J.; Talalay, P.

    1986-02-01

    Exposure of murine hepatoma (Hepa 1c1c7) cells to a variety of chemical agents known to protect animals against the neoplastic, mutagenic, and other toxic effects of chemical carcinogens results in dose- and time-dependent inductions of NAD(P)H:quinone reductase (EC 1.6.99.2). This enzyme protects against quinone toxicity by promoting obligatory two-electron reductions that divert quinones from oxidative cycling or direct interactions with critical nucleophiles. Quinone reductase levels are stable in culture, are easily measured, and are useful markers for the inductive effects of chemoprotective agents. The Hepa 1c1c7 system responds to chemoprotective compounds such as phenolic antioxidants /e.g., BHA (3(2)-tert-butyl-4-hydroxyanisole), BHT (3,5-di-tert-butyl-4-hydroxytoluene), and tert-butylhydroquinone/, lipophilic azo dyes belonging to the 1,1'-azonaphthalene, Sudan I (1-phenylazo-2-naphthol), and Sudan III (1-(4-phenylazophenylazo)-2-naphthol) families, polycyclic aromatic hydrocarbons, coumarin and various other lactones, flavonoids, and certain sulfur compounds (e.g., benzylisothiocyanate, dithiolthiones, and dithiocarbamates), all of which are recognized enzyme inducers and chemoprotectors in vivo. Quinone reductase induction in Hepa 1c1c7 cells therefore provides a simple, versatile, and reliable system for the evaluation of the potency, kinetics, and mechanism of action of anticarcinogens.

  12. NP1EC Degradation Pathways Under Oxic and Microxic Conditions

    SciTech Connect

    Montgomery-Brown, John; Li, Yongmei; Ding, Wang-Hsien; Mong, Gary M.; Campbell, James A.; Reinhard, Martin

    2008-03-22

    The degradation pathway of nonylphenol ethoxyacetic acid (NP1EC) and the conditions favoring CAP1EC formation were studied in aerobic microcosms constructed with soil from the Mesa soil aquifer treatment (SAT) facility (Arizona, USA) and pristine sediments from Coyote Creek (California, USA). In the Mesa microcosms, para-NP1EC was transformed to para-NP, before being rapidly transformed to nonyl alcohols via ipso-hydroxylation. While the formation of NP from APEMs has been observed by several researchers under anaerobic conditions, this is the first time the transient formation of NP from APEMs has been observed under aerobic conditions. Unlike the Mesa microcosms, large quantities of CAP1ECs were observed in the Coyote Creek microcosms. Initially, CA8P1ECs were the dominant metabolites, but as biodegradation continued, CA6P1ECs became the dominant metabolites. Compared to the CA8P1ECs, the number of CA6P1ECs peaks observed was small (<6) even though their concentrations were high. This suggests that several CA8P1ECs are degraded to only a few CA6P1EC isomers (i.e., the degradation pathway converges) or that some CA6P1EC metabolites are significantly more recalcitrant than others. The different biodegradation pathways observed in the Mesa and Coyote Creek microcosms result from the limited availability of dissolved oxygen in the Coyote Creek microcosms. In both sets of microcosms, the ortho isomers were transformed more slowly than the para isomers and in the Coyote Creek microcosms several ortho-CAP1ECs were observed. In addition, several unknown metabolites were observed in the Coyote Creek microcosms that were not seen in the abiotic or Mesa microcosms; these metabolites appear to be CAP1EC metabolites, have a -CH2-C6H4- fragment, and contain one carboxylic acid. Nitro-nonylphenol was observed in the Mesa microcosms, however, further experimentation illustrated that it was the product of an abiotic reaction between nitrite and nonylphenol under acidic conditions.

  13. Molecular cloning, expression profiles and characterization of a glutathione reductase in Hevea brasiliensis.

    PubMed

    Deng, Zhi; Zhao, Manman; Liu, Hui; Wang, Yuekun; Li, Dejun

    2015-11-01

    Glutathione reductase (GR; EC 1.8.1.7) is an important oxidoreductase that can protect organisms against various oxidative stresses. In this study, a new GR gene, named as HbGR2, was isolated from Hevea brasiliensis. The HbGR2 cDNA contained a 1674-bp open reading frame encoding 557 amino acids and the deduced HbGR2 protein showed high identities to the chloroplastic GRs from other plant species. HbGR2 was localized in the chloroplasts of tobacco mesophyll protoplasts. The cis-acting regulatory elements related to stress or hormone responses were predicted in the promoter region of HbGR2. The results from real-time RT-PCR analyses showed that HbGR2 was expressed throughout different tissues and developmental stages of leaves. Besides being related to tapping panel dryness (TPD), HbGR2 was regulated by several treatments including ethephon (ET), methyl jasmonate (MeJA), drought, low temperature, high salt, wounding and hydrogen peroxide (H2O2). The Escherichia coli (E. coli) cells overexpressing HbGR2 markedly increased their tolerance and survival at high concentrations of H2O2, suggesting that HbGR2 might play an important role in oxidative stress response in Hevea brasiliensis. PMID:26232647

  14. Molecular Cloning and Characterization of Two Genes Encoding Dihydroflavonol-4-Reductase from Populus trichocarpa

    PubMed Central

    Jia, Zhichun; Yang, Li; Sun, Yimin; Xiao, Xunyan; Song, Feng; Luo, Keming

    2012-01-01

    Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is a rate-limited enzyme in the biosynthesis of anthocyanins and condensed tannins (proanthocyanidins) that catalyzes the reduction of dihydroflavonols to leucoanthocyanins. In this study, two full-length transcripts encoding for PtrDFR1 and PtrDFR2 were isolated from Populus trichocarpa. Sequence alignment of the two PtrDFRs with other known DFRs reveals the homology of these genes. The expression profile of PtrDFRs was investigated in various tissues of P. trichocarpa. To determine their functions, two PtrDFRs were overexpressed in tobacco (Nicotiana tabacum) via Agrobacterium-mediated transformation. The associated color change in the flowers was observed in all 35S:PtrDFR1 lines, but not in 35S:PtrDFR2 lines. Compared to the wild-type control, a significantly higher accumulation of anthocyanins was detected in transgenic plants harboring the PtrDFR1. Furthermore, overexpressing PtrDFR1 in Chinese white poplar (P. tomentosa Carr.) resulted in a higher accumulation of both anthocyanins and condensed tannins, whereas constitutively expressing PtrDFR2 only improved condensed tannin accumulation, indicating the potential regulation of condensed tannins by PtrDFR2 in the biosynthetic pathway in poplars. PMID:22363429

  15. Trypanothione Reductase: A Target Protein for a Combined In Vitro and In Silico Screening Approach

    PubMed Central

    Garoff, Linnéa; Noack, Sandra; Krauth-Siegel, R. Luise; Selzer, Paul M.

    2015-01-01

    With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC50 values of <1 μM, were selected for determining the inhibitor constant. In first on parasites assays, three compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC50 values down to 2 μM. PMID:26042772

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

    PubMed Central

    Paritala, Hanumantharao; Carroll, Kate S.

    2013-01-01

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

  17. Sulfur Isotope Effects of Dissimilatory Sulfite Reductase

    PubMed Central

    Leavitt, William D.; Bradley, Alexander S.; Santos, André A.; Pereira, Inês A. C.; Johnston, David T.

    2015-01-01

    The precise interpretation of environmental sulfur isotope records requires a quantitative understanding of the biochemical controls on sulfur isotope fractionation by the principle isotope-fractionating process within the S cycle, microbial sulfate reduction (MSR). Here we provide the only direct observation of the major (34S/32S) and minor (33S/32S, 36S/32S) sulfur isotope fractionations imparted by a central enzyme in the energy metabolism of sulfate reducers, dissimilatory sulfite reductase (DsrAB). Results from in vitro sulfite reduction experiments allow us to calculate the in vitro DsrAB isotope effect in 34S/32S (hereafter, 34εDsrAB) to be 15.3 ± 2‰, 2σ. The accompanying minor isotope effect in 33S, described as 33λDsrAB, is calculated to be 0.5150 ± 0.0012, 2σ. These observations facilitate a rigorous evaluation of the isotopic fractionation associated with the dissimilatory MSR pathway, as well as of the environmental variables that govern the overall magnitude of fractionation by natural communities of sulfate reducers. The isotope effect induced by DsrAB upon sulfite reduction is a factor of 0.3–0.6 times prior indirect estimates, which have ranged from 25 to 53‰ in 34εDsrAB. The minor isotope fractionation observed from DsrAB is consistent with a kinetic or equilibrium effect. Our in vitro constraints on the magnitude of 34εDsrAB is similar to the median value of experimental observations compiled from all known published work, where 34εr−p = 16.1‰ (r–p indicates reactant vs. product, n = 648). This value closely matches those of MSR operating at high sulfate reduction rates in both laboratory chemostat experiments (34εSO4−H2S =  17.3 ± 1.5‰, 2σ) and in modern marine sediments (34εSO4−H2S =  17.3 ± 3.8‰). Targeting the direct isotopic consequences of a specific enzymatic processes is a fundamental step toward a biochemical foundation for reinterpreting the biogeochemical and geobiological sulfur isotope records in

  18. Sulfur Isotope Effects of Dissimilatory Sulfite Reductase.

    PubMed

    Leavitt, William D; Bradley, Alexander S; Santos, André A; Pereira, Inês A C; Johnston, David T

    2015-01-01

    The precise interpretation of environmental sulfur isotope records requires a quantitative understanding of the biochemical controls on sulfur isotope fractionation by the principle isotope-fractionating process within the S cycle, microbial sulfate reduction (MSR). Here we provide the only direct observation of the major ((34)S/(32)S) and minor ((33)S/(32)S, (36)S/(32)S) sulfur isotope fractionations imparted by a central enzyme in the energy metabolism of sulfate reducers, dissimilatory sulfite reductase (DsrAB). Results from in vitro sulfite reduction experiments allow us to calculate the in vitro DsrAB isotope effect in (34)S/(32)S (hereafter, [Formula: see text]) to be 15.3 ± 2‰, 2σ. The accompanying minor isotope effect in (33)S, described as [Formula: see text], is calculated to be 0.5150 ± 0.0012, 2σ. These observations facilitate a rigorous evaluation of the isotopic fractionation associated with the dissimilatory MSR pathway, as well as of the environmental variables that govern the overall magnitude of fractionation by natural communities of sulfate reducers. The isotope effect induced by DsrAB upon sulfite reduction is a factor of 0.3-0.6 times prior indirect estimates, which have ranged from 25 to 53‰ in (34)εDsrAB. The minor isotope fractionation observed from DsrAB is consistent with a kinetic or equilibrium effect. Our in vitro constraints on the magnitude of [Formula: see text] is similar to the median value of experimental observations compiled from all known published work, where (34)ε r-p = 16.1‰ (r-p indicates reactant vs. product, n = 648). This value closely matches those of MSR operating at high sulfate reduction rates in both laboratory chemostat experiments ([Formula: see text] 17.3 ± 1.5‰, 2σ) and in modern marine sediments ([Formula: see text] 17.3 ± 3.8‰). Targeting the direct isotopic consequences of a specific enzymatic processes is a fundamental step toward a biochemical foundation for reinterpreting the

  19. The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

    PubMed Central

    Wang, Lin; Goodey, Nina M; Benkovic, Stephen J; Kohen, Amnon

    2006-01-01

    Residues M42 and G121 of Escherichia coli dihydrofolate reductase (ecDHFR) are on opposite sides of the catalytic centre (15 and 19 Å away from it, respectively). Theoretical studies have suggested that these distal residues might be part of a dynamics network coupled to the reaction catalysed at the active site. The ecDHFR mutant G121V has been extensively studied and appeared to have a significant effect on rate, but only a mild effect on the nature of H-transfer. The present work examines the effect of M42W on the physical nature of the catalysed hydride transfer step. Intrinsic kinetic isotope effects (KIEs), their temperature dependence and activation parameters were studied. The findings presented here are in accordance with the environmentally coupled hydrogen tunnelling. In contrast to the wild-type (WT), fluctuations of the donor–acceptor distance were required, leading to a significant temperature dependence of KIEs and deflated intercepts. A comparison of M42W and G121V to the WT enzyme revealed that the reduced rates, the inflated primary KIEs and their temperature dependences resulted from an imperfect potential surface pre-arrangement relative to the WT enzyme. Apparently, the coupling of the enzyme's dynamics to the reaction coordinate was altered by the mutation, supporting the models in which dynamics of the whole protein is coupled to its catalysed chemistry. PMID:16873118

  20. Influence of N,N-dimethylaniline on the association of phenobarbital-induced cytochrome P-450 and NADPH-cytochrome c(P-450) reductase in a reconstituted rabbit liver microsomal enzyme system.

    PubMed

    Hlavica, P; Golly, I; Wolf, J

    1987-09-01

    N,N-Dimethylaniline when added to reaction mixtures provokes deviation from Michaelis-Menten law of the interaction kinetics of NADPH-cytochrome c(P-450) reductase (NADPH:ferrihaemoprotein oxidoreductase, EC 1.6.2.4) with highly purified phenobarbital-induced rabbit liver microsomal cytochrome P-450 (P-450LM2). This phenomenon is not associated with the low-to-high spin transition in the iron-coordination sphere of the haemoprotein, as elicited by the arylamine. Substrate-triggered departure from linearity of the kinetics is abolished by inclusion into the assay media of p-chloromercuribenzoate, hinting at a vital role in the process of thiols. Similarly, the parabolic progress curve (nH = 1.7) is transformed to a straight line (nH = 1.01) when the N-terminal reductase-binding domain in the P-450LM2 molecule is selectively blocked through covalent attachment of fluorescein isothiocyanate (FITC); such a modification does not alter the affinity of the haemoprotein for the amine substrate. Steady-state fluorescence polarization measurements reveal that N,N-dimethylaniline perturbs the motional properties of the fluorophore-bearing reductase-binding region, suggesting the induction of a conformational change. Summarizing these results, the data possibly indicate N,N-dimethylaniline-induced cooperativity in the association of reductase with P-450LM2. PMID:3113486

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

    PubMed

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

    2009-10-01

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

  2. A Method of EC Model Implementation Using Web Service Functions

    NASA Astrophysics Data System (ADS)

    Kurihara, Jun; Koizumi, Hisao; Ishikawa, Toshiyuki; Dasai, Takashi

    In recent years, advances in computer and communication technology and the associated rapid increase in the number of Internet users are encouraging advances in Electronic Commerce (EC). Business models of EC are being actively developed by many different enterprises and engineers, and implemented in many kinds of fields. Meanwhile Web services that reuse remote components over the Internet are drawing attention. Web services are based on SOAP/WSDL/UDDI and are given an important position as the infrastructure of the EC systems. The article analyzes the functions and structures of various business models, establishing the patterns of their distinctive and common features, and proposes a method of determining the implementation specifications of business models utilizing these patterns and Web service functions. This method has been applied to a parts purchasing system, which is a typical pattern of the B to B (Business to Business) EC applications. The article also discusses the results of evaluating this prototype system.

  3. Comparative Studies on the Induction and Inactivation of Nitrate Reductase in Corn Roots and Leaves 1

    PubMed Central

    Aslam, Muhammad; Oaks, Ann

    1976-01-01

    A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH2-nitrate reductase and NO3−-induced NADH-cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A × 182E). The three activities were induced in parallel in both tissues when NO3− was supplied. WO4= suppressed the induction of NADH- and FMNH2-nitrate reductase activities in root tips and leaves. The NO3−-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO4=. In leaves, on the other hand, there was a marked superinduction of the NO3−-induced NADH-cytochrome c reductase in the presence of WO4=. The half-life values of NADH-nitrate reductase and FMNH2-nitrate reductase measured by removing NO3− and adding WO4= to the medium, were 4 hours in root tips and 6 hours in excised leaves. Addition of NO3− in the induction medium together with WO4= gave partial protection of NADH-nitrate reductase and FMNH2-nitrate reductase activities in both root tips and leaves with a t0.5 of 6 and 8 hours, respectively. NO3− also reduced the loss of nitrate reductase activity from mature root sections. In the presence of cycloheximide, both NADH-nitrate reductase and NO3−-induced NADH-cytochrome c reductase activities were lost at similar rates in root tips. NO3− protected the loss of NO3−-induced NADH-cytochrome c reductase to the same extent as that of NADH-nitrate reductase. PMID:16659529

  4. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases

    NASA Astrophysics Data System (ADS)

    Cesaro, Patrizia; Cattaneo, Chiara; Bona, Elisa; Berta, Graziella; Cavaletto, Maria

    2015-09-01

    Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5-8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5-8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5-8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction.

  5. The Kinetics and Inhibition of the Enzyme Methemoglobin Reductase

    ERIC Educational Resources Information Center

    Splittgerber, A. G.; And Others

    1975-01-01

    Describes an undergraduate biochemistry experiment which involves the preparation and kinetics of an oxidation-reduction enzyme system, methemoglobin reductase. A crude enzyme extract is prepared and assayed spectrophotometrically. The enzyme system obeys Michaelis-Menton kinetics with respect to both substrate and the NADH cofactor. (MLH)

  6. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  7. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  8. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  9. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  10. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  11. Domain Evolution and Functional Diversification of Sulfite Reductases

    NASA Astrophysics Data System (ADS)

    Dhillon, Ashita; Goswami, Sulip; Riley, Monica; Teske, Andreas; Sogin, Mitchell

    2005-02-01

    Sulfite reductases are key enzymes of assimilatory and dissimilatory sulfur metabolism, which occur in diverse bacterial and archaeal lineages. They share a highly conserved domain "C-X5-C-n-C-X3-C" for binding siroheme and iron-sulfur clusters that facilitate electron transfer to the substrate. For each sulfite reductase cluster, the siroheme-binding domain is positioned slightly differently at the N-terminus of dsrA and dsrB, while in the assimilatory proteins the siroheme domain is located at the C-terminus. Our sequence and phylogenetic analysis of the siroheme-binding domain shows that sulfite reductase sequences diverged from a common ancestor into four separate clusters (aSir, alSir, dsr, and asrC) that are biochemically distinct; each serves a different assimilatory or dissimilatory role in sulfur metabolism. The phylogenetic distribution and functional grouping in sulfite reductase clusters (dsrA and dsrB vs. aSiR, asrC, and alSir) suggest that their functional diversification during evolution may have preceded the bacterial/archaeal divergence.

  12. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases

    PubMed Central

    Cesaro, Patrizia; Cattaneo, Chiara; Bona, Elisa; Berta, Graziella; Cavaletto, Maria

    2015-01-01

    Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5–8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5–8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5–8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction. PMID:26412036

  13. Characterization of mitochondrial thioredoxin reductase from C. elegans

    SciTech Connect

    Lacey, Brian M.; Hondal, Robert J. . E-mail: Robert.Hondal@uvm.edu

    2006-08-04

    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 {sub cat} of 610 min{sup -1} and a K {sub m} of 610 {mu}M using E. coli thioredoxin as substrate. The reported k {sub cat} is 25% of the k {sub 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.

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

    SciTech Connect

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

    1987-11-01

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

  15. Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation.

    PubMed

    Begara-Morales, Juan C; Sánchez-Calvo, Beatriz; Chaki, Mounira; Mata-Pérez, Capilla; Valderrama, Raquel; Padilla, María N; López-Jaramillo, Javier; Luque, Francisco; Corpas, Francisco J; Barroso, Juan B

    2015-09-01

    The ascorbate-glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO(-)) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO(-) and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO(-). The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO(-). These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO(-) or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate-glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement of

  16. Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation

    PubMed Central

    Begara-Morales, Juan C.; Sánchez-Calvo, Beatriz; Chaki, Mounira; Mata-Pérez, Capilla; Valderrama, Raquel; Padilla, María N.; Luque, Francisco; Corpas, Francisco J.; Barroso, Juan B.

    2015-01-01

    The ascorbate–glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO– and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO–. The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO–. These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO– or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate–glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement

  17. Measurement of nitrous oxide reductase activity in aquatic sediments

    SciTech Connect

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

    1986-01-01

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

  18. Nickel site of methane catalysis in the methyl reductase enzyme

    SciTech Connect

    Shelnutt, J.A.; Shiemke, A.K.; Scott, R.A.

    1988-01-01

    Methyl reductase is the enzyme of methanogenic bacteria that catalyzed the two-electron reduction of the methyl group of 2-(methylthio)ethanesulfonic acid (methyl-S-CoM) to methane and HS-CoM. The methyl group of methyl-S-CoM ultimately comes from the six-electron reduction of CO/sub 2/ by hydrogen, which also provides the reducing equivalents needed by methyl reductase. The nature of the catalytic site of methyl reductase is of current interest from the point of view of developing biomimetic C/sub 1/, chemistries directed toward methane synthesis and activation. In particular, Sandia is using molecular graphics and energy optimization techniques to design macromolecular catalysts that mimic the structure of sites of proteins that carry out C/sub 1/ chemistry. The goal is to produce catalysts whose function is the oxidation of low molecular weight hydrocarbon gases to generate liquid fuels or, alternatively, the reduction of abundant inorganic resources such as CO/sub 2/ to generate gaseous fuels. Unfortunately, the catalytic sites of many of the enzymes of interest, e.g., methyl reductase and methane monooxygenase, have not been characterized by X-ray crystallography and other structural techniques. With the goal of learning more about the structure of one of these naturally occurring sites of C/sub 1/ chemistry, we have obtained the first resonance Raman spectra of the nickel-macrocycle, called F/sub 430/, at the site of catalysis in methyl reductase. To help us structurally interpret the Raman spectra of the enzyme we have also obtained Raman spectra of solutions of the major forms of F/sub 430/ (salt-extracted and cytosol-free) at room temperature and at 77/degree/K and also, under similar solution conditions, spectra of a nickel-corphinoid derivative that is related to F/sub 430/.

  19. Characterization of recombinant glutathione reductase from the psychrophilic Antarctic bacterium Colwellia psychrerythraea.

    PubMed

    Ji, Mikyoung; Barnwell, Callie V; Grunden, Amy M

    2015-07-01

    Glutathione reductases catalyze the reduction of oxidized glutathione (glutathione disulfide, GSSG) using NADPH as the substrate to produce reduced glutathione (GSH), which is an important antioxidant molecule that helps maintain the proper reducing environment of the cell. A recombinant form of glutathione reductase from Colwellia psychrerythraea, a marine psychrophilic bacterium, has been biochemically characterized to determine its molecular and enzymatic properties. C. psychrerythraea glutathione reductase was shown to be a homodimer with a molecular weight of 48.7 kDa using SDS-PAGE, MALDI-TOF mass spectrometry and gel filtration. The C. psychrerythraea glutathione reductase sequence shows significant homology to that of Escherichia coli glutathione reductase (66 % identity), and it possesses the FAD and NADPH binding motifs, as well as absorption spectrum features which are characteristic of flavoenzymes such as glutathione reductase. The psychrophilic C. psychrerythraea glutathione reductase exhibits higher k cat and k cat/K m at lower temperatures (4 °C) compared to mesophilic Baker's yeast glutathione reductase. However, C. psychrerythraea glutathione reductase was able to complement an E. coli glutathione reductase deletion strain in oxidative stress growth assays, demonstrating the functionality of C. psychrerythraea glutathione reductase over a broad temperature range, which suggests its potential utility as an antioxidant enzyme in heterologous systems. PMID:26101017

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

    PubMed

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

    2003-12-12

    Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed. PMID:13129921

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

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

    DOEpatents

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

    2001-04-03

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

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

    DOEpatents

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

    2003-10-21

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

  4. A Method for the Separation and Partial Purification of the Three Forms of Nitrate Reductase Present in Wild-Type Soybean Leaves 1

    PubMed Central

    Streit, Luc; Martin, Barry A.; Harper, James E.

    1987-01-01

    A rapid and simple purification method was used to separate and purify nitrate reductases (NR) from Williams soybean leaves. Blue Sepharose columns were sequentially eluted with 50 millimolar NADPH and 50 millimolar NADH, thus separating NAD(P)H:NR from NADH:NRs. Subsequent purification of the collected peaks on a fast protein liquid chromatography-Mono Q column enabled separation of two NADH:NRs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the subunit relative molecular mass for all three NR forms (constitutive NAD(P)H:NR [pH 6.5], EC 1.6.6.2; constitutive NADH:NR [pH 6.5], EC not assigned; and inducible NADH:NR [pH 7.5], EC 1.6.6.1) was approximately 107 to 109 kilodaltons. All three NRs showed similar spectra with absorption maxima at 413 and 273 nanometers in the oxidized state, and with the characteristics of a cytochrome b type heme upon reduction with NADH (absorption maxima at 556, 527, and 424 nanometers). The technique developed provides an improved separation of the three NR forms from soybean leaves. The similarity of the NRs with regard to their cytochrome b556 type heme content and in relative molecular mass indicated that other differences must exist to account for the different kinetic and physical properties previously reported. Images Fig. 2 PMID:16665497

  5. Purine nucleoside phosphorylase as a cytosolic arsenate reductase.

    PubMed

    Gregus, Zoltán; Németi, Balázs

    2002-11-01

    The findings of the accompanying paper (Németi and Gregus, Toxicol: Sci. 70, 4-12) indicate that the arsenate (AsV) reductase activity of rat liver cytosol is due to an SH enzyme that uses phosphate (or its analogue, arsenate, AsV) and a purine nucleoside (guanosine or inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or inosine) + phosphate <--> guanine (or hypoxanthine) + ribose-1-phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII by rat liver cytosol. AsIII formed from AsV was quantified by HPLC-hydride generation-atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces AsV to AsIII, using AsV instead of phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 microM completely inhibited cytosolic AsV reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the AsV reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of AsV to AsIII in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or inosine). (4) AsV reductase activity of purified PNP, like the cytosolic AsV reductase activity, was inhibited by phosphate (a substrate of PNP alternative to AsV), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the AsV reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP

  6. Alpha- and EC-decay measurements of 257Rf

    NASA Astrophysics Data System (ADS)

    Heßberger, F. P.; Antalic, S.; Mistry, A. K.; Ackermann, D.; Andel, B.; Block, M.; Kalaninova, Z.; Kindler, B.; Kojouharov, I.; Laatiaoui, M.; Lommel, B.; Piot, J.; Vostinar, M.

    2016-07-01

    Alpha- and Electron capture (EC) decay properties of 257 Rf were investigated by measuring α - γ coincidences and correlations between conversion electrons (CE) emitted during the process of EC decay of 257Rf and α decays of the daughter isotope 257Lr. So far, previously unobserved α (8296 keV)- γ (557 keV) coincidences were measured and interpreted as decays of 257mRf ( 11/2-[725] into the 7/2-[743] level in 253No. A search of delayed coincidences between α particles and signals at E ≤ 1000 keV, which are interpreted as being due to CE emission, revealed a clear correlation between CE and α particles from the decay of 257Lr, which is regarded as a direct proof of the EC decay of 257gRf and 257mRf.

  7. Preliminary XCOV26 results for EC14012-1446

    NASA Astrophysics Data System (ADS)

    Provencal, J. L.; Thompson, S.; Montgomery, M.; Kanaan, A.; Shipman, H. L.; Dalessio, J.; Childers, D.; Clemens, C.; Rosen, R.; Henrique, P.; Kim, A.; Strickland, W.; Chandler, D.; Walter, B.; Watson, T. K.; Castanheira, B.; Wood, M.; Vennes, S.; Kepler, S. O.; Reed, M.; Nitta, A.; Kleinman, S. J.; Brown, T.; Kim, S.-L.; Sullivan, D.; Chen, Wen-Ping; Yang, M.; Shih, Chia-You; Jiang, X. J.; Sergeev, A. V.; Maksim, A.; Janulis, R.; Vats, H. O.; Baliyan, K. S.; Zola, S.; Baran, A.; Winiarski, M.; Ogloza, W.; Paparo, M.; Bognar, Z.; Papics, P.; Kilkenny, D.; Sefako, R.; Buckley, D.; Loaring, N.; Kniazev, A.; Silvotti, R.; Galleti, S.; Handler, G.; Nagel, T.; Vauclair, G.; Dolez, N.; Fremy, J. R.; Perez, J.; Almenara, J. M.; Fraga, L.

    2009-06-01

    EC14012-1446 is a hydrogen atmosphere (DA) white dwarf pulsator. Its rich pulsation spectrum displays a range of excited modes with complex multiplet structure, in addition to numerous combination frequencies. In April 2008, EC14012-1446 was the primary target of XCOV26. We obtained over 300 hrs of nearly continuous high speed photometry with the goal of using the nonlinear pulse shapes to empirically determine the parameters of the convection zone. The Fourier transform (FT) of the light curve contains power between 1000 to 4000 μHz, with the dominant peak at 1234 μHz. We find 13 independent frequencies distributed in 8 modes, as well as a myriad of combination frequencies. In the following, we present preliminary results and lay the groundwork for future investigation leading to light curve fitting of EC14012-1446.

  8. Decoding the Rich Pulsation Spectrum of EC 14012-1446

    NASA Astrophysics Data System (ADS)

    Bischoff-Kim, A.

    2013-12-01

    EC 14012-1446 is a ZZ Ceti star (DAV) that was the object of a Whole Earth Telescope run in 2008. The extended coverage run provided a detailed and well resolved period spectrum for the star, confirming and revealing twenty independent modes of vibration, including one triplet and a few more incomplete triplets. With a large number of modes (for pulsating white dwarfs) and good clues for some of the mode identifications from independent methods, EC 14012-1446 is a good candidate for “fast” asteroseismology, where we try to infer interior structure based on a minimal set of assumptions about stellar evolution. The method also allows some numerical experiments that test the validity of asteroseismic techniques used on white dwarfs. Here we experiment with using modified Echelle diagrams on the pulsation spectrum of EC 14012-1446 to aid mode identification.

  9. Measuring Eating Competence: Psychometric Properties and Validity of the ecSatter Inventory

    ERIC Educational Resources Information Center

    Lohse, Barbara; Satter, Ellyn; Horacek, Tanya; Gebreselassie, Tesfayi; Oakland, Mary Jane

    2007-01-01

    Objective: Assess validity of the ecSatter Inventory (ecSI) to measure eating competence (EC). Design: Concurrent administration of ecSI with validated measures of eating behaviors using on-line and paper-pencil formats. Setting: The on-line survey was completed by 370 participants; 462 completed the paper version. Participants: Participants…

  10. Involvement of nitrate reductase in auxin-induced NO synthesis

    PubMed Central

    Erdei, L

    2008-01-01

    It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.1 Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (NG-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation. PMID:19704423

  11. Involvement of nitrate reductase in auxin-induced NO synthesis.

    PubMed

    Kolbert, Zsuzsanna; Erdei, L

    2008-11-01

    It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.1 Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (N(G)-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation. PMID:19704423

  12. Methyltetrahydrofolate reductase polymorphism influences onset of Huntington's disease.

    PubMed

    Brune, N; Andrich, J; Gencik, M; Saft, C; Müller, Th; Valentin, S; Przuntek, H; Epplen, J T

    2004-01-01

    Onset of Huntington's disease (HD) negatively correlates with CAG repeat length of the HD gene, which encodes the protein huntingtin. This protein interacts with the homocysteine metabolizing enzyme cystathionine betasynthase (CBS). Objective of this study was to analyze the impact of CAG repeats, polymorphisms of various homocysteine metabolizing enzymes, like CBS, Methyltetrahydrofolate Reductase (MTHTR), Methionine Synthase Reductase (MSR) and methionine synthase (MS) on HD onset in 171 patients. The significant impact of CAG repeats on HD onset (chi2= 25.54, FG = 4, p<0.0001) with a significant correlation between both (R= -0.521, p=0.01) was obvious. HD patients with the homozygous MTHFR-1298-CC significantly (p = 0.024) earlier experienced HD symptoms. There was no influence demonstrable of CBS, MSR and MS. Determination of MTHFR polymorphisms and CAG repeats enables screening for subjects with putative early HD onset in order to study neuroprotective compounds in their efficacy to delay HD symptoms. PMID:15354395

  13. Genes and proteins of Escherichia coli (GenProtEc).

    PubMed

    Riley, M; Space, D B

    1996-01-01

    GenProtEc is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each. Also present are data on sequence similarities among E.coli proteins with PAM values, percent identity of amino acids, length of alignment and percent aligned. The database is available as a PKZip file by ftp from mbl.edu/pub/ecoli.exe. The program runs under MS-DOS on IMB-compatible machines. GenProtEc can also be accessed through the World Wide Web at URL http://mbl.edu/html/ecoli.html. PMID:8594596

  14. On the neutrinoless double β+/EC decays

    NASA Astrophysics Data System (ADS)

    Suhonen, Jouni

    2013-12-01

    The neutrinoless double positron-emission/electron-capture (0νβ+/EC) decays are studied for the magnitudes of the involved nuclear matrix elements (NMEs). Decays to the ground state, 0gs+, and excited 0+ states are discussed. The participant many-body wave functions are evaluated in the framework of the quasiparticle random-phase approximation (QRPA). Effective, G-matrix-derived nuclear forces are used in realistic single-particle model spaces. The channels β+β+, β+EC, and the resonant neutrinoless double electron capture (R0νECEC) are discussed.

  15. Flight evaluation of Spacelab 1 payload thermal/ECS interfaces

    NASA Technical Reports Server (NTRS)

    Ray, C. D.; Humphries, W. R.; Patterson, W. C.

    1984-01-01

    The Spacelab (SL-1) thermal/Environmental Control Systems (ECS) are discussed. Preflight analyses and flight data are compared in order to validate payload to Spacelab interfaces as well as corroborate modeling/analysis techniques. In doing so, a brief description of the Spacelab 1 payload configuration and the interactive Spacelab thermal/ECS systems are given. In particular, these interfaces address equipment cooling air, thermal and fluid conditions, humidity levels, both freon and water loop temperatures and load states, as well as passive radiant environment interfaces.

  16. On the neutrinoless double β{sup +}/EC decays

    SciTech Connect

    Suhonen, Jouni

    2013-12-30

    The neutrinoless double positron-emission/electron-capture (0νβ{sup +}/EC) decays are studied for the magnitudes of the involved nuclear matrix elements (NMEs). Decays to the ground state, 0{sub gs}{sup +}, and excited 0{sup +} states are discussed. The participant many-body wave functions are evaluated in the framework of the quasiparticle random-phase approximation (QRPA). Effective, G-matrix-derived nuclear forces are used in realistic single-particle model spaces. The channels β{sup +}β{sup +}, β{sup +}EC, and the resonant neutrinoless double electron capture (R0νECEC) are discussed.

  17. Structural Basis for Substrate Specificity in Human Monomeric Carbonyl Reductases

    PubMed Central

    El-Hawari, Yasser; Dunford, James E.; Kochan, Grazyna; Wsol, Vladimir; Martin, Hans-Joerg; Maser, Edmund; Oppermann, Udo

    2009-01-01

    Carbonyl reduction constitutes a phase I reaction for many xenobiotics and is carried out in mammals mainly by members of two protein families, namely aldo-keto reductases and short-chain dehydrogenases/reductases. In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids. One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum. A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date. Screening of a focused xenobiotic compound library revealed that CBR3 has narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further investigate structure-activity relationships between these enzymes we crystallized CBR3, performed substrate docking, site-directed mutagenesis and compared its kinetic features to CBR1. Despite high sequence similarities, the active sites differ in shape and surface properties. The data reveal that the differences in substrate specificity are largely due to a short segment of a substrate binding loop comprising critical residues Trp229/Pro230, Ala235/Asp236 as well as part of the active site formed by Met141/Gln142 in CBR1 and CBR3, respectively. The data suggest a minor role in xenobiotic metabolism for CBR3. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1. PMID:19841672

  18. Two fatty acyl reductases involved in moth pheromone biosynthesis.

    PubMed

    Antony, Binu; Ding, Bao-Jian; Moto, Ken'Ichi; Aldosari, Saleh A; Aldawood, Abdulrahman S

    2016-01-01

    Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective 'single pgFARs' produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a 'single reductase' can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

  19. Using chemical approaches to study selenoproteins - focus on thioredoxin reductases

    PubMed Central

    Hondal, Robert J.

    2009-01-01

    The study of selenocysteine-containing proteins is difficult due to the problems associated with the heterologous production of these proteins. These problems are due to the intricate recoding mechanism used by cells to translate the UGA codon as a sense codon for selenocysteine. The process is further complicated by the fact that eukaryotes and prokaryotes have different UGA recoding machineries. This review focuses on chemical approaches to produce selenoproteins and study the mechanism of selenoenzymes. The use of intein-mediated peptide ligation is discussed with respect to the production of the mammalian selenoenzymes thioredoxin reductase and selenoprotein R, also known as methionine sulfoxide reductase B1. New methods for removing protecting groups from selenocysteine post-synthesis and methods for selenosulfide/diselenide formation are also reviewed. Chemical approaches have also been used to study the enzymatic mechanism of thioredoxin reductase. The approach divides the enzyme into two modules, a large protein module lacking selenocysteine and a small, synthetic selenocysteine-containing peptide. Study of this semisynthetic enzyme has revealed three distinct enzymatic pathways that depend on the properties of the substrate. The enzyme utilizes a macromolecular mechanism for protein substrates, a second mechanism for small molecule substrates and a third pathway for selenium-containing substrates such as selenocystine. PMID:19406205

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

    PubMed

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

    2016-04-22

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

  1. Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro.

    PubMed

    Rockel, Peter; Strube, Frank; Rockel, Andra; Wildt, Juergen; Kaiser, Werner M

    2002-01-01

    NO (nitric oxide) production from sunflower plants (Helianthus annuus L.), detached spinach leaves (Spinacia oleracea L.), desalted spinach leaf extracts or commercial maize (Zea mays L.) leaf nitrate reductase (NR, EC 1.6.6.1) was continuously followed as NO emission into the gas phase by chemiluminescence detection, and its response to post-translational NR modulation was examined in vitro and in vivo. NR (purified or in crude extracts) in vitro produced NO at saturating NADH and nitrite concentrations at about 1% of its nitrate reduction capacity. The K(m) for nitrite was relatively high (100 microM) compared to nitrite concentrations in illuminated leaves (10 microM). NO production was competitively inhibited by physiological nitrate concentrations (K(i)=50 microM). Importantly, inactivation of NR in crude extracts by protein phosphorylation with MgATP in the presence of a protein phosphatase inhibitor also inhibited NO production. Nitrate-fertilized plants or leaves emitted NO into purified air. The NO emission was lower in the dark than in the light, but was generally only a small fraction of the total NR activity in the tissue (about 0.01-0.1%). In order to check for a modulation of NO production in vivo, NR was artificially activated by treatments such as anoxia, feeding uncouplers or AICAR (a cell permeant 5'-AMP analogue). Under all these conditions, leaves were accumulating nitrite to concentrations exceeding those in normal illuminated leaves up to 100-fold, and NO production was drastically increased especially in the dark. NO production by leaf extracts or intact leaves was unaffected by nitric oxide synthase inhibitors. It is concluded that in non-elicited leaves NO is produced in variable quantities by NR depending on the total NR activity, the NR activation state and the cytosolic nitrite and nitrate concentration. PMID:11741046

  2. Thiol–disulfide exchange is involved in the catalytic mechanism of peptide methionine sulfoxide reductase

    PubMed Central

    Lowther, W. Todd; Brot, Nathan; Weissbach, Herbert; Honek, John F.; Matthews, Brian W.

    2000-01-01

    Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) reverses the inactivation of many proteins due to the oxidation of critical methionine residues by reducing methionine sulfoxide, Met(O), to methionine. MsrA activity is independent of bound metal and cofactors but does require reducing equivalents from either DTT or a thioredoxin-regenerating system. In an effort to understand these observations, the four cysteine residues of bovine MsrA were mutated to serine in a series of permutations. An analysis of the enzymatic activity of the variants and their free sulfhydryl states by mass spectrometry revealed that thiol–disulfide exchange occurs during catalysis. In particular, the strictly conserved Cys-72 was found to be essential for activity and could form disulfide bonds, only upon incubation with substrate, with either Cys-218 or Cys-227, located at the C terminus. The significantly decreased activity of the Cys-218 and Cys-227 variants in the presence of thioredoxin suggested that these residues shuttle reducing equivalents from thioredoxin to the active site. A reaction mechanism based on the known reactivities of thiols with sulfoxides and the available data for MsrA was formulated. In this scheme, Cys-72 acts as a nucleophile and attacks the sulfur atom of the sulfoxide moiety, leading to the formation of a covalent, tetracoordinate intermediate. Collapse of the intermediate is facilitated by proton transfer and the concomitant attack of Cys-218 on Cys-72, leading to the formation of a disulfide bond. The active site is returned to the reduced state for another round of catalysis by a series of thiol—disulfide exchange reactions via Cys-227, DTT, or thioredoxin. PMID:10841552

  3. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    NASA Astrophysics Data System (ADS)

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils.

  4. Award of EC Television Prize for Broadcasts on Vocational Training.

    ERIC Educational Resources Information Center

    European Centre for the Development of Vocational Training, Berlin (West Germany).

    The European Centre for the Development of Vocational Training (CEDEFOP) is endeavoring to encourage television to provide more and better information on vocational and continuing education in the European Community (EC). Therefore, it held its first competition to award prizes for broadcasts presenting information on vocational training,…

  5. EcSL: Teaching Economics as a Second Language.

    ERIC Educational Resources Information Center

    Crowe, Richard

    Hazard Community College, in Kentucky, has implemented a new instructional methodology for economics courses called Economics as a Second Language (EcSL). This teaching approach, based on the theory of Rendigs Fel that the best model for learning economics is the foreign language classroom, utilizes strategies similar to those employed in…

  6. Draft genome sequence of Escherichia coli LCT-EC106.

    PubMed

    Li, Tianzhi; Pu, Fei; Yang, Rentao; Fang, Xiangqun; Wang, Junfeng; Guo, Yinghua; Chang, De; Su, Longxiang; Guo, Na; Jiang, Xuege; Zhao, Jiao; Liu, Changting

    2012-08-01

    Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the intestine of warm-blooded organisms. Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans. Here, we present the complete genome sequence of Escherichia coli LCT-EC106, which was isolated from CGMCC 1.2385. PMID:22843582

  7. Summary for the Theory Session at EC-18

    NASA Astrophysics Data System (ADS)

    Marushchenko, N. B.

    2015-03-01

    A brief review of the theory contributions presented in the EC-18 Workshop is given. The covered spectrum of topics is quite broad including the electron cyclotron wave propagation physics as well the results of numerical modelling of transport and current drive.

  8. Pharmacokinetics of budesonide (Entocort EC) capsules for Crohn's disease.

    PubMed

    Edsbäcker, Staffan; Andersson, Tommy

    2004-01-01

    This overview summarises available pharmacokinetic data on budesonide capsules (Entocort EC), approved for the treatment of mild-to-moderate active Crohn's disease involving the ileum and/or ascending colon and for prolongation of symptom control. Budesonide is a locally-acting glucocorticosteroid with an extensive, primarily hepatic, metabolism after oral administration. It is rapidly absorbed and biotransformed by cytochrome P450 (CYP) 3A to metabolites with negligible glucocorticoid activity. Entocort EC, a pH- and time-dependent oral formulation of budesonide, was developed to optimise drug delivery to the ileum and throughout the colon. Pharmaco-scintigraphic studies have confirmed that the Entocort EC formulation delays budesonide absorption and prolongs the rate of elimination but maintains complete absorption. This improves the delivery of budesonide to the intestinal lumen relative to a plain formulation. A low systemic availability of 9-21% indicates extensive first-pass elimination. Food appears to have little impact on the absorption of budesonide from Entocort EC capsules and the pharmacokinetics are dose-proportional between 3 and 15 mg. On average, systemic availability was 2.5-fold higher in patients with cirrhosis compared with healthy controls; however, mild liver impairment had little effect on systemic exposure. Pharmacokinetics appear unaffected by gender and age, although this has not been tested in younger children. Renal impairment is not expected to have an impact on the kinetics of Entocort EC. Budesonide is unlikely to inhibit the metabolism of other drugs, including CYP3A4 substrates, mainly because of the very low plasma concentrations obtained with the compound even after high doses of Entocort trade mark EC capsules. Strong CYP3A4 inhibitors, such as ketoconazole, will inhibit the metabolism of budesonide, resulting in several-fold increases in the area under the concentration-time curve of budesonide. Also, grapefruit juice intake

  9. Structure of the Molybdenum Site of EEcherichia Coli Trimethylamine N-Oxide Reductase

    SciTech Connect

    Zhang, L.; Nelson, K.Johnson; Rajagopalan, K.V.; George, G.N.

    2009-05-28

    We report a structural characterization of the molybdenum site of recombinant Escherichia coli trimethylamine N-oxide (TMAO) reductase using X-ray absorption spectroscopy. The enzyme active site shows considerable similarity to that of dimethyl sulfoxide (DMSO) reductase, in that, like DMSO reductase, the TMAO reductase active site can exist in multiple forms. Examination of the published crystal structure of TMAO oxidase from Shewanella massilia indicates that the postulated Mo coordination structure is chemically impossible. The presence of multiple active site structures provides a potential explanation for the anomalous features reported from the crystal structure.

  10. Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

    PubMed

    Belcourt, M F; Hodnick, W F; Rockwell, S; Sartorelli, A C

    1996-01-01

    Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins. PMID:8552660

  11. Familial Hypercholesterolemia: Identification of a Defect in the Regulation of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity Associated with Overproduction of Cholesterol

    PubMed Central

    Goldstein, Joseph L.; Brown, Michael S.

    1973-01-01

    The homozygous form of the autosomal dominant disorder, familial hypercholesterolemia, is characterized by the presence in children of profound hypercholesterolemia, cutaneous planar xanthomas, and rapidly progressive coronary vascular disease that usually results in death before age 30 years. Cultured skin fibroblasts from three unrelated subjects with this disorder showed 40- to 60-fold higher activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), the rate-controlling enzyme in cholesterol biosynthesis, when compared with fibroblasts of seven control subjects. Enhanced enzyme activity resulted from a complete absence of normal feedback suppression by low-density lipoproteins, which led to a marked overproduction of cholesterol by the mutant cells. The demonstration of apparently identical kinetic properties of the reductase activity of control and mutant cells, coupled with the evidence that this enzyme is normally regulated not by allosteric effectors but by alterations in enzyme synthesis and degradation, suggests that the primary genetic abnormality does not involve the structural gene for the enzyme itself, but a hitherto unidentified gene whose product is necessary for mediation of feedback control by lipoproteins. The fibroblasts of two obligate heterozygotes, the parents of one of the homozygotes, showed a pattern of enzyme regulation intermediate between that of controls and homozygotes. PMID:4355366

  12. Protein isotope effects in dihydrofolate reductase from Geobacillus stearothermophilus show entropic-enthalpic compensatory effects on the rate constant.

    PubMed

    Luk, Louis Y P; Ruiz-Pernía, J Javier; Dawson, William M; Loveridge, E Joel; Tuñón, Iñaki; Moliner, Vicent; Allemann, Rudolf K

    2014-12-10

    Catalysis by dihydrofolate reductase from the moderately thermophilic bacterium Geobacillus stearothermophilus (BsDHFR) was investigated by isotope substitution of the enzyme. The enzyme kinetic isotope effect for hydride transfer was close to unity at physiological temperatures but increased with decreasing temperatures to a value of 1.65 at 5 °C. This behavior is opposite to that observed for DHFR from Escherichia coli (EcDHFR), where the enzyme kinetic isotope effect increased slightly with increasing temperature. These experimental results were reproduced in the framework of variational transition-state theory that includes a dynamical recrossing coefficient that varies with the mass of the protein. Our simulations indicate that BsDHFR has greater flexibility than EcDHFR on the ps-ns time scale, which affects the coupling of the environmental motions of the protein to the chemical coordinate and consequently to the recrossing trajectories on the reaction barrier. The intensity of the dynamic coupling in DHFRs is influenced by compensatory temperature-dependent factors, namely the enthalpic barrier needed to achieve an ideal transition-state configuration with minimal nonproductive trajectories and the protein disorder that disrupts the electrostatic preorganization required to stabilize the transition state. Together with our previous studies of other DHFRs, the results presented here provide a general explanation why protein dynamic effects vary between enzymes. Our theoretical treatment demonstrates that these effects can be satisfactorily reproduced by including a transmission coefficient in the rate constant calculation, whose dependence on temperature is affected by the protein flexibility. PMID:25396728

  13. Differential Light Induction of Nitrate Reductases in Greening and Photobleached Soybean Seedlings 1

    PubMed Central

    Kakefuda, Genichi; Duke, Stanley H.; Duke, Stephen O.

    1983-01-01

    Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO3−, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO3− and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO3− and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO3−. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO3−, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO3− requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO3− requirement for induction were quite different. Km values for NO3− were identical for both nitrate reductases. PMID:16663185

  14. The effect of copper and gallium compounds on ribonucleotide reductase

    SciTech Connect

    Narasimhan, J.

    1992-01-01

    The mode of action of copper complexes (CuL and CuKTS) and gallium compounds (gallium nitrate and citrate) in cytotoxicity was studied. The effects of these agents on the enzyme ribonucleotide reductase was investigated by monitoring the tyrosyl free radical present in the active site of the enzyme through electron spin resonance (ESR) spectroscopy. Ribonucleotide reductase, a key enzyme in cellular proliferation, consists of two subunits. M1, a dimer of molecular weight 170,000 contains the substrate and effector binding sites. M2, a dimer of molecular weight 88,000, contains non-heme iron and tyrosyl free radical essential for the activity of the enzyme. In studies using copper complexes, the cellular oxidative chemistry was examined by ESR studies on adduct formation with membranes, and oxidation of thiols. Membrane thiols were oxidized through the reduction of the ESR signal of the thiol adduct and the analysis of sulfhydryl content. Using the radiolabel [sup 59]Fe, the inhibitory action of copper thiosemicarbazones on cellular iron uptake was shown. The inhibitory action of CuL on ribonucleotide reductase was shown by the quenching of the tyrosyl free radical on the M2 subunit. The hypothesis that gallium directly interacts with the M2 subunit of the enzyme and displaces the iron from it was proven. The tyrosyl free radical signal from cell lysates was inhibited by the direct addition of gallium compounds. Gallium content in the cells was measured by a fluorimetric method, to ensure the presence of sufficient amounts of gallium to compete with the iron in the M2 subunit. The enzyme activity, measured by the conversion of [sup 14]C-CDP to the labeled deoxy CDP, was inhibited by the addition of gallium nitrate in a cell free assay system. The immunoprecipitation studies of the [sup 59]Fe labeled M2 protein using the monoclonal antibody directed against this subunit suggested that gallium releases iron from the M2 subunit.

  15. Methylenetetrahydrofolate Reductase C677T: Hypoplastic Left Heart and Thrombosis.

    PubMed

    Spronk, Kimberly J; Olivero, Anthony D; Haw, Marcus P; Vettukattil, Joseph J

    2015-10-01

    The incidence of congenital heart defects is higher in infants with mutation of methylenetetrahydrofolate reductase (MTHFR) gene. The MTHFR C677T gene decreases the bioavailability of folate and increases plasma homocysteine, a risk factor for thrombosis. There have been no reported cases in the literature on the clinical implications of this procoagulable state in the setting of cyanotic heart disease, which itself has prothrombotic predisposition. Two patients with hypoplastic left heart syndrome developed postoperative thrombotic complications, both were homozygous for MTHFR C677T. We present these cases and highlight the implications of MTHFR mutation in the management of complex congenital heart disease. PMID:26467879

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

    PubMed

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

    2016-03-01

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

  17. Nanoscale Bio-Molecular Control Using EC-OWLS

    SciTech Connect

    Bearinger, J P; Voros, J; Hubbell, J A; Textor, M

    2002-11-20

    A recently developed technique termed ''Electrochemical Optical Waveguide Lightmode Spectroscopy'' (EC-OWLS) [1] combines evanescent-field optical sensing with electrochemical control of surface adsorption processes. Initial EC-OWLS investigations efficiently monitored molecular surface adsorption and layer thickness changes of an adsorbed polymer layer examined in situ as a function of potential applied to a waveguide1. A layer of indium tin oxide (ITO) served as both a high refractive index waveguide for optical sensing, and a conductive electrode; an electrochemical flow-through fluid cell incorporated working, reference and counter electrodes. Poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) served as a model, polycation adsorbate. Results indicate that adsorption and desorption of PLL-g-PEG from aqueous buffer are a function of applied potential, and that binding events subsequent to PLL-g-PEG functionalization are dependent on reorganization in the molecular adlayer.

  18. Computational Electromagnetic Modeling of SansEC(Trade Mark) Sensors

    NASA Technical Reports Server (NTRS)

    Smith, Laura J.; Dudley, Kenneth L.; Szatkowski, George N.

    2011-01-01

    This paper describes the preliminary effort to apply computational design tools to aid in the development of an electromagnetic SansEC resonant sensor composite materials damage detection system. The computational methods and models employed on this research problem will evolve in complexity over time and will lead to the development of new computational methods and experimental sensor systems that demonstrate the capability to detect, diagnose, and monitor the damage of composite materials and structures on aerospace vehicles.

  19. EC: an efficient error correction algorithm for short reads

    PubMed Central

    2015-01-01

    Background In highly parallel next-generation sequencing (NGS) techniques millions to billions of short reads are produced from a genomic sequence in a single run. Due to the limitation of the NGS technologies, there could be errors in the reads. The error rate of the reads can be reduced with trimming and by correcting the erroneous bases of the reads. It helps to achieve high quality data and the computational complexity of many biological applications will be greatly reduced if the reads are first corrected. We have developed a novel error correction algorithm called EC and compared it with four other state-of-the-art algorithms using both real and simulated sequencing reads. Results We have done extensive and rigorous experiments that reveal that EC is indeed an effective, scalable, and efficient error correction tool. Real reads that we have employed in our performance evaluation are Illumina-generated short reads of various lengths. Six experimental datasets we have utilized are taken from sequence and read archive (SRA) at NCBI. The simulated reads are obtained by picking substrings from random positions of reference genomes. To introduce errors, some of the bases of the simulated reads are changed to other bases with some probabilities. Conclusions Error correction is a vital problem in biology especially for NGS data. In this paper we present a novel algorithm, called Error Corrector (EC), for correcting substitution errors in biological sequencing reads. We plan to investigate the possibility of employing the techniques introduced in this research paper to handle insertion and deletion errors also. Software availability The implementation is freely available for non-commercial purposes. It can be downloaded from: http://engr.uconn.edu/~rajasek/EC.zip. PMID:26678663

  20. Summary of the experimental session EC-10 workshop

    SciTech Connect

    Lohr, J.

    1997-06-01

    This review summarizes a subset of the EC-10 presentations which had been assigned by the organizing committee identification tags beginning with EX. There were fourteen presentations in this group, seven oral and seven posters. Included among the oral presentations were two invited talks. With the exception of the review of plans for electron cyclotron waves in ITER, there were all reports of physics experiments or operational experience as opposed to accounts of hardware development.

  1. Present status of developing petroleum-substituting energy (EC)

    NASA Astrophysics Data System (ADS)

    1993-03-01

    The EC has had approximately 50% of its total energy demand supplied by imports from the exterior of the Community. Hence, it is getting important to develop oil-substituting renewable energy as well as to curtail the emission of carbon dioxide. In consideration of these situations, the results of investigation on the energy policy of the European Community are described. The policy comprises three courses: European Energy Charter, formation of an open European Community energy market, and environmental conservation. Particularly, concerning the reduction of carbon dioxide emission, the EC Council has decided to introduce carbon dioxide taxation so as to suppress the carbon dioxide emission in the year 2000 to the 1990 level. The arrangement for its introduction, however, encountered with difficulties because of the opposition of various countries other than the European Community and the industrial world of the European Community. Legislation of the investment promotion law for energy saving and the construction of infrastructure footing and an information network are ineffective due to the sluggish fuel price and economic recession. A plurality of EC member countries are advancing a comprehensive activity within the framework of the joint programs of research, development, and demonstration set for the renewable energy policy ensuring energy supply and environmental harmonization.

  2. Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent.

    PubMed Central

    Alberts, A W; Chen, J; Kuron, G; Hunt, V; Huff, J; Hoffman, C; Rothrock, J; Lopez, M; Joshua, H; Harris, E; Patchett, A; Monaghan, R; Currie, S; Stapley, E; Albers-Schonberg, G; Hensens, O; Hirshfield, J; Hoogsteen, K; Liesch, J; Springer, J

    1980-01-01

    Mevinolin, a fungal metabolite, was isolated from cultures of Aspergillus terreus. The structure and absolute configuration of mevinolini and its open acid form, mevinolinic acid, were determined by a combination of physical techniques. Mevinolin was shown to be 1,2,6,7,8,8a-hexahydro-beta, delta-dihydroxy-2,6-dimethyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-hepatanoic acid delta-lactone. Mevinolin in the hydroxy-acid form, mevinolinic acid, is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]; its Ki of 0.6 nM can be compared to 1.4 nM for the hydroxy acid form of the previously described related inhibitor, ML-236B (compactin, 6-demethylmevinolin). In the rat, orally administered sodium mevinolinate was an active inhibitor of cholesterol synthesis in an acute assay (50% inhibitory dose = 46 microgram/kg). Furthermore, it was shown that mevinolin was an orally active cholesterol-lowering agent in the dog. Treatment of dogs for 3 weeks with mevinolin at 8 mg/kg per day resulted in a 29.3 +/- 2.5% lowering of plasma cholesterol. PMID:6933445

  3. Inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase by mevinolin in familial hypercholesterolemia heterozygotes: effects on cholesterol balance.

    PubMed Central

    Grundy, S M; Bilheimer, D W

    1984-01-01

    Patients with heterozygous familial hypercholesterolemia (FH) have a deficiency of receptors for plasma low-density lipoprotein (LDL) that impairs removal of LDL from plasma. In these patients, mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase [mevalonate:NAD+ oxidoreductase (CoA-acylating), EC 1.1.1.88], increases receptors for LDL and decreases LDL concentrations. To determine whether mevinolin also causes severe decreases in total body synthesis of cholesterol, fecal excretions of neutral steroids and acidic steroids were determined in five FH heterozygotes before and during treatment with mevinolin. The drug produced an average decrease in plasma total cholesterol of 23% and in LDL cholesterol of 24%. Mevinolin caused a significant decrease in the output of neutral and acidic steroids in three patients, but it caused no alterations in two others. Changes in fecal output of steroids did not correlate with the degree of lowering of the patients' LDL-cholesterol level. In none of the patients did the output of fecal steroids fall below the values seen in normal subjects studied under similar conditions. One patient had a previous ileal exclusion operation and had a massive output of acidic steroids in the control period; mevinolin therapy caused a slight decrease in excretion of acidic steroids, but the output was still markedly above normal. We conclude that the LDL lowering action of mevinolin does not appear to require a severe decrease in cholesterol synthesis that might lead to depletion of vital body stores of cholesterol. PMID:6371816

  4. Synthesis and biological evaluation of novel inhibitors against 1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea.

    PubMed

    Chen, Haifeng; Han, Xinya; Qin, Nian; Wei, Lin; Yang, Yue; Rao, Li; Chi, Bo; Feng, Lingling; Ren, Yanliang; Wan, Jian

    2016-03-15

    1,3,8-Trihydroxynaphthalene reductase (3HNR) is an essential enzymes that is involved in fungal melanin biosynthesis. Based on the structural informations of active site of 3HNR, a series of β-nitrostyrene compounds were rationally designed and synthesized. The enzymatic activities of these compounds showed that most of them exhibited high inhibitory activities (<5.0 μM) against 3HNR; compound 3-2 exhibit the highest inhibitory activity (IC50=0.29 μM). In particular, some of these compounds had moderate fungicidal activity against Magnaporthe grisea. Compound 3-4 showed high in vivo activities against M. grisea (EC50=9.5 ppm). Furthermore, compound 3-2 was selected as a representative molecule, and the probable binding mode of this compound and the surrounding residues in the active site of 3HNR was elucidated by using molecular dock. The positive results suggest that β-nitrostyrene derivatives are most likely to be promising leads toward the discovery of novel agent of rice blast. PMID:26860927

  5. Cotton benzoquinone reductase: up-regulation during early fiber development and heterologous expression and characterization in Pichia pastoris.

    PubMed

    Turley, Rickie B; Taliercio, Earl

    2008-01-01

    Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme which catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2D-PAGE comparisons, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage but not in the fiberless line SL 1-7-1. These proteins were excised from the gel, partially sequenced and identified to be BR isoforms. PCR was used to amplify both full length coding regions of 609bp and once cloned, the restriction enzyme HindIII was used to distinguish the clones encoding the BR1 (one site) and BR2 (two sites) isoforms. Both deduced protein sequences had 203 residues which differed at 14 residues. The molecular mass and pIs were similar between the measured protein (2D-PAGE) and the theoretical protein (deduced). Heterologous proteins BR1 and BR2 were produced for further study by ligating the BR1 and BR2 clones in frame into the alpha-factor secretion sequence in pPICZalphaA vector and expressed with Pichia pastoris. Both BR1 and BR2 were approximately 26.5kDa and did enzymatically reduce 2,6-dimethoxybenzoquinone similar to the fungal BR. PMID:18534861

  6. A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase.

    PubMed

    Milhim, Mohammed; Gerber, Adrian; Neunzig, Jens; Hannemann, Frank; Bernhardt, Rita

    2016-08-10

    Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions. PMID:27238232

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Nahata, A; Dixit, V K

    2014-08-01

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

  9. Sequence and properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2.

    PubMed Central

    French, C E; Nicklin, S; Bruce, N C

    1996-01-01

    Pentaerythritol tetranitrate reductase, which reductively liberates nitrite from nitrate esters, is related to old yellow enzyme. Pentaerythritol tetranitrate reductase follows a ping-pong mechanism with competitive substrate inhibition by NADPH, is strongly inhibited by steroids, and is capable of reducing the unsaturated bond of 2-cyclohexen-1-one. PMID:8932320

  10. Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings

    NASA Technical Reports Server (NTRS)

    Warner, R. L.; Huffaker, R. C.

    1989-01-01

    Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.

  11. The effect of salts on the activity and stability of Escherichia coli and Haloferax volcanii dihydrofolate reductases.

    PubMed

    Wright, Donna B; Banks, Douglas D; Lohman, Jeremy R; Hilsenbeck, Jacqueline L; Gloss, Lisa M

    2002-10-18

    The extremely halophilic Archae require near-saturating concentrations of salt in the external environment and in their cytoplasm, potassium being the predominant intracellular cation. The proteins of these organisms have evolved to function in concentrations of salt that inactivate or precipitate homologous proteins from non-halophilic species. It has been proposed that haloadaptation is primarily due to clustering of acidic residues on the surface of the protein, and that these clusters bind networks of hydrated ions. The dihydrofolate reductases from Escherichia coli (ecDHFR) and two DHFR isozymes from Haloferax volcanii (hvDHFR1 and hvDHFR2) have been used as a model system to compare the effect of salts on a mesophilic and halophilic enzyme. The KCl-dependence of the activity and substrate affinity was investigated. ecDHFR is largely inactivated above 1M KCl, with no major effect on substrate affinity. hvDHFR1 and hvDHFR2 unfold at KCl concentrations below approximately 0.5M. Above approximately 1M, the KCl dependence of the hvDHFR activities can be attributed to the effect of salt on substrate affinity. The abilities of NaCl, KCl, and CsCl to enhance the stability to urea denaturation were determined, and similar efficacies of stabilization were observed for all three DHFR variants. The DeltaG degrees (H(2)O) values increased linearly with increasing KCl and CsCl concentrations. The increase of DeltaG degrees (H(2)O) as a function of the smallest cation, NaCl, is slightly curved, suggesting a minor stabilization from cation binding or screening of electrostatic repulsion. At their respective physiological ionic strengths, the DHFR variants exhibit similar stabilities. Salts stabilize ecDHFR and the hvDHFRs by a common mechanism, not a halophile-specific mechanism, such as the binding of hydrated salt networks. The primary mode of salt stabilization of the mesophilic and halophilic DHFRs appears to be through preferential hydration and the Hofmeister effect of

  12. Research on Intellectual Property Conflicts Identification in Knowledge Transferring among EC Enterprises

    NASA Astrophysics Data System (ADS)

    Su, Shibin

    As the lacks of existing research about intellectual property conflicts management of EC enterprise, the paper analysis the intellectual property conflicts in knowledge transferring among EC enterprises by intellectual property types, then, the paper makes research on intellectual property conflicts identification in knowledge transferring among EC enterprises, and gives relative assumption, meanwhile, the paper makes quantities identification of intellectual property conflicts in knowledge transferring among EC enterprises by evidential theory, finally, the paper gives the further research orientations.

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

    PubMed Central

    2008-01-01

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

  14. Life with too much polyprenol–polyprenol reductase deficiency

    PubMed Central

    Gründahl, J.E.H.; Guan, Z.; Rust, S.; Reunert, J.; Müller, B.; Du Chesne, I.; Zerres, K.; Rudnik-Schöneborn, S.; Ortiz-Brüchle, N.; Häusler, M.G.; Siedlecka, J.; Swiezewska, E.; Raetz, C.R.H.; Marquardt, T.

    2012-01-01

    Congenital disorders of glycosylation (CDG) are caused by a dysfunction of glycosylation, an essential step in the manufacturing process of glycoproteins. This paper focuses on a 6-year-old patient with a new type of CDG-I caused by a defect of the steroid 5α reductase type 3 gene (SRD5A3). The clinical features were psychomotor retardation, pathological nystagmus, slight muscular hypotonia and microcephaly. SRD5A3 was recently identified encoding the polyprenol reductase, an enzyme catalyzing the final step of the biosynthesis of dolichol, which is required for the assembly of the glycans needed for N-glycosylation. Although an early homozygous stop-codon (c.57G> A [W19X]) with no functional protein was found in the patient, about 70% of transferrin (Tf) was correctly glycosylated. Quantification of dolichol and unreduced polyprenol in the patient's fibroblasts demonstrated a high polyprenol/dolichol ratio with normal amounts of dolichol, indicating that high polyprenol levels might compete with dolichol for the initiation of N-glycan assembly but without supporting normal glycosylation and that there must be an alternative pathway for dolichol biosynthesis. PMID:22304929

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

    PubMed

    Khodashenas, Bahareh; Ghorbani, Hamid Reza

    2016-06-01

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

  16. New pteridine substrates for dihydropteridine reductase and horseradish peroxidase.

    PubMed Central

    Armarego, W L; Ohnishi, A; Taguchi, H

    1986-01-01

    The oxidation of 4,5-diaminopyrimidin-6(1H)-one, 5,6,7,8-tetrahydropteridin-4(3H)-one, its 6-methyl and cis-6,7-dimethyl derivatives, and 6-methyl- and cis-6-7-dimethyl-5,6,7,8-tetrahydropterins, by horseradish peroxidase/H2O2 is enzymic and follows Michaelis-Menten kinetics, and its Km and kcat. values were determined. This oxidation of 5,6,7,8-tetrahydropterins produces quinonoid dihydropterins of established structure, and they are known to be specific substrates for dihydropteridine reductase. By analogy the peroxidase/H2O2 oxidation of the 5,6,7,8-tetrahydropteridin-4(3H)-ones should produce similar quinonoid dihydro species. The quinonoid species derived from 5,6,7,8-tetrahydropteridin-4(3H)-one and its 6-methyl and cis-6,7-dimethyl derivatives are shown to be viable substrates for human brain dihydropteridine reductase, and apparent Km and Vmax. values are reported. PMID:3718470

  17. Two fatty acyl reductases involved in moth pheromone biosynthesis

    PubMed Central

    Antony, Binu; Ding, Bao-Jian; Moto, Ken’Ichi; Aldosari, Saleh A.; Aldawood, Abdulrahman S.

    2016-01-01

    Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective ‘single pgFARs’ produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a ‘single reductase’ can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

  18. Azotobacter vinelandii NADPH:ferredoxin reductase cloning, sequencing, and overexpression.

    PubMed

    Isas, J M; Yannone, S M; Burgess, B K

    1995-09-01

    Azotobacter vinelandii ferredoxin I (AvFdI) controls the expression of another protein that was originally designated Protein X. Recently we reported that Protein X is a NADPH-specific flavoprotein that binds specifically to FdI (Isas, J.M., and Burgess, B.K. (1994) J. Biol. Chem. 269, 19404-19409). The gene encoding this protein has now been cloned and sequenced. Protein X is 33% identical and has an overall 53% similarity with the fpr gene product from Escherichia coli that encodes NADPH:ferredoxin reductase. On the basis of this similarity and the similarity of the physical properties of the two proteins, we now designate Protein X as A. vinelandii NADPH:ferredoxin reductase and its gene as the fpr gene. The protein has been overexpressed in its native background in A. vinelandii by using the broad host range multicopy plasmid, pKT230. In addition to being regulated by FdI, the fpr gene product is overexpressed when A. vinelandii is grown under N2-fixing conditions even though the fpr gene is not preceded by a nif specific promoter. By analogy to what is known about fpr expression in E. coli, we propose that FdI may exert its regulatory effect on fpr by interacting with the SoxRS regulon. PMID:7673160

  19. SORGOdb: Superoxide Reductase Gene Ontology curated DataBase

    PubMed Central

    2011-01-01

    Background Superoxide reductases (SOR) catalyse the reduction of superoxide anions to hydrogen peroxide and are involved in the oxidative stress defences of anaerobic and facultative anaerobic organisms. Genes encoding SOR were discovered recently and suffer from annotation problems. These genes, named sor, are short and the transfer of annotations from previously characterized neelaredoxin, desulfoferrodoxin, superoxide reductase and rubredoxin oxidase has been heterogeneous. Consequently, many sor remain anonymous or mis-annotated. Description SORGOdb is an exhaustive database of SOR that proposes a new classification based on domain architecture. SORGOdb supplies a simple user-friendly web-based database for retrieving and exploring relevant information about the proposed SOR families. The database can be queried using an organism name, a locus tag or phylogenetic criteria, and also offers sequence similarity searches using BlastP. Genes encoding SOR have been re-annotated in all available genome sequences (prokaryotic and eukaryotic (complete and in draft) genomes, updated in May 2010). Conclusions SORGOdb contains 325 non-redundant and curated SOR, from 274 organisms. It proposes a new classification of SOR into seven different classes and allows biologists to explore and analyze sor in order to establish correlations between the class of SOR and organism phenotypes. SORGOdb is freely available at http://sorgo.genouest.org/index.php. PMID:21575179

  20. Recessive congenital methaemoglobinaemia: cytochrome b(5) reductase deficiency.

    PubMed

    Percy, Melanie J; Lappin, Terry R

    2008-05-01

    Some 60 years ago, Quentin Gibson reported the first hereditary disorder involving an enzyme when he deduced that familial methaemoglobinaemia was caused by an enzymatic lesion associated with the glycolysis pathway in red blood cells. This disorder, now known as recessive congenital methaemoglobinaemia (RCM), is caused by NADH-cytochrome b5 reductase (cb(5)r) deficiency. Two distinct clinical forms, types I and II, have been recognized, both characterized by cyanosis from birth. In type II, the cyanosis is accompanied by neurological impairment and reduced life expectancy. Cytochrome b(5) reductase is composed of one FAD and one NADH binding domain linked by a hinge region. It is encoded by the CYB5R3 (previously known as DIA1) gene and more than 40 mutations have been described, some of which are common to both types of RCM. Mutations associated with type II tend to cause incorrect splicing, disruption of the active site or truncation of the protein. At present the description of the sequence variants of cb(5)r in the literature is confusing, due to the use of two conventions which differ by one codon position. Herein we propose a new system for nomenclature of cb(5)r based on recommendations of the Human Genome Variation Society. The development of a heterologous expression system has allowed the impact of naturally occurring variants of cb(5)r to be assessed and has provided insight into the function of cb(5)r. PMID:18318771

  1. Retrospective approach to methylenetetrahydrofolate reductase mutations in children.

    PubMed

    Özer, Işıl; Özçetin, Mustafa; Karaer, Hatice; Kurt, Semiha G; Şahin, Şemsettin

    2011-07-01

    Methylenetetrahydrofolate reductase reduces methyltetrahydrofolate, a cosubstrate in the remethylation of homocysteine, from methylenetetrahydrofolate. Congenital defects, hematologic tumors, and intrauterine growth retardation can occur during childhood. This study evaluated clinical and laboratory treatment approaches in children diagnosed with methylenetetrahydrofolate reductase mutations. Our group included 23 boys and 14 girls, aged 103.4 ± 70.8 months S.D. Clinical findings of patients and homocysteine, vitamin B12, folate, hemogram, electroencephalography, cranial magnetic resonance imaging, and echocardiography data were evaluated in terms of treatment approach. Our patients' findings included vitamin B12 at 400.4 ± 224.6 pg/mL S.D. (normal range, 300-700 pg/mL), folate at 10.1 ± 4.5 ng/mL S.D. (normal range, 1.8-9 ng/mL), and homocysteine at 8.4 ± 4.7 μmol/L S.D. (normal range, 5.5-17 μmol/L). Eighty-eight percent of patients demonstrated clinical findings. In comparisons involving categorical variables between groups, χ(2) tests were used. No relationship was evident between mutation type, laboratory data, and clinical severity. All mothers who had MTHFR mutations and had babies with sacral dimples had taken folate supplements during pregnancy. To avoid the risk of neural tube defects, pregnant women with a MTHFR mutation may require higher than normally recommended doses of folic acid supplementation for optimum health. PMID:21723457

  2. The Role of Thioredoxin Reductases in Brain Development

    PubMed Central

    Soerensen, Jonna; Jakupoglu, Cemile; Beck, Heike; Förster, Heidi; Schmidt, Jörg; Schmahl, Wolfgang; Schweizer, Ulrich

    2008-01-01

    The thioredoxin-dependent system is an essential regulator of cellular redox balance. Since oxidative stress has been linked with neurodegenerative disease, we studied the roles of thioredoxin reductases in brain using mice with nervous system (NS)-specific deletion of cytosolic (Txnrd1) and mitochondrial (Txnrd2) thioredoxin reductase. While NS-specific Txnrd2 null mice develop normally, mice lacking Txnrd1 in the NS were significantly smaller and displayed ataxia and tremor. A striking patterned cerebellar hypoplasia was observed. Proliferation of the external granular layer (EGL) was strongly reduced and fissure formation and laminar organisation of the cerebellar cortex was impaired in the rostral portion of the cerebellum. Purkinje cells were ectopically located and their dendrites stunted. The Bergmann glial network was disorganized and showed a pronounced reduction in fiber strength. Cerebellar hypoplasia did not result from increased apoptosis, but from decreased proliferation of granule cell precursors within the EGL. Of note, neuron-specific inactivation of Txnrd1 did not result in cerebellar hypoplasia, suggesting a vital role for Txnrd1 in Bergmann glia or neuronal precursor cells. PMID:18350150

  3. Synthesis and metabolism of inhibitors of ribonucleotide reductase

    SciTech Connect

    Smith, F.T.

    1985-01-01

    In an effort to prepare more effective inhibitors of ribo-nucleotide reductase a series of 2-substituted-4,6-dihydroxypyrimidines was prepared via the appropriately substituted benzamidine. None of the compounds exhibited in vivo activity against L1210 leukemia. No further testing was performed. In order to investigate the metabolism of 3,4-dihydroxybenzohydroxamic acid, a known inhibitor of ribonucleotide reductase, radiolabeled 3,4-dihydroxybenzohydroxamic acid was synthesized by a modification of the procedure of Pichat and Tostain. /sup 14/C-3,4-Dihydroxybenzoic acid was converted to the methyl ester and subsequently reacted with hydroxylamine to give the hydroxamic acid. /sup 14/C-3,4-Dihydroxybenzohydroxamic acid was given i.p. to Sprague-Dawley rats. Excretion occurred mainly (72%) via the urine. HPLC coupled with GC/MS analyses showed that the compound was excreted mainly unchanged. The compound was metabolized to 3,4-dihydroxybenzamide, 4-methoxy-3-hydroxybenzohydroxamic acid, and 4-hydroxy-3-methoxybenzohydroxamic acid. HPLC analysis also showed the lack of formation of any glucuronide or sulfate conjugates through either the hydroxamic acid or catechol functionalities.

  4. Interspecific variation for thermal dependence of glutathione reductase in sainfoin.

    PubMed

    Kidambi, S P; Mahan, J R; Matches, A G

    1990-05-01

    Understanding the biochemical and physiological consequences of species variation would expedite improvement in agronomically useful genotypes of sainfoin (Onobrychis spp.) Information on variation among sainfoin species is lacking on thermal dependence of glutathione reductase (B.C. 1.6.4.2.), which plays an important role in the protection of plants from both high and low temperature stresses by preventing harmful oxidation of enzymes and membranes. Our objective was to investigate the interspecific variation for thermal dependency of glutathione reductase in sainfoin. Large variation among species was found for: (i) the minimum apparent Km (0.4-2.5 μM NADPH), (ii) the temperature at which the minimum apparent Km was observed (15°-5°C), and (iii) the thermal kinetic windows (2°-30°C width) over a 15°-45°C temperature gradient. In general, tetraploid species had narrower (≤17°C) thermal kinetic windows than did diploid species (∼30°C), with one exception among the diploids. Within the tetraploid species, the cultivars of O. viciifolia had a broader thermal kinetic window (≥7°C) than the plant introduction (PI 212241, >2 °C) itself. PMID:24226572

  5. A mutant of barley lacking NADH-hydroxypyruvate reductase

    SciTech Connect

    Blackwell, R.; Lea, P. )

    1989-04-01

    A mutant of barley, LaPr 88/29, deficient in peroxisomal NADH-hydroxypyruvate reductase (HPR) activity has been identified. Compared to the wild type the activities of NADH-HPR and NADPH-HPR were severely reduced but the mutant was still capable of fixing CO{sub 2} at rates equivalent to 75% of that of the wild type in air. Although lacking an enzyme in the main photorespiratory pathway, there appeared to be little disruption to photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C) serine were similar in both mutant and wild type. LaPr 88/29 has been used to show that NADH-glyoxylate reductase (GR) and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-HPR activity is due to the NADH-HPR enzyme. Immunological studies, using antibodies raised against spinach HPR, have shown that the NADH-dependent enzyme protein is absent in LaPr 88/29 but there appears to be enhanced synthesis of the NADPH-dependent enzyme protein.

  6. Carbonyl reductase of dog liver: purification, properties, and kinetic mechanism.

    PubMed

    Hara, A; Nakayama, T; Deyashiki, Y; Kariya, K; Sawada, H

    1986-01-01

    A carbonyl reductase has been extracted into 0.5 M KCl from dog liver and purified to apparent homogeneity by a three-step procedure consisting of chromatography on CM-Sephadex, Matrex green A, and Sephadex G-100 in high-ionic-strength buffers. The enzyme is a dimer composed of two identical subunits of molecular weight 27,000. The pH optimum is 5.5 and the isoelectric point of the enzyme is 9.3. The enzyme reduces aromatic ketones and aldehydes; the aromatic ketones with adjacent medium alkyl chains are the best substrates. Quinones, ketosteroids, prostaglandins, and aliphatic carbonyl compounds are poor or inactive substrates for the enzyme. As a cofactor the enzyme utilizes NADPH, the pro-S hydrogen atom of which is transferred to the substrate. Two moles of NADPH bind to one mole of the enzyme molecule, causing a blue shift and enhancement of the cofactor fluorescence. The reductase reaction is reversible and the equilibrium constant determined at pH 7.0 is 12.8. Steady-state kinetic measurements in both directions suggest that the reaction proceeds through a di-iso ordered bi-bi mechanism. PMID:3511844

  7. An electron-bifurcating caffeyl-CoA reductase.

    PubMed

    Bertsch, Johannes; Parthasarathy, Anutthaman; Buckel, Wolfgang; Müller, Volker

    2013-04-19

    A low potential electron carrier ferredoxin (E0' ≈ -500 mV) is used to fuel the only bioenergetic coupling site, a sodium-motive ferredoxin:NAD(+) oxidoreductase (Rnf) in the acetogenic bacterium Acetobacterium woodii. Because ferredoxin reduction with physiological electron donors is highly endergonic, it must be coupled to an exergonic reaction. One candidate is NADH-dependent caffeyl-CoA reduction. We have purified a complex from A. woodii that contains a caffeyl-CoA reductase and an electron transfer flavoprotein. The enzyme contains three subunits encoded by the carCDE genes and is predicted to have, in addition to FAD, two [4Fe-4S] clusters as cofactor, which is consistent with the experimental determination of 4 mol of FAD, 9 mol of iron, and 9 mol of acid-labile sulfur. The enzyme complex catalyzed caffeyl-CoA-dependent oxidation of reduced methyl viologen. With NADH as donor, it catalyzed caffeyl-CoA reduction, but this reaction was highly stimulated by the addition of ferredoxin. Spectroscopic analyses revealed that ferredoxin and caffeyl-CoA were reduced simultaneously, and a stoichiometry of 1.3:1 was determined. Apparently, the caffeyl-CoA reductase-Etf complex of A. woodii uses the novel mechanism of flavin-dependent electron bifurcation to drive the endergonic ferredoxin reduction with NADH as reductant by coupling it to the exergonic NADH-dependent reduction of caffeyl-CoA. PMID:23479729

  8. Properties of seleno-methionine substituted assimilatory nitrate reductase

    SciTech Connect

    Solomonson, L.P.; Barber, M.J. )

    1991-03-11

    Assimilatory NADH:nitrate reductase contains FAD, heme and Mo-pterin arranged in an NADH{yields}FAD{yields}heme{yields}Mo-Pterin{yields}NO{sub 3} electron transfer sequence. A functional Mo-pterin center is essential for all nitrate-reducing activities. To assess the possible functional role of Met, a Se-Met substituted NR was obtained by addition of Se-Met to ammonia-grown Chlorella cells prior to induction of NR activity. Increase in NADH:dehydrogenase partial activities and nitrate reductase protein proceeded normally following induction but little or no nitrate-reducing activity was expressed. This effect was observed with as little as 10{sup {minus}5} Se-Met and was prevented by a 10-fold excess of Met. A less pronounced effect was observed with 10{sup {minus}4}M Se-Cys. The purified Se-Met substituted enzyme exhibited the same apparent physical size, spectral properties and NADH dehydrogenase activities as control NR but was devoid of nitrate-reducing activities. These results suggest that one or more Met residues are essential for the catalytic function of the molybdo-pterin center of assimilatory NR.

  9. Predicted EC50 and EC95 of Remifentanil for Smooth Removal of a Laryngeal Mask Airway Under Propofol Anesthesia

    PubMed Central

    Yoo, Ji Young; Kwak, Hyun Jeong; Lee, Kyung Cheon; Kim, Go Wun

    2015-01-01

    Purpose The purpose of this study was to determine the effect-site concentration (Ce) of remifentanil in 50% of patients (EC50) and 95% of patients (EC95) for smooth laryngeal mask airway (LMA) removal in adults under propofol and remifentanil anesthesia. Materials and Methods Twenty-five patients of ASA physical status I-II and ages 18-60 years who were to undergo minor gynecological or orthopedic surgery were assessed in this study. Anesthesia was induced and maintained with propofol and remifentanil target-controlled infusion (TCI). Remifentanil was maintained at a predetermined Ce during the emergence period. The modified Dixon's up-and-down method was used to determine the remifentanil concentration, starting from 1.0 ng/mL (step size of 0.2 ng/mL). Successful removal of the LMA was regarded as absence of coughing/gagging, clenched teeth, gross purposeful movements, breath holding, laryngospasm, or desaturation to SpO2<90%. Results The mean±SD Ce of remifentanil for smooth LMA removal after propofol anesthesia was 0.83±0.16 ng/mL. Using isotonic regression with a bootstrapping approach, the estimated EC50 and EC95 of remifentanil Ce were 0.91 ng/mL [95% confidence interval (CI), 0.77-1.07 ng/mL] and 1.35 ng/mL (95% CI, 1.16-1.38 ng/mL), respectively. Conclusion Our results showed that remifentanil TCI at an established Ce is a reliable technique for achieving safe and smooth emergence without coughing, laryngospasm, or other airway reflexes. PMID:26069139

  10. Pinpointing a Mechanistic Switch Between Ketoreduction and "Ene" Reduction in Short-Chain Dehydrogenases/Reductases.

    PubMed

    Lygidakis, Antonios; Karuppiah, Vijaykumar; Hoeven, Robin; Ní Cheallaigh, Aisling; Leys, David; Gardiner, John M; Toogood, Helen S; Scrutton, Nigel S

    2016-08-01

    Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (-)-menthone:(-)-menthol reductase and (-)-menthone:(+)-neomenthol reductase, and the "ene" reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases. PMID:27411040

  11. Natural variation in arsenate tolerance identifies an arsenate reductase in Arabidopsis thaliana.

    PubMed

    Sánchez-Bermejo, Eduardo; Castrillo, Gabriel; del Llano, Bárbara; Navarro, Cristina; Zarco-Fernández, Sonia; Martinez-Herrera, Dannys Jorge; Leo-del Puerto, Yolanda; Muñoz, Riansares; Cámara, Carmen; Paz-Ares, Javier; Alonso-Blanco, Carlos; Leyva, Antonio

    2014-01-01

    The enormous amount of environmental arsenic was a major factor in determining the biochemistry of incipient life forms early in the Earth's history. The most abundant chemical form in the reducing atmosphere was arsenite, which forced organisms to evolve strategies to manage this chemical species. Following the great oxygenation event, arsenite oxidized to arsenate and the action of arsenate reductases became a central survival requirement. The identity of a biologically relevant arsenate reductase in plants nonetheless continues to be debated. Here we identify a quantitative trait locus that encodes a novel arsenate reductase critical for arsenic tolerance in plants. Functional analyses indicate that several non-additive polymorphisms affect protein structure and account for the natural variation in arsenate reductase activity in Arabidopsis thaliana accessions. This study shows that arsenate reductases are an essential component for natural plant variation in As(V) tolerance. PMID:25099865

  12. Inhibition of rat steroid 5 alpha-reductase (isozyme 1) by suramin.

    PubMed

    Taylor, M F; Bhattacharyya, A K; Collins, D C

    1995-07-01

    In this study, we show the inhibition of rat steroid 5 alpha-reductase (isozyme 1) by suramin. The enzyme activity decreased in a dose-dependent manner as suramin concentrations increased with the calculated drug dose required for 50% inhibition (at 5 microM testosterone and 200 microM NADPH) being 13 microM. Suramin showed non-competitive inhibition of 5 alpha-reductase with respect to testosterone (KT1 = 2.4 microM) and competitive inhibition with respect to NADPH (KiNADPH = 220 nM). Furthermore, suramin and NADP+, but not NAD+, protected 5 alpha-reductase from labeling by 2-azido-NADP+, a photoactive probe which has recently been used to identify the NADPH binding domain of 5 alpha-reductase. These results suggest that suramin inhibits rat steroid 5 alpha-reductase (isozyme 1) at the level of NADPH binding to the enzyme. PMID:7482629

  13. 77 FR 8722 - Airworthiness Directives; Eurocopter Deutschland Model EC135 Helicopters

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-15

    ... Deutschland Model EC135 Helicopters AGENCY: Federal Aviation Administration (FAA), DOT. ACTION: Final rule. SUMMARY: We are adopting a new airworthiness directive (AD) for Eurocopter Deutschland (ECD) Model EC135... include an AD that would apply to the Eurocopter Deutschland (ECD) Model EC135 helicopters. That NPRM...

  14. Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint.

    PubMed

    Turner, Glenn W; Davis, Edward M; Croteau, Rodney B

    2012-06-01

    Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation. The menthone reductase responsible for the ultimate pathway reduction step, menthone-menthol reductase (MMR), has been characterized and found to share significant sequence similarity with its counterpart reductase, a menthone-neomenthol reductase, which catalyzes a minor enzymatic reaction associated with oil maturation. Further, the menthone reductases share significant sequence similarity with the temporally separate and mechanistically different isopiperitenone reductase (IPR). Here we present immunocytochemical localizations for these reductases using a polyclonal antibody raised against menthone-menthol reductase. The polyclonal antibody used for this study showed little specificity between these three reductases, but by using it for immunostaining of tissues of different ages we were able to provisionally separate staining of an early biosynthetic enzyme, IPR, found in young, immature leaves from that of the oil maturation enzyme, MMR, found in older, mature leaves. Both reductases were localized to the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes, and were absent from all other cell types examined. PMID:22170164

  15. Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

    PubMed Central

    Cohen, G; Yanko, M; Mislovati, M; Argaman, A; Schreiber, R; Av-Gay, Y; Aharonowitz, Y

    1993-01-01

    The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated. Images PMID:8349555

  16. Functional complementation of a nitrate reductase defective mutant of a green alga Dunaliella viridis by introducing the nitrate reductase gene.

    PubMed

    Sun, Yu; Gao, Xiaoshu; Li, Qiyun; Zhang, Qingqi; Xu, Zhengkai

    2006-08-01

    Nitrate reductase (NR) catalyzes NAD (P) H dependent reduction of nitrate to nitrite. Transformation systems have been established in several species of green algae by nitrate reductase gene functional complementation. In this report, an endogenous NR cDNA (3.4 kb) and a genomic fragment (14.6 kb) containing the NR gene (DvNIA1) were isolated from the D. viridis cDNA and genomic libraries respectively. Southern blot and Northern blot analyses showed that this gene exists as a single copy in D. viridis and is induced by nitrate. To obtain a NR defective mutant as a recipient strain, D. viridis cells were treated with a chemical mutagen and then cultured on a chlorate-containing plate to enrich chlorate tolerant mutants. Southern analysis showed that one isolate, B14, had a deletion in the DvNIA1 gene region. Using electroporation conditions determined in this laboratory, plasmid pDVNR containing the intact DvNIA1 gene has been electroporated into the defective mutant B14. Strains retaining a nitrate assimilation phenotype were obtained from nitrate plates after spreading the electroporated cells. In some individual strains, transcription of the introduced gene was detected. NR activity in these strains was slightly higher than that in the defective B14 cell, but excretion of nitrite into culture media was almost as high as that of the wild-type cell. Possible episomal presence of the introduced DNA in D. viridis is discussed. PMID:16797881

  17. Curcumin is a tight-binding inhibitor of the most efficient human daunorubicin reductase--Carbonyl reductase 1.

    PubMed

    Hintzpeter, Jan; Hornung, Jan; Ebert, Bettina; Martin, Hans-Jörg; Maser, Edmund

    2015-06-01

    Curcumin is a major component of the plant Curcuma longa L. It is traditionally used as a spice and coloring in foods and is an important ingredient in curry. Curcuminoids have anti-oxidant and anti-inflammatory properties and gained increasing attention as potential neuroprotective and cancer preventive compounds. In the present study, we report that curcumin is a potent tight-binding inhibitor of human carbonyl reductase 1 (CBR1, Ki=223 nM). Curcumin acts as a non-competitive inhibitor with respect to the substrate 2,3-hexandione as revealed by plotting IC50-values against various substrate concentrations and most likely as a competitive inhibitor with respect to NADPH. Molecular modeling supports the finding that curcumin occupies the cofactor binding site of CBR1. Interestingly, CBR1 is one of the most effective human reductases in converting the anthracycline anti-tumor drug daunorubicin to daunorubicinol. The secondary alcohol metabolite daunorubicinol has significantly reduced anti-tumor activity and shows increased cardiotoxicity, thereby limiting the clinical use of daunorubicin. Thus, inhibition of CBR1 may increase the efficacy of daunorubicin in cancer tissue and simultaneously decrease its cardiotoxicity. Western-blots demonstrated basal expression of CBR1 in several cell lines. Significantly less daunorubicin reduction was detected after incubating A549 cell lysates with increasing concentrations of curcumin (up to 60% less with 50 μM curcumin), suggesting a beneficial effect in the co-treatment of anthracycline anti-tumor drugs together with curcumin. PMID:25541467

  18. Polymorphisms in the methylene tetrahydrofolate reductase and methionine synthase reductase genes and their correlation with unexplained recurrent spontaneous abortion susceptibility.

    PubMed

    Zhu, L

    2015-01-01

    We aimed to explore the correlation between unexplained recurrent spontaneous abortion and polymorphisms in the methylene tetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes. A case control study was conducted in 118 patients with unexplained recurrent spontaneous abortion (abortion group) and 174 healthy women (control group). The genetic material was extracted from the oral mucosal epithelial cells obtained from all subjects. The samples were subjected to fluorescence quantitative PCR to detect the single nucleotide polymorphisms (SNPs) in the MTHFR (C677T and A1298C) and MTRR (A66G) gene loci. The distribution frequency (18/118, 15.3%) of the MTHFR 677TT genotype was significantly higher in the abortion group (χ2 = 11.006, P = 0.004) than in the control group (2/174, 1.1%); on the other hand, the distribution frequency of the MTHFR A1298C genotype did not significantly differ between the abortion and control groups (χ(2) = 0.441, P = 0.507). The distribution frequency of the MTRR A66G genotype was also significantly higher in the abortion group (14/118, 11.9%; χ(2) = 10.503, P = 0.005) than in the control group (8/174, 4.6%). The MTHFR C677T and MTRR A66G polymorphisms are significantly correlated with the occurrence of spontaneous abortion. PMID:26345779

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

    PubMed Central

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

    2015-01-01

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

  20. The nitrate-sensing NasST system regulates nitrous oxide reductase and periplasmic nitrate reductase in Bradyrhizobium japonicum.

    PubMed

    Sánchez, Cristina; Itakura, Manabu; Okubo, Takashi; Matsumoto, Takashi; Yoshikawa, Hirofumi; Gotoh, Aina; Hidaka, Masafumi; Uchida, Takafumi; Minamisawa, Kiwamu

    2014-10-01

    The soybean endosymbiont Bradyrhizobium japonicum is able to scavenge the greenhouse gas N2O through the N2O reductase (Nos). In previous research, N2O emission from soybean rhizosphere was mitigated by B. japonicum Nos(++) strains (mutants with increased Nos activity). Here, we report the mechanism underlying the Nos(++) phenotype. Comparative analysis of Nos(++) mutant genomes showed that mutation of bll4572 resulted in Nos(++) phenotype. bll4572 encodes NasS, the nitrate (NO3(-))-sensor of the two-component NasST regulatory system. Transcriptional analyses of nosZ (encoding Nos) and other genes from the denitrification process in nasS and nasST mutants showed that, in the absence of NO3(-) , nasS mutation induces nosZ and nap (periplasmic nitrate reductase) via nasT. NO3(-) addition dissociated the NasS-NasT complex in vitro, suggesting the release of the activator NasT. Disruption of nasT led to a marked decrease in nosZ and nap transcription in cells incubated in the presence of NO3(-). Thus, although NasST is known to regulate the NO3(-)-mediated response of NO3(-) assimilation genes in bacteria, our results show that NasST regulates the NO3(-) -mediated response of nosZ and napE genes, from the dissimilatory denitrification pathway, in B. japonicum. PMID:24947409

  1. Generation of poly-β-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice.

    PubMed

    Tsuda, Hirohisa; Shiraki, Mari; Inoue, Eri; Saito, Terumi

    2016-08-20

    It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) (14)C-PHB generated from 2-(14)C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1-10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination. PMID:27372278

  2. Evaluation of the size segregation of elemental carbon (EC) emission in Europe: influence on the simulation of EC long-range transportation

    NASA Astrophysics Data System (ADS)

    Chen, Ying; Cheng, Ya-Fang; Nordmann, Stephan; Birmili, Wolfram; Denier van der Gon, Hugo A. C.; Ma, Nan; Wolke, Ralf; Wehner, Birgit; Sun, Jia; Spindler, Gerald; Mu, Qing; Pöschl, Ulrich; Su, Hang; Wiedensohler, Alfred

    2016-02-01

    Elemental Carbon (EC) has a significant impact on human health and climate change. In order to evaluate the size segregation of EC emission in the EUCAARI inventory and investigate its influence on the simulation of EC long-range transportation in Europe, we used the fully coupled online Weather Research and Forecasting/Chemistry model (WRF-Chem) at a resolution of 2 km focusing on a region in Germany, in conjunction with a high-resolution EC emission inventory. The ground meteorology conditions, vertical structure and wind pattern were well reproduced by the model. The simulations of particle number and/or mass size distributions were evaluated with observations at the central European background site Melpitz. The fine mode particle concentration was reasonably well simulated, but the coarse mode was substantially overestimated by the model mainly due to the plume with high EC concentration in coarse mode emitted by a nearby point source. The comparisons between simulated EC and Multi-angle Absorption Photometers (MAAP) measurements at Melpitz, Leipzig-TROPOS and Bösel indicated that the coarse mode EC (ECc) emitted from the nearby point sources might be overestimated by a factor of 2-10. The fraction of ECc was overestimated in the emission inventory by about 10-30 % for Russia and 5-10 % for Eastern Europe (e.g., Poland and Belarus). This incorrect size-dependent EC emission results in a shorter atmospheric life time of EC particles and inhibits the long-range transport of EC. A case study showed that this effect caused an underestimation of 20-40 % in the EC mass concentration in Germany under eastern wind pattern.

  3. Savannah River Site ECS-2 tests uncertainty report

    SciTech Connect

    Wilkins, S.C.; Larson, R.A.

    1990-07-01

    This document presents a measurement uncertainty analysis for the instruments used in the ECS-2 test series conducted for the Savannah River Site at the Idaho National Engineering Laboratory. The tests are a series of downflow dryout heat transfer experiments designed to support computer code development and verification in setting limits for the Savannah River Production reactors. The measurements include input current, voltage, and power; air and water flows, fluid and metal temperatures, and absolute and differential pressures. An analysis of the data acquisition system as it relates to these measurements is also included. 18 refs., 6 figs., 12 tabs.

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

    PubMed Central

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

    1997-01-01

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

  5. Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants.

    PubMed Central

    Benveniste, I; Lesot, A; Hasenfratz, M P; Durst, F

    1989-01-01

    Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals. Images Fig. 5. PMID:2499315

  6. Steroid 5β-Reductase from Leaves of Vitis vinifera: Molecular Cloning, Expression, and Modeling.

    PubMed

    Ernst, Mona; Munkert, Jennifer; Campa, Manuela; Malnoy, Mickael; Martens, Stefan; Müller-Uri, Frieder

    2015-11-25

    A steroid 5β-reductase gene corresponding to the hypothetical protein LOC100247199 from leaves of Vitis vinifera (var. 'Chardonnay') was cloned and overexpressed in Escherichia coli. The recombinant protein showed 5β-reductase activity when progesterone was used as a substrate. The reaction was stereoselective, producing only 5β-products such as 5β-pregnane-3,20-dione. Other small substrates (terpenoids and enones) were also accepted as substrates, indicating the highly promiscuous character of the enzyme class. Our results show that the steroid 5β-reductase gene, encoding an orthologous enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in leaves of the cardenolide-free plant V. vinifera. We emphasize the fact that, on some occasions, different reductases (e.g., progesterone 5β-reductase and monoterpenoid reductase) can also use molecules that are similar to the final products as a substrate. Therefore, in planta, the different reductases may contribute to the immense number of diverse small natural products finally leading to the flavor of wine. PMID:26537436

  7. Divergent evolution of protein conformational dynamics in dihydrofolate reductase.

    PubMed

    Bhabha, Gira; Ekiert, Damian C; Jennewein, Madeleine; Zmasek, Christian M; Tuttle, Lisa M; Kroon, Gerard; Dyson, H Jane; Godzik, Adam; Wilson, Ian A; Wright, Peter E

    2013-11-01

    Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about the evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, Escherichia coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary-sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics following a pattern of divergent evolution that is tuned by cellular environment. PMID:24077226

  8. Structure of a bacterial homologue of vitamin K epoxide reductase

    SciTech Connect

    Li, Weikai; Schulman, Sol; Dutton, Rachel J.; Boyd, Dana; Beckwith, Jon; Rapoport, Tom A.

    2010-03-19

    Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain {gamma}-carboxylation of many blood coagulation factors. Here, we report the 3.6 {angstrom} crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.

  9. Go Green: The Anti-Inflammatory Effects of Biliverdin Reductase

    PubMed Central

    Wegiel, Barbara; Otterbein, Leo E.

    2012-01-01

    Biliverdin (BV) has emerged as a cytoprotective and important anti-inflammatory molecule. Conversion of BV to bilirubin (BR) is catalyzed by biliverdin reductase (BVR) and is required for the downstream signaling and nuclear localization of BVR. Recent data by others and us make clear that BVR is a critical regulator of innate immune responses resulting from acute insult and injury and moreover, that a lack of BVR results in an enhanced proinflammatory phenotype. In macrophages, BVR is regulated by its substrate BV which leads to activation of the PI3K–Akt-IL-10 axis and inhibition of TLR4 expression via direct binding of BVR to the TLR4 promoter. In this review, we will summarize recent findings on the role of BVR and the bile pigments in inflammation in context with its activity as an enzyme, receptor, and transcriptional regulator. PMID:22438844

  10. Genetic Evidence for a Molybdopterin-Containing Tellurate Reductase

    PubMed Central

    Theisen, Joanne; Zylstra, Gerben J.

    2013-01-01

    The genetic identity and cofactor composition of the bacterial tellurate reductase are currently unknown. In this study, we examined the requirement of molybdopterin biosynthesis and molybdate transporter genes for tellurate reduction in Escherichia coli K-12. The results show that mutants deleted of the moaA, moaB, moaE, or mog gene in the molybdopterin biosynthesis pathway lost the ability to reduce tellurate. Deletion of the modB or modC gene in the molybdate transport pathway also resulted in complete loss of tellurate reduction activity. Genetic complementation by the wild-type sequences restored tellurate reduction activity in the mutant strains. These findings provide genetic evidence that tellurate reduction in E. coli involves a molybdoenzyme. PMID:23475618

  11. Pulse radiolysis studies on superoxide reductase from Treponema pallidum.

    PubMed

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

    2001-05-25

    Superoxide reductases (SORs) are small metalloenzymes, which catalyze reduction of O2*- to H2O2. The reaction of the enzyme from Treponema pallidum with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bi-molecular reaction of the ferrous center with O2, with a rate constant of 6 x 10 (8) M(-1) s(-1). A first intermediate is formed which is converted to a second one with a slower rate constant of 4800 s(-1). This latter value is 10 times higher than the corresponding one previously reported in the case of SOR from Desulfoarculus baarsii. The reconstituted spectra for the two intermediates are consistent with formation of transient iron-peroxide species. PMID:11377434

  12. Divergent evolution of protein conformational dynamics in dihydrofolate reductase

    PubMed Central

    Bhabha, Gira; Ekiert, Damian C.; Jennewein, Madeleine; Zmasek, Christian M.; Tuttle, Lisa M.; Kroon, Gerard; Dyson, H. Jane; Godzik, Adam; Wilson, Ian A.; Wright, Peter E.

    2013-01-01

    Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, E. coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics, following a pattern of divergent evolution that is tuned by the cellular environment. PMID:24077226

  13. Mechanism of inhibition of ribonucleotide reductase with motexafin gadolinium (MGd)

    SciTech Connect

    Zahedi Avval, Farnaz; Berndt, Carsten; Pramanik, Aladdin; Holmgren, Arne

    2009-02-13

    Motexafin gadolinium (MGd) is an expanded porphyrin anticancer agent which selectively targets tumor cells and works as a radiation enhancer, with promising results in clinical trials. Its mechanism of action is oxidation of intracellular reducing molecules and acting as a direct inhibitor of mammalian ribonucleotide reductase (RNR). This paper focuses on the mechanism of inhibition of RNR by MGd. Our experimental data present at least two pathways for inhibition of RNR; one precluding subunits oligomerization and the other direct inhibition of the large catalytic subunit of the enzyme. Co-localization of MGd and RNR in the cytoplasm particularly in the S-phase may account for its inhibitory properties. These data can elucidate an important effect of MGd on the cancer cells with overproduction of RNR and its efficacy as an anticancer agent and not only as a general radiosensitizer.

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

    PubMed

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

    2006-05-19

    Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins. PMID:16600295

  15. Thioredoxin reductase 1 suppresses adipocyte differentiation and insulin responsiveness

    PubMed Central

    Peng, Xiaoxiao; Giménez-Cassina, Alfredo; Petrus, Paul; Conrad, Marcus; Rydén, Mikael; Arnér, Elias S. J.

    2016-01-01

    Recently thioredoxin reductase 1 (TrxR1), encoded by Txnrd1, was suggested to modulate glucose and lipid metabolism in mice. Here we discovered that TrxR1 suppresses insulin responsiveness, anabolic metabolism and adipocyte differentiation. Immortalized mouse embryonic fibroblasts (MEFs) lacking Txnrd1 (Txnrd1−/−) displayed increased metabolic flux, glycogen storage, lipogenesis and adipogenesis. This phenotype coincided with upregulated PPARγ expression, promotion of mitotic clonal expansion and downregulation of p27 and p53. Enhanced Akt activation also contributed to augmented adipogenesis and insulin sensitivity. Knockdown of TXNRD1 transcripts accelerated adipocyte differentiation also in human primary preadipocytes. Furthermore, TXNRD1 transcript levels in subcutaneous adipose tissue from 56 women were inversely associated with insulin sensitivity in vivo and lipogenesis in their isolated adipocytes. These results suggest that TrxR1 suppresses anabolic metabolism and adipogenesis by inhibition of intracellular signaling pathways downstream of insulin stimulation. PMID:27346647

  16. 5-Alpha-Reductase Inhibitors and Combination Therapy.

    PubMed

    Füllhase, Claudius; Schneider, Marc P

    2016-08-01

    By inhibiting the conversion from testosterone to dihydrotestosterone 5-Alpha reductase inhibitors (5ARIs) are able to hinder prostatic growth, shrink prostate volumes, and improve BPH-related LUTS. 5ARIs are particularly beneficial for patients with larger prostates (>30-40ml). Generally the side effects of 5ARI treatment are mild, and according to the FORTA classification 5ARIs are suitable for frail elderly. 5ARI / alpha-blocker (AB) combination therapy showed the best symptomatic outcome and risk reduction for clinical progression. Combining Phosphodieseterase type 5 inhbibitors (PDE5Is) with 5ARIs counteracts the negative androgenic sexual side effects of 5ARIs, and simultaneously combines their synergistic effects on LUTS. PMID:27476125

  17. Substrate specificity of an aflatoxin-metabolizing aldehyde reductase.

    PubMed Central

    Ellis, E M; Hayes, J D

    1995-01-01

    The enzyme from rat liver that reduces aflatoxin B1-dialdehyde exhibits a unique catalytic specificity distinct from that of other aldo-keto reductases. This enzyme, designated AFAR, displays high activity towards dicarbonyl-containing compounds with ketone groups on adjacent carbon atoms; 9,10-phenanthrenequinone, acenaphthenequinone and camphorquinone were found to be good substrates. Although AFAR can also reduce aromatic and aliphatic aldehydes such as succinic semialdehyde, it is inactive with glucose, galactose and xylose. The enzyme also exhibits low activity towards alpha,beta-unsaturated carbonyl-containing compounds. Determination of the apparent Km reveals that AFAR has highest affinity for 9,10-phenanthrenequinone and succinic semialdehyde, and low affinity for glyoxal and DL-glyceraldehyde. PMID:8526867

  18. Selective depletion of vascular EC-SOD augments chronic hypoxic pulmonary hypertension

    PubMed Central

    Woods, Crystal; Taylor, Joann M.; Benninger, Richard K. P.; Johnson, Richard D.; Villegas, Leah R.; Stenmark, Kurt R.; Harrison, David G.; Majka, Susan M.; Irwin, David; Farrow, Kathryn N.

    2014-01-01

    Excess superoxide has been implicated in pulmonary hypertension (PH). We previously found lung overexpression of the antioxidant extracellular superoxide dismutase (EC-SOD) attenuates PH and pulmonary artery (PA) remodeling. Although comprising a small fraction of total SOD activity in most tissues, EC-SOD is abundant in arteries. We hypothesize that the selective loss of vascular EC-SOD promotes hypoxia-induced PH through redox-sensitive signaling pathways. EC-SODloxp/loxp × Tgcre/SMMHC mice (SMC EC-SOD KO) received tamoxifen to conditionally deplete smooth muscle cell (SMC)-derived EC-SOD. Mice were exposed to hypobaric hypoxia for 35 days, and PH was assessed by right ventricular systolic pressure measurements and right ventricle hypertrophy. Vascular remodeling was evaluated by morphometric analysis and two-photon microscopy for collagen. We examined cGMP content and soluble guanylate cyclase expression and activity in lung, lung phosphodiesterase 5 (PDE5) expression and activity, and expression of endothelial nitric oxide synthase and GTP cyclohydrolase-1 (GTPCH-1), the rate-limiting enzyme in tetrahydrobiopterin synthesis. Knockout of SMC EC-SOD selectively decreased PA EC-SOD without altering total lung EC-SOD. PH and vascular remodeling induced by chronic hypoxia was augmented in SMC EC-SOD KO. Depletion of SMC EC-SOD did not impact content or activity of lung soluble guanylate cyclase or PDE5, yet it blunted the hypoxia-induced increase in cGMP. Although total eNOS was not altered, active eNOS and GTPCH-1 decreased with hypoxia only in SMC EC-SOD KO. We conclude that the localized loss of PA EC-SOD augments chronic hypoxic PH. In addition to oxidative inactivation of NO, deletion of EC-SOD seems to reduce eNOS activity, further compromising pulmonary vascular function. PMID:25326578

  19. Selective depletion of vascular EC-SOD augments chronic hypoxic pulmonary hypertension.

    PubMed

    Nozik-Grayck, Eva; Woods, Crystal; Taylor, Joann M; Benninger, Richard K P; Johnson, Richard D; Villegas, Leah R; Stenmark, Kurt R; Harrison, David G; Majka, Susan M; Irwin, David; Farrow, Kathryn N

    2014-12-01

    Excess superoxide has been implicated in pulmonary hypertension (PH). We previously found lung overexpression of the antioxidant extracellular superoxide dismutase (EC-SOD) attenuates PH and pulmonary artery (PA) remodeling. Although comprising a small fraction of total SOD activity in most tissues, EC-SOD is abundant in arteries. We hypothesize that the selective loss of vascular EC-SOD promotes hypoxia-induced PH through redox-sensitive signaling pathways. EC-SOD(loxp/loxp) × Tg(cre/SMMHC) mice (SMC EC-SOD KO) received tamoxifen to conditionally deplete smooth muscle cell (SMC)-derived EC-SOD. Mice were exposed to hypobaric hypoxia for 35 days, and PH was assessed by right ventricular systolic pressure measurements and right ventricle hypertrophy. Vascular remodeling was evaluated by morphometric analysis and two-photon microscopy for collagen. We examined cGMP content and soluble guanylate cyclase expression and activity in lung, lung phosphodiesterase 5 (PDE5) expression and activity, and expression of endothelial nitric oxide synthase and GTP cyclohydrolase-1 (GTPCH-1), the rate-limiting enzyme in tetrahydrobiopterin synthesis. Knockout of SMC EC-SOD selectively decreased PA EC-SOD without altering total lung EC-SOD. PH and vascular remodeling induced by chronic hypoxia was augmented in SMC EC-SOD KO. Depletion of SMC EC-SOD did not impact content or activity of lung soluble guanylate cyclase or PDE5, yet it blunted the hypoxia-induced increase in cGMP. Although total eNOS was not altered, active eNOS and GTPCH-1 decreased with hypoxia only in SMC EC-SOD KO. We conclude that the localized loss of PA EC-SOD augments chronic hypoxic PH. In addition to oxidative inactivation of NO, deletion of EC-SOD seems to reduce eNOS activity, further compromising pulmonary vascular function. PMID:25326578

  20. Evidence for a hexaheteromeric methylenetetrahydrofolate reductase in Moorella thermoacetica.

    PubMed

    Mock, Johanna; Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Thauer, Rudolf K

    2014-09-01

    Moorella thermoacetica can grow with H₂ and CO₂, forming acetic acid from 2 CO₂ via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown. PMID:25002540

  1. Fatty acyl-CoA reductases of birds

    PubMed Central

    2011-01-01

    Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis. PMID:22151413

  2. The modulation of carbonyl reductase 1 by polyphenols.

    PubMed

    Boušová, Iva; Skálová, Lenka; Souček, Pavel; Matoušková, Petra

    2015-01-01

    Carbonyl reductase 1 (CBR1), an enzyme belonging to the short-chain dehydrogenases/reductases family, has been detected in all human tissues. CBR1 catalyzes the reduction of many xenobiotics, including important drugs (e.g. anthracyclines, nabumetone, bupropion, dolasetron) and harmful carbonyls and quinones. Moreover, it participates in the metabolism of a number of endogenous compounds and it may play a role in certain pathologies. Plant polyphenols are not only present in many human food sources, but are also a component of many popular dietary supplements and herbal medicines. Many studies reviewed herein have demonstrated the potency of certain flavonoids, stilbenes and curcuminoids in the inhibition of the activity of CBR1. Interactions of these polyphenols with transcriptional factors, which regulate CBR1 expression, have also been reported in several studies. As CBR1 plays an important role in drug metabolism as well as in the protection of the organism against potentially harmful carbonyls, the modulation of its expression/activity may have significant pharmacological and/or toxicological consequences. Some polyphenols (e.g. luteolin, apigenin and curcumin) have been shown to be very potent CBR1 inhibitors. The inhibition of CBR1 seems useful regarding the increased efficacy of anthracycline therapy, but it may cause the worse detoxification of reactive carbonyls. Nevertheless, all known information about the interactions of polyphenols with CBR1 have only been based on the results of in vitro studies. With respect to the high importance of CBR1 and the frequent consumption of polyphenols, in vivo studies would be very helpful for the evaluation of risks/benefits of polyphenol interactions with CBR1. PMID:26415702

  3. Characterization of the nitric oxide reductase from Thermus thermophilus

    PubMed Central

    Schurig-Briccio, Lici A.; Venkatakrishnan, Padmaja; Hemp, James; Bricio, Carlos; Berenguer, José; Gennis, Robert B.

    2013-01-01

    Nitrous oxide (N2O) is a powerful greenhouse gas implicated in climate change. The dominant source of atmospheric N2O is incomplete biological dentrification, and the enzymes responsible for the release of N2O are NO reductases. It was recently reported that ambient emissions of N2O from the Great Boiling Spring in the United States Great Basin are high, and attributed to incomplete denitrification by Thermus thermophilus and related bacterial species [Hedlund BP, et al. (2011) Geobiology 9(6)471–480]. In the present work, we have isolated and characterized the NO reductase (NOR) from T. thermophilus. The enzyme is a member of the cNOR family of enzymes and belongs to a phylogenetic clade that is distinct from previously examined cNORs. Like other characterized cNORs, the T. thermophilus cNOR consists of two subunits, NorB and NorC, and contains a one heme c, one Ca2+, a low-spin heme b, and an active site consisting of a high-spin heme b and FeB. The roles of conserved residues within the cNOR family were investigated by site-directed mutagenesis. The most important and unexpected result is that the glutamic acid ligand to FeB is not essential for function. The E211A mutant retains 68% of wild-type activity. Mutagenesis data and the pattern of conserved residues suggest that there is probably not a single pathway for proton delivery from the periplasm to the active site that is shared by all cNORs, and that there may be multiple pathways within the T. thermophilus cNOR. PMID:23858452

  4. Isolation and characterization of nitric oxide reductase from Paracoccus halodenitrificans.

    PubMed

    Sakurai, N; Sakurai, T

    1997-11-11

    Nitric oxide reductase was isolated from the membrane fraction of a denitrifying bacterium, Paracoccus halodenitrificans, in the presence of n-dodecyl beta-D-maltoside. A relatively simple and effective procedure to purify NO reductase using DEAE-Toyopearl and hydroxyapatite (ceramic) chromatographies has been developed. The enzyme consisted of two subunits with molecular masses of 20 and 42 kDa associated with the c-type heme and two b-type hemes, respectively. The optical and magnetic circular dichroism (MCD) spectra of the oxidized (as isolated) and reduced enzymes indicated that the heme c is in the low-spin state and the hemes b are in the high- and low-spin states. The EPR spectrum also showed the presence of the split high-spin component (g perpendicular = 6.6, 6.0) and two low spin components (gz,y,x = 2.96, 2.26, 1.46, gz = 3.59). Although the presence of an extra iron was suggested from atomic absorption spectroscopy, a non-heme iron could not be detected by colorimetric titrations using ferene and 2-(5-nitro-2-pyridylazo)- 5-(N-propyl-N-sulfopropylamino)phenolate (PAPS). One of the extra signals at g = 4.3 and 2.00 might come from a non-heme iron, while they may originate from an adventitious iron and a certain nonmetallic radical, respectively. When CO acted on the reduced enzyme, both of the low-spin hemes were not affected, and when NO acted on the reduced enzyme, the optical and MCD spectra were of a mixture of the oxidized and reduced enzymes. Consequently, the reduction of NO was supposed to take place at the high-spin heme b. The heme c and the low-spin heme b centers were considered to function as electron mediators during the intermolecular and intramolecular processes. PMID:9374857

  5. Bacteriophage T4 Virion Baseplate Thymidylate Synthetase and Dihydrofolate Reductase

    PubMed Central

    Kozloff, L. M.; Lute, M.; Crosby, L. K.

    1977-01-01

    Additional evidence is presented that both the phage T4D-induced thymidylate synthetase (gp td) and the T4D-induced dihydrofolate reductase (gp frd) are baseplate structural components. With regard to phage td it has been found that: (i) low levels of thymidylate synthetase activity were present in highly purified preparations of T4D ghost particles produced after infection with td+, whereas particles produced after infection with td− had no measurable enzymatic activity; (ii) a mutation of the T4D td gene from tdts to td+ simultaneously produced a heat-stable thymidylate synthetase enzyme and heat-stable phage particles (it should be noted that the phage baseplate structure determines heat lability); (iii) a recombinant of two T4D mutants constructed containing both tdts and frdts genes produced particles whose physical properties indicate that these two molecules physically interact in the baseplate. With regard to phage frd it has been found that two spontaneous revertants each of two different T4D frdts mutants to frd+ not only produced altered dihydrofolate reductases but also formed phage particles with heat sensitivities different from their parents. Properties of T4D particles produced after infection with parental T4D mutants presumed to have a deletion of the td gene and/or the frd gene indicate that these particles still retain some characteristics associated with the presence of both the td and the frd molecules. Furthermore, the particles produced by the deletion mutants have been found to be physically different from the parent particles. PMID:894793

  6. Identification of imine reductase-specific sequence motifs.

    PubMed

    Fademrecht, Silvia; Scheller, Philipp N; Nestl, Bettina M; Hauer, Bernhard; Pleiss, Jürgen

    2016-05-01

    Chiral amines are valuable building blocks for the production of a variety of pharmaceuticals, agrochemicals and other specialty chemicals. Only recently, imine reductases (IREDs) were discovered which catalyze the stereoselective reduction of imines to chiral amines. Although several IREDs were biochemically characterized in the last few years, knowledge of the reaction mechanism and the molecular basis of substrate specificity and stereoselectivity is limited. To gain further insights into the sequence-function relationships, the Imine Reductase Engineering Database (www.IRED.BioCatNet.de) was established and a systematic analysis of 530 putative IREDs was performed. A standard numbering scheme based on R-IRED-Sk was introduced to facilitate the identification and communication of structurally equivalent positions in different proteins. A conservation analysis revealed a highly conserved cofactor binding region and a predominantly hydrophobic substrate binding cleft. Two IRED-specific motifs were identified, the cofactor binding motif GLGxMGx5 [ATS]x4 Gx4 [VIL]WNR[TS]x2 [KR] and the active site motif Gx[DE]x[GDA]x[APS]x3 {K}x[ASL]x[LMVIAG]. Our results indicate a preference toward NADPH for all IREDs and explain why, despite their sequence similarity to β-hydroxyacid dehydrogenases (β-HADs), no conversion of β-hydroxyacids has been observed. Superfamily-specific conservations were investigated to explore the molecular basis of their stereopreference. Based on our analysis and previous experimental results on IRED mutants, an exclusive role of standard position 187 for stereoselectivity is excluded. Alternatively, two standard positions 139 and 194 were identified which are superfamily-specifically conserved and differ in R- and S-selective enzymes. Proteins 2016; 84:600-610. © 2016 Wiley Periodicals, Inc. PMID:26857686

  7. Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

    PubMed Central

    Mock, Johanna; Wang, Shuning; Huang, Haiyan; Kahnt, Jörg

    2014-01-01

    Moorella thermoacetica can grow with H2 and CO2, forming acetic acid from 2 CO2 via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown. PMID:25002540

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

    PubMed Central

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

    1999-01-01

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

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

    PubMed

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

    1999-10-01

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

  10. Developmental regulation of ecdysone receptor (EcR) and EcR-controlled gene expression during pharate-adult development of honeybees (Apis mellifera)

    PubMed Central

    Mello, Tathyana R. P.; Aleixo, Aline C.; Pinheiro, Daniel G.; Nunes, Francis M. F.; Bitondi, Márcia M. G.; Hartfelder, Klaus; Barchuk, Angel R.; Simões, Zilá L. P.

    2014-01-01

    Major developmental transitions in multicellular organisms are driven by steroid hormones. In insects, these, together with juvenile hormone (JH), control development, metamorphosis, reproduction and aging, and are also suggested to play an important role in caste differentiation of social insects. Here, we aimed to determine how EcR transcription and ecdysteroid titers are related during honeybee postembryonic development and what may actually be the role of EcR in caste development of this social insect. In addition, we expected that knocking-down EcR gene expression would give us information on the participation of the respective protein in regulating downstream targets of EcR. We found that in Apis mellifera females, EcR-A is the predominantly expressed variant in postembryonic development, while EcR-B transcript levels are higher in embryos, indicating an early developmental switch in EcR function. During larval and pupal stages, EcR-B expression levels are very low, while EcR-A transcripts are more variable and abundant in workers compared to queens. Strikingly, these transcript levels are opposite to the ecdysteroid titer profile. 20-hydroxyecdysone (20E) application experiments revealed that low 20E levels induce EcR expression during development, whereas high ecdysteroid titers seem to be repressive. By means of RNAi-mediated knockdown (KD) of both EcR transcript variants we detected the differential expression of 234 poly-A+ transcripts encoding genes such as CYPs, MRJPs and certain hormone response genes (Kr-h1 and ftz-f1). EcR-KD also promoted the differential expression of 70 miRNAs, including highly conserved ones (e.g., miR-133 and miR-375), as well honeybee-specific ones (e.g., miR-3745 and miR-3761). Our results put in evidence a broad spectrum of EcR-controlled gene expression during postembryonic development of honeybees, revealing new facets of EcR biology in this social insect. PMID:25566327

  11. Developmental regulation of ecdysone receptor (EcR) and EcR-controlled gene expression during pharate-adult development of honeybees (Apis mellifera).

    PubMed

    Mello, Tathyana R P; Aleixo, Aline C; Pinheiro, Daniel G; Nunes, Francis M F; Bitondi, Márcia M G; Hartfelder, Klaus; Barchuk, Angel R; Simões, Zilá L P

    2014-01-01

    Major developmental transitions in multicellular organisms are driven by steroid hormones. In insects, these, together with juvenile hormone (JH), control development, metamorphosis, reproduction and aging, and are also suggested to play an important role in caste differentiation of social insects. Here, we aimed to determine how EcR transcription and ecdysteroid titers are related during honeybee postembryonic development and what may actually be the role of EcR in caste development of this social insect. In addition, we expected that knocking-down EcR gene expression would give us information on the participation of the respective protein in regulating downstream targets of EcR. We found that in Apis mellifera females, EcR-A is the predominantly expressed variant in postembryonic development, while EcR-B transcript levels are higher in embryos, indicating an early developmental switch in EcR function. During larval and pupal stages, EcR-B expression levels are very low, while EcR-A transcripts are more variable and abundant in workers compared to queens. Strikingly, these transcript levels are opposite to the ecdysteroid titer profile. 20-hydroxyecdysone (20E) application experiments revealed that low 20E levels induce EcR expression during development, whereas high ecdysteroid titers seem to be repressive. By means of RNAi-mediated knockdown (KD) of both EcR transcript variants we detected the differential expression of 234 poly-A(+) transcripts encoding genes such as CYPs, MRJPs and certain hormone response genes (Kr-h1 and ftz-f1). EcR-KD also promoted the differential expression of 70 miRNAs, including highly conserved ones (e.g., miR-133 and miR-375), as well honeybee-specific ones (e.g., miR-3745 and miR-3761). Our results put in evidence a broad spectrum of EcR-controlled gene expression during postembryonic development of honeybees, revealing new facets of EcR biology in this social insect. PMID:25566327

  12. Steroidal pyrazolines evaluated as aromatase and quinone reductase-2 inhibitors for chemoprevention of cancer.

    PubMed

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

    2012-05-01

    The aromatase and quinone reductase-2 inhibition of synthesized heterocyclic pyrazole derivatives fused with steroidal structure for chemoprevention of cancer is reported herein. All compounds were interestingly less toxic than the reference drug (Cyproterone(®)). The aromatase inhibitory activities of these compounds were much more potent than the lead compound resveratrol, which has an IC(50) of 80 μM. In addition, all the compounds displayed potent quinone reductase-2 inhibition. Initially the acute toxicity of the compounds was assayed via the determination of their LD(50). The aromatase and quinone reductase-2 inhibitors resulting from this study have potential value in the treatment and prevention of cancer. PMID:22361454

  13. The use of 5-alpha reductase inhibitors for the prevention of prostate cancer.

    PubMed

    Yu, Eun-mi; El-Ayass, Walid; Aragon-Ching, Jeanny B

    2010-07-01

    The use of 5-alpha-reductase inhibitors has been studied not only in benign prostatic hyperplasia, but as a chemopreventive strategy in prostate cancer. Both finasteride and dutasteride, 5 alpha-reductase inhibitors (5ARI), have been shown to decrease the risk of prostate cancer. The results of the REDUCE trial using the dual alpha-reductase isoenzyme inhibitor dutasteride, has recently been published by Andriole et al. in the New England Journal of Medicine. Certain considerations regarding its use and applicability to men with high risk of developing prostate cancer are herein discussed. PMID:20574153

  14. Cloning and characterization of the methyl coenzyme M reductase genes from Methanobacterium thermoautotrophicum.

    PubMed Central

    Bokranz, M; Bäumner, G; Allmansberger, R; Ankel-Fuchs, D; Klein, A

    1988-01-01

    The genes coding for methyl coenzyme M reductase were cloned from a genomic library of Methanobacterium thermoautotrophicum Marburg into Escherichia coli by using plasmid expression vectors. When introduced into E. coli, the reductase genes were expressed, yielding polypeptides identical in size to the three known subunits of the isolated enzyme, alpha, beta, and gamma. The polypeptides also reacted with the antibodies raised against the respective enzyme subunits. In M. thermoautotrophicum, the subunits are encoded by a gene cluster whose transcript boundaries were mapped. Sequence analysis revealed two more open reading frames of unknown function located between two of the methyl coenzyme M reductase genes. Images PMID:2448287

  15. Comparative hydrogen-deuterium exchange for a mesophilic vs thermophilic dihydrofolate reductase at 25 °C: identification of a single active site region with enhanced flexibility in the mesophilic protein.

    PubMed

    Oyeyemi, Olayinka A; Sours, Kevin M; Lee, Thomas; Kohen, Amnon; Resing, Katheryn A; Ahn, Natalie G; Klinman, Judith P

    2011-09-27

    The technique of hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been applied to a mesophilic (E. coli) dihydrofolate reductase under conditions that allow direct comparison to a thermophilic (B. stearothermophilus) ortholog, Ec-DHFR and Bs-DHFR, respectively. The analysis of hydrogen-deuterium exchange patterns within proteolytically derived peptides allows spatial resolution, while requiring a series of controls to compare orthologous proteins with only ca. 40% sequence identity. These controls include the determination of primary structure effects on intrinsic rate constants for HDX as well as the use of existing 3-dimensional structures to evaluate the distance of each backbone amide hydrogen to the protein surface. Only a single peptide from the Ec-DHFR is found to be substantially more flexible than the Bs-DHFR at 25 °C in a region located within the protein interior at the intersection of the cofactor and substrate-binding sites. The surrounding regions of the enzyme are either unchanged or more flexible in the thermophilic DHFR from B. stearothermophilus. The region with increased flexibility in Ec-DHFR corresponds to one of two regions previously proposed to control the enthalpic barrier for hydride transfer in Bs-DHFR [Oyeyemi et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 10074]. PMID:21859100

  16. An ethoxyquin-inducible aldehyde reductase from rat liver that metabolizes aflatoxin B1 defines a subfamily of aldo-keto reductases.

    PubMed Central

    Ellis, E M; Judah, D J; Neal, G E; Hayes, J D

    1993-01-01

    Protection of liver against the toxic and carcinogenic effects of aflatoxin B1 (AFB1) can be achieved through the induction of detoxification enzymes by chemoprotectors such as the phenolic antioxidant ethoxyquin. We have cloned and sequenced a cDNA encoding an aldehyde reductase (AFB1-AR), which is expressed in rat liver in response to dietary ethoxyquin. Expression of the cDNA in Escherichia coli and purification of the recombinant enzyme reveals that the protein exhibits aldehyde reductase activity and is capable of converting the protein-binding dialdehyde form of AFB1-dihydrodiol to the nonbinding dialcohol metabolite. We show that the mRNA encoding this enzyme is markedly elevated in the liver of rats fed an ethoxyquin-containing diet, correlating with acquisition of resistance to AFB1. AFB1-AR represents the only carcinogen-metabolizing aldehyde reductase identified to date that is induced by a chemoprotector. Alignment of the amino acid sequence of AFB1-AR with other known and putative aldehyde reductases shows that it defines a subfamily within the aldo-keto reductase superfamily. Images Fig. 2 Fig. 3 Fig. 4 PMID:8234296

  17. Metabolism of the Synthetic Progestogen Norethynodrel by Human Ketosteroid Reductases of the Aldo-Keto Reductase Superfamily

    PubMed Central

    Jin, Yi; Duan, Ling; Chen, Mo; Penning, Trevor M; Kloosterboer, Helenius J.

    2012-01-01

    Human ketosteroid reductases of the aldo-keto reductase (AKR) superfamily, i.e. AKR1C1-4, are implicated in the biotransformation of synthetic steroid hormones. Norethynodrel (NOR, 17α-ethynyl-17β-hydroxy-estra-5(10)-en-3-one), the progestin component of the first marketed oral contraceptive, is known to undergo rapid and extensive metabolism to 3α- and 3β-hydroxy metabolites. The ability of the four human AKR1C enzymes to catalyze the metabolism of NOR has now been characterized. AKR1C1 and AKR1C2 almost exclusively converted NOR to 3β-hydroxy NOR, while AKR1C3 gave 3β-hydroxy NOR as the main product and AKR1C4 predominantly formed 3α-hydroxy NOR. Individual AKR1C enzymes also displayed distinct kinetic properties in the reaction of NOR. In contrast, norethindrone (NET), the Δ4-isomer of NOR and the most commonly used synthetic progestin, was not a substrate for the AKR1C enzymes. NOR is also structurally identical to the hormone replacement therapeutic tibolone (TIB), except TIB has a methyl group at the 7α-position. Product profiles and kinetic parameters for the reduction of NOR catalyzed by each individual AKR1C isoform were identical to those for the reduction of TIB catalyzed by the respective isoform. These data suggest that the presence of the 7α-methyl group has a minimal effect on the stereochemical outcome of the reaction and kinetic behavior of each enzyme. Results indicate a role of AKR1C in the hepatic and peripheral metabolism of NOR to 3α- and 3β-hydroxy NOR and provide insights into the differential pharmacological properties of NOR, NET and TIB. PMID:22210085

  18. Methylenetetrahydrofolate reductase and methionine synthase reductase gene polymorphisms and protection from microvascular complications in adolescents with type 1 diabetes.

    PubMed

    Wiltshire, Esko J; Mohsin, Fauzia; Chan, Albert; Donaghue, Kim C

    2008-08-01

    Folate status has been associated with endothelial dysfunction in adolescents with type 1 diabetes, and elevated total plasma homoocyst(e)ine (tHcy) is a risk for vascular disease in the non-diabetic population. Polymorphisms in genes involved in folate and homocysteine metabolism are implicated in vascular disease. We aimed to determine whether polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes are risk factors for early microvascular disease in a large group of adolescents with type 1 diabetes. Four hundred and eighty adolescents were screened annually for retinopathy and microalbuminuria for a median of 4 yr. Molecular analysis for the polymorphisms 677C-->T, 1298A-->C in MTHFR, and 66A-->G in MTRR was performed. The MTRR 66GG genotype reduced the risk for elevated albumin excretion rate (AER) (OR 0.47, CI 0.25, 0.88, p = 0.018) and showed a trend to reduced risk for microalbuminuria (OR 0.27, CI 0.06-1.21, p = 0.09). Survival without elevated AER was increased with the MTRR 66GG genotype (12.4 vs. 9.7 yr, p = 0.04) and with the MTHFR 1298CC genotype (15.2 vs. 10.2 yr, p = 0.007). Conversely, survival without retinopathy was reduced with the MTHFR 677TT and MTRR 66GG combined genotype (6.2 vs. 10.2 yr, p = 0.015). The MTRR 66GG and MTHFR 1298 CC genotypes may confer protection against early nephropathy, possibly because they are associated with lower tHcy. The MTHFR 677 TT was only related to earlier onset retinopathy in combination with MTRR 66GG. PMID:18774994

  19. The subunit composition of human extracellular superoxide dismutase (EC-SOD) regulate enzymatic activity

    PubMed Central

    Petersen, Steen V; Valnickova, Zuzana; Oury, Tim D; Crapo, James D; Chr Nielsen, Niels; Enghild, Jan J

    2007-01-01

    Background Human extracellular superoxide dismutase (EC-SOD) is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space. We have previously shown that the EC-SOD subunit exists in two distinct folding variants based on differences in the disulfide bridge pattern (Petersen SV, Oury TD, Valnickova Z, Thøgersen IB, Højrup P, Crapo JD, Enghild JJ. Proc Natl Acad Sci USA. 2003;100(24):13875–80). One variant is enzymatically active (aEC-SOD) while the other is inactive (iEC-SOD). The EC-SOD subunits are associated into covalently linked dimers through an inter-subunit disulfide bridge creating the theoretical possibility of 3 dimers (aa, ai or ii) with different antioxidant potentials. We have analyzed the quaternary structure of the endogenous EC-SOD disulfide-linked dimer to investigate if these dimers in fact exist. Results The analyses of EC-SOD purified from human tissue show that all three dimer combinations exist including two homo-dimers (aa and ii) and a hetero-dimer (ai). Because EC-SOD is a tetramer the dimers may combine to generate 5 different mature EC-SOD molecules where the specific activity of each molecule is determined by the ratio of aEC-SOD and iEC-SOD subunits. Conclusion This finding shows that the aEC-SOD and iEC-SOD subunits combine in all 3 possible ways supporting the presence of tetrameric enzymes with variable enzymatic activity. This variation in enzymatic potency may regulate the antioxidant level in the extracellular space and represent a novel way of modulating enzymatic activity. PMID:17937792

  20. Redesigning alcohol dehydrogenases/reductases for more efficient biosynthesis of enantiopure isomers.

    PubMed

    Zhang, Rongzhen; Xu, Yan; Xiao, Rong

    2015-12-01

    Alcohol dehydrogenases/reductases predominantly catalyze the asymmetric biosynthesis of optically pure stereoisomers because of their unique chiral constitutions. The enantioselectivities of alcohol dehydrogenases/reductases are substrate- and cofactor-dependent, and therefore they usually catalyze specific reactions with high enantioselectivity under physiological conditions; this may not be suitable for asymmetric biosynthesis with non-natural substrates or non-natural cofactors, and under nonphysiological conditions. It is therefore necessary to modify alcohol dehydrogenases/reductases using various redesigning tools such as directed evolution and rational design, and their combinations, as well as engineering enzyme modules for more efficient production of "non-natural" products. In this article, progress in these aspects of alcohol dehydrogenase/reductase design is reviewed, and future challenges are discussed. PMID:26320091

  1. Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line

    SciTech Connect

    Kaufman, R.J.; Schimke, R.T.

    1981-12-01

    During stepwise increases in the methotrexate concentration in culture medium, the authors selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). The authors studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.

  2. 5α-reductase inhibitors, antiviral and anti-tumor activities of some steroidal cyanopyridinone derivatives.

    PubMed

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

    2012-01-01

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

  3. "EC to go" takes off at Maryland sites.

    PubMed

    2000-03-01

    Baltimore-based Planned Parenthood of Maryland and the Baltimore City Health Department have joined forces in "EC to Go," which distributes free emergency contraceptive pills (ECPs) through the seven affiliate sites of Planned Parenthood and the three family planning centers of the city. The distribution program was started in October 1999 and funds were provided by an undisclosed area foundation. Although the program is still in its infancy, it has recorded some 800 prescriptions of ECPs in the last fiscal year, and 600 prescriptions have been logged in just the first 6 months of the current fiscal year. To inform the public about the program, Planned Parenthood developed newspaper advertisements, a 60-second radio spot, and coupon distributions, all of which emphasize the fact that emergency contraception is a higher dose of birth control, which can prevent pregnancy if taken within 72 hours of unprotected sex. PMID:12349550

  4. Design and synthesis of hepatoselective, pyrrole-based HMG-CoA reductase inhibitors.

    PubMed

    Pfefferkorn, Jeffrey A; Song, Yuntao; Sun, Kuai-Lin; Miller, Steven R; Trivedi, Bharat K; Choi, Chulho; Sorenson, Roderick J; Bratton, Larry D; Unangst, Paul C; Larsen, Scott D; Poel, Toni-Jo; Cheng, Xue-Min; Lee, Chitase; Erasga, Noe; Auerbach, Bruce; Askew, Valerie; Dillon, Lisa; Hanselman, Jeffrey C; Lin, Zhiwu; Lu, Gina; Robertson, Andrew; Olsen, Karl; Mertz, Thomas; Sekerke, Catherine; Pavlovsky, Alexander; Harris, Melissa S; Bainbridge, Graeme; Caspers, Nicole; Chen, Huifen; Eberstadt, Matthias

    2007-08-15

    This manuscript describes the design and synthesis of a series of pyrrole-based inhibitors of HMG-CoA reductase for the treatment of hypercholesterolemia. Analogs were optimized using structure-based design and physical property considerations resulting in the identification of 44, a hepatoselective HMG-CoA reductase inhibitor with excellent acute and chronic efficacy in a pre-clinical animal models. PMID:17574412

  5. Isolation of Assimilatory- and Dissimilatory-Type Sulfite Reductases from Desulfovibrio vulgaris

    PubMed Central

    Lee, Jin-Po; LeGall, Jean; Peck, Harry D.

    1973-01-01

    Bisulfite reductase (desulfoviridin) and an assimilatory sulfite reductase have been purified from extracts of Desulfovibrio vulgaris. The bisulfite reductase has absorption maxima at 628, 580, 408, 390, and 279 nm, and a molecular weight of 226,000 by sedimentation equilibrium, and was judged to be free of other proteins by disk electrophoresis and ultracentrifugation. On gels, purified bisulfite reductase exhibited two green bands which coincided with activity and protein. The enzyme appears to be a tetramer but was shown to have two different types of subunits having molecular weights of 42,000 and 50,000. The chromophore did not form an alkaline ferrohemochromogen, was not reduced with dithionite or borohydride, and did not form a spectrally visible complex with CO. The assimilatory sulfite reductase has absorption maxima at 590, 545, 405 and 275 nm and a molecular weight of 26,800, and appears to consist of a single polypeptide chain as it is not dissociated into subunits by sodium dodecyl sulfate. By disk electrophoresis, purified sulfite reductase exhibited a single greenish-brown band which coincided with activity and protein. The sole product of the reduction was sulfide, and the chromophore was reduced by borohydride in the presence of sulfite. Carbon monoxide reacted with the reduced chromophore but it did not form a typical pyridine ferrohemochromogen. Thiosulfate, trithionate, and tetrathionate were not reduced by either enzyme preparation. In the presence of 8 M urea, the spectrum of bisulfite reductase resembles that of the sulfite reductase, thus suggesting a chemical relationship between the two chromophores. Images PMID:4725615

  6. Completion Report for Well ER-EC-1

    SciTech Connect

    Townsend, M.J.

    2000-12-01

    Well ER-EC-1 was drilled for the U.S. Department of Energy, Nevada Operations Office in support of the Nevada Environmental Restoration Project at the Nevada Test Site, Nye County, Nevada. This well was drilled in the spring of 1999 as part of the U.S. Department of Energy's hydrogeologic investigation well program in the Western Pahute Mesa - Oasis Valley region just west of the Test Site. A 44.5-centimeter surface hole was drilled and cased off to the depth 675.1 meters below the surface. The hole diameter was then decreased to 31.1 centimeters for drilling to a total depth of 1,524.0 meters. A preliminary composite, static, water level was measured at the depth of approximately 566.3 meters prior to installation of the completion string. One completion string with three isolated, slotted intervals was installed in the well. Detailed lithologic descriptions with preliminary stratigraphic assignments are included in the report. These are based on composite drill cuttings collected every 3 meters and 31 sidewall samples taken at various depths below 680 meters, supplemented by geophysical log data. Detailed chemical and mineralogical studies of rock samples are in progress. The well penetrated Tertiary-age lava and tuff of the Timber Mountain Group, the Paintbrush Group, the Calico Hills Formation, the Crater Flat Group, and the Volcanics of Quartz Mountain. The preliminary geologic interpretation of data from Well ER-EC-1 indicates the presence of a structural trough or bench filled with a thick section of post-Rainier Mesa lava. These data also suggest that this site is located on a buried structural ridge that may separate the Silent Canyon and Timber Mountain caldera complexes.

  7. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    PubMed Central

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils. PMID:24435070

  8. "Subversive" substrates for the enzyme trypanothione disulfide reductase: alternative approach to chemotherapy of Chagas disease.

    PubMed Central

    Henderson, G B; Ulrich, P; Fairlamb, A H; Rosenberg, I; Pereira, M; Sela, M; Cerami, A

    1988-01-01

    The trypanosomatid flavoprotein disulfide reductase, trypanothione reductase, is shown to catalyze one-electron reduction of suitably substituted naphthoquinone and nitrofuran derivatives. A number of such compounds have been chemically synthesized, and a structure-activity relationship has been established; the enzyme is most active with compounds that contain basic functional groups in side-chain residues. The reduced products are readily reoxidized by molecular oxygen and thus undergo classical enzyme-catalyzed redox cycling. In addition to their ability to act as substrates for trypanothione reductase, the compounds are also shown to effectively inhibit enzymatic reduction of the enzyme's physiological substrate, trypanothione disulfide. Under aerobic conditions, trypanothione reductase is not inactivated by these redox-cycling substrates, whereas under anaerobic conditions the nitrofuran compounds cause irreversible inactivation of the enzyme. When tested for biological activity against Trypanosoma cruzi trypomastigotes, many of the test compounds were trypanocidal, and this activity correlated with their relative ability to act as substrates for trypanothione reductase. The activity of the enzyme with these redox-cycling derivatives constitutes a subversion of its normal antioxidant role within the cell. For this reason these compounds may be termed "subversive" substrates for trypanothione reductase. PMID:3135548

  9. The role of glutathione reductase and related enzymes on cellular redox homoeostasis network.

    PubMed

    Couto, Narciso; Wood, Jennifer; Barber, Jill

    2016-06-01

    In this review article we examine the role of glutathione reductase in the regulation, modulation and maintenance of cellular redox homoeostasis. Glutathione reductase is responsible for maintaining the supply of reduced glutathione; one of the most abundant reducing thiols in the majority of cells. In its reduced form, glutathione plays key roles in the cellular control of reactive oxygen species. Reactive oxygen species act as intracellular and extracellular signalling molecules and complex cross talk between levels of reactive oxygen species, levels of oxidised and reduced glutathione and other thiols, and antioxidant enzymes such as glutathione reductase determine the most suitable conditions for redox control within a cell or for activation of programmed cell death. Additionally, we discuss the translation and expression of glutathione reductase in a number of organisms including yeast and humans. In yeast and human cells, a single gene expresses more than one form of glutathione reductase, destined for residence in the cytoplasm or for translocation to different organelles; in plants, however, two genes encoding this protein have been described. In general, insects and kinetoplastids (a group of protozoa, including Plasmodia and Trypanosoma) do not express glutathione reductase or glutathione biosynthetic enzymes. Instead, they express either the thioredoxin system or the trypanothione system. The thioredoxin system is also present in organisms that have the glutathione system and there may be overlapping functions with cross-talk between the two systems. Finally we evaluate therapeutic targets to overcome oxidative stress associated cellular disorders. PMID:26923386

  10. X-ray structure of trypanothione reductase from Crithidia fasciculata at 2. 4- angstrom resolution

    SciTech Connect

    Kuriyan, J.; Xiangpeng Kong; Krishna, T.S.R.; Murgolo, N.J.; Field, H.; Cerami, A.; Henderson, G.B. ); Sweet, R.M. )

    1991-10-01

    Trypanosomes and related protozoan parasites lack glutathione reductase and possess instead a closely related enzyme that serves as the reductant of a bis(glutathione)-spermidien conjugate, trypanothione. The human and parasite enzymes have mutually exclusive substrate specificities, providing a route for the design of therapeutic agents by specific inhibition of the parasite enzyme. The authors report here the three-dimensional structure of trypanothione reductase from Crithidia fasciculata and show that it closely resembles the structure of human glutathione reductase. In particular, the core structure surrounding the catalytic machinery is almost identical in the two enzymes. However, significant differences are found at the substrate binding sites. A cluster of basic residues in glutathione reductase is replaced by neutral, hydrophobic, or acidic residues in trypanothione reductase, consistent with the nature of the spermidine linkage and the change in overall charge of the substrate from {minus}2 to +1, respectively. The binding site is more open in trypanothione reductase due to rotations of about 4{degree} in the domains that form in site, with relative shifts of as much as 2-3 {angstrom} in residues that can interact with potential inhibitors and complement previous modeling and mutagenesis studies on the two enzymes.

  11. Characterization of anaerobic sulfite reduction by Salmonella typhimurium and purification of the anaerobically induced sulfite reductase

    SciTech Connect

    Hallenbeck, P.C. ); Clark, M.A.; Barrett, E.L. )

    1989-06-01

    Mutants of Salmonella typhimurium that lack the biosynthetic sulfite reductase (cysI and cysJ mutants) retain the ability to reduce sulfite for growth under anaerobic conditions. Here we report studies of sulfite reduction by a cysI mutant of S. typhimurium and purification of the associated anaerobic sulfite reductase. Sulfite reduction for anaerobic growth did not require a reducing atmosphere but was prevented by an argon atmosphere contaminated with air (<0.33%). It was also prevented by the presence of 0.1 mM nitrate. Anaerobic growth in liquid minimal medium, but not on agar, was found to require additions of trace amounts (10{sup {minus}7} M) of cysteine. Spontaneous mutants that grew under the argon contaminated with air also lost the requirement for 10{sup {minus}7}M cysteine for anaerobic growth in liquid. A role for sulfite reduction in anaerobic energy generation was contraindicated by the findings that sulfite reduction did not improve cell yields, and anaerobic sulfite reductase activity was greatest during the stationary phase of growth. Sulfite reductase was purified from the cytoplasmic fraction of the anaerobically grown cysI mutant and was purified 190-fold. The most effective donor in crude extracts was NADH. NADHP and methyl viologen were, respectively, 40 and 30% as effective as NADH. Oxygen reversibly inhibited the enzyme. The anaerobic sulfite reductase showed some resemblance to the biosynthetic sulfite reductase, but apparently it has a unique, as yet unidentified function.

  12. The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

    PubMed Central

    Profant, Deborah A.; Roberts, Christopher J.; Koning, Ann J.; Wright, Robin L.

    1999-01-01

    In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using β-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain. PMID:10512876

  13. Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase.

    PubMed Central

    Fransen, M; Van Veldhoven, P P; Subramani, S

    1999-01-01

    To elucidate unknown mammalian peroxisomal enzymes and functions, we subjected M13 phage expressing fusions between the gene encoding protein VI and a rat liver cDNA library to an immunoaffinity selection process in vitro (biopanning) with the use of antibodies raised against peroxisomal subfractions. In an initial series of biopanning experiments, four different cDNA clones were obtained. These cDNA species encoded two previously identified peroxisomal enzymes, catalase and urate oxidase, and two novel proteins that contained a C-terminal peroxisomal targeting signal (PTS1). A primary structure analysis of these novel proteins revealed that one, ending in the tripeptide AKL, is homologous to the yeast peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34; DCR), an enzyme required for the degradation of unsaturated fatty acids, and that the other, ending in the tripeptide SRL, is a putative member of the short-chain dehydrogenase/reductase (SDR) family, with three isoforms. Green fluorescent protein (GFP) fusions encoding GFP-DCR-AKL, GFP-DCR, GFP-SDR-SRL and GFP-SDR were expressed in mammalian cells. The analysis of the subcellular location of the recombinant fusion proteins confirmed the peroxisomal localization of GFP-DCR-AKL and GFP-SDR-SRL, as well as the functionality of the PTS1. That the AKL protein is indeed an NADPH-dependent DCR was demonstrated by showing DCR activity of the bacterially expressed protein. These results demonstrate at the molecular level that mammalian peroxisomes do indeed contain a DCR. In addition, the results presented here indicate that the protein VI display system is suitable for the isolation of rare cDNA clones from cDNA libraries and that this technology facilitates the identification of novel peroxisomal proteins. PMID:10333503

  14. Corn leaf nitrate reductase - A nontoxic alternative to cadmium for photometric nitrate determinations in water samples by air-segmented continuous-flow analysis

    USGS Publications Warehouse

    Patton, C.J.; Fischer, A.E.; Campbell, W.H.; Campbell, E.R.

    2002-01-01

    Development, characterization, and operational details of an enzymatic, air-segmented continuous-flow analytical method for colorimetric determination of nitrate + nitrite in natural-water samples is described. This method is similar to U.S. Environmental Protection Agency method 353.2 and U.S. Geological Survey method 1-2545-90 except that nitrate is reduced to nitrite by soluble nitrate reductase (NaR, EC 1.6.6.1) purified from corn leaves rather than a packed-bed cadmium reactor. A three-channel, air-segmented continuous-flow analyzer-configured for simultaneous determination of nitrite (0.020-1.000 mg-N/L) and nitrate + nitrite (0.05-5.00 mg-N/L) by the nitrate reductase and cadmium reduction methods-was used to characterize analytical performance of the enzymatic reduction method. At a sampling rate of 90 h-1, sample interaction was less than 1% for all three methods. Method detection limits were 0.001 mg of NO2- -N/L for nitrite, 0.003 mg of NO3-+ NO2- -N/L for nitrate + nitrite by the cadmium-reduction method, and 0.006 mg of NO3- + NO2- -N/L for nitrate + nitrite by the enzymatic-reduction method. Reduction of nitrate to nitrite by both methods was greater than 95% complete over the entire calibration range. The difference between the means of nitrate + nitrite concentrations in 124 natural-water samples determined simultaneously by the two methods was not significantly different from zero at the p = 0.05 level.

  15. Identification of the OsOPR7 gene encoding 12-oxophytodienoate reductase involved in the biosynthesis of jasmonic acid in rice.

    PubMed

    Tani, Tomoyuki; Sobajima, Hiroyuki; Okada, Kazunori; Chujo, Tetsuya; Arimura, Shin-Ichi; Tsutsumi, Nobuhiro; Nishimura, Mikio; Seto, Hideharu; Nojiri, Hideaki; Yamane, Hisakazu

    2008-02-01

    Enzyme 12-oxophytodienoate (OPDA) reductase (EC1.3.1.42), which is involved in the biosynthesis of jasmonic acid (JA), catalyses the reduction of 10, 11-double bonds of OPDA to yield 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0). The rice OsOPR1 gene encodes OPDA reductase (OPR) converting (-)-cis-OPDA preferentially, rather than (+)-cis-OPDA, a natural precursor of JA. Here, we provide evidence that an OPR family gene in rice chromosome 8, designated OsOPR7, encodes the enzyme involved in the JA biosynthesis. Recombinant OsOPR7-His protein efficiently catalysed the reduction of both enantiomers of cis-OPDA, similar to the OPR3 protein in Arabidopsis thaliana (L.) Heynh. The expression of OsOPR7 mRNA was induced and reached maximum levels within 0.5 h of mechanical wounding and drought stress, and the endogenous JA level started to increase in accordance with the increase in OsOPR7 expression. The GFP-OsOPR7 fusion protein was detected exclusively in peroxisomes in onion epidermal cells. Furthermore, complementation analysis using an Arabidopsis opr3 mutant indicated that the OsOPR7 gene, but not OsOPR1, was able to complement the phenotypes of male sterility in the mutant caused by JA deficiency, and that JA production in the opr3 mutant was also restored by the expression of the OsOPR7 gene. We conclude that the OsOPR7 gene encodes the enzyme catalysing the reduction of natural (+)-cis-OPDA for the JA biosynthesis in rice. PMID:17938955

  16. Effect of Elastic Compression Stocking (ECS) on Leg Veins During 2G Centrifugation

    NASA Astrophysics Data System (ADS)

    Arbeille, Ph.; Kaspransky, R.

    2008-06-01

    Objective: evaluate the calf vein response to hypergravity, and check the efficiency of elastic compression stocking (ECS) in preventing their distension. Method: Tibial (Tib csa) and Gastrocnemius (Gast csa) vein cross section area were investigated by echography. The subject was submitted to (a) 10 min stand test (ST), (b) 2G centrifugation for 2 min, (c) 10 min ST, with and without ECS. Results: Centrifugation at 2G induced a higher vein distension for both Gast and Tib vein compare to ST. At 2G centrifugation, ECS reduced the amplitude of the csa increase and limited the max vein csa to the ST value without ECS.

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

    PubMed Central

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

    1998-01-01

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

  18. Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis

    PubMed Central

    Cha, Mee-Kyung; Bae, Yoo-Jeen; Kim, Kyu-Jeong; Park, Byung-Joon; Kim, Il-Han

    2015-01-01

    AIM: To identify alkyl hydroperoxide reductase subunit C (AhpC) homologs in Bacillus subtilis (B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria. METHODS: Two AhpC homologs (AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues (C37S, C47S, C166S, C37/47S, C37/166S, C47/166S, and C37/47/166S for AhpC_H1; C52S, C169S, and C52/169S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahpC genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined. RESULTS: Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis

  19. Molecular Cloning and Characterization of Three Genes Encoding Dihydroflavonol-4-Reductase from Ginkgo biloba in Anthocyanin Biosynthetic Pathway

    PubMed Central

    Hua, Cheng; Linling, Li; Shuiyuan, Cheng; Fuliang, Cao; Feng, Xu; Honghui, Yuan; Conghua, Wu

    2013-01-01

    Dihydroflavonol-4-reductase (DFR, EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Three DFR cDNA clones (designated GbDFRs) were isolated from the gymnosperm Ginkgo biloba. The deduced GbDFR proteins showed high identities to other plant DFRs, which form three distinct DFR families. Southern blot analysis showed that the three GbDFRs each belong to a different DFR family. Phylogenetic tree analysis revealed that the GbDFRs share the same ancestor as other DFRs. The expression of the three recombinant GbDFRs in Escherichia coli showed that their actual protein sizes were in agreement with predictions from the cDNA sequences. The recombinant proteins were purified and their activity was analyzed; both GbDFR1 and GbDFR3 could catalyze dihydroquercetin conversion to leucocyanidin, while GbDFR2 catalyzed dihydrokaempferol conversion to leucopelargonidin. qRT-PCR showed that the GbDFRs were expressed in a tissue-specific manner, and transcript accumulation for the three genes was highest in young leaves and stamens. These transcription patterns were in good agreement with the pattern of anthocyanin accumulation in G.biloba. The expression profiles suggested that GbDFR1 and GbDFR2 are mainly involved in responses to plant hormones, environmental stress and damage. During the annual growth cycle, the GbDFRs were significantly correlated with anthocyanin accumulation in leaves. A fitted linear curve showed the best model for relating GbDFR2 and GbDFR3 with anthocyanin accumulation in leaves. GbDFR1 appears to be involved in environmental stress response, while GbDFR3 likely has primary functions in the synthesis of anthocyanins. These data revealed unexpected properties and differences in three DFR proteins from a single species. PMID:23991027

  20. Molecular and Biochemical Analysis of Two cDNA Clones Encoding Dihydroflavonol-4-Reductase from Medicago truncatula1

    PubMed Central

    Xie, De-Yu; Jackson, Lisa A.; Cooper, John D.; Ferreira, Daneel; Paiva, Nancy L.

    2004-01-01

    Dihydroflavonol-4-reductase (DFR; EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Two DFR cDNA clones (MtDFR1 and MtDFR2) were isolated from the model legume Medicago truncatula cv Jemalong. Both clones were functionally expressed in Escherichia coli, confirming that both encode active DFR proteins that readily reduce taxifolin (dihydroquercetin) to leucocyanidin. M. truncatula leaf anthocyanins were shown to be cyanidin-glucoside derivatives, and the seed coat proanthocyanidins are known catechin and epicatechin derivatives, all biosynthesized from leucocyanidin. Despite high amino acid similarity (79% identical), the recombinant DFR proteins exhibited differing pH and temperature profiles and differing relative substrate preferences. Although no pelargonidin derivatives were identified in M. truncatula, MtDFR1 readily reduced dihydrokaempferol, consistent with the presence of an asparagine residue at a location known to determine substrate specificity in other DFRs, whereas MtDFR2 contained an aspartate residue at the same site and was only marginally active on dihydrokaempferol. Both recombinant DFR proteins very efficiently reduced 5-deoxydihydroflavonol substrates fustin and dihydrorobinetin, substances not previously reported as constituents of M. truncatula. Transcript accumulation for both genes was highest in young seeds and flowers, consistent with accumulation of condensed tannins and leucoanthocyanidins in these tissues. MtDFR1 transcript levels in developing leaves closely paralleled leaf anthocyanin accumulation. Overexpression of MtDFR1 in transgenic tobacco (Nicotiana tabacum) resulted in visible increases in anthocyanin accumulation in flowers, whereas MtDFR2 did not. The data reveal unexpected properties and differences in two DFR proteins from a single species. PMID:14976232

  1. Kinetic, thermodynamic and X-ray structural insights into the interaction of melatonin and analogues with quinone reductase 2

    SciTech Connect

    Calamini, Barbara; Santarsiero, Bernard D.; Boutin, Jean A.; Mesecar, Andrew D.

    2008-09-12

    Melatonin exerts its biological effects through at least two transmembrane G-protein-coupled receptors, MT1 and MT2, and a lower-affinity cytosolic binding site, designated MT3. MT3 has recently been identified as QR2 (quinone reductase 2) (EC 1.10.99.2) which is of significance since it links the antioxidant effects of melatonin to a mechanism of action. Initially, QR2 was believed to function analogously to QR1 in protecting cells from highly reactive quinones. However, recent studies indicate that QR2 may actually transform certain quinone substrates into more highly reactive compounds capable of causing cellular damage. Therefore it is hypothesized that inhibition of QR2 in certain cases may lead to protection of cells against these highly reactive species. Since melatonin is known to inhibit QR2 activity, but its binding site and mode of inhibition are not known, we determined the mechanism of inhibition of QR2 by melatonin and a series of melatonin and 5-hydroxytryptamine (serotonin) analogues, and we determined the X-ray structures of melatonin and 2-iodomelatonin in complex with QR2 to between 1.5 and 1.8 {angstrom} (1 {angstrom} = 0.1 nm) resolution. Finally, the thermodynamic binding constants for melatonin and 2-iodomelatonin were determined by ITC (isothermal titration calorimetry). The kinetic results indicate that melatonin is a competitive inhibitor against N-methyldihydronicotinamide (K{sub i} = 7.2 {mu}M) and uncompetitive against menadione (K{sub i} = 92 {mu}M), and the X-ray structures shows that melatonin binds in multiple orientations within the active sites of the QR2 dimer as opposed to an allosteric site. These results provide new insights into the binding mechanisms of melatonin and analogues to QR2.

  2. Severe scoliosis in a patient with severe methylenetetrahydrofolate reductase deficiency.

    PubMed

    Munoz, Tatiana; Patel, Jinesh; Badilla-Porras, Ramses; Kronick, Jonathan; Mercimek-Mahmutoglu, Saadet

    2015-01-01

    Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare autosomal recessively inherited inborn error of folate metabolism. We report a new patient with severe MTHFR deficiency who presented at age 4 months with early onset severe scoliosis associated with severe hypotonia. Markedly decreased MTHFR enzyme activity (0.3 nmoles CHO/mg protein/h; reference range>9) and compound heterozygous mutations (c. 1304T>C; p.Phe435Ser and c.1539dup; p.Glu514Argfs∗24) in the MTHFR gene confirmed the diagnosis. She was treated with vitamin B12, folic acid and betaine supplementation and showed improvements in her developmental milestones and hypotonia. To the best of our knowledge, this is the first patient with MTHFR deficiency reported with severe early onset scoliosis. Despite the late diagnosis and treatment initiation, she showed favorable short-term neurodevelopmental outcome. This case suggests that homocysteine measurement should be included in the investigations of patients with developmental delay, hypotonia and scoliosis within first year of life prior to organizing genetic investigations. PMID:24726568

  3. 5alpha-reductase: history and clinical importance.

    PubMed

    Marks, Leonard S

    2004-01-01

    The treatment of men with symptomatic benign prostatic hyperplasia (BPH) has shifted dramatically from surgery to drug therapy over the past decade. The revolution in BPH treatment began with the discovery of congenital 5alpha-reductase (5AR) deficiency, leading to the appreciation of 2 different androgenic hormones: testosterone, which mediates overt masculinization in the adult male, and dihydrotestosterone (DHT), which mediates prostatic growth, acne, facial beard, and male pattern baldness. Inhibition of DHT in adults results in prostatic shrinkage and symptomatic relief in many men, without the side effects seen with conventional androgen-deprivation therapy. The 5AR inhibitor drugs (finasteride and the dual inhibitor, dutasteride) are able to ablate the accumulation of intraprostatic DHT, the mechanism most responsible for prostate growth and maintenance. Not only may these drugs relieve symptoms, but they may also alter the natural history of the BPH process. Future indications for the 5ARI drugs could include chemoprevention of prostate cancer, prophylaxis of BPH-related complications, and treatment of BPH-associated hematuria. PMID:16985920

  4. Glyoxalase 1 and glutathione reductase 1 regulate anxiety in mice.

    PubMed

    Hovatta, Iiris; Tennant, Richard S; Helton, Robert; Marr, Robert A; Singer, Oded; Redwine, Jeffrey M; Ellison, Julie A; Schadt, Eric E; Verma, Inder M; Lockhart, David J; Barlow, Carrolee

    2005-12-01

    Anxiety and fear are normal emotional responses to threatening situations. In human anxiety disorders--such as panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, social phobia, specific phobias and generalized anxiety disorder--these responses are exaggerated. The molecular mechanisms involved in the regulation of normal and pathological anxiety are mostly unknown. However, the availability of different inbred strains of mice offers an excellent model system in which to study the genetics of certain behavioural phenotypes. Here we report, using a combination of behavioural analysis of six inbred mouse strains with quantitative gene expression profiling of several brain regions, the identification of 17 genes with expression patterns that correlate with anxiety-like behavioural phenotypes. To determine if two of the genes, glyoxalase 1 and glutathione reductase 1, have a causal role in the genesis of anxiety, we performed genetic manipulation using lentivirus-mediated gene transfer. Local overexpression of these genes in the mouse brain resulted in increased anxiety-like behaviour, while local inhibition of glyoxalase 1 expression by RNA interference decreased the anxiety-like behaviour. Both of these genes are involved in oxidative stress metabolism, linking this pathway with anxiety-related behaviour. PMID:16244648

  5. Catalytic Cycle of Human Glutathione Reductase Near 1 Å Resolution

    SciTech Connect

    Berkholz, Donald S.; Faber, H. Richard; Savvides, Savvas N.; Karplus, P. Andrew

    2008-09-08

    Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione reductase (GR) at nominal resolutions between 1.1 and 0.95 {angstrom} that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity -- a net deviation of 53 deg. across five residues. But this apparent strain is not a factor in catalysis, as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be due to the protein environment but instead due to partial synchrotron reduction of the flavin by the synchrotron beam. Finally, of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds.

  6. Expression analysis of dihydroflavonol 4-reductase genes in Petunia hybrida.

    PubMed

    Chu, Y X; Chen, H R; Wu, A Z; Cai, R; Pan, J S

    2015-01-01

    Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside. PMID:25966276

  7. Correlated Protein Motion Measurements of Dihydrofolate Reductase Crystals

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2014-03-01

    We report the first direct measurements of the long range structural vibrational modes in dihydrofolate reductase (DHFR). DHFR is a universal housekeeping enzyme that catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetra-hydrofolate, with the aid of coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). This crucial enzymatic role as the target for anti-cancer [methotrexate (MTX)], and other clinically useful drugs, has made DHFR a long-standing target of enzymological studies. The terahertz (THz) frequency range (5-100 cm-1), corresponds to global correlated protein motions. In our lab we have developed Crystal Anisotropy Terahertz Microscopy (CATM), which directly measures these large scale intra-molecular protein vibrations, by removing the relaxational background of the solvent and residue side chain librational motions. We demonstrate narrowband features in the anisotropic absorbance for mouse DHFR with the ligand binding of NADPH and MTX single crystals as well as Escherichia coli DHFR with the ligand binding of NADPH and MTX single crystals. This work is supported by NSF grant MRI2 grant DBI2959989.

  8. Structural basis and mechanism of enoyl reductase inhibition by triclosan.

    PubMed

    Stewart, M J; Parikh, S; Xiao, G; Tonge, P J; Kisker, C

    1999-07-23

    The enoyl-acyl carrier protein reductase (ENR) is involved in bacterial fatty acid biosynthesis and is the target of the antibacterial diazaborine compounds and the front-line antituberculosis drug isoniazid. Recent studies suggest that ENR is also the target for the broad-spectrum biocide triclosan. The 1.75 A crystal structure of EnvM, the ENR from Escherichia coli, in complex with triclosan and NADH reveals that triclosan binds specifically to EnvM. These data provide a molecular mechanism for the antibacterial activity of triclosan and substantiate the hypothesis that its activity results from inhibition of a specific cellular target rather than non-specific disruption of the bacterial cell membrane. This has important implications for the emergence of drug-resistant bacteria, since triclosan is an additive in many personal care products such as toothpastes, mouthwashes and soaps. Based on this structure, rational design of triclosan derivatives is possible which might be effective against recently identified triclosan-resistant bacterial strains. PMID:10398587

  9. A second target of benzamide riboside: dihydrofolate reductase.

    PubMed

    Roussel, Breton; Johnson-Farley, Nadine; Kerrigan, John E; Scotto, Kathleen W; Banerjee, Debabrata; Felczak, Krzysztof; Pankiewicz, Krzysztof W; Gounder, Murugesan; Lin, HongXia; Abali, Emine Ercikan; Bertino, Joseph R

    2012-11-01

    Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth. PMID:22954684

  10. Alkyl hydroperoxide reductase: a candidate Helicobacter pylori vaccine.

    PubMed

    O'Riordan, Avril A; Morales, Veronica Athie; Mulligan, Linda; Faheem, Nazia; Windle, Henry J; Kelleher, Dermot P

    2012-06-01

    Helicobacter pylori (H. pylori) is the most important etiological agent of chronic active gastritis, peptic ulcer disease and gastric cancer. The aim of this study was to evaluate the efficacy of alkyl hydroperoxide reductase (AhpC) and mannosylated AhpC (mAhpC) as candidate vaccines in the C57BL/6J mouse model of H. pylori infection. Recombinant AhpC was cloned, over-expressed and purified in an unmodified form and was also engineered to incorporate N and C-terminal mannose residues when expressed in the yeast Pichia pastoris. Mice were immunized systemically and mucosally with AhpC and systemically with mAhpC prior to challenge with H. pylori. Serum IgG responses to AhpC were determined and quantitative culture was used to determine the efficacy of vaccination strategies. Systemic prophylactic immunization with AhpC/alum and mAhpC/alum conferred protection against infection in 55% and 77.3% of mice, respectively. Mucosal immunization with AhpC/cholera toxin did not protect against infection and elicited low levels of serum IgG in comparison with systemic immunization. These data support the use of AhpC as a potential vaccine candidate against H. pylori infection. PMID:22512976

  11. Solvent effects on catalysis by Escherichia coli dihydrofolate reductase.

    PubMed

    Loveridge, E Joel; Tey, Lai-Hock; Allemann, Rudolf K

    2010-01-27

    Hydride transfer catalyzed by dihydrofolate reductase (DHFR) has been described previously within an environmentally coupled model of hydrogen tunneling, where protein motions control binding of substrate and cofactor to generate a tunneling ready conformation and modulate the width of the activation barrier and hence the reaction rate. Changes to the composition of the reaction medium are known to perturb protein motions. We have measured kinetic parameters of the reaction catalyzed by DHFR from Escherichia coli in the presence of various cosolvents and cosolutes and show that the dielectric constant, but not the viscosity, of the reaction medium affects the rate of reaction. Neither the primary kinetic isotope effect on the reaction nor its temperature dependence were affected by changes to the bulk solvent properties. These results are in agreement with our previous report on the effect of solvent composition on catalysis by DHFR from the hyperthermophile Thermotoga maritima. However, the effect of solvent on the temperature dependence of the kinetic isotope effect on hydride transfer catalyzed by E. coli DHFR is difficult to explain within a model, in which long-range motions couple to the chemical step of the reaction, but may indicate the existence of a short-range promoting vibration or the presence of multiple nearly isoenergetic conformational substates of enzymes with similar but distinct catalytic properties. PMID:20047317

  12. Molecular Cloning of Complementary DNA Encoding Maize Nitrite Reductase

    PubMed Central

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

    1988-01-01

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

  13. Chromate reductase activity in Streptomyces sp. MC1.

    PubMed

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

    2010-02-01

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

  14. Crystal structure studies on sulfur oxygenase reductase from Acidianus tengchongensis

    SciTech Connect

    Li Mei; Chen Zhiwei; Zhang Pingfeng; Pan Xiaowei; Jiang Chengying; An Xiaomin; Liu Shuangjiang; Chang Wenrui

    2008-05-09

    Sulfur oxygenase reductase (SOR) simultaneously catalyzes oxidation and reduction of elemental sulfur to produce sulfite, thiosulfate, and sulfide in the presence of molecular oxygen. In this study, crystal structures of wild type and mutants of SOR from Acidianus tengchongensis (SOR-AT) in two different crystal forms were determined and it was observed that 24 identical SOR monomers form a hollow sphere. Within the icosatetramer sphere, the tetramer and trimer channels were proposed as the paths for the substrate and products, respectively. Moreover, a comparison of SOR-AT with SOR-AA (SOR from Acidianus ambivalens) structures showed that significant differences existed at the active site. Firstly, Cys31 is not persulfurated in SOR-AT structures. Secondly, the iron atom is five-coordinated rather than six-coordinated, since one of the water molecules ligated to the iron atom in the SOR-AA structure is lost. Consequently, the binding sites of substrates and a hypothetical catalytic process of SOR were proposed.

  15. 5α-Reductase: History and Clinical Importance

    PubMed Central

    Marks, Leonard S

    2004-01-01

    The treatment of men with symptomatic benign prostatic hyperplasia (BPH) has shifted dramatically from surgery to drug therapy over the past decade. The revolution in BPH treatment began with the discovery of congenital 5α-reductase (5AR) deficiency, leading to the appreciation of 2 different androgenic hormones: testosterone, which mediates overt masculinization in the adult male, and dihydrotestosterone (DHT), which mediates prostatic growth, acne, facial beard, and male pattern baldness. Inhibition of DHT in adults results in prostatic shrinkage and symptomatic relief in many men, without the side effects seen with conventional androgen-deprivation therapy. The 5AR inhibitor drugs (finasteride and the dual inhibitor, dutasteride) are able to ablate the accumulation of intraprostatic DHT, the mechanism most responsible for prostate growth and maintenance. Not only may these drugs relieve symptoms, but they may also alter the natural history of the BPH process. Future indications for the 5ARI drugs could include chemoprevention of prostate cancer, prophylaxis of BPH-related complications, and treatment of BPH-associated hematuria. PMID:16985920

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

    PubMed

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

    2015-10-01

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

  17. Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase

    PubMed Central

    Chatelle, Claire; Kraemer, Stéphanie; Ren, Guoping; Chmura, Hannah; Marechal, Nils; Boyd, Dana; Roggemans, Caroline; Ke, Na; Riggs, Paul; Bardwell, James

    2015-01-01

    Abstract Aims: Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state. Results: We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. Innovation and Conclusions: Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization. Antioxid. Redox Signal. 23, 945–957. PMID:26191605

  18. Imexon enhances gemcitabine cytotoxicity by inhibition of ribonucleotide reductase

    PubMed Central

    Roman, Nicholas O.; Samulitis, Betty K.; Wisner, Lee; Landowski, Terry H.; Dorr, Robert T.

    2010-01-01

    Purpose Gemcitabine (GEM) is currently the standard first line treatment for pancreatic cancer; however, the overall survival of patients with this disease remains poor. Imexon is a pro-oxidant small molecule which produced a high response rate in combination with GEM in a phase I trial in pancreatic cancer. In this study, we investigate the combination of GEM with a novel redox-active agent, imexon, in vitro and in vivo. Methods Median effect analysis was used for in vitro combination cytotoxicity. The effect of imexon on GEM metabolism and uptake into cells and into DNA and effects on ribonucleotide reductase (RNR) were examined in vitro. The pharmacokinetics and antitumor efficacy of the imexon/GEM combination was evaluated in mouse models. Results In three human pancreatic cancer lines, there was additivity for the imexon/GEM combination. There was significantly greater efficacy for the drug combination in Panc-1 xenograft tumors. A pharmacokinetic study in mice showed a near doubling in the AUC of imexon when GEM was co-administered, with no effect of imexon on GEM's pharmacokinetic disposition. In vitro, imexon did not alter GEM's metabolism or uptake into DNA, but significantly inhibited RNR, and this effect was greater when combined with GEM. Conclusions These results suggest that the interaction between imexon and GEM may be due to complimentary inhibition of RNR plus an enhanced exposure to imexon when the GEM is administered in vivo. This combination is currently being tested in a randomized phase II trial in pancreatic cancer. PMID:20339847

  19. Regulation and degradation of HMGCo-A reductase.

    PubMed

    Panda, T; Devi, V Amutha

    2004-12-01

    The enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) controls the biosynthesis of cholesterol. Hypercholesterolemia and atherosclerosis are critical health risk factors. One way of controlling these risk factors is to manipulate regulation as well as degradation of HMGR. At present, a class of compounds called statins, which are HMGR inhibitors, are used for the treatment of hypercholesterolemia. However, statins suffer major setbacks as their use produces more adverse reactions than the desirable one of inhibiting the enzyme. Genetically engineered forms of HMGR are also studied in primitive life forms like bacteria, but detailed investigation of this enzyme in human systems is certainly required. Extensive studies have been made on the regulatory aspects of this enzyme, but no breakthrough is conspicuous in the clinical background to find an alternative treatment for hypercholesterolemia. The immediate need is to find an alternate way of regulating degradation of the enzyme. This review presents the importance of regulation and degradation of the HMGR enzyme in different systems to gain possible insight into alternative schemes for regulating this enzyme and, if these exist, the feasibility of extending them same to studies in mammalian systems. A high degree of similarity exists between mammalian and yeast HMGR. Detailed studies reported on the regulation and degradation of the yeast enzyme also throw more light on the mammalian system, leading to a better understanding of ways of controlling hypercholesterolemia. PMID:15558272

  20. Mutation Update and Review of Severe Methylenetetrahydrofolate Reductase Deficiency.

    PubMed

    Froese, D Sean; Huemer, Martina; Suormala, Terttu; Burda, Patricie; Coelho, David; Guéant, Jean-Louis; Landolt, Markus A; Kožich, Viktor; Fowler, Brian; Baumgartner, Matthias R

    2016-05-01

    Severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is caused by mutations in the MTHFR gene and results in hyperhomocysteinemia and varying severity of disease, ranging from neonatal lethal to adult onset. Including those described here, 109 MTHFR mutations have been reported in 171 families, consisting of 70 missense mutations, 17 that primarily affect splicing, 11 nonsense mutations, seven small deletions, two no-stop mutations, one small duplication, and one large duplication. Only 36% of mutations recur in unrelated families, indicating that most are "private." The most common mutation is c.1530A>G (numbered from NM_005957.4, p.Lys510 = ) causing a splicing defect, found in 13 families; the most common missense mutation is c.1129C>T (p.Arg377Cys) identified in 10 families. To increase disease understanding, we report enzymatic activity, detected mutations, and clinical onset information (early, <1 year; or late, >1 year) for all published patients available, demonstrating that patients with early onset have less residual enzyme activity than those presenting later. We also review animal models, diagnostic approaches, clinical presentations, and treatment options. This is the first large review of mutations in MTHFR, highlighting the wide spectrum of disease-causing mutations. PMID:26872964

  1. Loss of quinone reductase 2 function selectively facilitates learning behaviors.

    PubMed

    Benoit, Charles-Etienne; Bastianetto, Stephane; Brouillette, Jonathan; Tse, YiuChung; Boutin, Jean A; Delagrange, Philippe; Wong, TakPan; Sarret, Philippe; Quirion, Rémi

    2010-09-22

    High levels of reactive oxygen species (ROS) are associated with deficits in learning and memory with age as well as in Alzheimer's disease. Using DNA microarray, we demonstrated the overexpression of quinone reductase 2 (QR2) in the hippocampus in two models of learning deficits, namely the aged memory impaired rats and the scopolamine-induced amnesia model. QR2 is a cytosolic flavoprotein that catalyzes the reduction of its substrate and enhances the production of damaging activated quinone and ROS. QR2-like immunostaining is enriched in cerebral structures associated with learning behaviors, such as the hippocampal formation and the temporofrontal cortex of rat, mouse, and human brains. In cultured rat embryonic hippocampal neurons, selective inhibitors of QR2, namely S26695 and S29434, protected against menadione-induced cell death by reversing its proapoptotic action. S26695 (8 mg/kg) also significantly inhibited scopolamine-induced amnesia. Interestingly, adult QR2 knock-out mice demonstrated enhanced learning abilities in various tasks, including Morris water maze, object recognition, and rotarod performance test. Other behaviors related to anxiety (elevated plus maze), depression (forced swim), and schizophrenia (prepulse inhibition) were not affected in QR2-deficient mice. Together, these data suggest a role for QR2 in cognitive behaviors with QR2 inhibitors possibly representing a novel therapeutic strategy toward the treatment of learning deficits especially observed in the aged brain. PMID:20861374

  2. Direct electrochemistry of nitrate reductase from the fungus Neurospora crassa.

    PubMed

    Kalimuthu, Palraj; Ringel, Phillip; Kruse, Tobias; Bernhardt, Paul V

    2016-09-01

    We report the first direct (unmediated) catalytic electrochemistry of a eukaryotic nitrate reductase (NR). NR from the filamentous fungus Neurospora crassa, is a member of the mononuclear molybdenum enzyme family and contains a Mo, heme and FAD cofactor which are involved in electron transfer from NAD(P)H to the (Mo) active site where reduction of nitrate to nitrite takes place. NR was adsorbed on an edge plane pyrolytic graphite (EPG) working electrode. Non-turnover redox responses were observed in the absence of nitrate from holo NR and three variants lacking the FAD, heme or Mo cofactor. The FAD response is due to dissociated cofactor in all cases. In the presence of nitrate, NR shows a pronounced cathodic catalytic wave with an apparent Michaelis constant (KM) of 39μM (pH7). The catalytic cathodic current increases with temperature from 5 to 35°C and an activation enthalpy of 26kJmol(-1) was determined. In spite of dissociation of the FAD cofactor, catalytically activity is maintained. PMID:27060250

  3. Constitutive nitrate reductase expression and inhibition in winged bean

    SciTech Connect

    Wu, Shenchuan; Harper, J.E. )

    1990-05-01

    It was found that NO{sub 3}{sup {minus}} had no effect on winged bean nitrate reductase activity (NRA). Similar NRA was expressed in plants grown on NO{sub 3}{sup {minus}}, urea, NH{sub 4}{sup +}, and nil N. This indicated that the primary NR expressed in winged bean was constitutive, rather than substrate-inducible. Maximum NRA in winged bean was obtained in the light. KClO{sub 3} was capable of inhibiting NRA of leaves if added to the root growth medium or to the NR assay medium, indicating possible competition with NO{sub 3}{sup {minus}} at the reduction site. While it has previously been shown that either cycloheximide alone, or both cycloheximide and chloramphenicol impair the synthesis of NR protein, our data unexpectedly demonstrated that cycloheximide had little effect on NRA, whereas chloramphenicol greatly inhibited the expression of NRA in winged bean. One interpretation is that chloroplasts may influence the activity and/or synthesis of constitutive NR proteins.

  4. Fasciola gigantica thioredoxin glutathione reductase: Biochemical properties and structural modeling.

    PubMed

    Gupta, Ankita; Kesherwani, Manish; Velmurugan, Devadasan; Tripathi, Timir

    2016-08-01

    Platyhelminth thioredoxin glutathione reductase (TGR) is a multifunctional enzyme that crosstalk between the conventional thioredoxin (Trx) and glutathione (GSH) system. It has been validated as a potential drug target in blood flukes. In the present study, we have performed a biochemical study on Fasciola gigantica TGR with substrates DTNB and GSSG. The Michaelis constant (Km) with DTNB was found to be 4.34±0.12μM while it was 61.15±1.50μM with GSSG. The kinetic results were compared with the TGR activities of other helminths. FgTGR showed typical hysteretic behavior with GSSG as other TGRs. We also described a homology-based structure of FgTGR. The cofactors (NADPH and FAD) and substrates (GSSG and DTNB) were docked, and two possible binding sites for substrates were identified in a single chain. The substrates were found to bind more favorably in the second site of TrxR domains. We also presented the first report on binding interaction of DTNB with a TGR. DTNB forms H-bond with His204 and Arg450 of chain A, Sec597, and Gly598 from chain B, salt-bridge with Lys124, and numerous other hydrophobic interactions. Helminth TGR represents an important enzyme in the redox and antioxidant system; hence, its inhibition can be used as an effective strategy against liver flukes. PMID:27112978

  5. Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase

    PubMed Central

    Reddish, Michael J.; Vaughn, Morgan B.; Fu, Rong; Dyer, R. Brian

    2016-01-01

    Enzymes are known to change among several conformational states during turnover. The role of such dynamic structural changes in catalysis is not fully understood. The influence of dynamics in catalysis can be inferred, but not proven, by comparison of equilibrium structures of protein variants and protein–ligand complexes. A more direct way to establish connections between protein dynamics and the catalytic cycle is to probe the kinetics of specific protein motions in comparison to progress along the reaction coordinate. We have examined the enzyme model system dihydrofolate reductase (DHFR) from Escherichia coli with tryptophan fluorescence-probed temperature-jump spectroscopy. We aimed to observe the kinetics of the ligand binding and ligand-induced conformational changes of three DHFR complexes to establish the relationship among these catalytic steps. Surprisingly, in all three complexes, the observed kinetics do not match a simple sequential two-step process. Through analysis of the relationship between ligand concentration and observed rate, we conclude that the observed kinetics correspond to the ligand binding step of the reaction and a noncoupled enzyme conformational change. The kinetics of the conformational change vary with the ligand's identity and presence but do not appear to be directly related to progress along the reaction coordinate. These results emphasize the need for kinetic studies of DHFR with highly specific spectroscopic probes to determine which dynamic events are coupled to the catalytic cycle and which are not. PMID:26901612

  6. Aerobic degradation of 2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase

    SciTech Connect

    French, C.E.; Bruce, N.C.; Nicklin, S.

    1998-08-01

    Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.

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

    SciTech Connect

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

    2006-12-01

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

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

    PubMed Central

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

    1995-01-01

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

  9. Modeling the effects of a speed limit reduction on traffic-related elemental carbon (EC) concentrations and population exposure to EC

    NASA Astrophysics Data System (ADS)

    Lefebvre, W.; Fierens, F.; Trimpeneers, E.; Janssen, S.; Van de Vel, K.; Deutsch, F.; Viaene, P.; Vankerkom, J.; Dumont, G.; Vanpoucke, C.; Mensink, C.; Peelaerts, W.; Vliegen, J.

    2011-01-01

    Despite the scarce effect of speed limit reduction measures on total mass PM 10 and PM 2.5 concentrations, it is shown that the effect on the probably more hazardous component elemental carbon (EC) is more important which means that, from the viewpoint of health benefit, speed reductions during smog episodes may well be justified. Especially in the very dense highway network in Flanders with a 60% share of diesel cars (the highest in Europe) a speed limit reduction from 120 to 90 km/h during winter smog episodes on selected sections of Flemish highways leads to a significant decrease of the EC concentrations near those highways. Key findings are that the decrease in EC depends on the distance from the highways. In the direct vicinity of the highways, a decrease compared to the base-line scenario where no speed limit changes were implemented of up to 30% of the EC concentrations is modeled. Within a distance of 1500 m of the concerned highway sections there is an average decrease in EC of 0.18 μg m -3 affecting about 1 million people living in these areas. When the speed limit reduction measure is in force, the EC exposure of about 300,000 people decreases by at least 5% and 7500 people experience a decrease of 15% or more.

  10. Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency

    SciTech Connect

    Goyette, P.; Frosst, P.; Rosenblatt, D.S.; Rozen. R.

    1995-05-01

    5-Methyltetrahydrofolate, the major form of folate in plasma, is a carbon donor for the remethylation of homocysteine to methionine. This form of folate is generated from 5,10-methylenetetrahydrofolate through the action of 5,10-methylenetetrahydrofolate reductase (MTHFR), a cytosolic flavoprotein. Patients with an autosomal recessive severe deficiency of MTHFR have homocystinuria and a wide range of neurological and vascular disturbances. We have recently described the isolation of a cDNA for MTHFR and the identification of two mutations in patients with severe MTHFR deficiency. We report here the characterization of seven novel mutations in this gene: six missense mutations and a 5{prime} splice-site defect that activates a cryptic splice in the coding sequence. We also present a preliminary analysis of the relationship between genotype and phenotype for all nine mutations identified thus far in this gene. A nonsense mutation and two missense mutations (proline to leucine and threonine to methionine) in the homozygous state are associated with extremely low activity (0%-3%) and onset of symptoms within the 1st year of age. Other missense mutations (arginine to cysteine and arginine to glutamine) are associated with higher enzyme activity and later onset of symptoms. 19 refs., 4 figs., 2 tabs.

  11. Evidence for an Inactivating System of Nitrate Reductase in Hordeum vulgare L. during Darkness That Requires Protein Synthesis 1

    PubMed Central

    Travis, R. L.; Jordan, W. R.; Huffaker, R. C.

    1969-01-01

    The disappearance of nitrate reductase activity in leaves of Hordeum vulgare L. during darkness was inhibited by cycloheximide, actinomycin D, and low temperature. Thus, protein synthesis was probably required for the disappearance of nitrate reductase in the dark. Since chloramphenicol did not affect the rate of loss of activity, the degradation or inactivation apparently required protein synthesis by the cytoplasmic ribosomal system. Consistent with this observation, nitrate reductase is also reportedly located in the cytoplasm. Thus, the amount of nitrate reductase activity present in leaves of barley may be controlled by a balance between activating and inactivating systems. PMID:16657182

  12. SPME-GC-EC of chlorinated paraffins in water

    SciTech Connect

    Webster, G.R.B.; Sarna, L.P.; Thomas, D.A.

    1995-12-31

    Contamination of aquatic ecosystems with chlorinated organic contaminants is an increasing toxicological problem. Chlorinated paraffins (CPs) are a class of compounds which are used as fire retardants in paints and as high pressure lubricants and are classified as Priority Toxic Substances under the Canadian Environmental Protection Act (CEPA). As the largest group of high molecular weight chlorinated hydrocarbons produced in Western Europe and North America, CPs have recently come under increased regulatory scrutiny in Canada, the USA, and Europe, because of concerns about their environmental persistence, possible adverse effects on terrestrial and aquatic organisms, and potential carcinogenicity to humans. In both the monitoring of such contamination and the determination of the success of remediation methods, straightforward and inexpensive analytical methodology increases the ease of environmental assessment and facilitates regulatory enforcement. CPs have been considered to be incapable of being analyzed by GC. The authors report the solid phase microextraction (SPME) based analysis of CPs by GC using electron capture (EC) detection. Both synthetic C{sub 10} standards and fractionated commercially derived material have been analyzed by this method in water.

  13. Assessment of the ITER EC Upper Launcher Performance

    NASA Astrophysics Data System (ADS)

    Figini, Lorenzo; Farina, Daniela; Poli, Emanuele; Sauter, Olivier; Bruschi, Alessandro; Goodman, Timothy; Moro, Alessandro; Platania, Paola; Sozzi, Carlo; Cavinato, Mario; Saibene, Gabriella; Henderson, Mark

    2015-03-01

    The 24 MW ITER Electron Cyclotron (EC) Heating and Current Drive (H&CD) system, operating at 170 GHz, consists of one Equatorial (EL) and four Upper Launchers (UL). The main task of the UL will be the control of Magneto-Hydrodynamic (MHD) activity such as Neoclassical Tearing Modes (NTMs) at the q=3/2 and q=2 surfaces, but it will also be needed for current profile tailoring in advanced scenarios and to assist plasma break-down and L- to H-mode transition. Moreover, it is required to be e.ective both when ITER will operate at nominal and reduced magnetic field magnitude. Here the performance of the UL is assessed through the study of the full temporal evolution of di.erent scenarios, including the reference ITER 15MA H-mode plasma, a half-field case at 2.65T, and a steady state scenario. The ECCD efficiency has been evaluated for a wide range of injection angles, deriving the optimal angles and the power required for NTMs stabilization, as well as the steering range necessary to reach the rational surfaces during all the phases of the discharge. The steering sensitivity to shifts of the target or aiming errors has been estimated too. The result is an assessment of the UL design requirements to achieve the desired functionalities, which, together with the engineering limits, will be used to drive the optimization and finalization of the UL design.

  14. 76 FR 27956 - Airworthiness Directives; Eurocopter Deutschland Model EC135 Helicopters

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-13

    ... Deutschland Model EC135 Helicopters AGENCY: Federal Aviation Administration (FAA), DOT. ACTION: Notice of... Deutschland (ECD) Model EC135 helicopters. This proposed AD results from a mandatory continuing airworthiness...: You may send comments by any of the following methods: Federal eRulemaking Portal: Go to...

  15. Multidomain sumoylation of the ecdysone receptor (EcR) from Drosophila melanogaster.

    PubMed

    Seliga, Justyna; Bielska, Katarzyna; Wieczorek, Elżbieta; Orłowski, Marek; Niedenthal, Rainer; Ożyhar, Andrzej

    2013-11-01

    The 20-hydroxyecdysone receptor (EcR) is a transcription factor belonging to the nuclear receptor superfamily. Together with the ultraspiracle nuclear receptor (Usp) it coordinates critical biological processes in insects such as development and reproduction. EcR and its ligands are used in commercially available ecdysone-inducible expression systems and are considered to be artificial gene switches with potential therapeutic applications. However, the regulation of EcR action is still unclear, especially in mammals and as far as posttranslational modifications are concerned. Up until now, there has been no study on EcR sumoylation. Using bioinformatic predictors, a Ubc9 fusion-directed sumoylation system and mutagenesis experiments, we present EcR as a new target of SUMO1 and SUMO3 modification. Our research revealed that EcR undergoes isoform-specific multisumoylation. The pattern of modification remains unchanged in the presence of the ligand and the dimerization partner. The SUMO acceptor sites are located in the DNA-binding domain and the ligand-binding domain that both exhibit structural plasticity. We also demonstrated the existence of a sumoylation site in the F region and EcRA-A/B region, both revealing characteristics of intrinsically disordered regions. The consequences of modification and the resulting impact on conformation and function may be especially crucial for the disordered sequences in these two areas. The isoform-specificity of sumoylation may explain the differences in the transcriptional activity of EcR isoforms. PMID:23727127

  16. 40 CFR Table 2 of Subpart Ec to... - Toxic Equivalency Factors

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 6 2010-07-01 2010-07-01 false Toxic Equivalency Factors 2 Table 2 of Subpart Ec to Part 60 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS... Ec to Part 60—Toxic Equivalency Factors Dioxin/furan congener Toxic equivalency factor...

  17. 40 CFR Table 2 of Subpart Ec to... - Toxic Equivalency Factors

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 6 2011-07-01 2011-07-01 false Toxic Equivalency Factors 2 Table 2 of Subpart Ec to Part 60 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS... Ec to Part 60—Toxic Equivalency Factors Dioxin/furan congener Toxic equivalency factor...

  18. Program Unit Funding: A Handbook for ECS Operators in the 2002/2003 School Year.

    ERIC Educational Resources Information Center

    Alberta Learning, Edmonton. Special Education Branch.

    This handbook is written specifically for Early Childhood Services (ECS) operators in Alberta, Canada, applying for Program Unit Funding for students with severe disabilities. It is also designed to enhance the understanding of how assistance is provided to ECS children with severe disabilities by teachers, special needs assistants, parents, and…

  19. Governors' Top Education Issues: 2015 State of the State Addresses. ECS Education Trends

    ERIC Educational Resources Information Center

    Aragon, Stephanie; Rowland, Julie

    2015-01-01

    Education Commission of the States (ECS) strives to keep its constituency apprised of education policy trends across the states. To provide a comprehensive overview of educational priorities outlined by governors, ECS summarized the education proposals and accomplishments detailed in every 2015 State of the State address delivered to date. Each…

  20. EC Research Programmes--Information and Influence (or, How to Lobby in Brussels).

    ERIC Educational Resources Information Center

    Bode, Christian

    1991-01-01

    Research projects within the European Community (EC) have used different strategies and instruments for information dissemination and lobbying on national and international levels. The scientific community must have stronger autonomous representation in Brussels as an equal partner of the EC bureaucracy, similar to that in some member states.…