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Sample records for atp-induced ribonucleotide reductase

  1. Automated mass action model space generation and analysis methods for two-reactant combinatorially complex equilibriums: An analysis of ATP-induced ribonucleotide reductase R1 hexamerization data

    Directory of Open Access Journals (Sweden)

    Radivoyevitch Tomas

    2009-12-01

    Full Text Available Abstract Background Ribonucleotide reductase is the main control point of dNTP production. It has two subunits, R1, and R2 or p53R2. R1 has 5 possible catalytic site states (empty or filled with 1 of 4 NDPs, 5 possible s-site states (empty or filled with ATP, dATP, dTTP or dGTP, 3 possible a-site states (empty or filled with ATP or dATP, perhaps two possible h-site states (empty or filled with ATP, and all of this is folded into an R1 monomer-dimer-tetramer-hexamer equilibrium where R1 j-mers can be bound by variable numbers of R2 or p53R2 dimers. Trillions of RNR complexes are possible as a result. The problem is to determine which are needed in models to explain available data. This problem is intractable for 10 reactants, but it can be solved for 2 and is here for R1 and ATP. Results Thousands of ATP-induced R1 hexamerization models with up to three (s, a and h ATP binding sites per R1 subunit were automatically generated via hypotheses that complete dissociation constants are infinite and/or that binary dissociation constants are equal. To limit the model space size, it was assumed that s-sites are always filled in oligomers and never filled in monomers, and to interpret model terms it was assumed that a-sites fill before h-sites. The models were fitted to published dynamic light scattering data. As the lowest Akaike Information Criterion (AIC of the 3-parameter models was greater than the lowest of the 2-parameter models, only models with up to 3 parameters were fitted. Models with sums of squared errors less than twice the minimum were then partitioned into two groups: those that contained no occupied h-site terms (508 models and those that contained at least one (1580 models. Normalized AIC densities of these two groups of models differed significantly in favor of models that did not include an h-site term (Kolmogorov-Smirnov p -15; consistent with this, 28 of the top 30 models (ranked by AICs did not include an h-site term and 28

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

    Science.gov (United States)

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

    2016-06-01

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

  3. Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

    DEFF Research Database (Denmark)

    Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie; Fleck, Oliver; Watson, Adam T; Poitelea, Marius; O'Shea, Charlotte; Chahwan, Charly; Holmberg, Christian; Kragelund, Birthe B; Nielsen, Olaf; Osborne, Mark; Carr, Antony M; Liu, Cong

    2010-01-01

    The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and...

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

    Science.gov (United States)

    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

  5. Aspects of Ribonucleotide Reductase Regulation and Genome Stability

    DEFF Research Database (Denmark)

    Nielsen, Helena Berner Nedergaard

    In all living cells, synthesis of the DNA building blocks, deoxyribonucleoside triphosphates (dNTPs), is tightly regulated to ensure a precise DNA replication to maintain genomic stability. Ribonucleotide reductase (RNR) is the enzyme responsible for reducing ribonucleotides to their deoxy forms....... RNR consists of two subunits: R1 and R2, both of which are essential. The activity of RNR is strictly regulated to control the dNTP pool, both by allosteric feedback control and transcriptional and translational controls. Four inhibitory proteins of RNR have been identified in yeast: Spd1 in fission...... yeast, and Sml1, Hug1, and Dif1 in budding yeast. An elevated, as well as a reduced dNTP pool is shown to lead to an increase in spontaneous mutation rates, hence regulation of RNR is very important in order to maintain genomic stability. No human inhibitory proteins have yet been identified to regulate...

  6. The effect of copper and gallium compounds on ribonucleotide reductase

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    OpenAIRE

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

    2012-01-01

    The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRN...

  8. Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization

    Energy Technology Data Exchange (ETDEWEB)

    Fairman, James Wesley; Wijerathna, Sanath Ranjan; Ahmad, Md Faiz; Xu, Hai; Nakano, Ryo; Jha, Shalini; Prendergast, Jay; Welin, R. Martin; Flodin, Susanne; Roos, Annette; Nordlund, Pär; Li, Zongli; Walz, Thomas; Dealwis, Chris Godfrey (Case Western); (Harvard-Med); (Karolinska); (Tennessee-K)

    2011-07-25

    Ribonucleotide reductase (RR) is an {alpha}{sub n}{beta}{sub n} (RR1-RR2) complex that maintains balanced dNTP pools by reducing NDPs to dNDPs. RR1 is the catalytic subunit, and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1. Here, we report the first X-ray structures of human RR1 bound to TTP alone, dATP alone, TTP-GDP, TTP-ATP, and TTP-dATP. These structures provide insights into regulation of RR by ATP or dATP. At physiological dATP concentrations, RR1 forms inactive hexamers. We determined the first X-ray structure of the RR1-dATP hexamer and used single-particle electron microscopy to visualize the {alpha}{sub 6}-{beta}{beta}'-dATP holocomplex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data indicate a mechanism for regulating RR activity by dATP-induced oligomerization.

  9. Ribonucleotide reductase and cancer: biological mechanisms and targeted therapies.

    Science.gov (United States)

    Aye, Y; Li, M; Long, M J C; Weiss, R S

    2015-04-16

    Accurate DNA replication and repair is essential for proper development, growth and tumor-free survival in all multicellular organisms. A key requirement for the maintenance of genomic integrity is the availability of adequate and balanced pools of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Notably, dNTP pool alterations lead to genomic instability and have been linked to multiple human diseases, including mitochondrial disorders, susceptibility to viral infection and cancer. In this review, we discuss how a key regulator of dNTP biosynthesis in mammals, the enzyme ribonucleotide reductase (RNR), impacts cancer susceptibility and serves as a target for anti-cancer therapies. Because RNR-regulated dNTP production can influence DNA replication fidelity while also supporting genome-protecting DNA repair, RNR has complex and stage-specific roles in carcinogenesis. Nevertheless, cancer cells are dependent on RNR for de novo dNTP biosynthesis. Therefore, elevated RNR expression is a characteristic of many cancers, and an array of mechanistically distinct RNR inhibitors serve as effective agents for cancer treatment. The dNTP metabolism machinery, including RNR, has been exploited for therapeutic benefit for decades and remains an important target for cancer drug development. PMID:24909171

  10. Methyl-hydroxylamine as an efficacious antibacterial agent that targets the ribonucleotide reductase enzyme

    OpenAIRE

    Julián Gómez, Esther; Baelo, Aida; Gavaldà Santapau, Juan; Torrents Serra, Eduard

    2015-01-01

    The emergence of multidrug-resistant bacteria has encouraged vigorous efforts to develop antimicrobial agents with new mechanisms of action. Ribonucleotide reductase (RNR) is a key enzyme in DNA replication that acts by converting ribonucleotides into the corresponding deoxyribonucleotides, which are the building blocks of DNA replication and repair. RNR has been extensively studied as an ideal target for DNA inhibition, and several drugs that are already available on the market are used for ...

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

    Science.gov (United States)

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

    2012-09-01

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

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

    Science.gov (United States)

    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

  13. Loss of the tyrosyl radical in mouse ribonucleotide reductase by (-)-epicatechin

    International Nuclear Information System (INIS)

    The flavonoid (-)-epicatechin was previously demonstrated to interfere with tyrosine nitration by peroxynitrite [Biochem. Biophys. Res. Commun. 285 (2001) 782]. This effect was hypothesized to be based upon an interaction of epicatechin with a transiently generated tyrosyl radical. In the present study, using electron paramagnetic resonance, we demonstrate that (-)-epicatechin is capable of destabilizing the tyrosyl radical of the mouse ribonucleotide reductase R2 component. First-order rate constants for the disappearance of tyrosyl radical signals were 1 x 10-4 and 2 x 10-4 s-1for epicatechin and hydroxyurea, a well-known tyrosyl radical scavenger, respectively. In keeping with scavenging the ribonucleotide reductase tyrosyl radical, cellular production of deoxyribonucleotides and DNA synthesis were impaired by (-)-epicatechin in normal human keratinocytes and in human squamous carcinoma cells

  14. A Fluorimetric Readout Reporting the Kinetics of Nucleotide-Induced Human Ribonucleotide Reductase Oligomerization

    OpenAIRE

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

    2014-01-01

    Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typicall...

  15. Single-Cell Analysis of Ribonucleotide Reductase Transcriptional and Translational Response to DNA Damage

    OpenAIRE

    Mazumder, Aprotim; Tummler, Katja; Bathe, Mark; Samson, Leona D.

    2013-01-01

    The ribonucleotide reductase (RNR) enzyme catalyzes an essential step in the production of deoxyribonucleotide triphosphates (dNTPs) in cells. Bulk biochemical measurements in synchronized Saccharomyces cerevisiae cells suggest that RNR mRNA production is maximal in late G1 and S phases; however, damaged DNA induces RNR transcription throughout the cell cycle. But such en masse measurements reveal neither cell-to-cell heterogeneity in responses nor direct correlations between transcript and p...

  16. GENERATION OF THE GLYCYL RADICAL OF THE ANAEROBIC ESCHERICHIA-COLI RIBONUCLEOTIDE REDUCTASE REQUIRES A SPECIFIC ACTIVATING ENZYME

    NARCIS (Netherlands)

    SUN, XY; ELIASSON, R; PONTIS, E; ANDERSSON, J; BUIST, G; SJOBERG, BM; REICHARD, P

    1995-01-01

    The anaerobic ribonucleotide reductase from Escherichia coli contains a glycyl radical as part of its polypeptide structure. The radical is generated by an enzyme system present in E. coli. The reductase is coded for by the nrdD gene located at 96 min. Immediately downstream, we now find an open rea

  17. Coenzyme B12 Controls Transcription of the Streptomyces Class Ia Ribonucleotide Reductase nrdABS Operon via a Riboswitch Mechanism†

    OpenAIRE

    Borovok, Ilya; Gorovitz, Batia; Schreiber, Rachel; Aharonowitz, Yair; Cohen, Gerald

    2006-01-01

    Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomycetes contain genes coding for two RNRs. The class Ia RNR is oxygen dependent, and the class II RNR is oxygen independent and requires coenzyme B12. Either RNR is sufficient for vegetative growth. We show here that the Streptomyces coelicolor M145 nrdABS genes encoding the class Ia RNR are regulated by coenzyme B12. The 5′-untrans...

  18. Tangled up in knots – Structures of inactivated forms of E. coli class Ia Ribonucleotide Reductase

    OpenAIRE

    Zimanyi, Christina M.; Ando, Nozomi; Brignole, Edward J; Asturias, Francisco J; Stubbe, JoAnne; Drennan, Catherine L.

    2012-01-01

    Successful targets for anti-cancer drugs such as clofarabine and gemcitabine, ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4β4 rings. Here, we present the first X-ray structure of gemcitabine-inhibited E. coli RNR and show that the previously described α4β4 rings can interlock to form an unprecedented (α4β4)2 megacomplex. This complex is also see...

  19. Caracemide, a site-specific irreversible inhibitor of protein R1 of Escherichia coli ribonucleotide reductase

    DEFF Research Database (Denmark)

    Larsen, I. K.; Cornett, Claus; Karlsson, M.; Sahlin, M.; Sjoberg, B. M.

    1992-01-01

    The anticancer drug caracemide, N-acetyl-N,O-di(methylcarbamoyl)hydroxylamine, and one of its degradation products, N-acetyl-O-methylcarbamoyl-hydroxylamine, were found to inhibit the enzyme ribonucleotide reductase of Escherichia coli by specific interaction with its larger component protein R1...... caracemide inactivation. These results indicate that caracemide inactivates R1 by covalent modification at the substrate-binding site. By analogy with the known interaction between caracemide and acetylcholinesterase or choline acetyltransferase, we propose that the modification of R1 occurs at an activated...... cysteine or serine residue in the active site of the enzyme....

  20. Targeting the Large Subunit of Human Ribonucleotide Reductase for Cancer Chemotherapy

    Directory of Open Access Journals (Sweden)

    Prem Singh Kaushal

    2011-10-01

    Full Text Available Ribonucleotide reductase (RR is a crucial enzyme in de novo DNA synthesis, where it catalyses the rate determining step of dNTP synthesis. RRs consist of a large subunit called RR1 (α, that contains two allosteric sites and one catalytic site, and a small subunit called RR2 (β, which houses a tyrosyl free radical essential for initiating catalysis. The active form of mammalian RR is an anbm hetero oligomer. RR inhibitors are cytotoxic to proliferating cancer cells. In this brief review we will discuss the three classes of RR, the catalytic mechanism of RR, the regulation of the dNTP pool, the substrate selection, the allosteric activation, inactivation by ATP and dATP, and the nucleoside drugs that target RR. We will also discuss possible strategies for developing a new class of drugs that disrupts the RR assembly.

  1. Large and small subunits of the Aujeszky's disease virus ribonucleotide reductase: nucleotide sequence and putative structure.

    Science.gov (United States)

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

    1994-09-13

    We determined the entire DNA sequence of two adjacent open reading frames of Aujeszky's disease virus encoding ribonucleotide reductase genes with the intergenic sequence of 9 bp. From the sequence analysis we deduce that ORFs encode large and small subunits, with sizes of 835 and 303 amino acids, respectively. Amino acid sequence comparison of ADV RR2 with that of equine herpesvirus type 1, bovine herpesvirus type 1, HSV-1 and varicella zoster virus revealed that 48% of amino acids represent clusters of residues conserved in all compared sequences. In the N-terminal part ADV RR1 shows low homology to the RR1 of other herpesviruses. Rest of the RR1 protein contains highly conserved amino acid sequences divided by blocks of low homology. PMID:8086454

  2. dNTP deficiency induced by HU via inhibiting ribonucleotide reductase affects neural tube development

    International Nuclear Information System (INIS)

    Highlights: • Murine NTDs were successfully induced by means of hydroxyurea (HU). • The impairment of dNTP was induced via inhibition of ribonucleotide reductase. • dNTP deficiency induced by HU caused defective DNA synthesis and repair. • Abnormal apoptosis and proliferation induced by HU affected neural tube development. - Abstract: Exposure to environmental toxic chemicals in utero during the neural tube development period can cause developmental disorders. To evaluate the disruption of neural tube development programming, the murine neural tube defects (NTDs) model was induced by interrupting folate metabolism using methotrexate in our previous study. The present study aimed to examine the effects of dNTP deficiency induced by hydroxyurea (HU), a specific ribonucleotide reductase (RNR) inhibitor, during murine neural tube development. Pregnant C57BL/6J mice were intraperitoneally injected with various doses of HU on gestation day (GD) 7.5, and the embryos were checked on GD 11.5. RNR activity and deoxynucleoside triphosphate (dNTP) levels were measured in the optimal dose. Additionally, DNA damage was examined by comet analysis and terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) assay. Cellular behaviors in NTDs embryos were evaluated with phosphorylation of histone H3 (PH-3) and caspase-3 using immunohistochemistry and western blot analysis. The results showed that NTDs were observed mostly with HU treatment at an optimal dose of 225 mg/kg b/w. RNR activity was inhibited and dNTP levels were decreased in HU-treated embryos with NTDs. Additionally, increased DNA damage, decreased proliferation, and increased caspase-3 were significant in NTDs embryos compared to the controls. Results indicated that HU induced murine NTDs model by disturbing dNTP metabolism and further led to the abnormal cell balance between proliferation and apoptosis

  3. Identification of a hydroxide ligand at the iron center of ribonucleotide reductase by resonance Raman spectroscopy

    International Nuclear Information System (INIS)

    The resonance Raman spectrum of protein B2 of ribonucleotide reductase from Escherichia coli shows several features related to its oxo-bridged binuclear iron center. A peak at 492 cm-1 is assigned to the symmetric stretch of the Fe-O-Fe moiety on the basis of its 13-cm-1 shift to lower energy upon 18O substitution. The 18O species shows an additional peak at 731 cm-1, which is a good candidate for the asymmetric stretch of the Fe-O-Fe moiety. Its exact location in the 16O species is obscured by the presence of a protein tryptophan vibration at 758 cm-1. A third resonance-enhanced peak at 598 cm-1 is identified as an Fe-OH vibration on the basis of its 24-cm-1 shift to lower energy in H218O, its 2-cm-1 shift to lower energy in D2O, and its pH-dependent intensity. A hydrogen-bonded μ-oxo bridge similar to that in hemerythrin is suggested by the unusually low frequency for the Fe-O-Fe symmetric stretch and the 3-cm-1 shift to higher energy of nu/sub s/ (Fe-O-Fe), an Fe-O-Fe angle of 1380 can be calculated. This small angle suggests that the iron center consists of a tribridged core as in hemerythrin. A model for the binuclear iron center of ribonucleotide reductase is presented in which the hydroxide ligand sites provide an explanation for the half-of-sites reactivity of the enzyme

  4. Peroxo-Type Intermediates in Class I Ribonucleotide Reductase and Related Binuclear Non-Heme Iron Enzymes

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta; Bell, Caleb B.; Clay, MIchael D.;

    2009-01-01

    We have performed a systematic study of chemically possible peroxo-type intermediates occurring in the non-heme di-iron enzyme class la ribonucleotide reductase, using spectroscopically calibrated computational chemistry. Density functional computations of equilibrium structures, Fe-O and O...... carboxylate conformations occurring during the O-2 reaction of this class of non-heme iron enzymes. Our procedure identifies and characterizes various possible candidates for peroxo intermediates experimentally observed along the ribonucleotide reductase dioxygen activation reaction. The study explores how...... a proton to a terminal carboxylate. ligand in the site which increases the electron affinity and triggers electron transfer to form X. Both pathways provide a mechanism for the activation of peroxy intermediates in binuclear non-heme iron enzymes for reactivity. The studies further show that water...

  5. Methyl-hydroxylamine as an efficacious antibacterial agent that targets the ribonucleotide reductase enzyme.

    Directory of Open Access Journals (Sweden)

    Esther Julián

    Full Text Available The emergence of multidrug-resistant bacteria has encouraged vigorous efforts to develop antimicrobial agents with new mechanisms of action. Ribonucleotide reductase (RNR is a key enzyme in DNA replication that acts by converting ribonucleotides into the corresponding deoxyribonucleotides, which are the building blocks of DNA replication and repair. RNR has been extensively studied as an ideal target for DNA inhibition, and several drugs that are already available on the market are used for anticancer and antiviral activity. However, the high toxicity of these current drugs to eukaryotic cells does not permit their use as antibacterial agents. Here, we present a radical scavenger compound that inhibited bacterial RNR, and the compound's activity as an antibacterial agent together with its toxicity in eukaryotic cells were evaluated. First, the efficacy of N-methyl-hydroxylamine (M-HA in inhibiting the growth of different Gram-positive and Gram-negative bacteria was demonstrated, and no effect on eukaryotic cells was observed. M-HA showed remarkable efficacy against Mycobacterium bovis BCG and Pseudomonas aeruginosa. Thus, given the M-HA activity against these two bacteria, our results showed that M-HA has intracellular antimycobacterial activity against BCG-infected macrophages, and it is efficacious in partially disassembling and inhibiting the further formation of P. aeruginosa biofilms. Furthermore, M-HA and ciprofloxacin showed a synergistic effect that caused a massive reduction in a P. aeruginosa biofilm. Overall, our results suggest the vast potential of M-HA as an antibacterial agent, which acts by specifically targeting a bacterial RNR enzyme.

  6. The Dimanganese(II) Site of Bacillus subtilis Class Ib Ribonucleotide Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Boal, Amie K.; Cotruvo, Jr., Joseph A.; Stubbe, JoAnne; Rosenzweig, Amy C. (MIT); (NWU)

    2014-10-02

    Class Ib ribonucleotide reductases (RNRs) use a dimanganese-tyrosyl radical cofactor, Mn{sub 2}{sup III}-Y{sm_bullet}, in their homodimeric NrdF ({beta}2) subunit to initiate reduction of ribonucleotides to deoxyribonucleotides. The structure of the Mn{sub 2}{sup II} form of NrdF is an important component in understanding O{sub 2}-mediated formation of the active metallocofactor, a subject of much interest because a unique flavodoxin, NrdI, is required for cofactor assembly. Biochemical studies and sequence alignments suggest that NrdF and NrdI proteins diverge into three phylogenetically distinct groups. The only crystal structure to date of a NrdF with a fully ordered and occupied dimanganese site is that of Escherichia coli Mn{sub 2}{sup II}-NrdF, prototypical of the enzymes from actinobacteria and proteobacteria. Here we report the 1.9 {angstrom} resolution crystal structure of Bacillus subtilis Mn{sub 2}{sup II}-NrdF, representative of the enzymes from a second group, from Bacillus and Staphylococcus. The structures of the metal clusters in the {beta}2 dimer are distinct from those observed in E. coli Mn{sub 2}{sup II}-NrdF. These differences illustrate the key role that solvent molecules and protein residues in the second coordination sphere of the Mn{sub 2}{sup II} cluster play in determining conformations of carboxylate residues at the metal sites and demonstrate that diverse coordination geometries are capable of serving as starting points for Mn{sub 2}{sup III}-Y{sm_bullet} cofactor assembly in class Ib RNRs.

  7. Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms

    OpenAIRE

    Liu, Cong; Powell, Kelly A.; Mundt, Kirsten; Wu, LeJung; Carr, Antony M.; Caspari, Thomas

    2003-01-01

    The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two sepa...

  8. Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): A combined experimental and theoretical study

    OpenAIRE

    Popović-Bijelić, Ana; Kowol, Christian R.; Lind, Maria E S; Luo, Jinghui; Himo, Fahmi; Enyedy, Éva A.; Arion, Vladimir B.; Gräslund, Astrid

    2011-01-01

    Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is currently the most promising chemotherapeutic compound among the class of α-N-heterocyclic thiosemicarbazones. Here we report further insights into the mechanism(s) of anticancer drug activity and inhibition of mouse ribonucleotide reductase (RNR) by Triapine. In addition to the metal-free ligand, its iron(III), gallium(III), zinc(II) and copper (II) complexes were studied, aiming to correlate their cytotoxic activities wi...

  9. RNA-dependent inhibition of ribonucleotide reductase is a major pathway for 5-azacytidine activity in acute myeloid leukemia

    OpenAIRE

    Aimiuwu, Josephine; Wang, Hongyan; Chen, Ping; Xie, Zhiliang; Wang, Jiang, 1959-; Liu, Shujun; Klisovic, Rebecca; Mims, Alice; Blum, William; Marcucci, Guido; Chan, Kenneth K.

    2012-01-01

    5-Azacytidine (5-azaC) is an azanucleoside approved for myelodysplastic syndrome. Approximately 80%-90% of 5-azaC is believed to be incorporated into RNA, which disrupts nucleic acid and protein metabolism leading to apoptosis. A smaller fraction (10%-20%) of 5-azaC inhibits DNA methylation and synthesis through conversion to decitabine triphosphate and subsequent DNA incorporation. However, its precise mechanism of action remains unclear. Ribonucleotide reductase (RR) is a highly regulated e...

  10. Tangled Up in Knots: Structures of Inactivated Forms of E. coli Class Ia Ribonucleotide Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Zimanyi, Christina M.; Ando, Nozomi; Brignole, Edward J.; Asturias, Francisco J.; Stubbe, JoAnne; Drennan, Catherine L. (MIT); (Scripps)

    2012-10-23

    Ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair and are successful targets for anticancer drugs such as clofarabine and gemcitabine. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into {alpha}{sub 4}{beta}{sub 4} rings. Here, we present the first X-ray structure of a gemcitabine-inhibited E. coli RNR and show that the previously described {alpha}{sub 4}{beta}{sub 4} rings can interlock to form an unprecedented ({alpha}{sub 4}{beta}{sub 4}){sub 2} megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNRs as well as present a mechanism for the formation of these unusual structures.

  11. A novel assay revealed that ribonucleotide reductase is functionally important for interstrand DNA crosslink repair.

    Science.gov (United States)

    Fujii, Naoaki; Evison, Benjamin J; Actis, Marcelo L; Inoue, Akira

    2015-11-01

    Cells have evolved complex biochemical pathways for DNA interstrand crosslink (ICL) removal. Despite the chemotherapeutic importance of ICL repair, there have been few attempts to identify which mechanistic DNA repair inhibitor actually inhibits ICL repair. To identify such compounds, a new and robust ICL repair assay was developed using a novel plasmid that contains synthetic ICLs between a CMV promoter region that drives transcription and a luciferase reporter gene, and an SV40 origin of replication and the large T antigen (LgT) gene that enables self-replication in mammalian cells. In a screen against compounds that are classified as inhibitors of DNA repair or synthesis, the reporter generation was exquisitely sensitive to ribonucleotide reductase (RNR) inhibitors such as gemcitabine and clofarabine, but not to inhibitors of PARP, ATR, ATM, Chk1, and others. The effect was observed also by siRNA downregulation of RNR. Moreover, the reporter generation was also particularly sensitive to 3-AP, a non-nucleoside RNR inhibitor, but not significantly sensitive to DNA replication stressors, suggesting that the involvement of RNR in ICL repair is independent of incorporation of a nucleotide RNR inhibitor into DNA to induce replication stress. The reporter generation from a modified version of the plasmid that lacks the LgT-SV40ori motif was also adversely affected by RNR inhibitors, further indicating a role for RNR in ICL repair that is independent of DNA replication. Intriguingly, unhooking of cisplatin-ICL from nuclear DNA was significantly inhibited by low doses of gemcitabine, suggesting an unidentified functional role for RNR in the process of ICL unhooking. The assay approach could identify other molecules essential for ICLR in quantitative and flexible manner. PMID:26462050

  12. Synergistic reduction of HIV-1 infectivity by 5-azacytidine and inhibitors of ribonucleotide reductase.

    Science.gov (United States)

    Rawson, Jonathan M O; Roth, Megan E; Xie, Jiashu; Daly, Michele B; Clouser, Christine L; Landman, Sean R; Reilly, Cavan S; Bonnac, Laurent; Kim, Baek; Patterson, Steven E; Mansky, Louis M

    2016-06-01

    Although many compounds have been approved for the treatment of human immunodeficiency type-1 (HIV-1) infection, additional anti-HIV-1 drugs (particularly those belonging to new drug classes) are still needed due to issues such as long-term drug-associated toxicities, transmission of drug-resistant variants, and development of multi-class resistance. Lethal mutagenesis represents an antiviral strategy that has not yet been clinically translated for HIV-1 and is based on the use of small molecules to induce excessive levels of deleterious mutations within the viral genome. Here, we show that 5-azacytidine (5-aza-C), a ribonucleoside analog that induces the lethal mutagenesis of HIV-1, and multiple inhibitors of the enzyme ribonucleotide reductase (RNR) interact in a synergistic fashion to more effectively reduce the infectivity of HIV-1. In these drug combinations, RNR inhibitors failed to significantly inhibit the conversion of 5-aza-C to 5-aza-2'-deoxycytidine, suggesting that 5-aza-C acts primarily as a deoxyribonucleoside even in the presence of RNR inhibitors. The mechanism of antiviral synergy was further investigated for the combination of 5-aza-C and one specific RNR inhibitor, resveratrol, as this combination improved the selectivity index of 5-aza-C to the greatest extent. Antiviral synergy was found to be primarily due to the reduced accumulation of reverse transcription products rather than the enhancement of viral mutagenesis. To our knowledge, these observations represent the first demonstration of antiretroviral synergy between a ribonucleoside analog and RNR inhibitors, and encourage the development of additional ribonucleoside analogs and RNR inhibitors with improved antiretroviral activity. PMID:27117260

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

    Directory of Open Access Journals (Sweden)

    Asuncion Valenzuela MM

    2015-02-01

    Full Text Available Malyn M Asuncion Valenzuela, Imilce Castro, Amber Gonda, Carlos J Diaz Osterman, Jessica M Jutzy, Jonathan R Aspe, Salma Khan, Jonathan W Neidigh, Nathan R Wall Center for Health Disparities and Molecular Medicine, Division of Biochemistry, Department of Basic Sciences, Loma Linda University, Loma Linda, CA, USA Abstract: New agent development, mechanistic understanding, and combinatorial partnerships with known and novel modalities continue to be important in the study of pancreatic cancer and its improved treatment. In this study, known antimetabolite drugs such as gemcitabine (ribonucleotide reductase inhibitor and 5-fluorouracil (thymidylate synthase inhibitor were compared with novel members of these two drug families in the treatment of a chemoresistant pancreatic cancer cell line PANC-1. Cellular survival data, along with protein and messenger ribonucleic acid expression for survivin, XIAP, cIAP1, and cIAP2, were compared from both the cell cytoplasm and from exosomes after single modality treatment. While all antimetabolite drugs killed PANC-1 cells in a time- and dose-dependent manner, neither family significantly altered the cytosolic protein level of the four inhibitors of apoptosis (IAPs investigated. Survivin, XIAP, cIAP1, and cIAP2 were found localized to exosomes where no significant difference in expression was recorded. This inability for significant and long-lasting expression may be a reason why pancreatic cancer lacks responsiveness to these and other cancer-killing agents. Continued investigation is required to determine the responsibilities of these IAPs in their role in chemoresistance in pancreatic adenocarcinoma. Keywords: IAPs, exosomes, pancreatic cancer, antimetabolites, gemcitabine, cladribine, hydroxyurea, 5-fluorodeoxyuridine, 5-fluorouracil

  14. Biochemical Characterization of the Split Class II Ribonucleotide Reductase from Pseudomonas aeruginosa.

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    Mikael Crona

    Full Text Available The opportunistic pathogen Pseudomonas aeruginosa can grow under both aerobic and anaerobic conditions. Its flexibility with respect to oxygen load is reflected by the fact that its genome encodes all three existing classes of ribonucleotides reductase (RNR: the oxygen-dependent class I RNR, the oxygen-indifferent class II RNR, and the oxygen-sensitive class III RNR. The P. aeruginosa class II RNR is expressed as two separate polypeptides (NrdJa and NrdJb, a unique example of a split RNR enzyme in a free-living organism. A split class II RNR is also found in a few closely related γ-Proteobacteria. We have characterized the P. aeruginosa class II RNR and show that both subunits are required for formation of a biologically functional enzyme that can sustain vitamin B12-dependent growth. Binding of the B12 coenzyme as well as substrate and allosteric effectors resides in the NrdJa subunit, whereas the NrdJb subunit mediates efficient reductive dithiol exchange during catalysis. A combination of activity assays and activity-independent methods like surface plasmon resonance and gas phase electrophoretic macromolecule analysis suggests that the enzymatically active form of the enzyme is a (NrdJa-NrdJb2 homodimer of heterodimers, and a combination of hydrogen-deuterium exchange experiments and molecular modeling suggests a plausible region in NrdJa that interacts with NrdJb. Our detailed characterization of the split NrdJ from P. aeruginosa provides insight into the biochemical function of a unique enzyme known to have central roles in biofilm formation and anaerobic growth.

  15. Inhibition of hepatitis B virus replication by targeting ribonucleotide reductase M2 protein.

    Science.gov (United States)

    Liu, Xia; Xu, Zhijian; Hou, Chuanwei; Wang, Meng; Chen, Xinhuan; Lin, Qinghui; Song, Rui; Lou, Meng; Zhu, Lijun; Qiu, Yunqing; Chen, Zhi; Yang, Chunhao; Zhu, Weiliang; Shao, Jimin

    2016-03-01

    Chronic hepatitis B virus (HBV) infection is a key factor for hepatocellular carcinoma worldwide. Ribonucleotide reductase (RR) regulates the deoxyribonucleoside triphosphates biosynthesis and serves as a target for anti-cancer therapy. Here, we demonstrate that RR is essential for HBV replication and the viral covalently-closed-circular DNA (cccDNA) synthesis in host liver cells. By performing computer-assisted virtual screening against the crystal structure of RR small subunit M2 (RRM2), osalmid, was identified as a potential RRM2-targeting compound. Osalmid was shown to be 10-fold more active in inhibiting RR activity than hydroxyurea, and significantly inhibited HBV DNA and cccDNA synthesis in HepG2.2.15 cells. In contrast, hydroxyurea and the RR large subunit (RRM1)-inhibitory drug gemcitabine showed little selective activity against HBV replication. In addition, osalmid also was shown to possess potent activity against a 3TC-resistant HBV strain, suggesting utility in treating drug-resistant HBV infections. Interestingly, osalmid showed synergistic effects with lamivudine (3TC) in vitro and in vivo without significant toxicity, and was shown to inhibit RR activity in vivo, thus verifying its in vivo function. Furthermore, 4-cyclopropyl-2-fluoro-N-(4-hydroxyphenyl) benzamide (YZ51), a novel derivative of osalmid, showed higher efficacy than osalmid with more potent RR inhibitory activity. These results suggest that RRM2 might be targeted for HBV inhibition, and the RRM2-targeting compound osalmid and its derivative YZ51 could be a novel class of anti-HBV candidates with potential use for hepatitis B and HBV-related HCC treatment. PMID:26774458

  16. Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Small Subunit Localization in Response to Iron Deficiency.

    Science.gov (United States)

    Sanvisens, Nerea; Romero, Antonia M; Zhang, Caiguo; Wu, Xiaorong; An, Xiuxiang; Huang, Mingxia; Puig, Sergi

    2016-04-29

    Ribonucleotide reductase (RNR) is an essential iron-dependent enzyme that catalyzes deoxyribonucleotide synthesis in eukaryotes. Living organisms have developed multiple strategies to tightly modulate RNR function to avoid inadequate or unbalanced deoxyribonucleotide pools that cause DNA damage and genome instability. Yeast cells activate RNR in response to genotoxic stress and iron deficiency by facilitating redistribution of its small heterodimeric subunit Rnr2-Rnr4 from the nucleus to the cytoplasm, where it forms an active holoenzyme with large Rnr1 subunit. Dif1 protein inhibits RNR by promoting nuclear import of Rnr2-Rnr4. Upon DNA damage, Dif1 phosphorylation by the Dun1 checkpoint kinase and its subsequent degradation enhances RNR function. In this report, we demonstrate that Dun1 kinase triggers Rnr2-Rnr4 redistribution to the cytoplasm in response to iron deficiency. We show that Rnr2-Rnr4 relocalization by low iron requires Dun1 kinase activity and phosphorylation site Thr-380 in the Dun1 activation loop, but not the Dun1 forkhead-associated domain. By using different Dif1 mutant proteins, we uncover that Dun1 phosphorylates Dif1 Ser-104 and Thr-105 residues upon iron scarcity. We observe that the Dif1 phosphorylation pattern differs depending on the stimuli, which suggests different Dun1 activating pathways. Importantly, the Dif1-S104A/T105A mutant exhibits defects in nucleus-to-cytoplasm redistribution of Rnr2-Rnr4 by iron limitation. Taken together, these results reveal that, in response to iron starvation, Dun1 kinase phosphorylates Dif1 to stimulate Rnr2-Rnr4 relocalization to the cytoplasm and promote RNR function. PMID:26970775

  17. Mechanism-based inhibition of a mutant Escherichia coli ribonucleotide reductase (cysteine-225----serine) by its substrate CDP.

    OpenAIRE

    Mao, S. S.; Johnston, M I; Bollinger, J M; Stubbe, J.

    1989-01-01

    The B1 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1) has been overexpressed using the pT7-5/pGP1-2 system developed by Tabor and Richardson [Tabor, S. & Richardson, C. (1985) Proc. Natl. Acad. Sci. USA 82, 1074-1078]. This method has allowed the preparation of two mutant B1 subunits in which two of the four thiols postulated to be within the active site of the enzyme, Cys-225 and Cys-759, have been changed to serines. Incubation of the [Ser225]B1 mutant with the B2 subuni...

  18. Clofarabine 5′-di and -triphosphates inhibit human ribonucleotide reductase by altering the quaternary structure of its large subunit

    OpenAIRE

    Aye, Yimon; Stubbe, JoAnne

    2011-01-01

    Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of α- and β-subunits that form active αnβm (n, m = 2 or 6) complexes. α binds NDP substrates (CDP, UDP, ADP, and GDP, C site) as well as ATP and dNTPs (dATP, dGTP, TTP) allosteric effectors that control enzyme activity (A site) and substrate specificity (S site). Clofarabine (ClF), an adenosine analog, is used in the treatment of refractory leukemias. Its mode of cytotoxicity is...

  19. Clofarabine Targets the Large Subunit (α) of Human Ribonucleotide Reductase in Live Cells by Assembly into Persistent Hexamers

    OpenAIRE

    Aye, Yimon; Brignole, Edward J; Long, Marcus J. C.; Chittuluru, Johnathan; Drennan, Catherine L.; Asturias, Francisco J; Stubbe, JoAnne

    2012-01-01

    Clofarabine (ClF) is a drug used in the treatment of leukemia. One of its primary targets is human ribonucleotide reductase (hRNR), a dual-subunit, (α2)m(β2)n, regulatory enzyme indispensable in de novo dNTP synthesis. We report that in live mammalian cells, ClF targets hRNR by converting its α-subunit into kinetically-stable hexamers. We established mammalian expression platforms that enabled isolation of functional α and characterization of its altered oligomeric associations in response to...

  20. Regulators of ribonucleotide reductase inhibit Ty1 mobility in saccharomyces cerevisiae

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    O'Donnell John P

    2010-11-01

    Full Text Available Abstract Background Ty1 is a long terminal repeat retrotransposon of Saccharomyces cerevisiae, with a replication cycle similar to retrovirus replication. Structurally, Ty1 contains long terminal repeat (LTR regions flanking the gag and pol genes that encode for the proteins that enable Ty1 mobility. Reverse transcriptase produces Ty1 complementary (cDNA that can either be integrated back into the genome by integrase or recombined into the yeast genome through homologous recombination. The frequency of Ty1 mobility is temperature sensitive, with optimum activity occurring at 24-26°C. Results In this study, we identified two host genes that when deleted allow for high temperature Ty1 mobility: RFX1 and SML1. The protein products of these genes are both negative regulators of the enzyme ribonucleotide reductase, a key enzyme in regulating deoxyribonucleotide triphosphate (dNTP levels in the cell. Processing of Ty1 proteins is defective at high temperature, and processing is not improved in either rfx1 or sml1 deletion strains. Ty1 mobility at high temperature is mediated by homologous recombination of Ty1 cDNA to Ty1 elements within the yeast genome. We quantified cDNA levels in wild type, rfx1 and sml1 deletion background strains at different temperatures. Southern blot analysis demonstrated that cDNA levels were not markedly different between the wild type and mutant strains as temperatures increased, indicating that the increased Ty1 mobility is not a result of increased cDNA synthesis in the mutant strains. Homologous recombination efficiency was increased in both rfx1 and sml1 deletion strains at high temperatures; the rfx1 deletion strain also had heightened homologous recombination efficiency at permissive temperatures. In the presence of the dNTP reducing agent hydroxyurea at permissive temperatures, Ty1 mobility was stimulated in the wild type and sml1 deletion strains but not in the rfx1 deletion strain. Mobility frequency was greatly

  1. Bacillus subtilis class Ib ribonucleotide reductase is a dimanganese(III)-tyrosyl radical enzyme.

    Science.gov (United States)

    Zhang, Yan; Stubbe, Joanne

    2011-06-28

    Bacillus subtilis class Ib ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, providing the building blocks for DNA replication and repair. It is composed of two proteins: α (NrdE) and β (NrdF). β contains the metallo-cofactor, essential for the initiation of the reduction process. The RNR genes are organized within the nrdI-nrdE-nrdF-ymaB operon. Each protein has been cloned, expressed, and purified from Escherichia coli. As isolated, recombinant NrdF (rNrdF) contained a diferric-tyrosyl radical [Fe(III)(2)-Y(•)] cofactor. Alternatively, this cluster could be self-assembled from apo-rNrdF, Fe(II), and O(2). Apo-rNrdF loaded using 4 Mn(II)/β(2), O(2), and reduced NrdI (a flavodoxin) can form a dimanganese(III)-Y(•) [Mn(III)(2)-Y(•)] cofactor. In the presence of rNrdE, ATP, and CDP, Mn(III)(2)-Y(•) and Fe(III)(2)-Y(•) rNrdF generate dCDP at rates of 132 and 10 nmol min(-1) mg(-1), respectively (both normalized for 1 Y(•)/β(2)). To determine the endogenous cofactor of NrdF in B. subtilis, the entire operon was placed behind a Pspank(hy) promoter and integrated into the B. subtilis genome at the amyE site. All four genes were induced in cells grown in Luria-Bertani medium, with levels of NrdE and NrdF elevated 35-fold relative to that of the wild-type strain. NrdE and NrdF were copurified in a 1:1 ratio from this engineered B. subtilis. The visible, EPR, and atomic absorption spectra of the purified NrdENrdF complex (eNrdF) exhibited characteristics of a Mn(III)(2)-Y(•) center with 2 Mn/β(2) and 0.5 Y(•)/β(2) and an activity of 318-363 nmol min(-1) mg(-1) (normalized for 1 Y(•)/β(2)). These data strongly suggest that the B. subtilis class Ib RNR is a Mn(III)(2)-Y(•) enzyme. PMID:21561096

  2. A hot oxidant, 3-NO2Y122 radical, unmasks conformational gating in ribonucleotide reductase.

    Science.gov (United States)

    Yokoyama, Kenichi; Uhlin, Ulla; Stubbe, JoAnne

    2010-11-01

    Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y(•)) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y(122)(•)→W(48)→Y(356) within β to Y(731)→Y(730)→C(439) within α). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO(2)Y) has recently been incorporated site-specifically in place of Y(122) in β2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO(2)Y(•)) and was inactive. In the present paper we show that incubation of apo-Y(122)NO(2)Y-β2 with Fe(2+) and O(2) generates a diferric-NO(2)Y(•) that has a half-life of 40 s at 25 °C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO(2)Y(•)/β2 and then mixed with α2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO(2)Y(•) is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s(-1)), that dCDP is produced at 107 s(-1), and that a new Y(•) is produced at 97 s(-1). Studies with pathway mutants suggest that the new Y(•) is predominantly located at 356 in β2. In consideration of these data and the crystal structure of Y(122)NO(2)Y-β2, a mechanism for PCET uncoupling in NO(2)Y(•)-RNR is proposed. PMID:20929229

  3. Expression, purification, crystallization and preliminary X-ray analysis of ORF60, the small subunit (R2) of ribonucleotide reductase from Kaposi's sarcoma-associated herpesvirus (KSHV)

    OpenAIRE

    Gurmu, Daniel; Dahlroth, Sue-Li; Haas, Juergen; Nordlund, Par; Erlandsen, Heidi

    2010-01-01

    Ribonucleotide reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides, which are the building blocks of DNA. The enzyme is present in all life forms as well as in some large DNA viruses such as herpesviruses. The alpha-herpesviruses and gamma-herpesviruses encode two class Ia RNR subunits, R1 and R2, while the beta-herpesvirus subfamily only encode an inactive R1 subunit. Here, the crystallization of the R2 subunit of RNR encoded by the ORF60 gene from the oncov...

  4. Mechanism of ribonucleotide reductase from Herpes simplex virus type 1. Evidence for 3' carbon-hydrogen bond cleavage and inactivation by nucleotide analogs

    International Nuclear Information System (INIS)

    Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of [3'-3H]ADP, [3'-H]UDP, and [5-3H] UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of [3'-3H]ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of [beta-32P]ClUDP with the reductase resulted in the production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase

  5. Mechanism-based inhibition of a mutant Escherichia coli ribonucleotide reductase (cysteine-225 → serine) by its substrate CDP

    International Nuclear Information System (INIS)

    The B1 subunit of Escherichia coli ribonucleotide reductase has been overexpressed using the pT7-5/pGP1-2 system developed by Tabor and Richardson. This method has allowed the preparation of two mutant B1 subunits in which two of the four thiols postulated to be within the active site of the enzyme, Cys-225 and Cys-759, have been changed to serines. Incubation of the [Ser225]B1 mutant with the B2 subunit, [U-14C]CDP, and the allosteric effector ATP results in production of cytosine, destruction of the tyrosyl radical in B2, radiolabeling of the protein, and cleavage of the B1 subunit into two pieces of 26 and 61.5 kDa. This process is independent of the identity of reductant. The [Ser759]B1 mutant reduces CDP in the presence of thioredoxin/thioredoxin reductase at 7.7% the rate of wild-type B1. When dithiothreitol is utilized as reductant, however, the rate of CDP reduction with [Ser759]B1 is identical to that observed with wild type

  6. Studies of ribonucleotide reductase in crucian carp-an oxygen dependent enzyme in an anoxia tolerant vertebrate.

    Directory of Open Access Journals (Sweden)

    Guro K Sandvik

    Full Text Available The enzyme ribonucleotide reductase (RNR catalyzes the conversion of ribonucleotides to deoxyribonucleotides, the precursors for DNA. RNR requires a thiyl radical to activate the substrate. In RNR of eukaryotes (class Ia RNR, this radical originates from a tyrosyl radical formed in reaction with oxygen (O(2 and a ferrous di-iron center in RNR. The crucian carp (Carassius carassius is one of very few vertebrates that can tolerate several months completely without oxygen (anoxia, a trait that enables this fish to survive under the ice in small ponds that become anoxic during the winter. Previous studies have found indications of cell division in this fish after 7 days of anoxia. This appears nearly impossible, as DNA synthesis requires the production of new deoxyribonucleotides and therefore active RNR. We have here characterized RNR in crucian carp, to search for adaptations to anoxia. We report the full-length sequences of two paralogs of each of the RNR subunits (R1i, R1ii, R2i, R2ii, p53R2i and p53R2ii, obtained by cloning and sequencing. The mRNA levels of these subunits were measured with quantitative PCR and were generally well maintained in hypoxia and anoxia in heart and brain. We also report maintained or increased mRNA levels of the cell division markers proliferating cell nuclear antigen (PCNA, brain derived neurotrophic factor (BDNF and Ki67 in anoxic hearts and brains. Electron paramagnetic resonance (EPR measurements on in vitro expressed crucian carp R2 and p53R2 proteins gave spectra similar to mammalian RNRs, including previously unpublished human and mouse p53R2 EPR spectra. However, the radicals in crucian carp RNR small subunits, especially in the p53R2ii subunit, were very stable at 0°C. A long half-life of the tyrosyl radical during wintertime anoxia could allow for continued cell division in crucian carp.

  7. Redox-dependent structural coupling between the α2 and β2 subunits in E. coli ribonucleotide reductase.

    Science.gov (United States)

    Offenbacher, Adam R; Watson, R Atlee; Pagba, Cynthia V; Barry, Bridgette A

    2014-03-20

    Ribonucleotide reductase (RNR) catalyzes the production of deoxyribonucleotides in all cells. In E. coli class Ia RNR, a transient α2β2 complex forms when a ribonucleotide substrate, such as CDP, binds to the α2 subunit. A tyrosyl radical (Y122O•)-diferric cofactor in β2 initiates substrate reduction in α2 via a long-distance, proton-coupled electron transfer (PCET) process. Here, we use reaction-induced FT-IR spectroscopy to describe the α2β2 structural landscapes, which are associated with dATP and hydroxyurea (HU) inhibition. Spectra were acquired after mixing E. coli α2 and β2 with a substrate, CDP, and the allosteric effector, ATP. Isotopic chimeras, (13)Cα2β2 and α2(13)Cβ2, were used to define subunit-specific structural changes. Mixing of α2 and β2 under turnover conditions yielded amide I (C═O) and II (CN/NH) bands, derived from each subunit. The addition of the inhibitor, dATP, resulted in a decreased contribution from amide I bands, attributable to β strands and disordered structures. Significantly, HU-mediated reduction of Y122O• was associated with structural changes in α2, as well as β2. To define the spectral contributions of Y122O•/Y122OH in the quaternary complex, (2)H4 labeling of β2 tyrosines and HU editing were performed. The bands of Y122O•, Y122OH, and D84, a unidentate ligand to the diferric cluster, previously identified in isolated β2, were observed in the α2β2 complex. These spectra also provide evidence for a conformational rearrangement at an additional β2 tyrosine(s), Yx, in the α2β2/CDP/ATP complex. This study illustrates the utility of reaction-induced FT-IR spectroscopy in the study of complex enzymes. PMID:24606240

  8. Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.

    Directory of Open Access Journals (Sweden)

    Ane B Tomter

    Full Text Available Ribonucleotide reductase (RNR catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g(1-value of 2.0090 for the tyrosyl radical was extracted. This g(1-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν(7a = 1500 cm(-1 was found to be insensitive to deuterium-oxide exchange. Additionally, the (18O-sensitive Fe-O-Fe symmetric stretching (483 cm(-1 of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g(1-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011 J Biol Chem 286: 33053-33060 indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-21

    As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration and

  10. RNA-dependent inhibition of ribonucleotide reductase is a major pathway for 5-azacytidine activity in acute myeloid leukemia

    Science.gov (United States)

    Aimiuwu, Josephine; Wang, Hongyan; Chen, Ping; Xie, Zhiliang; Wang, Jiang; Liu, Shujun; Klisovic, Rebecca; Mims, Alice; Blum, William; Marcucci, Guido

    2012-01-01

    5-Azacytidine (5-azaC) is an azanucleoside approved for myelodysplastic syndrome. Approximately 80%-90% of 5-azaC is believed to be incorporated into RNA, which disrupts nucleic acid and protein metabolism leading to apoptosis. A smaller fraction (10%-20%) of 5-azaC inhibits DNA methylation and synthesis through conversion to decitabine triphosphate and subsequent DNA incorporation. However, its precise mechanism of action remains unclear. Ribonucleotide reductase (RR) is a highly regulated enzyme comprising 2 subunits, RRM1 and RRM2, that provides the deoxyribonucleotides required for DNA synthesis/repair. In the present study, we found for the first time that 5-azaC is a potent inhibitor of RRM2 in leukemia cell lines, in a mouse model, and in BM mononuclear cells from acute myeloid leukemia (AML) patients. 5-azaC–induced RRM2 gene expression inhibition involves its direct RNA incorporation and an attenuated RRM2 mRNA stability. Therefore, 5-azaC causes a major perturbation of deoxyribonucleotide pools. We also demonstrate herein that the initial RR-mediated 5-azaC conversion to decitabine is terminated through its own inhibition. In conclusion, we identify RRM2 as a novel molecular target of 5-azaC in AML. Our findings provide a basis for its more widespread clinical use either alone or in combination. PMID:22517893

  11. DNA damage induced by the anticodon nuclease from a Pichia acaciae killer strain is linked to ribonucleotide reductase depletion.

    Science.gov (United States)

    Wemhoff, Sabrina; Klassen, Roland; Meinhardt, Friedhelm

    2016-02-01

    Virus like element (VLE) encoded killer toxins of Pichia acaciae and Kluyveromyces lactis kill target cells through anticodon nuclease (ACNase) activity directed against tRNA(Gln) and tRNA(Glu) respectively. Not only does tRNA cleavage disable translation, it also affects DNA integrity as well. Consistent with DNA damage, which is involved in toxicity, target cells' mutation frequencies are elevated upon ACNase exposure, suggesting a link between translational integrity and genome surveillance. Here, we analysed whether ACNase action impedes the periodically and highly expressed S-phase specific ribonucleotide reductase (RNR) and proved that RNR expression is severely affected by PaT. Because RNR catalyses the rate-limiting step in dNTP synthesis, mutants affected in dNTP synthesis were scrutinized with respect to ACNase action. Mutations elevating cellular dNTPs antagonized the action of both the above ACNases, whereas mutations lowering dNTPs aggravated toxicity. Consistently, prevention of tRNA cleavage in elp3 or trm9 mutants, which both affect the wobble uridine modification of the target tRNA, suppressed the toxin hypersensitivity of a dNTP synthesis mutant. Moreover, dNTP synthesis defects exacerbated the PaT ACNase sensitivity of cells defective in homologous recombination, proving that dNTP depletion is responsible for subsequent DNA damage. PMID:26247322

  12. A tyrosine-tryptophan dyad and radical-based charge transfer in a ribonucleotide reductase-inspired maquette

    Science.gov (United States)

    Pagba, Cynthia V.; McCaslin, Tyler G.; Veglia, Gianluigi; Porcelli, Fernando; Yohannan, Jiby; Guo, Zhanjun; McDaniel, Miranda; Barry, Bridgette A.

    2015-12-01

    In class 1a ribonucleotide reductase (RNR), a substrate-based radical is generated in the α2 subunit by long-distance electron transfer involving an essential tyrosyl radical (Y122O.) in the β2 subunit. The conserved W48 β2 is ~10 Å from Y122OH; mutations at W48 inactivate RNR. Here, we design a beta hairpin peptide, which contains such an interacting tyrosine-tryptophan dyad. The NMR structure of the peptide establishes that there is no direct hydrogen bond between the phenol and the indole rings. However, electronic coupling between the tyrosine and tryptophan occurs in the peptide. In addition, downshifted ultraviolet resonance Raman (UVRR) frequencies are observed for the radical state, reproducing spectral downshifts observed for β2. The frequency downshifts of the ring and CO bands are consistent with charge transfer from YO. to W or another residue. Such a charge transfer mechanism implies a role for the β2 Y-W dyad in electron transfer.

  13. A reduction in ribonucleotide reductase activity slows down the chromosome replication fork but does not change its localization.

    Directory of Open Access Journals (Sweden)

    Ingvild Odsbu

    Full Text Available BACKGROUND: It has been proposed that the enzymes of nucleotide biosynthesis may be compartmentalized or concentrated in a structure affecting the organization of newly replicated DNA. Here we have investigated the effect of changes in ribonucleotide reductase (RNR activity on chromosome replication and organization of replication forks in Escherichia coli. METHODOLOGY/PRINCIPAL FINDINGS: Reduced concentrations of deoxyribonucleotides (dNTPs obtained by reducing the activity of wild type RNR by treatment with hydroxyurea or by mutation, resulted in a lengthening of the replication period. The replication fork speed was found to be gradually reduced proportionately to moderate reductions in nucleotide availability. Cells with highly extended C periods showed a "delay" in cell division i.e. had a higher cell mass. Visualization of SeqA structures by immunofluorescence indicated no change in organization of the new DNA upon moderate limitation of RNR activity. Severe nucleotide limitation led to replication fork stalling and reversal. Well defined SeqA structures were not found in situations of extensive replication fork repair. In cells with stalled forks obtained by UV irradiation, considerable DNA compaction was observed, possibly indicating a reorganization of the DNA into a "repair structure" during the initial phase of the SOS response. CONCLUSION/SIGNIFICANCE: The results indicate that the replication fork is slowed down in a controlled manner during moderate nucleotide depletion and that a change in the activity of RNR does not lead to a change in the organization of newly replicated DNA. Control of cell division but not control of initiation was affected by the changes in replication elongation.

  14. Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): A combined experimental and theoretical study

    Science.gov (United States)

    Popović-Bijelić, Ana; Kowol, Christian R.; Lind, Maria E.S.; Luo, Jinghui; Himo, Fahmi; Enyedy, Éva A.; Arion, Vladimir B.; Gräslund, Astrid

    2012-01-01

    Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is currently the most promising chemotherapeutic compound among the class of α-N-heterocyclic thiosemicarbazones. Here we report further insights into the mechanism(s) of anticancer drug activity and inhibition of mouse ribonucleotide reductase (RNR) by Triapine. In addition to the metal-free ligand, its iron(III), gallium(III), zinc(II) and copper (II) complexes were studied, aiming to correlate their cytotoxic activities with their effects on the diferric/tyrosyl radical center of the RNR enzyme in vitro. In this study we propose for the first time a potential specific binding pocket for Triapine on the surface of the mouse R2 RNR protein. In our mechanistic model, interaction with Triapine results in the labilization of the diferric center in the R2 protein. Subsequently the Triapine molecules act as iron chelators. In the absence of external reductants, and in presence of the mouse R2 RNR protein, catalytic amounts of the iron(III)–Triapine are reduced to the iron(II)–Triapine complex. In the presence of an external reductant (dithiothreitol), stoichiometric amounts of the potently reactive iron (II)–Triapine complex are formed. Formation of the iron(II)–Triapine complex, as the essential part of the reaction outcome, promotes further reactions with molecular oxygen, which give rise to reactive oxygen species (ROS) and thereby damage the RNR enzyme. Triapine affects the diferric center of the mouse R2 protein and, unlike hydroxyurea, is not a potent reductant, not likely to act directly on the tyrosyl radical. PMID:21955844

  15. Composition and Structure of the Inorganic Core of Relaxed Intermediate X(Y122F) of Escherichia coli Ribonucleotide Reductase.

    Science.gov (United States)

    Doan, Peter E; Shanmugam, Muralidharan; Stubbe, JoAnne; Hoffman, Brian M

    2015-12-16

    Activation of the diferrous center of the β2 (R2) subunit of the class 1a Escherichia coli ribonucleotide reductases by reaction with O2 followed by one-electron reduction yields a spin-coupled, paramagnetic Fe(III)/Fe(IV) intermediate, denoted X, whose identity has been sought by multiple investigators for over a quarter of a century. To determine the composition and structure of X, the present study has applied (57)Fe, (14,15)N, (17)O, and (1)H electron nuclear double resonance (ENDOR) measurements combined with quantitative measurements of (17)O and (1)H electron paramagnetic resonance line-broadening studies to wild-type X, which is very short-lived, and to X prepared with the Y122F mutant, which has a lifetime of many seconds. Previous studies have established that over several seconds the as-formed X(Y122F) relaxes to an equilibrium structure. The present study focuses on the relaxed structure. It establishes that the inorganic core of relaxed X has the composition [(OH(-))Fe(III)-O-Fe(IV)]: there is no second inorganic oxygenic bridge, neither oxo nor hydroxo. Geometric analysis of the (14)N ENDOR data, together with recent extended X-ray absorption fine structure measurements of the Fe-Fe distance (Dassama, L. M.; et al. J. Am. Chem. Soc. 2013, 135, 16758), supports the view that X contains a "diamond-core" Fe(III)/Fe(IV) center, with the irons bridged by two ligands. One bridging ligand is the oxo bridge (OBr) derived from O2 gas. Given the absence of a second inorganic oxygenic bridge, the second bridging ligand must be protein derived, and is most plausibly assigned as a carboxyl oxygen from E238. PMID:26636616

  16. A Single Conserved Residue Mediates Binding of the Ribonucleotide Reductase Catalytic Subunit RRM1 to RRM2 and Is Essential for Mouse Development

    DEFF Research Database (Denmark)

    Specks, Julia; Lecona, Emilio; Lopez-Contreras, Andres J.;

    2015-01-01

    The ribonucleotide reductase (RNR) complex, composed of a catalytic subunit (RRM1) and a regulatory subunit (RRM2), is thought to be a rate-limiting enzymatic complex for the production of nucleotides. In humans, the Rrm1 gene lies at 11p15.5, a tumor suppressor region, and RRM1 expression in......, despite being viable, leads to increased interaction of the RNR complex with its allosteric inhibitor Sml1. In contrast to yeast, homozygous mutant mice carrying the Rrm1 mutation (Rrm1WG/WG) are not viable, even at the earliest embryonic stages. Proteomic analyses failed to identify proteins that...

  17. Antitumor and radiosensitizing effects of (E)-2'-Deoxy-2'-(Fluoromethylene) cytidine, a novel inhibior of ribonucleotide diphosphate reductase on human colon carcinoma xenografts in nude mice.

    OpenAIRE

    Sun, Lin-Quan; Li, Ye-Xiong; Guillou, Louis; Mirimanoff, René-Olivier; Coucke, Philippe

    1997-01-01

    Antitumor and radiosensitizing effects of (E).2'-deoxy.2'-(fluromethyl ene) cytidine (FMdC), a novel inhibitor of ribonucleotide reductase, were evaluated on nude mice bearing s.c. xenografts and liver metastases of a human colon carcinoma. FMdC given once daily or twice weekly has a dose-dependent antitumor effect. The maximum tolerated dose In the mice was reached with 10 mgi'kg applied daily over 12 days. Twice weekly administration of FMdC reduced its toxicity but lowere...

  18. RNRdb, a curated database of the universal enzyme family ribonucleotide reductase, reveals a high level of misannotation in sequences deposited to Genbank

    Directory of Open Access Journals (Sweden)

    Poole Anthony M

    2009-12-01

    Full Text Available Abstract Background Ribonucleotide reductases (RNRs catalyse the only known de novo pathway for deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. While ribonucleotide reduction has a single evolutionary origin, significant differences between RNRs nevertheless exist, notably in cofactor requirements, subunit composition and allosteric regulation. These differences result in distinct operational constraints (anaerobicity, iron/oxygen dependence and cobalamin dependence, and form the basis for the classification of RNRs into three classes. Description In RNRdb (Ribonucleotide Reductase database, we have collated and curated all known RNR protein sequences with the aim of providing a resource for exploration of RNR diversity and distribution. By comparing expert manual annotations with annotations stored in Genbank, we find that significant inaccuracies exist in larger databases. To our surprise, only 23% of protein sequences included in RNRdb are correctly annotated across the key attributes of class, role and function, with 17% being incorrectly annotated across all three categories. This illustrates the utility of specialist databases for applications where a high degree of annotation accuracy may be important. The database houses information on annotation, distribution and diversity of RNRs, and links to solved RNR structures, and can be searched through a BLAST interface. RNRdb is accessible through a public web interface at http://rnrdb.molbio.su.se. Conclusion RNRdb is a specialist database that provides a reliable annotation and classification resource for RNR proteins, as well as a tool to explore distribution patterns of RNR classes. The recent expansion in available genome sequence data have provided us with a picture of RNR distribution that is more complex than believed only a few years ago; our database indicates that RNRs of all three classes are found across all three cellular domains. Moreover, we find a

  19. Arrest of cell cycle by inhibition of ribonucleotide reductase induces accumulation of NAD+ by Mn2+-supplemented growth of Corynebacterium ammoniagenes.

    Science.gov (United States)

    Abbouni, Bouziane; Elhariry, Hesham M; Auling, Georg

    2003-01-01

    Cell division of the wild type strain Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 which requires 1 microM Mn2+ for balanced growth was inhibited by addition of 20 mM hydroxyurea (HU) or 10 mM p-methoxyphenol (MP) to a Mn2+-supplemented fermentation medium at an appropriate time. Scanning electron microscopy (SEM) showed a restricted elongation characteristic of arrest of the cell cycle in coryneform bacteria. The cultures treated with HU or MP had, respectively, a fourfold or sixfold enhanced accumulation of NAD+ by a salvage biosynthetic pathway. An assay of nucleotide-permeable cells for ribonucleotide reductase activity using [3H-CDP] as substrate revealed a pre-early and complete decline of DNA precursor biosynthesis not found in the untreated control. Overproduction of NAD+ is an alternative to the conventional fermentation process using Mn2+ deficiency. A simple model is presented to discuss the metabolic regulation of the new process based on the presence of a manganese ribonucleotide reductase (Mn-RNR) in the producing strain. PMID:12882290

  20. Role of Arginine 293 and Glutamine 288 in Communication between Catalytic and Allosteric Sites in Yeast Ribonucleotide Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Ahmad, Md. Faiz; Kaushal, Prem Singh; Wan, Qun; Wijerathna, Sanath R.; An, Xiuxiang; Huang, Mingxia; Dealwis, Chris Godfrey (Case Western); (Colorado)

    2012-11-01

    Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 ({alpha}) that contains the catalytic site and RR2 ({beta}) that houses a diferric-tyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-type enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP-CDP [AMPPNP, adenosine 5-({beta},{gamma}-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.

  1. Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli α2 ribonucleotide reductase.

    Science.gov (United States)

    Nick, Thomas U; Lee, Wankyu; Kossmann, Simone; Neese, Frank; Stubbe, JoAnne; Bennati, Marina

    2015-01-14

    Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y122•, Escherichia coli) in subunit β of a cysteine (C439) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y122 ⇆ Y356 in β to Y731 ⇆ Y730 ⇆ C439 in α); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y356-β, Y731- and Y730-α, and each protein was incubated with the appropriate second subunit β(α), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y•) in the active α2β2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH2Y•s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. (2)H electron-nuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH2Y730•, one to NH2Y731• and none to NH2Y356•. Similar experiments with double mutants α-NH2Y730/C439A and α-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within α. The implications of these observations for the PCET process within α and at the interface are discussed. PMID:25516424

  2. Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer.

    Science.gov (United States)

    Hsieh, Y-Y; Chou, C-J; Lo, H-L; Yang, P-M

    2016-01-01

    Colorectal cancer (CRC) is the second leading cause of cancer-related death in males and females in the world. It is of immediate importance to develop novel therapeutics. Human ribonucleotide reductase (RRM1/RRM2) has an essential role in converting ribonucleoside diphosphate to 2'-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. RRM2 is a prognostic biomarker and predicts poor survival of CRC. In addition, increased RRM2 activity is associated with malignant transformation and tumor cell growth. Bioinformatics analyses show that RRM2 was overexpressed in CRC and might be an attractive target for treating CRC. Therefore, we attempted to search novel RRM2 inhibitors by using a gene expression signature-based approach, connectivity MAP (CMAP). The result predicted GW8510, a cyclin-dependent kinase inhibitor, as a potential RRM2 inhibitor. Western blot analysis indicated that GW8510 inhibited RRM2 expression through promoting its proteasomal degradation. In addition, GW8510 induced autophagic cell death. In addition, the sensitivities of CRC cells to GW8510 were associated with the levels of RRM2 and endogenous autophagic flux. Taken together, our study indicates that GW8510 could be a potential anti-CRC agent through targeting RRM2. PMID:27551518

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

    International Nuclear Information System (INIS)

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

  4. Herpes Simplex Virus 1 (HSV-1) and HSV-2 Mediate Species-Specific Modulations of Programmed Necrosis through the Viral Ribonucleotide Reductase Large Subunit R1

    Science.gov (United States)

    Yu, Xiaoliang; Li, Yun; Chen, Qin; Su, Chenhe; Zhang, Zili; Yang, Chengkui; Hu, Zhilin; Hou, Jue; Zhou, Jinying; Gong, Ling; Jiang, Xuejun

    2015-01-01

    ABSTRACT Receptor-interacting protein kinase 3 (RIP3) and its substrate mixed-lineage kinase domain-like protein (MLKL) are core regulators of programmed necrosis. The elimination of pathogen-infected cells by programmed necrosis acts as an important host defense mechanism. Here, we report that human herpes simplex virus 1 (HSV-1) and HSV-2 had opposite impacts on programmed necrosis in human cells versus their impacts in mouse cells. Similar to HSV-1, HSV-2 infection triggered programmed necrosis in mouse cells. However, neither HSV-1 nor HSV-2 infection was able to induce programmed necrosis in human cells. Moreover, HSV-1 or HSV-2 infection in human cells blocked tumor necrosis factor (TNF)-induced necrosis by preventing the induction of an RIP1/RIP3 necrosome. The HSV ribonucleotide reductase large subunit R1 was sufficient to suppress TNF-induced necrosis, and its RIP homotypic interaction motif (RHIM) domain was required to disrupt the RIP1/RIP3 complex in human cells. Therefore, this study provides evidence that HSV has likely evolved strategies to evade the host defense mechanism of programmed necrosis in human cells. IMPORTANCE This study demonstrated that infection with HSV-1 and HSV-2 blocked TNF-induced necrosis in human cells while these viruses directly activated programmed necrosis in mouse cells. Expression of HSV R1 suppressed TNF-induced necrosis of human cells. The RHIM domain of R1 was essential for its association with human RIP3 and RIP1, leading to disruption of the RIP1/RIP3 complex. This study provides new insights into the species-specific modulation of programmed necrosis by HSV. PMID:26559832

  5. Ribonucleotide reduction and the possible role of cobalamin in evolution.

    Science.gov (United States)

    Dickman, S R

    1977-12-29

    The biological pathways of ribonucleotide reduction are briefly reviewed. The hypothesis is presented that reduction of ribonucleoside triphosphates to their deoxynucleotide analogs through the mediation of vitamin B12 or a similar corrinoid preceded and was necessary for the subsequent development of a DNA-type genome. There are two known biological systems for ribonucleotide reduction: (1) The ribonucleoside diphosphate reduction system which utilizes a nonheme iron ribonucleotide reductase enzyme, thioredoxin and its reductase, and NADPH. This enzyme complex is found in most bacteria, some higher organisms, and in all animals. (2) The ribonucleoside triphosphate reduction system which utilizes adenosyl cobalamin, ribonucleotide reductase and either thioredoxin or a disulfhydryl compound. The cobalamin-dependent reductase is restricted to a few species of bacteria and blue-gree algae. This system is considered more primitive than the iron reductase one based on their differences in distribution, components, and products. PMID:599575

  6. Two distinct mechanisms of inactivation of the class Ic ribonucleotide reductase from Chlamydia trachomatis by hydroxyurea: implications for the protein gating of intersubunit electron transfer.

    Science.gov (United States)

    Jiang, Wei; Xie, Jiajia; Varano, Paul T; Krebs, Carsten; Bollinger, J Martin

    2010-06-29

    Catalysis by a class I ribonucleotide reductase (RNR) begins when a cysteine (C) residue in the alpha(2) subunit is oxidized to a thiyl radical (C(*)) by a cofactor approximately 35 A away in the beta(2) subunit. In a class Ia or Ib RNR, a stable tyrosyl radical (Y(*)) is the C oxidant, whereas a Mn(IV)/Fe(III) cluster serves this function in the class Ic enzyme from Chlamydia trachomatis (Ct). It is thought that, in either case, a chain of Y residues spanning the two subunits mediates C oxidation by forming transient "pathway" Y(*)s in a multistep electron transfer (ET) process that is "gated" by the protein so that it occurs only in the ready holoenzyme complex. The drug hydroxyurea (HU) inactivates both Ia/b and Ic beta(2) subunits by reducing their C oxidants. Reduction of the stable cofactor Y(*) (Y122(*)) in Escherichia coli class Ia beta(2) is faster in the presence of alpha(2) and a substrate (CDP), leading to speculation that HU might intercept a transient ET pathway Y(*) under these turnover conditions. Here we show that this mechanism is one of two that are operant in HU inactivation of the Ct enzyme. HU reacts with the Mn(IV)/Fe(III) cofactor to give two distinct products: the previously described homogeneous Mn(III)/Fe(III)-beta(2) complex, which forms only under turnover conditions (in the presence of alpha(2) and the substrate), and a distinct, diamagnetic Mn/Fe cluster, which forms approximately 900-fold less rapidly as a second phase in the reaction under turnover conditions and as the sole outcome in the reaction of Mn(IV)/Fe(III)-beta(2) only. Formation of Mn(III)/Fe(III)-beta(2) also requires (i) either Y338, the subunit-interfacial ET pathway residue of beta(2), or Y222, the surface residue that relays the "extra electron" to the Mn(IV)/Fe(IV) intermediate during activation of beta(2) but is not part of the catalytic ET pathway, and (ii) W51, the cofactor-proximal residue required for efficient ET between either Y222 or Y338 and the cofactor

  7. Evidence That the [beta] Subunit of Chlamydia trachomatis Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(IV)/Iron(III) Cofactor in Site 1

    Energy Technology Data Exchange (ETDEWEB)

    Dassama, Laura M.K.; Boal, Amie K.; Krebs, Carsten; Rosenzweig, Amy C.; Bollinger, Jr., J. Martin (NWU); (Penn)

    2014-10-02

    The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the {beta} subunit oxidizes a cysteine residue {approx}35 {angstrom} away in the {alpha} subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the Mn{sup IV} ion of a Mn{sup IV}/Fe{sup III} cluster, which assembles in a reaction between O{sub 2} and the Mn{sup II}/Fe{sup II} complex of {beta}. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn{sup IV} ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn{sup IV} ion most likely resides in this site (i.e., {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}), but a subsequent computational study favored its occupation of site 2 ({sup 1}Fe{sup III}/{sup 2}Mn{sup IV}). In this work, we have sought to resolve the location of the Mn{sup IV} ion in Ct RNR-{beta} by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily Mn{sup IV}/Fe{sup III} clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both {sup 1}Mn{sup II}/{sup 2}Fe{sup II} and {sup 1}Fe{sup II}/{sup 2}Mn{sup II} complexes are competent to react with O{sub 2} to produce the corresponding oxidized states. However, with diminished Mn{sup II} loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these 'low-Mn' samples on a per-Mn basis implies that the {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}-{beta} is at least the more active of the two oxidized forms and may be the only active form.

  8. In vivo examination of hydroxyurea and the novel ribonucleotide reductase inhibitors trimidox and didox in combination with the reverse transcriptase inhibitor abacavir: suppression of retrovirus-induced immunodeficiency disease.

    Science.gov (United States)

    Sumpter, L Ryan; Inayat, Mohammed S; Yost, Erin E; Duvall, William; Hagan, Espen; Mayhew, Christopher N; Elford, Howard L; Gallicchio, Vincent S

    2004-06-01

    Inhibition of ribonucleotide reductase (RR) has gained attention as a potential strategy for HIV-1 therapy through the success of hydroxyurea (HU) to potentiate the activity of the nucleoside reverse transcriptase inhibitor (NRTI) didanosine (ddI) in clinical trials. However, the use of HU has been limited by its development of hematopoietic toxicity. In this study, the novel RR inhibitors didox (DX; 3,4-dihydroxybenzohydroxamic acid), and trimidox (TX; 3,4,5-trihydroxybenzamidoxime) were evaluated along with HU for anti-retroviral efficacy in LPBM5-induced retro-viral disease (MAIDS) both as monotherapeutic regimens and in combination with the guanine containing NRTI abacavir (ABC). Anti-retroviral drug efficacy was determined by measuring inhibition of splenomegaly, hypergammaglobulinemia, and splenic levels of proviral DNA. In this study, all RRIs tested showed the ability to improve the efficacy of ABC in the MAIDS model by reducing splenomegaly, hypergammaglobulinemia, and splenic proviral DNA levels. PMID:15130534

  9. Expression, purification, crystallization and preliminary X-ray analysis of ORF60, the small subunit (R2) of ribonucleotide reductase from Kaposi’s sarcoma-associated herpesvirus (KSHV)

    International Nuclear Information System (INIS)

    Crystals of the R2 subunit from the oncovirus Kaposi’s sarcoma-associated γ-herpesvirus (KSHV) were obtained by the use of in situ proteolysis. The crystals diffracted to 2.0 Å resolution and belonged to space group P21. Ribonucleotide reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides, which are the building blocks of DNA. The enzyme is present in all life forms as well as in some large DNA viruses such as herpesviruses. The α-herpesviruses and γ-herpesviruses encode two class Ia RNR subunits, R1 and R2, while the β-herpesvirus subfamily only encode an inactive R1 subunit. Here, the crystallization of the R2 subunit of RNR encoded by the ORF60 gene from the oncovirus Kaposi’s sarcoma-associated γ-herpesvirus (KSHV) is reported. These are the first crystals of a viral R2 subunit; the use of in situ proteolysis with chymotrypsin and the addition of hexamine cobalt(III) chloride that were necessary to obtain crystals are described. Optimization of the crystallization conditions yielded crystals that diffracted to 2.0 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 63.9, b = 71.2, c = 71.8 Å, α = 90, β = 106.7, γ = 90°. The data set collected was 95.3% complete, with an Rmerge of 9.6%. There are two molecules in the asymmetric unit, corresponding to a solvent content of 43.4%

  10. Comparison of the expression of human equilibrative nucleotide transporter 1 (hENT1) and ribonucleotide reductase subunit M1 (RRM1) genes in seven non-Hodgkin lymphoma cell lines.

    Science.gov (United States)

    Zhao, H B; Zhang, X F; Shi, F; Zhang, M Z; Xue, W L

    2016-01-01

    We investigated the variability in the expression of human equilibrative nucleoside transporter 1 (hENT1) and ribonucleotide reductase subunit M1 (RRM1) in non-Hodgkin lymphoma cell lines. hENT1 and RRM1 mRNA expression levels in natural killer (NK) cells and seven non-Hodgkin lymphoma cell lines (YTS, SNK-6, Jeko-1, ly-1, Raji, Karpas, and Jurket) were studied using reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) and the results were compared using the Student t-test. mRNA expression of hENT1 was detectable in YTS, SNK-6, Jeko-1, ly-1, Raji, Karpas, Jurket, and NK cells, which revealed variability in gene expression. There were significant differences in the mRNA expression values of hENT1 (P = 0.021) and RRM1 (P = 0.002) compared to those in NK cells. mRNA expression of both hENT1 and RRM1 was closely associated with non-Hodgkin lymphoma cell proliferation. Differential expression analysis of hENT1 and RRM1 in non-Hodgkin lymphoma cell lines may provide novel drug leads for precision medicine. PMID:27173327

  11. A PHASE I STUDY OF THE NOVEL RIBONUCLEOTIDE REDUCTASE INHIBITOR 3-AMINOPYRIDINE-2-CARBOXALDEHYDE THIOSEMICARBAZONE (3-AP, TRIAPINE®) IN COMBINATION WITH THE NUCLEOSIDE ANALOG FLUDARABINE FOR PATIENTS WITH REFRACTORY ACUTE LEUKEMIAS AND AGGRESSIVE MYELOPROLIFERATIVE DISORDERS

    Science.gov (United States)

    Karp, Judith E.; Giles, Francis J.; Gojo, Ivana; Morris, Lawrence; Greer, Jacqueline; Johnson, Bonny; Thein, Mya; Sznol, Mario; Low, Jennifer

    2009-01-01

    Triapine® is a potent ribonucleotide reductase (RR) inhibitor that depletes intracellular deoxyribonculeotide pools, especially dATP. We designed a Phase I trial of Triapine followed by the adenosine analog fludarabine in adults with refractory acute leukemias and aggressive myeloproliferative disorders (MPD). Two schedules were examined: A. Triapine 105mg/m2/day over 4 hours followed by fludarabine daily × 5 (24 patients, fludarabine 15–30 mg/m2/dose); B. Triapine 200mg/m2 over 24 hours followed by 5 days of fludarabine 30 mg/m2/day (9 patients). Complete and partial responses (CR,PR) occurred in Schedule A (5/24, 21%), with CR occurring at the 2 highest fludarabine doses (2/12, 17%). In contrast, no CR or PR occurred in Schedule B. Four of the 5 responses occurred in patients with underlying MPD (4/14, 29%). Drug-related toxicities included fever and metabolic acidosis. Triapine 105 mg/m2 followed by fludarabine 30mg/m2 daily × 5 is active in refractory myeloid malignancies and warrants continuing study for patients with aggressive MPD. PMID:17640728

  12. Site-specific insertion of 3-aminotyrosine into subunit alpha2 of E. coli ribonucleotide reductase: direct evidence for involvement of Y730 and Y731 in radical propagation.

    Science.gov (United States)

    Seyedsayamdost, Mohammad R; Xie, Jianming; Chan, Clement T Y; Schultz, Peter G; Stubbe, JoAnne

    2007-12-01

    E. coli ribonucleotide reductase (RNR) catalyzes the production of deoxynucleotides using complex radical chemistry. Active RNR is composed of a 1:1 complex of two subunits: alpha2 and beta2. Alpha2 binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide reduction. Beta2 contains the stable diiron tyrosyl radical (Y122.) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 A between the Y122 in beta2 and C439 in the active site of alpha2. A docking model of alpha2beta2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, aromatic amino acid radical intermediates, including Y730 and Y731 in alpha2. In this study the function of residues Y730 and Y731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-alpha2 and Y731NH2Y-alpha2 have been generated with high fidelity in yields of 4-6 mg/g of cell paste. These mutants have been examined by stopped flow UV-vis and EPR spectroscopies in the presence of beta2, CDP, and ATP. The results reveal formation of an NH2Y radical (NH2Y730. or NH2Y731.) in a kinetically competent fashion. Activity assays demonstrate that both NH2Y-alpha2s make deoxynucleotides. These results show that the NH2Y. can oxidize C439 suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within alpha2. The observed NH2Y. may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover. PMID:17990884

  13. The Balancing Act of Ribonucleotides in DNA.

    Science.gov (United States)

    Cerritelli, Susana M; Crouch, Robert J

    2016-05-01

    The abundance of ribonucleotides in DNA remained undetected until recently because they are efficiently removed by the ribonucleotide excision repair (RER) pathway, a process similar to Okazaki fragment (OF) processing after incision by Ribonuclease H2 (RNase H2). All DNA polymerases incorporate ribonucleotides during DNA synthesis. How many, when, and why they are incorporated has been the focus of intense work during recent years by many labs. In this review, we discuss recent advances in ribonucleotide incorporation by eukaryotic DNA polymerases that suggest an evolutionarily conserved role for ribonucleotides in DNA. We also review the data that indicate that removal of ribonucleotides has an important role in maintaining genome stability. PMID:26996833

  14. ATP-induced [Ca2+]i changes and depolarization in GH3 cells

    OpenAIRE

    Chung, Hae Sook; Park, Kyu Sang; Cha, Seung Kyu; Kong, In Deok; Lee, Joong Woo

    2000-01-01

    Extracellular ATP is a neurotransmitter and mediates a variety of responses. In the endocrine system, there are data suggesting a physiological role for ATP in Ca2+ signalling and hormone secretion. However, the ATP receptor subtype involved has not been clearly elucidated in GH3 cells, a rat anterior pituitary cell line.BzATP- and ATP-induced [Ca2+]i responses had EC50 values of 18 and 651 μM, respectively. The maximal response to ATP was only 59±8% of that for BzATP. The BzATP-induced [Ca2+...

  15. 核糖核苷酸还原酶M1、细胞核增殖抗原在基底细胞样乳腺癌组织的表达及意义%Expression and clinical significance of ribonucleotide reductase M1 and proliferation cell nuclear antigen in bassal-like carcinoma of breast in young female patient

    Institute of Scientific and Technical Information of China (English)

    祁旦巳; 周伶俐; 高宝辉; 张旭彤

    2015-01-01

    Objective To study the role of ribonucleotide reductase M 1 (RRM 1) and proliferation cell nuclear antigen (Ki-67) in the pathogenesis mechanism of bassal-like breast carcinoma (BLBC) in young female patient.Methods The clinical data of 52 young female patients (≤35 years old) treated in the Second Affiliated Hospital of Wenzhou Medical University, from June 2009 to May 2014 were analyzed.Of these patients, 22 cases of BLBC and 30 cases of non-BLBC were picked out based on immunohistochemisy, the proteins of RRM1 and Ki-67 were detected in BLBC and non-BLBC cases.Results The positive expression rate of RRM1 in BLBC organization was significantly lower than the non-BLBC group (22.7% vs.86.7% , P < 0.05);The positive expression rate of Ki-67 in BLBC organization was significantly higher than the non-BLBC group (95.5% vs.66.7%, P < 0.05).Conclusion The expression of RRM1 and Ki-67 in BLBC organizations with non-BLBC showed a negative correlation has important significance in prognostic judgment of breast cancer And maybe BLBC for sensitive to chemotherapy drugs gemcitabine.%目的 探讨核糖核苷酸还原酶M1(RRM1)及细胞核增殖抗原(Ki-67)在青年女性患者基底细胞样乳腺癌(BLBC)及非基底细胞样乳腺癌中的表达.方法 收集青年乳腺癌患者(年龄≤35岁)的石蜡标本,根据免疫组织化学结果选出基底细胞样乳腺癌22例,并随机选择非基底细胞样乳腺癌30例,通过免疫组织化学链霉菌抗生物素蛋白-过氧化物酶(SP)法检测RRM1及Ki-67在肿瘤中的表达.结果 RRM1在BLBC组织中的表达阳性率为22.7% (5/22),显著低于非BLBC组织的86.7% (26/30,P<0.05);Ki-67在BLBC组织中表达阳性率为95.5% (21/22),显著高于非BLBC组织的66.7% (20/30,P<0.05).结论 RRM1与Ki-67在BLBC组织与非BLBC组织中的表达呈负相关,检测RRM1与Ki-67有助于判断乳腺癌的预后,并且BLBC可能对代谢类化疗药物吉西他滨敏感.

  16. Eugenol Inhibits ATP-induced P2X Currents in Trigeminal Ganglion Neurons

    OpenAIRE

    Li, Hai Ying; Lee, Byung Ky; Kim, Joong Soo; Jung, Sung Jun; Oh, Seog Bae

    2008-01-01

    Eugenol is widely used in dentistry to relieve pain. We have recently demonstrated voltage-gated Na+ and Ca2+ channels as molecular targets for its analgesic effects, and hypothesized that eugenol acts on P2X3, another pain receptor expressed in trigeminal ganglion (TG), and tested the effects of eugenol by whole-cell patch clamp and Ca2+ imaging techniques. In the present study, we investigated whether eugenol would modulate 5'-triphosphate (ATP)-induced currents in rat TG neurons and P2X3-e...

  17. Saffron reduces ATP-induced retinal cytotoxicity by targeting P2X7 receptors.

    Science.gov (United States)

    Corso, Lucia; Cavallero, Anna; Baroni, Debora; Garbati, Patrizia; Prestipino, Gianfranco; Bisti, Silvia; Nobile, Mario; Picco, Cristiana

    2016-03-01

    P2X7-type purinergic receptors are distributed throughout the nervous system where they contribute to physiological and pathological functions. In the retina, this receptor is found in both inner and outer cells including microglia modulating signaling and health of retinal cells. It is involved in retinal neurodegenerative disorders such as retinitis pigmentosa and age-related macular degeneration (AMD). Experimental studies demonstrated that saffron protects photoreceptors from light-induced damage preserving both retinal morphology and visual function and improves retinal flicker sensitivity in AMD patients. To evaluate a possible interaction between saffron and P2X7 receptors (P2X7Rs), different cellular models and experimental approaches were used. We found that saffron positively influences the viability of mouse primary retinal cells and photoreceptor-derived 661W cells exposed to ATP, and reduced the ATP-induced intracellular calcium increase in 661W cells. Similar results were obtained on HEK cells transfected with recombinant rat P2X7R but not on cells transfected with rat P2X2R. Finally, patch-clamp experiments showed that saffron inhibited cationic currents in HEK-P2X7R cells. These results point out a novel mechanism through which saffron may exert its protective role in neurodegeneration and support the idea that P2X7-mediated calcium signaling may be a crucial therapeutic target in the treatment of neurodegenerative diseases. PMID:26739703

  18. Differential modulation of ATP-induced calcium signalling by A1 and A2 adenosine receptors in cultured cortical astrocytes

    OpenAIRE

    Alloisio, Susanna; Cugnoli, Carlo; Ferroni, Stefano; Nobile, Mario

    2004-01-01

    Despite the accumulating evidence that under various pathological conditions the extracellular elevation of adenine-based nucleotides and nucleosides plays a key role in the control of astroglial reactivity, how these signalling molecules interact in the regulation of astrocyte function is still largely elusive.The action of the nucleoside adenosine in the modulation of the intracellular calcium signalling ([Ca2+]i) elicited by adenosine 5′-triphosphate (ATP)-induced activation of P2 purinoce...

  19. N114S mutation causes loss of ATP-induced aggregation of human phosphoribosylpyrophosphate synthetase 1

    International Nuclear Information System (INIS)

    This study examined recombinant wild-type human phosphoribosylpyrophosphate synthetase 1 (wt-PRS1, EC 2.7.6.1) and the point mutant Asn114Ser PRS1 (N114S-Mutant) in cells of a patient with primary gout. Dynamic light-scattering and sedimentation velocity experiments indicated that the monomeric wt-PRS1 in solution was assembled into hexamers after adding the substrate ATP. However, this ATP-induced aggregation effect was not observed with N114S-Mutant, which has a 50% higher enzymatic activity than that of wt-PRS1. Synchrotron radiation circular dichroism spectroscopy revealed that the point mutation causes an increase of α-helix content and a decrease of turn content. Examination of the crystal structure of wt-PRS1 indicated that 12 hydrogen bonds formed by 6 pairs of N114 and D139 have an important role in stabilizing the hexamer. We suggest that the substitution of S114 for N114 in N114S-Mutant leads to the rupture of 12 hydrogen bonds and breakage of the PO43- allosteric site where PO43- functions as a fixer of the ATP-binding loop. Therefore, we consider that formation of the hexamer as the structural basis of the ADP allosteric inhibition is greatly weakened by the N114S mutation, and that alteration of the ATP-binding loop conformation is the key factor in the increased activity of N114S-Mutant. These two factors could be responsible for the high level of activity of N114S-Mutant in this patient.

  20. Ribonucleotide reduction - horizontal transfer of a required function spans all three domains

    Directory of Open Access Journals (Sweden)

    Torrents Eduard

    2010-12-01

    Full Text Available Abstract Background Ribonucleotide reduction is the only de novo pathway for synthesis of deoxyribonucleotides, the building blocks of DNA. The reaction is catalysed by ribonucleotide reductases (RNRs, an ancient enzyme family comprised of three classes. Each class has distinct operational constraints, and are broadly distributed across organisms from all three domains, though few class I RNRs have been identified in archaeal genomes, and classes II and III likewise appear rare across eukaryotes. In this study, we examine whether this distribution is best explained by presence of all three classes in the Last Universal Common Ancestor (LUCA, or by horizontal gene transfer (HGT of RNR genes. We also examine to what extent environmental factors may have impacted the distribution of RNR classes. Results Our phylogenies show that the Last Eukaryotic Common Ancestor (LECA possessed a class I RNR, but that the eukaryotic class I enzymes are not directly descended from class I RNRs in Archaea. Instead, our results indicate that archaeal class I RNR genes have been independently transferred from bacteria on two occasions. While LECA possessed a class I RNR, our trees indicate that this is ultimately bacterial in origin. We also find convincing evidence that eukaryotic class I RNR has been transferred to the Bacteroidetes, providing a stunning example of HGT from eukaryotes back to Bacteria. Based on our phylogenies and available genetic and genomic evidence, class II and III RNRs in eukaryotes also appear to have been transferred from Bacteria, with subsequent within-domain transfer between distantly-related eukaryotes. Under the three-domains hypothesis the RNR present in the last common ancestor of Archaea and eukaryotes appears, through a process of elimination, to have been a dimeric class II RNR, though limited sampling of eukaryotes precludes a firm conclusion as the data may be equally well accounted for by HGT. Conclusions Horizontal gene

  1. Synergistic augmentation of ATP-induced interleukin-6 production by arsenite in HaCaT cells.

    Science.gov (United States)

    Sumi, Daigo; Asao, Masashi; Okada, Hideta; Yogi, Kuniko; Miyataka, Hideki; Himeno, Seiichiro

    2016-06-01

    Chronic arsenic exposure causes cutaneous diseases such as hyperkeratosis and skin cancer. However, little information has been available regarding the molecular mechanisms underlying these symptoms. Because extracellular ATP and interleukin-6 (IL-6) are involved in pathological aspects of cutaneous diseases, we examined whether sodium arsenite (As(III)) affects ATP-induced IL-6 production in human epidermal keratinocyte HaCaT cells. The results showed that the addition of As(III) into the medium of HaCaT cells dose dependently increased the production of IL-6 induced by extracellular ATP, although As(III) alone had no effect on IL-6 production. To elucidate the mechanism of the synergistic effect of As(III) on IL-6 production by extracellular ATP, we next examined the phosphorylation of p38, ERK and epidermal growth factor receptor (EGFR), since we found that these signaling molecules were stimulated by exposure to extracellular ATP. The results indicated that ATP-induced phosphorylation of p38, ERK and EGFR was synergistically enhanced by co-exposure to As(III). To clarify the mechanisms underlying the enhanced phosphorylation of p38, ERK and EGFR by As(III), we explored two possible mechanisms: the inhibition of extracellular ATP degradation and the inhibition of protein tyrosine phosphatases (PTPs) activity by As(III). The degradation of extracellular ATP was not changed by As(III), whereas the activity of PTPs was significantly inhibited by As(III). Our results suggest that As(III) augments ATP-induced IL-6 production in HaCaT cells through enhanced phosphorylation of the EGFR and p38/ERK pathways, which is associated with the inhibition of PTPs activity. PMID:26104857

  2. ATP induced vasodilatation and purinergic receptors in the human leg: roles of nitric oxide, prostaglandins and adenosine

    DEFF Research Database (Denmark)

    Mortensen, Stefan P; Gonzalez-Alonso, Jose; Bune, Laurids; Saltin, Bengt; Pilegaard, Henriette; Hellsten, Ylva

    2009-01-01

    -arterial infusion of ATP (0.45-2.45 micromol/min; mean+/-SEM) in 19 healthy, male subjects with and without co-infusion of NG-mono-methyl-L-arginine (L-NMMA; NO formation inhibitor; 12.3+/-0.3 mg/min), indomethacin (INDO; prostaglandin formation blocker; 613+/-12 microg/min) and/or theophylline (adenosine receptor...... was associated with a parallel lowering in leg vascular conductance and cardiac output and a compensatory increase in leg O2 extraction. Infusion of theophylline did not alter the ATP induced leg hyperemia or systemic variables. Real time PCR analysis of the mRNA content from the vastus lateralus...

  3. Extracellular ATP induces spikes in cytosolic free Ca(2+) but not in NADPH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca(2+) concentration, we simultaneously measured Ca(2+) oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...... damped Ca(2+) spikes with a period of 15s and slower irregular spikes with a period greater than 50s. Spikes in Ca(2+) occurred in the absence of Ca(2+) influx, but the amplitude was damped by inhibition of Ca(2+) influx. Using the oxidation of hydroethidine as a cytosolic marker of oxidant production......, we show that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca(2+) but, despite subsequent spikes in Ca(2+) concentration, no oscillations in oxidase activity could be...

  4. Extracellular ATP induces spikes in cytosolic free Ca2+ but not in NADH oxidase activity in neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Olsen, Lars Folke; Hallett, Maurice B.

    2011-01-01

    In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca2+ concentration, we simultaneously measured Ca2+ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast...... damped Ca2+ spikes with a period of 15 s and slower irregular spikes with a period greater than 50 s. Spikes in Ca2+ occurred in the absence of Ca2+ influx, but the amplitude was damped by inhibition of Ca2+ influx. Using the oxidation of hydroethidine as a cytosolic marker of oxidant production, we show...... that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca2+ but, despite subsequent spikes in Ca2+ concentration, no oscillations in oxidase activity could be detected. ATP...

  5. Structure-function analysis of ribonucleotide bypass by B family DNA replicases

    Energy Technology Data Exchange (ETDEWEB)

    Clausen, Anders R.; Murray, Michael S.; Passer, Andrew R.; Pedersen, Lars C.; Kunkel, Thomas A. [NIH

    2013-11-01

    Ribonucleotides are frequently incorporated into DNA during replication, they are normally removed, and failure to remove them results in replication stress. This stress correlates with DNA polymerase (Pol) stalling during bypass of ribonucleotides in DNA templates. Here we demonstrate that stalling by yeast replicative Pols δ and ε increases as the number of consecutive template ribonucleotides increases from one to four. The homologous bacteriophage RB69 Pol also stalls during ribonucleotide bypass, with a pattern most similar to that of Pol ε. Crystal structures of an exonuclease-deficient variant of RB69 Pol corresponding to multiple steps in single ribonucleotide bypass reveal that increased stalling is associated with displacement of Tyr391 and an unpreferred C2´-endo conformation for the ribose. Even less efficient bypass of two consecutive ribonucleotides in DNA correlates with similar movements of Tyr391 and displacement of one of the ribonucleotides along with the primer-strand DNA backbone. These structure–function studies have implications for cellular signaling by ribonucleotides, and they may be relevant to replication stress in cells defective in ribonucleotide excision repair, including humans suffering from autoimmune disease associated with RNase H2 defects.

  6. Sinorhizobium meliloti requires a cobalamin-dependent ribonucleotide reductase for symbiosis with its plant host

    OpenAIRE

    Taga, Michiko E.; Walker, Graham C.

    2010-01-01

    Vitamin B[subscript 12] (cobalamin) is a critical cofactor for animals and protists, yet its biosynthesis is limited to prokaryotes. We previously showed that the symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti requires cobalamin to establish a symbiotic relationship with its plant host, Medicago sativa (alfalfa). Here, the specific requirement for cobalamin in the S. meliloti–alfalfa symbiosis was investigated. Of the three known cobalamin-dependent enzymes in S. melilo...

  7. Extracellular ATP-induced nuclear Ca{sup 2+} transient is mediated by inositol 1,4,5-trisphosphate receptors in mouse pancreatic {beta}-cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zheng; Li, Zhengzheng; Peng, Gong; Chen, Xiaoli; Yin, Wenxuan [National Laboratory of Biomacromolecules, Institute of Biophysics of Chinese Academy of Sciences, 15 Datun Rd., Beijing 100101 (China); Kotlikoff, Michael I. [Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (United States); Yuan, Zeng-qiang, E-mail: zqyuan@sun5.ibp.ac.cn [National Laboratory of Biomacromolecules, Institute of Biophysics of Chinese Academy of Sciences, 15 Datun Rd., Beijing 100101 (China); Ji, Guangju, E-mail: gj28@ibp.ac.cn [National Laboratory of Biomacromolecules, Institute of Biophysics of Chinese Academy of Sciences, 15 Datun Rd., Beijing 100101 (China)

    2009-05-01

    Extracellular ATP (eATP) induces an intracellular Ca{sup 2+} transient by activating phospholipase C (PLC)-associated P2X4 purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent Ca{sup 2+} release from intracellular stores in mouse pancreatic {beta}-cells. Using laser scanning confocal microscopy, Ca{sup 2+} indicator fluo-4 AM, and the cell permeable nuclear indicator Hoechst 33342, we examined the properties of eATP-induced Ca{sup 2+} release in pancreatic {beta}-cell nuclei. eATP induced a higher nuclear Ca{sup 2+} transient in pancreatic {beta}-cell nuclei than in the cytosol. After pretreatment with thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca{sup 2+}-ATPase (SERCA) pumps, the amplitude of eATP-induced Ca{sup 2+} transients in the nucleus was still much higher than those in the cytosol. This effect of eATP was not altered by inhibition of either the plasma membrane Ca{sup 2+}-ATPase (PMCA) or the plasma membrane Na{sup +}/Ca{sup 2+} exchanger (NCX) by LaCl{sub 3} or by replacement of Na{sup +} with N-Methyl-Glucosamine. eATP-induced nuclear Ca{sup 2+} transients were abolished by a cell-permeable IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), but were not blocked by the ryanodine receptor (RyR) antagonist ryanodine. Immunofluorescence studies showed that IP3Rs are expressed on the nuclear envelope of pancreatic {beta}-cells. These results indicate that eATP triggers nuclear Ca{sup 2+} transients by mobilizing a nuclear Ca{sup 2+} store via nuclear IP3Rs.

  8. Inflammatory responses are not sufficient to cause delayed neuronal death in ATP-induced acute brain injury.

    Directory of Open Access Journals (Sweden)

    Hey-Kyeong Jeong

    Full Text Available BACKGROUND: Brain inflammation is accompanied by brain injury. However, it is controversial whether inflammatory responses are harmful or beneficial to neurons. Because many studies have been performed using cultured microglia and neurons, it has not been possible to assess the influence of multiple cell types and diverse factors that dynamically and continuously change in vivo. Furthermore, behavior of microglia and other inflammatory cells could have been overlooked since most studies have focused on neuronal death. Therefore, it is essential to analyze the precise roles of microglia and brain inflammation in the injured brain, and determine their contribution to neuronal damage in vivo from the onset of injury. METHODS AND FINDINGS: Acute neuronal damage was induced by stereotaxic injection of ATP into the substantia nigra pars compacta (SNpc and the cortex of the rat brain. Inflammatory responses and their effects on neuronal damage were investigated by immunohistochemistry, electron microscopy, quantitative RT-PCR, and stereological counting, etc. ATP acutely caused death of microglia as well as neurons in a similar area within 3 h. We defined as the core region the area where both TH(+ and Iba-1(+ cells acutely died, and as the penumbra the area surrounding the core where Iba-1(+ cells showed activated morphology. In the penumbra region, morphologically activated microglia arranged around the injury sites. Monocytes filled the damaged core after neurons and microglia died. Interestingly, neither activated microglia nor monocytes expressed iNOS, a major neurotoxic inflammatory mediator. Monocytes rather expressed CD68, a marker of phagocytic activity. Importantly, the total number of dopaminergic neurons in the SNpc at 3 h (∼80% of that in the contralateral side did not decrease further at 7 d. Similarly, in the cortex, ATP-induced neuron-damage area detected at 3 h did not increase for up to 7 d. CONCLUSIONS: Different cellular

  9. ATP induces guinea pig gallbladder smooth muscle excitability via the P2Y4 receptor and COX-1 activity.

    Science.gov (United States)

    Bartoo, Aaron C; Nelson, Mark T; Mawe, Gary M

    2008-06-01

    The purpose of this study was to elucidate the mechanisms by which ATP increases guinea pig gallbladder smooth muscle (GBSM) excitability. We evaluated changes in membrane potential and action potential (AP) frequency in GBSM by use of intracellular recording. Application of ATP (100 microM) caused membrane depolarization and a significant increase in AP frequency that were not sensitive to block by tetrodotoxin (0.5 microM). The nonselective P2 antagonist, suramin (100 microM), blocked the excitatory response, resulting in decreased AP frequency in the presence of ATP. The excitatory response to ATP was not altered by pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid (30 microM), a nonselective P2X antagonist. UTP also caused membrane depolarization and increased AP frequency, with a similar dose-response relationship as ATP. RT-PCR demonstrated that the P2Y(4), but not P2Y(2), receptor subtype is expressed in guinea pig gallbladder muscularis. ATP induced excitation was blocked by indomethacin (10 microM) and the cyclooxygenase (COX)-1 inhibitor SC-560 (300 nM), but not the COX-2 inhibitor nimesulide (500 nM). These data suggest that ATP stimulates P2Y(4) receptors within the gallbladder muscularis and, in turn, stimulate prostanoid production via COX-1 leading to increased excitability of GBSM. PMID:18436624

  10. [Interaction of Ag+ ions with ribonucleotides of canonical bases].

    Science.gov (United States)

    Sorokin, V A; Valeev, V A; Gladchenko, G O; Sysa, I V; Degtiar, M V; Volchok, I V; Blagoĭ, Iu P

    1999-01-01

    The interaction of Ag+ ions with ribonucleotides of canonical bases in aqueous solution was studied by differential UV spectroscopy. Atoms coordinating silver ions (N7, O6 of guanosine 5'-monophosphate, N3, O2 of cytidine 5'-monophosphate, N7, N1, N3 of adenosine 5'-monophosphate and N3 of uridine 5'-monophosphate) and the binding constants characterizing the formation of appropriate complexes were determined. The differences in the relative affinity of Ag+ ions for the atoms of nucleotide bases correlate with the potential on them. PMID:10418671

  11. The properties of the ATP-induced depolarization and current in single cells isolated from the guinea-pig urinary bladder.

    OpenAIRE

    Inoue, R.; Brading, A. F.

    1990-01-01

    1. The actions of exogenously applied ATP were investigated with the whole-cell patch clamp method in single cells isolated from guinea-pig urinary bladder with a modified concentration jump technique. 2. Rapid application of ATP (threshold ca. 100 nM) depolarized the cell membrane with superimposition of action potentials which was followed by transient hyperpolarization. In the presence of D600, the amplitude of the ATP-induced depolarization was a function of the ATP concentration (EC50: 0...

  12. Defective removal of ribonucleotides from DNA promotes systemic autoimmunity

    Science.gov (United States)

    Günther, Claudia; Kind, Barbara; Reijns, Martin A.M.; Berndt, Nicole; Martinez-Bueno, Manuel; Wolf, Christine; Tüngler, Victoria; Chara, Osvaldo; Lee, Young Ae; Hübner, Norbert; Bicknell, Louise; Blum, Sophia; Krug, Claudia; Schmidt, Franziska; Kretschmer, Stefanie; Koss, Sarah; Astell, Katy R.; Ramantani, Georgia; Bauerfeind, Anja; Morris, David L.; Cunninghame Graham, Deborah S.; Bubeck, Doryen; Leitch, Andrea; Ralston, Stuart H.; Blackburn, Elizabeth A.; Gahr, Manfred; Witte, Torsten; Vyse, Timothy J.; Melchers, Inga; Mangold, Elisabeth; Nöthen, Markus M.; Aringer, Martin; Kuhn, Annegret; Lüthke, Kirsten; Unger, Leonore; Bley, Annette; Lorenzi, Alice; Isaacs, John D.; Alexopoulou, Dimitra; Conrad, Karsten; Dahl, Andreas; Roers, Axel; Alarcon-Riquelme, Marta E.; Jackson, Andrew P.; Lee-Kirsch, Min Ae

    2014-01-01

    Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2–associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage–associated pathways in the initiation of autoimmunity. PMID:25500883

  13. Ribonucleotide and Ribonucleoside Determination by Ambient Pressure Ion Mobility Spectrometry (IMS)

    OpenAIRE

    Kanu, Abu B.; Hampikian, Greg; Brandt, Simon D; Hill, Herbert H.

    2009-01-01

    Reduced mobilities, resolving powers and detection limits for 12 ribonucleotides and 4 ribonucleosides were measured by ambient pressure electrospray ionization ion mobility spectrometry (ESI-IMS). With the instrument used in this study it was possible to separate some of these compounds within the mixtures. In addition, the detection limits reported for the ribonucleotides and ribonucleosides ranged from 15 to 300 picomoles whereas resolving power ranged from 41 to 56 suggesting that ambient...

  14. In Situ Imidazole Activation of Ribonucleotides for Abiotic RNA Oligomerization Reactions

    Science.gov (United States)

    Burcar, Bradley T.; Jawed, Mohsin; Shah, Hari; McGown, Linda B.

    2015-06-01

    The hypothesis that RNA played a significant role in the origin of life requires effective and efficient abiotic pathways to produce RNA oligomers. The most successful abiotic oligomerization reactions to date have utilized high-energy, modified, or pre-activated ribonucleotides to generate strands of RNA up to 50-mers in length. In spite of their success, these modifications and pre-activation reactions significantly alter the ribonucleotides in ways that are highly unlikely to have occurred on a prebiotic Earth. This research seeks to address this problem by exploring an aqueous based method for activating the canonical ribonucleotides in situ using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and imidazole. The reactions were run with and without a montmorillonite clay catalyst and compared to reactions that used ribonucleotides that were pre-activated with imidazole. The effects of pH and ribonucleotide concentration were also investigated. The results demonstrate the ability of in situ activation of ribonucleotides to generate linear RNA oligomers in solution, providing an alternative route to produce RNA for use in prebiotic Earth scenarios.

  15. Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA.

    OpenAIRE

    Batey, R T; Inada, M; Kujawinski, E; Puglisi, J D; Williamson, J. R.

    1992-01-01

    A general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs is described. Bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides. These are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare RNAs for NMR studies. For 15N-labeling, E.coli is grown on 15N-ammonium sulfate, whereas for 13C-labeling, Methylophilus methylotro...

  16. Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand

    OpenAIRE

    Yasukawa, Takehiro; Reyes, Aurelio; Cluett, Tricia J.; Yang, Ming-Yao; Bowmaker, Mark; Jacobs, Howard T.; Holt, Ian J.

    2006-01-01

    Using two-dimensional agarose gel electrophoresis, we show that mitochondrial DNA (mtDNA) replication of birds and mammals frequently entails ribonucleotide incorporation throughout the lagging strand (RITOLS). Based on a combination of two-dimensional agarose gel electrophoretic analysis and mapping of 5′ ends of DNA, initiation of RITOLS replication occurs in the major non-coding region of vertebrate mtDNA and is effectively unidirectional. In some cases, conversion of nascent RNA strands t...

  17. MgATP-induced conformational changes in the iron protein from Azotobacter vinelandii, as studied by small-angle x-ray scattering.

    Science.gov (United States)

    Chen, L; Gavini, N; Tsuruta, H; Eliezer, D; Burgess, B K; Doniach, S; Hodgson, K O

    1994-02-01

    Small angle x-ray scattering experiments have been carried out on the purified iron proteins of nitrogenase from wild-type Azotobacter vinelandii and from a Nif- mutant strain, A. vinelandii UW91 (which has an A157S mutation). This study was designed to investigate the influence of MgATP and MgADP binding on the protein structure in solution. For the wild-type protein, the binding of MgATP induces a significant conformational change that is observed as a decrease of about 2.0 A in the radius of gyration. In contrast, the binding of MgADP to the wild-type iron protein does not detectably affect the radius of gyration. In the absence of nucleotides, the radius of gyration for the UW91 mutant is indistinguishable from that of the wild-type. However, unlike for the wild-type protein, the radius of gyration of the UW91 iron protein is unaffected by the addition of MgATP. We have previously shown that the UW91 iron protein has a normal [4Fe-4S] cluster and MgATP binding ability but that it is completely blocked for electron transfer and MgATP hydrolysis (Gavini, N., and Burgess, B. K. (1992) J. Biol. Chem. 267, 21179-21186). These x-ray scattering measurements suggest that a conformation different from that of the native state is therefore required for the iron protein to perform electron transfer to the MoFe protein. These results also support the hypothesis that Ala-157 is crucial for the iron protein to establish the electron-transfer-favored conformation induced by MgATP binding. PMID:8106367

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

    Science.gov (United States)

    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

  19. Genetics Home Reference: sepiapterin reductase deficiency

    Science.gov (United States)

    Skip to main content Your Guide to Understanding Genetic Conditions Enable Javascript for addthis links to activate. ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions sepiapterin reductase deficiency sepiapterin reductase ...

  20. Fatty acyl-CoA reductase

    Energy Technology Data Exchange (ETDEWEB)

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

  1. Positive interactive radiosensitization observed in vitro with the combination of two nucleoside analogs (E)-2'-deoxy-2'-(fluoromethylene) cytidine (FMdC) and iododeoxyuridine (IdUrd).

    OpenAIRE

    Coucke, Philippe; Cottin, E.; Azria, D; Martineau, P.; Adamer, F; Decosterd, L-A; Buchegger, F; Do, H-P

    2004-01-01

    (E)-20-Deoxy-20-(fluoromethylene) cytidine (FMdC), an inhibitor of ribonucleotide diphosphate reductase (RR), is a potent radiation-sensitiser acting through alterations in the deoxyribonucleoside triphosphate (dNTP) pool in the de novo pathway to DNA synthesis. The activity of thymidine kinase (TK), a key enzyme in the ‘salvage pathway’, is known to increase in response to a lowering of dATP induced by FMdC. Nucleoside analogues such as iododeoxyuridine (IdUrd) are incorporated into DNA a...

  2. Phase I study of the ribonucleotide reductase inhibitor 3-aminopyridine-2-carboxaldehyde-thiosemicarbazone (3-AP) in combination with high dose cytarabine in patients with advanced myeloid leukemia

    Science.gov (United States)

    Larson, Richard A.; Gajria, Devika; Dolan, M. Eileen; Delaney, Shannon M.; Karrison, Theodore G.; Ratain, Mark J.; Stock, Wendy

    2014-01-01

    Summary Purpose This Phase I dose escalation study was based on the hypothesis that the addition of 3-aminopyridine-2-carboxaldehyde-thiosemicarbazone (3-AP) to cytarabine would enhance cytarabine cytotoxicity. The primary objective of the study was to establish the maximum tolerated dose of 3-AP when given in combination with a fixed dose of cytarabine. Experimental design Twenty-five patients with relapsed or refractory myeloid leukemia were enrolled to three dose levels of 3-AP. Cytarabine was administered as a 2 h infusion at a fixed dose of 1,000 mg/m2/day for 5 consecutive days. Escalating doses of 3-AP as a 2 h infusion were administered on days 2 through 5. The 3-AP infusion preceded the start of the cytarabine infusion by 4 h. Results In general, the toxicities observed with the combination were similar to the expected toxicity profile for cytarabine when utilized as a single agent at this dose and schedule. However, two of three patients developed dose-limiting methemoglobinemia at the highest 3-AP dose studied (100 mg/m2). Transient reversible methemoglobinemia was documented in 11 of 15 patients enrolled at the 75 mg/m2 dose level. Objective evidence of clinical activity was observed in four patients. Conclusions The combination of 3-AP and cytarabine given on this schedule is feasible in advanced myeloid leukemia. The recommended Phase II dose is 75 mg/m2/day of 3-AP on days 2–5 given prior to cytarabine administered at a dose of 1,000 mg/m2/day over 5 consecutive days. Methemoglobinemia is a common toxicity of this combination and requires close monitoring. PMID:18217206

  3. Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase.

    Science.gov (United States)

    Deis, Siobhan M; Doshi, Arpit; Hou, Zhanjun; Matherly, Larry H; Gangjee, Aleem; Dann, Charles E

    2016-08-16

    Pemetrexed and methotrexate are antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo[2,3-d]pyrimidine antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition. PMID:27439469

  4. Identification, Characterization, and Classification of Genes Encoding Perchlorate Reductase

    OpenAIRE

    Bender, Kelly S.; Shang, Ching; Chakraborty, Romy; Belchik, Sara M.; Coates, John D.; Achenbach, Laurie A.

    2005-01-01

    The reduction of perchlorate to chlorite, the first enzymatic step in the bacterial reduction of perchlorate, is catalyzed by perchlorate reductase. The genes encoding perchlorate reductase (pcrABCD) in two Dechloromonas species were characterized. Sequence analysis of the pcrAB gene products revealed similarity to α- and β-subunits of microbial nitrate reductase, selenate reductase, dimethyl sulfide dehydrogenase, ethylbenzene dehydrogenase, and chlorate reductase, all of which are type II m...

  5. Sepiapterin Reductase Deficiency: Mimic of Cerebral Palsy

    OpenAIRE

    J Gordon Millichap

    2012-01-01

    Researchers at University of California at San Diego, and 22 other US national and international centers studied the clinical, biochemical, and molecular findings in a cohort of 38 patients with sepiapterin reductase deficiency (SRD).

  6. Multiple aldehyde reductases of human brain.

    Science.gov (United States)

    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

  7. Whence flavins? Redox-active ribonucleotides link metabolism and genome repair to the RNA world.

    Science.gov (United States)

    Nguyen, Khiem Van; Burrows, Cynthia J

    2012-12-18

    lowered one-electron reduction potential and a red-shifted adsorption spectrum that facilitates excited-state electron transfer in a spectral window that does not produce cyclobutane pyrimidine dimers. Curiously, the best candidate for a primordial flavin is a base damage product, 8-oxo-7,8-dihydroguanine (8-oxoGua or "OG"). Other redox-active ribonucleotides include 5-hydroxycytidine and 5-hydroxyuridine, which display some of the characteristics of flavins, but might also behave like NADH. PMID:23054469

  8. MicroRNA modulation induced by AICA ribonucleotide in J1 mouse ES cells.

    Directory of Open Access Journals (Sweden)

    Xiaoyan Shi

    Full Text Available ES cells can propagate indefinitely, maintain self-renewal, and differentiate into almost any cell type of the body. These properties make them valuable in the research of embryonic development, regenerative medicine, and organ transplantation. MicroRNAs (miRNAs are considered to have essential functions in the maintenance and differentiation of embryonic stem cells (ES cells. It was reported that, strong external stimuli, such as a transient low-pH and hypoxia stress, were conducive to the formation of induced pluripotent stem cells (iPS cells. AICA ribonucleotide (AICAR is an AMP-activated protein kinase activator, which can let cells in the state of energy stress. We have demonstrated that AICAR can maintain the pluripotency of J1 mouse ES cells through modulating protein expression in our previous research, but its effects on ES cell miRNA expression remain unknown. In this study, we conducted small RNA high-throughput sequencing to investigate AICAR influence on J1 mouse ES cells by comparing the miRNA expression patterns of the AICAR-treated cells and those without treatment. The result showed that AICAR can significantly modulate the expression of multiple miRNAs, including those have crucial functions in ES cell development. Some differentially expressed miRNAs were selected and confirmed by real-time PCR. For the differently expressed miRNAs identified, further study was conducted regarding the pluripotency and differentiation associated miRNAs with their targets. Moreover, miR-134 was significantly down-regulated after AICAR treatment, and this was suggested to be directly associated with the up-regulated pluripotency markers, Nanog and Sox2. Lastly, Myc was significantly down-regulated after AICAR treatment; therefore, we predicted miRNAs that may target Myc and identified that AICAR induced up-regulation of miR-34a, 34b, and 34c can repress Myc expression in J1 mouse ES cells. Taken together, our study provide a new mechanism for

  9. Structural and mechanistic insights on nitrate reductases.

    Science.gov (United States)

    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

  10. Respiratory arsenate reductase as a bidirectional enzyme

    International Nuclear Information System (INIS)

    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.

  11. Respiratory arsenate reductase as a bidirectional enzyme

    Science.gov (United States)

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

    2009-01-01

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

  12. 21 CFR 864.7375 - Glutathione reductase assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864... reductase assay. (a) Identification. A glutathione reductase assay is a device used to determine the... fluorescence and photometry. The results of this assay are used in the diagnosis of liver disease,...

  13. Characterization of the chlorate reductase from Pseudomonas chloritidismutans

    NARCIS (Netherlands)

    Wolterink, A.F.W.M.; Schiltz, E.; Hagedoorn, P.L.; Hagen, W.R.; Kengen, S.W.M.; Stams, A.J.M.

    2003-01-01

    A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P. chloritidismutans reduced only chlorate and bromate. Differences were al

  14. Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase: a new approach in PCR-mediated analysis of mRNA sequences.

    OpenAIRE

    Schmidt, W. M.; Mueller, M W

    1996-01-01

    Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase is a polymerase chain reaction (PCR)-mediated technique that was developed to facilitate cloning and direct sequence analysis of complete 5'-terminal unknown coding regions of rare RNA molecules. In contrast with standard tailing protocols using dNTPs as the substrate, ribo-tailing of cDNA ends is easily controllable, self-limited (from two to four rNMP incorporations) and highly efficient (>98%). B...

  15. SCREENING OF HMG CO A REDUCTASE INHIBITOR PRODUCING MARINE ACTINOMYCETES

    OpenAIRE

    SRINU, PHANI BHUSHAN,MOGES, SRILAKSHMI, SANKAR, PRABHAKAR,LAKSHMINARAYANA

    2013-01-01

    The objective of the present study was screening of 3-hydroxy-3- methyl glutaryl Co A (HMG CoA) reductase inhibitor producing marine actinomycetes. A total of 65 morphologically different actinomycetes were screened for HMG CoA reductase inhibitor production in a two stage submerged fermentation and evaluated for HMG CoA reductase inhibitor activity by agar diffusion and thin layer chromatography technique using lovostatin as a control. Among 65 marine Actinomycete strains, only one strain pr...

  16. Characterization of the chlorate reductase from Pseudomonas chloritidismutans

    OpenAIRE

    Wolterink, A.F.W.M.; Schiltz, E; Hagedoorn, P.L.; Hagen, W.R.; Kengen, S.W.M.; Stams, A.J.M.

    2003-01-01

    A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P. chloritidismutans reduced only chlorate and bromate. Differences were also found in N-terminal sequences, molecular weight, and subunit composition. Metal analysis and electron paramagnetic resonance measurements showed the presence of iron and molybdenum, which are al...

  17. Characterization of the Chlorate Reductase from Pseudomonas chloritidismutans

    OpenAIRE

    2003-01-01

    A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P. chloritidismutans reduced only chlorate and bromate. Differences were also found in N-terminal sequences, molecular weight, and subunit composition. Metal analysis and electron paramagnetic resonance measurements showed the presence of iron and molybdenum, which are al...

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

    International Nuclear Information System (INIS)

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

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

    International Nuclear Information System (INIS)

    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 ·/FMNH2 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

  20. Genetic identification of a respiratory arsenate reductase

    OpenAIRE

    Saltikov, Chad W.; Newman, Dianne K.

    2003-01-01

    For more than a decade, it has been recognized that arsenate [H2AsO41-; As(V)] can be used by microorganisms as a terminal electron acceptor in anaerobic respiration. Given the toxicity of arsenic, the mechanistic basis of this process is intriguing, as is its evolutionary origin. Here we show that a two-gene cluster (arrAB; arsenate respiratory reduction) in the bacterium Shewanella sp. strain ANA-3 specifically confers respiratory As(V) reductase activity. Mutants with in-frame deletions of...

  1. Membrane-associated chromate reductase activity from Enterobacter cloacae.

    OpenAIRE

    P. C. Wang; Mori, T.; Toda, K.; Ohtake, H

    1990-01-01

    Washed cells of Enterobacter cloacae HO1 reduced hexavalent chromium (chromate: CrO4(2-) anaerobically. Chromate reductase activity was preferentially associated with the membrane fraction of the cells. Right-side-out membrane vesicles prepared from E. cloacae cells showed high chromate reductase activities when ascorbate-reduced phenazine methosulfate was added as an electron donor.

  2. Canopy and seasonal profiles of nitrate reductase in soybeans

    Energy Technology Data Exchange (ETDEWEB)

    Harper, J.E.; Hageman, R.H.

    1972-01-01

    Nitrate reductase activity of soybeans (Glycine max L. Merr.) was evaluated in soil plots and outdoor hydroponic gravel culture systems throughout the growing season. Nitrate reductase profiles within the plant canopy were also established. Mean activity per gram fresh weight per hour of the entire plant canopy was highest in the seedling stage while total activity (activity per gram fresh weight per hour times the total leaf weight) reached a maximum when plants were in the full bloom to midpod fill stage. Nitrate reductase activity per gram fresh weight per hour was highest in the uppermost leaf just prior to full expansion and declined with leaf positions lower in the canopy. Total nitrate reductase activity per leaf was also highest in the uppermost fully expanded leaf during early growth stages. Maximum total activity shifted to leaf positions lower in the plant canopy with later growth stages. Nitrate reductase activity of soybeans grown in hydroponic systems was significantly higher than activity of adjacent soil grown plants at later growth stages, which suggested that under normal field conditions the potential for nitrate utilization may not be realized. Nitrate reductase activity per gram fresh weight per hour and nitrate content were positively correlated over the growing season with plants grown in either soil or solution culture. Computations based upon the nitrate reductase assay of plants grown in hydroponics indicated that from 1.7 to 1.8 grams N could have been supplied to the plant via the nitrate reductase process. 11 references, 9 figures, 3 tables.

  3. ERYTHROCYTE AND PLASMA-CHOLESTEROL ESTER LONG-CHAIN POLYUNSATURATED FATTY-ACIDS OF LOW-BIRTH-WEIGHT BABIES FED PRETERM FORMULA WITH AND WITHOUT RIBONUCLEOTIDES - COMPARISON WITH HUMAN-MILK

    NARCIS (Netherlands)

    WOLTIL, HA; VANBEUSEKOM, CM; SIEMENSMA, AD; POLMAN, HA; MUSKIET, FAJ; OKKEN, A

    1995-01-01

    We investigated whether a regular formula for premature infants (pre) supplemented with ribonucleotides (pre+RN) raises erythrocyte and plasma cholesterol ester (CE) long-chain polyunsaturated fatty acids (LCPUFAs) of low-birthweight babies (less than or equal to 2.50 kg) compared with their breast-

  4. Structure and expression of human dihydropteridine reductase

    International Nuclear Information System (INIS)

    Dihydropteridine reductase catalyzes the NADH-mediated reduction of quinonoid dihydrobiopterin and is an essential component of the pterindependent aromatic amino acid hydroxylating systems. A cDNA for human DHPR was isolated from a human liver cDNA library in the vector λgt11 using a monospecific antibody against sheep DHPR. The nucleic acid sequence and amino acid sequence of human DHPR were determined from a full-length clone. A 112 amino acid sequence of sheep DHPR was obtained by sequencing purified sheep DHPR. This sequence is highly homologous to the predicted amino acid sequence of the human protein. Gene transfer of the recombinant human DHPR into COS cells leads to expression of DHPR enzymatic activity. These results indicate that the cDNA clone identified by antibody screening is an authentic and full-length cDNA for human DHPR

  5. Nitrite Reductase Activity in Engineered Azurin Variants.

    Science.gov (United States)

    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

  6. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases

    OpenAIRE

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

    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...

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

    OpenAIRE

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

    2012-01-01

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

  8. An overview on 5alpha-reductase inhibitors.

    Science.gov (United States)

    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

  9. Binding of Fidarestat Stereoisomers with Aldose Reductase

    Directory of Open Access Journals (Sweden)

    Dae-Sil Lee

    2006-11-01

    Full Text Available The stereospecificity in binding to aldose reductase (ALR2 of two fidarestat {6-fluoro-2',5'-dioxospiro[chroman-4,4'-imidazolidine]-2-carboxamide} stereoisomers [(2S,4Sand (2R,4S] has been investigated by means of molecular dynamics simulations using freeenergy integration techniques. The difference in the free energy of binding was found to be2.0 ± 1.7 kJ/mol in favour of the (2S,4S-form, in agreement with the experimentalinhibition data. The relative mobilities of the fidarestats complexed with ALR2 indicate alarger entropic penalty for hydrophobic binding of (2R,4S-fidarestat compared to (2S,4S-fidarestat, partially explaining its lower binding affinity. The two stereoisomers differmainly in the orientation of the carbamoyl moiety with respect to the active site and rotationof the bond joining the carbamoyl substituent to the ring. The detailed structural andenergetic insights obtained from out simulations allow for a better understanding of thefactors determining stereospecific inhibitor-ALR2 binding in the EPF charges model.

  10. Aldose reductase inhibitory compounds from Xanthium strumarium.

    Science.gov (United States)

    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

  11. Aldose reductase mediates retinal microglia activation.

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Yeon Bok Kim

    2014-01-01

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

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

    Science.gov (United States)

    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

  14. The role of biliverdin reductase in colorectal cancer

    International Nuclear Information System (INIS)

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

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

    DEFF Research Database (Denmark)

    Farver, Ole; Brunori, Maurizio; Cutruzzolà, Francesca;

    2009-01-01

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

  16. Aldo keto reductases 1B in endocrinology and metabolism

    Directory of Open Access Journals (Sweden)

    AntoineMartinez

    2012-08-01

    Full Text Available The aldose reductase (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 aldose reductase function in non-diabetic condition remains unresolved. Aldose reductase-like enzymes (AKR1B10, Akr1b7 and Akr1b8 are highly related isoforms often co-expressed with bona fide aldose reductase, 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 aldose reductase shows ubiquitous expression, aldose reductase-like proteins exhibit tissue-specific patterns of expression. We focused on 3 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

  17. Expression and site-directed mutagenesis of human dihydrofolate reductase

    International Nuclear Information System (INIS)

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 → Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by α-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme

  18. Effect of vanadium on nitrate reductase activity in tomato leaves

    OpenAIRE

    J. Buczek

    2015-01-01

    The activity of nitrate reductase in cell-free extracts from tomato leaves is completely inhibited by 100 μM NaVO3 or VOCl2. In experiments in vivo vanadium ions inhibit the activity of the enzyme in 50 to 60 per cent. Addition of l mM vanadium to the medium on which tomato seedlings are grown causes after 24 h almost complete inhibition of nitrate reductase activity in cell-free extracts of the enzyme. Inhibition with vanadium may be abolished in experiments in vitro if the extract is treate...

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

    DEFF Research Database (Denmark)

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

    1998-01-01

    The intramolecular electron transfer (ET) between the type 1 Cu(I) and the type 2 Cu(II) sites of Alcaligenes xylosoxidans dissimilatory nitrite reductase (AxNiR) has been studied in order to compare it with the analogous process taking place in ascorbate oxidase (AO). This internal process is......(I) and the trinuclear copper centre in ascorbate oxidase, and the characteristics of the internal ET processes of these enzymes are compared. The data are consistent with the faster ET observed in nitrite reductase arising from a more advantageous entropy of activation when compared with ascorbate...

  20. Artificial electron donors for nitrate and nitrite reductases usable as mediators in amperometric biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Strehlitz, B. (Umweltforschungszentrum Leipzig-Halle GmbH, Leipzig (Germany)); Gruendig, B. (Institut fuer Chemo- und Biosensorik, Muenster-Roxel (Germany)); Vorlop, K.D. (Bundesforschungsanstalt fuer Landwirtschaft, Braunschweig (Germany). Inst. fuer Technologie); Bartholmes, P. (Witten-Herdecke Univ., Witten (Germany). Inst. fuer Biochemie); Kotte, H. (Umweltforschungszentrum Leipzig-Halle GmbH, Leipzig (Germany)); Stottmeister, U. (Umweltforschungszentrum Leipzig-Halle GmbH, Leipzig (Germany))

    1994-07-01

    Various nitrate and nitrite reductases are capable of accepting electrons from artificial donors. Combining these redox active donors with an amperometric redox electrode which is covered with an immobilized layer of such a nitrate or nitrite reductase, new enzyme sensors can be created for the detection of nitrate or nitrite, respectively. A range of suitable electron donors for nitrate reductases and nitrite reductase from different sources have been selected and characterized by electrochemical methods. (orig.)

  1. Inhibition of Albendazole and Oxfendazole on the Activity of Fumaric Reductase in Cysticercus cellulosae

    Institute of Scientific and Technical Information of China (English)

    GAO Xue-jun; LI Qing-zhang; LI Xia

    2004-01-01

    The activity of fumaric reductase in Cysticercus cellulosae tissue homogenate with albendazole and oxfendazole individually was detected. Results showed that the two kinds of drugs both could inhabite the activity of fumaric reductase. The results indicate that the mechanism of action of benzimidazole carbamate drugs is probably inhabiting the complex of fumaric reductase noncompetently, thus lead to the exhaostion of energy and death.

  2. Sepiapterin reductase deficiency an autosomal recessive DOPA-responsive dystonia

    NARCIS (Netherlands)

    N.G. Abeling; M. Duran; H.D. Bakker; L. Stroomer; B. Thony; N. Blau; J. Booij; B.T. Poll-The

    2006-01-01

    The diagnosis of a 14-year-old girl with a new homoallelic mutation in the sepiapterin reductase (SR) gene is reported. Initially she presented at the age of 2 with hypotonia and mild cognitive developmental delay, and was diagnosed as having mild methylmalonic aciduria, which was recently identifie

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

    Science.gov (United States)

    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.

  4. Bidirectional catalysis by copper-containing nitrite reductase

    NARCIS (Netherlands)

    Wijma, HJ; Canters, GW; de Vries, S; Verbeet, MP

    2004-01-01

    The copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the oxidation of nitric oxide to nitrite, the reverse of its physiological reaction. Thermodynamic and kinetic constants with the physiological electron donor pseudoazurin were determined for both directions

  5. Thioredoxin reductase from s. Coelicolor as a drug target

    Czech Academy of Sciences Publication Activity Database

    Koháryová, M.; Brynda, Jiří; Řezáčová, Pavlína; Kollárová, M.

    2015-01-01

    Roč. 282, Suppl 1 (2015), s. 396. ISSN 1742-464X. [Congress of the Federation of European Biochemical Societies (FEBS) - The Biochemical Basis of Life /40./. 04.07.2015-09.07.2015, Berlin] Institutional support: RVO:61388963 ; RVO:68378050 Keywords : thioredoxin reductase * protein structure * drugs Subject RIV: CE - Biochemistry

  6. Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene

    DEFF Research Database (Denmark)

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

    1989-01-01

    Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi...

  7. Fatty Acyl-CoA Reductase 1 Deficiency

    Directory of Open Access Journals (Sweden)

    Charles N Swisher

    2015-01-01

    Full Text Available Investigators from Erlangen, Germany; Calgary, CA; and Kafranbel, Syria, identified mutations in the gene, fatty acyl-CoA reductase 1 (FAR1 deficiency, adding to three other genes involved in plasmalogen biosynthesis, in two families affected by severe intellectual disability, early-onset epilepsy, microcephaly, congenital cataracts, growth retardation, and spasticity.

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

    Science.gov (United States)

    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.

  9. Management of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone-induced methemoglobinemia

    OpenAIRE

    Kunos, Charles A.; Radivoyevitch, Tomas; Ingalls, Stephen T.; Hoppel, Charles L.

    2012-01-01

    The anticancer agent 3-aminopyridine-2-carboxaldehyde thiosemicarbazone is a ribonucleotide reductase inhibitor. It inactivates ribonucleotide reductase by disrupting an iron-stabilized radical in ribonucleotide reductase's small subunits, M2 and M2b (p53R2). Unfortunately, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone also alters iron II (Fe2+) in hemoglobin. This creates Fe3+ methemoglobin that does not deliver oxygen. Fe2+ in hemoglobin normally auto-oxidizes to inactive Fe3+ methemog...

  10. Rapid Identification of Aldose Reductase Inhibitory Compounds from Perilla frutescens

    OpenAIRE

    Soon Sung Lim; Ji Hun Paek; Kuk Hyun Shin; Young-Hee Kang; Jae-Yong Lee

    2013-01-01

    The ethyl acetate (EtOAc) soluble fraction of methanol extracts of Perilla frutescens (P. frutescens) inhibits aldose reductase (AR), the key enzyme in the polyol pathway. Our investigation of inhibitory compounds from the EtOAc soluble fraction of P. frutescens was followed by identification of the inhibitory compounds by a combination of HPLC microfractionation and a 96-well enzyme assay. This allowed the biological activities to be efficiently matched with selected HPLC peaks. Structural a...

  11. Determination of plasma gluthatione reductase enzyme activity in osteoporotic women

    OpenAIRE

    Sadeghi N; Oveisi M.R.; Jannat B.; Hajimahmoodi M; Jamshidi A.R; Sajadian Z.

    2008-01-01

    Background: Osteoporosis is a disease of high prevalence with increased bone loss. Free radicals have been proved to be involved in bone resorption. Glutathione reductase (GR) plays an essential role in cell defense against reactive oxygen metabolites by sustaining the reduced status of an important antioxidant, glutathione. In the present study GR activity of plasma as an antioxidant enzyme in relation to Bone Mineral Density (BMD) was investigated.Material and Method: GR activity was measur...

  12. Glutathione reductase: solvent equilibrium and kinetic isotope effects

    International Nuclear Information System (INIS)

    Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D2O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456

  13. Internal electron transfer within mitochondrial succinate-cytochrome C reductase

    International Nuclear Information System (INIS)

    Internal electron transfer within succinate-cytochrome C reductase from pigeon breast muscle mitochondria was followed by the pulse radiolytic technique. The electron equivalent is transferred from an unknown donor to b type cytochrome(s), in a first order process with a rate constant of: 660 +- 150s-1. This process might be the rate determining step of electron transfer in mitochondria, since it is similar in rate to the turnover number of the mitochondrial respiratory chain

  14. Aldo-keto reductase (AKR) superfamily: Genomics and annotation

    OpenAIRE

    Mindnich Rebekka D; Penning Trevor M

    2009-01-01

    Abstract Aldo-keto reductases (AKRs) are phase I metabolising enzymes that catalyse the reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)-dependent reduction of carbonyl groups to yield primary and secondary alcohols on a wide range of substrates, including aliphatic and aromatic aldehydes and ketones, ketoprostaglan-dins, ketosteroids and xenobiotics. In so doing they functionalise the carbonyl group for conjugation (phase II enzyme reactions). Although functionally diverse, AK...

  15. Phosphoglycerate kinase acts in tumour angiogenesis as a disulphide reductase

    Science.gov (United States)

    Lay, Angelina J.; Jiang, Xing-Mai; Kisker, Oliver; Flynn, Evelyn; Underwood, Anne; Condron, Rosemary; Hogg, Philip J.

    2000-12-01

    Disulphide bonds in secreted proteins are considered to be inert because of the oxidizing nature of the extracellular milieu. An exception to this rule is a reductase secreted by tumour cells that reduces disulphide bonds in the serine proteinase plasmin. Reduction of plasmin initiates proteolytic cleavage in the kringle 5 domain and release of the tumour blood vessel inhibitor angiostatin. New blood vessel formation or angiogenesis is critical for tumour expansion and metastasis. Here we show that the plasmin reductase isolated from conditioned medium of fibrosarcoma cells is the glycolytic enzyme phosphoglycerate kinase. Recombinant phosphoglycerate kinase had the same specific activity as the fibrosarcoma-derived protein. Plasma of mice bearing fibrosarcoma tumours contained several-fold more phosphoglycerate kinase, as compared with mice without tumours. Administration of phosphoglycerate kinase to tumour-bearing mice caused an increase in plasma levels of angiostatin, and a decrease in tumour vascularity and rate of tumour growth. Our findings indicate that phosphoglycerate kinase not only functions in glycolysis but is secreted by tumour cells and participates in the angiogenic process as a disulphide reductase.

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

    Science.gov (United States)

    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

  17. Cloning and sequence of the human adrenodoxin reductase gene

    International Nuclear Information System (INIS)

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

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

    OpenAIRE

    Jolanta Marciniak; Grażyna Kłobus; Józef Buczek; Tadeusz Stefaniak; Jarosław Mazur

    2014-01-01

    Soluble nitrate reductase from cucumber roots (Cucumis sativus L.) was isolated and purified with blue-Sepharose 4B. Specific antibodies against the NR protein were raised by immunization of a goat. Using polyclonal antibodies anti-NR properties of the nitrate reductase from various cucumber tissues were examined. Experiments showed difference in immuno-logical properties of nitrate reductase (NR) from cotyledon roots and leaves.

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

    Directory of Open Access Journals (Sweden)

    Jolanta Marciniak

    2014-02-01

    Full Text Available Soluble nitrate reductase from cucumber roots (Cucumis sativus L. was isolated and purified with blue-Sepharose 4B. Specific antibodies against the NR protein were raised by immunization of a goat. Using polyclonal antibodies anti-NR properties of the nitrate reductase from various cucumber tissues were examined. Experiments showed difference in immuno-logical properties of nitrate reductase (NR from cotyledon roots and leaves.

  20. Histochemical Localization of Glutathione Dependent NBT-Reductase in Mouse Skin

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective Localization of the glutathione dependent Nitroblue tetrazolium (NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated. Methods The fresh frozen tissue sections (8m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. Results  The activity of the NBT-reductase in mouse skin has been found to be localized in the areas rich in glutathione and actively proliferating area of the skin. Conclusion The activity of the NBT-reductase seems to be dependent on the glutathione contents.

  1. biliverdin. Is there a role for biliverdin reductase?

    Directory of Open Access Journals (Sweden)

    AndreasDaiber

    2012-03-01

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

  2. Autoimmunity in Membranous Nephropathy Targets Aldose Reductase and SOD2

    OpenAIRE

    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 biopsi...

  3. Applications of Carboxylic Acid Reductases in Oleaginous Microbes

    Energy Technology Data Exchange (ETDEWEB)

    Resch, Michael G.; Linger, Jeffrey; McGeehan, John; Tyo, Keith; Beckham, Gregg

    2016-04-24

    Carboxylic acid reductases (CARs) are recently emerging reductive enzymes for the direct production of aldehydes from biologically-produced carboxylic acids. Recent work has demonstrated that these powerful enzymes are able to reduce a very broad range of volatile- to long-chain fatty acids as well as aromatic acids. Here, we express four CAR enzymes from different fungal origins to test their activity against fatty acids commonly produced in oleaginous microbes. These in vitro results will inform metabolic engineering strategies to conduct mild biological reduction of carboxylic acids in situ, which is conventionally done via hydrotreating catalysis at high temperatures and hydrogen pressures.

  4. Mediated electrochemistry of dimethyl sulfoxide reductase promoted by carbon nanotubes

    Institute of Scientific and Technical Information of China (English)

    BERNHARDT; Paul; V

    2010-01-01

    Mediated electrochemistry of dimethyl sulfoxide reductase from Rhodobacter capsulatus (DMSOR) which is immobilized on a bare glassy carbon (GC) electrode and a carbon nanotube (CNT)-modified GC electrode was studied using the Co complex (trans-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine)cobalt(III) ([Co(trans-diammac)] +) as a mediator.The cyclic voltammograms of different electrodes were carried out at different substrate (DMSO) concentrations.The results demonstrated that the catalytic current was increased by employing CNT as a promoter.

  5. Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

    OpenAIRE

    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 f...

  6. Pulse radiolysis studies on superoxide reductase from Treponema pallidum.

    OpenAIRE

    Nivière, V; Lombard, M.; Fontecave, M.; Houée-Levin, C

    2001-01-01

    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 ...

  7. Methylenetetrahydrofolate reductase (MTHFR) deficiency presenting as a rash.

    LENUS (Irish Health Repository)

    Crushell, Ellen

    2012-09-01

    We report on the case of a 2-year-old girl recently diagnosed with Methylenetetrahydrofolate reductase (MTHFR) deficiency who originally presented in the neonatal period with a distinctive rash. At 11 weeks of age she developed seizures, she had acquired microcephaly and developmental delay. The rash deteriorated dramatically following commencement of phenobarbitone; both rash and seizures abated following empiric introduction of pyridoxine and folinic acid as treatment of possible vitamin responsive seizures. We postulate that phenobarbitone in combination with MTHFR deficiency may have caused her rash to deteriorate and subsequent folinic acid was helpful in treating the rash and preventing further acute neurological decline as commonly associated with this condition.

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

    Science.gov (United States)

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

    Science.gov (United States)

    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 ...

  10. Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription

    DEFF Research Database (Denmark)

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

    1992-01-01

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

  11. Functions of Flavin Reductase and Quinone Reductase in 2,4,6-Trichlorophenol Degradation by Cupriavidus necator JMP134▿

    OpenAIRE

    Belchik, Sara Mae; Xun, Luying

    2007-01-01

    The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH2, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for t...

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

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-02-01

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

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

    OpenAIRE

    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 ...

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

    Science.gov (United States)

    Khodashenas, Bahareh; Ghorbani, Hamid Reza

    2016-06-01

    Today, the synthesis of silver nanoparticles (Ag NPs) is very common since it has many applications in different areas. The synthesis of these nanoparticles is done by means of physical, chemical, or biological methods. However, due to its inexpensive and environmentally friendly features, the biological method is more preferable. In the present study, using nitrate reductase enzyme available in the Escherichia coli (E. coli) bacterium, the biosynthesis of Ag NPs was investigated. In addition, the activity of the nitrate reductase enzyme was optimised by changing its cultural conditions, and the effects of silver nitrate (AgNO3) 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 (AgNO3) with 1, 5, 10, and 20 cc supernatant and 435 nm for 0.01M AgNO3 with 20 cc supernatant. In this study, it was found that there is a direct relationship between the AgNO3 concentration and the size of produced Ag NPs. PMID:27256897

  15. Azotobacter vinelandii NADPH:ferredoxin reductase cloning, sequencing, and overexpression.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  17. Pinpointing a Mechanistic Switch Between Ketoreduction and "Ene" Reduction in Short-Chain Dehydrogenases/Reductases.

    Science.gov (United States)

    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

  18. Trypanothione Reductase: A Viable Chemotherapeutic Target for Antitrypanosomal and Antileishmanial Drug Design

    Directory of Open Access Journals (Sweden)

    M. Omar F. Khan

    2007-01-01

    Full Text Available Trypanosomiasis and leishmaniasis are two debilitating disease groups caused by parasites of Trypanosoma and Leishmania spp. and affecting millions of people worldwide. A brief outline of the potential targets for rational drug design against these diseases are presented, with an emphasis placed on the enzyme trypanothione reductase. Trypanothione reductase was identified as unique to parasites and proposed to be an effective target against trypanosomiasis and leishmaniasis. The biochemical basis of selecting this enzyme as a target, with reference to the simile and contrast to human analogous enzyme glutathione reductase, and the structural aspects of its active site are presented. The process of designing selective inhibitors for the enzyme trypanothione reductase has been discussed. An overview of the different chemical classes of inhibitors of trypanothione reductase with their inhibitory activities against the parasites and their prospects as future chemotherapeutic agents are briefl y revealed.

  19. Functional complementation of a nitrate reductase defective mutant of a green alga Dunaliella viridis by introducing the nitrate reductase gene.

    Science.gov (United States)

    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

  20. Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

    Science.gov (United States)

    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

  1. Structure of a bacterial homologue of vitamin K epoxide reductase

    Energy Technology Data Exchange (ETDEWEB)

    Li, Weikai; Schulman, Sol; Dutton, Rachel J.; Boyd, Dana; Beckwith, Jon; Rapoport, Tom A. (Harvard-Med); (HHMI)

    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.

  2. Two methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms, schizophrenia and bipolar disorder

    DEFF Research Database (Denmark)

    Jönsson, Erik G; Larsson, Kristina; Vares, Maria;

    2008-01-01

    disorder. In a replication attempt the MTHFR C677T and A1298C SNPs were analyzed in three Scandinavian schizophrenia case-control samples. In addition, Norwegian patients with bipolar disorder were investigated. There were no statistically significant allele or genotype case-control differences. The...... present Scandinavian results do not verify previous associations between the putative functional MTHFR gene polymorphisms and schizophrenia or bipolar disorder. However, when combined with previous studies in meta-analyses there is still evidence for association between the MTHFR C677T polymorphism and......Recent meta-analyses of the methylenetetrahydrofolate reductase gene (MTHFR) have suggested association between two of its functional single gene polymorphisms (SNPs; C677T and A1298C) and schizophrenia. Studies have also suggested association between MTHFR C677T and A1298C variation and bipolar...

  3. Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).

    Science.gov (United States)

    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

  4. Pulse radiolysis studies on superoxide reductase from Treponema pallidum

    CERN Document Server

    Nivière, V; Fontecave, M; Houée-Levin, C

    2015-01-01

    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.

  5. Two methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms, schizophrenia and bipolar disorder

    DEFF Research Database (Denmark)

    Jönsson, Erik G; Larsson, Kristina; Vares, Maria; Hansen, Thomas; Wang, August G; Djurovic, Srdjan; Rønningen, Kjersti S; Andreassen, Ole A; Agartz, Ingrid; Werge, Thomas; Terenius, Lars; Hall, Håkan

    disorder. In a replication attempt the MTHFR C677T and A1298C SNPs were analyzed in three Scandinavian schizophrenia case-control samples. In addition, Norwegian patients with bipolar disorder were investigated. There were no statistically significant allele or genotype case-control differences. The...... present Scandinavian results do not verify previous associations between the putative functional MTHFR gene polymorphisms and schizophrenia or bipolar disorder. However, when combined with previous studies in meta-analyses there is still evidence for association between the MTHFR C677T polymorphism and......Recent meta-analyses of the methylenetetrahydrofolate reductase gene (MTHFR) have suggested association between two of its functional single gene polymorphisms (SNPs; C677T and A1298C) and schizophrenia. Studies have also suggested association between MTHFR C677T and A1298C variation and bipolar...

  6. Isolation and characterization of nitric oxide reductase from Paracoccus halodenitrificans.

    Science.gov (United States)

    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

  7. Identification of imine reductase-specific sequence motifs.

    Science.gov (United States)

    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

  8. Fatty acyl-CoA reductases of birds

    Directory of Open Access Journals (Sweden)

    Hellenbrand Janine

    2011-12-01

    Full Text Available Abstract Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba, domestic chicken (Gallus gallus domesticus and domestic goose (Anser anser domesticus. Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.

  9. Evidence for a hexaheteromeric methylenetetrahydrofolate reductase in Moorella thermoacetica.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  11. The use of 5-alpha reductase inhibitors for the prevention of prostate cancer.

    Science.gov (United States)

    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

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from...... Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan...

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

    Science.gov (United States)

    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

  14. Steroidal pyrazolines evaluated as aromatase and quinone reductase-2 inhibitors for chemoprevention of cancer.

    Science.gov (United States)

    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

  15. Association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and age of onset in schizophrenia

    DEFF Research Database (Denmark)

    Vares, Maria; Saetre, Peter; Deng, Hong;

    2010-01-01

    Different lines of evidence indicate that methylenetetrahydrofolate reductase (MTHFR) functional gene polymorphisms, causative in aberrant folate-homocysteine metabolism, are associated with increased vulnerability to several heritable developmental disorders. Opposing views are expressed...

  16. Histochemical Localization of Glutathione Dependent NBT—Reductase in Mouse Skin

    Institute of Scientific and Technical Information of China (English)

    YOESHWERSHUKLA

    2001-01-01

    Objective:Localization of the glutathione dependent Nitroblue tetrazolium(NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated.Methods:The fresh frozen tissue sections(8m thickness)were prepared and incuated in medium containing NBT,reduced glutathione(GSH) and Phosphate uffer,The staining for GSH was performed with mercury orange.Results:The activity of the NBT-reductase in mouse skin has een found to be localized in the areas rich in glutatione and actively proliferating area of the skin.Conclusion:The activity of the NBT-reductase seems to be dependent on the glutatione contents.

  17. Redesigning alcohol dehydrogenases/reductases for more efficient biosynthesis of enantiopure isomers.

    Science.gov (United States)

    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

  18. 5α-reductase inhibitors, antiviral and anti-tumor activities of some steroidal cyanopyridinone derivatives.

    Science.gov (United States)

    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

  19. Comparison of the Stereospecificity and Immunoreactivity of NADH-Ferricyanide Reductases in Plant Membranes

    OpenAIRE

    Fredlund, Kenneth M.; Struglics, André; Widell, Susanne; ASKERLUND, Per; Kader, Jean-Claude; Møller, Ian M.

    1994-01-01

    The substrate stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane and peroxisomal membrane of potato (Solanum tuberosum L.) tubers, spinach (Spinacea oleracea L.) leaf plasma membrane, and red beetroot (Beta vulgaris L.) tonoplast were all specific for the [beta]-hydrogen of NADH, whereas the reductases in wheat root (Triticum aestivum L.) endoplasmic reticulum and potato tuber outer mitochondrial membrane were both [alpha]-hydrogen specific. In all...

  20. Nucleotide sequence of a cyanobacterial nifH gene coding for nitrogenase reductase

    OpenAIRE

    Mevarech, Moshe; Rice, Douglas; Haselkorn, Robert

    1980-01-01

    The nucleotide sequence of nifH, the structural gene for nitrogenase reductase (component II or Fe protein of nitrogenase) from the cyanobacterium Anabaena 7120 has been determined. Also reported are 194 bases of the 5′-flanking sequence and 170 bases of the 3′-flanking sequence. The predicted amino acid sequence was compared with that determined for the complete nitrogenase reductase of Clostridium pasteurianum and the cysteine-containing peptides of the protein from Azotobacter vinelandii. ...

  1. Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean

    OpenAIRE

    Cheng, Qun; Li, Ninghui; Dong, Lidong; Zhang, Dayong; Fan, Sujie; Jiang, Liangyu; Wang, Xin; Xu, Pengfei; Zhang, Shuzhen

    2015-01-01

    Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatmen...

  2. Design and synthesis of hepatoselective, pyrrole-based HMG-CoA reductase inhibitors.

    Science.gov (United States)

    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

  3. Protein Method for Investigating Mercuric Reductase Gene Expression in Aquatic Environments

    OpenAIRE

    Ogunseitan, O A

    1998-01-01

    A colorimetric assay for NADPH-dependent, mercuric ion-specific oxidoreductase activity was developed to facilitate the investigation of mercuric reductase gene expression in polluted aquatic ecosystems. Protein molecules extracted directly from unseeded freshwater and samples seeded with Pseudomonas aeruginosa PU21(Rip64) were quantitatively assayed for mercuric reductase activity in microtiter plates by stoichiometric coupling of mercuric ion reduction to a colorimetric redox chain through ...

  4. Molecular pharmacology and antitumor activity of palmarumycin based inhibitors of thioredoxin reductase.

    OpenAIRE

    Powis, Garth; Wipf, Peter; Lynch, Stephen M.; Birmingham, Anne; Kirkpatrick, D. Lynn

    2006-01-01

    The cytosolic thioredoxin (Trx) redox system comprising Trx-1 and the NADPH dependent thioredoxin reductase -1 reductase (TrxR-1) is an important regulator of cell growth and survival. Trx-1 is overexpressed in many human tumors where it is associated with increased cell proliferation, decreased apoptosis and decreased patient survival. We hypothesized that TrxR-1 provides a target to inhibit the activity of overexpressed Trx-1 for the development of novel anticancer agents. We found that the...

  5. Soil acidity as affecting micronutrients concentration, nitrato reductase enzyme activity and yield in upland rice plants

    OpenAIRE

    Edemar Moro; Carlos Alexandre Costa Crusciol; Heitor Cantarella; Adriano Stephan Nascente; Adriana Lima Moro; Fernando Broetto

    2013-01-01

    The lowest grain yield of rice under no-tillage system (NTS) in relation to the conventional system may be due to the predominance nitrate in the soil and the low nitrate reductase activity. Another reason may be caused by micronutrient deficiency because of superficially soil acidity corrections. Therefore, the objective of this study was to evaluate the changes caused by soil pH in the N forms in the soil, micronutrients concentration in rice plants, nitrate reductase activity, yield of ric...

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

    OpenAIRE

    M.T. Goodarzi; Zal, F; M. Malakooti; M. R. Safari S. Sadeghian

    2006-01-01

    Aldose reductase is a critical enzyme in the polyol pathway that plays an important role in diabetes mellitus. Inhibition of the activity of this enzyme can prevent cataract in diabetic patients’lenses. In this study the inhibitory effect of two flavonoids, quercetin and naringin, in the activity of aldose reductase in streptozotocin-induced diabetic and healthy rats were investigated. Thirty male rats were divided in six groups. The first, second and third group were healthy rats that receiv...

  7. Glutathione Reductase/Glutathione Is Responsible for Cytotoxic Elemental Sulfur Tolerance via Polysulfide Shuttle in Fungi*

    OpenAIRE

    Sato, Ikuo; Shimatani, Kanami; Fujita, Kensaku; Abe, Tsuyoshi; Shimizu, Motoyuki; Fujii, Tatsuya; Hoshino, Takayuki; Takaya, Naoki

    2011-01-01

    Fungi that can reduce elemental sulfur to sulfide are widely distributed, but the mechanism and physiological significance of the reaction have been poorly characterized. Here, we purified elemental sulfur-reductase (SR) and cloned its gene from the elemental sulfur-reducing fungus Fusarium oxysporum. We found that NADPH-glutathione reductase (GR) reduces elemental sulfur via glutathione as an intermediate. A loss-of-function mutant of the SR/GR gene generated less sulfide from elemental sulf...

  8. Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors.

    OpenAIRE

    Subramani, S.; Mulligan, R.; Berg, P

    1981-01-01

    A mouse complementary deoxyribonucleic acid segment coding for the enzyme dihydrofolate reductase has been cloned in two general classes of vectors containing simian virus 40 deoxyribonucleic acid: (i) those that can be propagated as virions in permissive cells and (ii) those that can be introduced into and maintained stably in various mammalian cells. Both types of vectors express the mouse dihydrofolate reductase by using signals supplied by simian virus 40 deoxyribonucleic acid sequences. ...

  9. Interaction of Product Analogs with the Active Site of Rhodobacter sphaeroides Dimethylsulfoxide Reductase

    OpenAIRE

    George, Graham N.; Nelson, Kimberly Johnson; Harris, Hugh H.; Doonan, Christian J.; Rajagopalan, K V

    2007-01-01

    We report a structural characterization using X-ray absorption spectroscopy of Rhodobacter sphaeroides dimethylsulfoxide (DMSO) reductase reduced with trimethylarsine, and show that this is structurally analogous to the physiologically relevant dimethylsulfide-reduced DMSO reductase. Our data unambiguously indicate that these species should be regarded as formal MoIV species, and indicate a classical coordination complex of trimethylarsine oxide, with no special structural distortions. The si...

  10. In Silico Docking studies of Aldose Reductase Inhibitory activity of selected Flavonoids

    OpenAIRE

    Muthuswamy Umamaheswari; C. S. Aji; Kuppusamy Asokkumar; Thirumalaisamy Sivashanmugam; Varadharajan Subhadradevi; Puliyath Jagannath; Arumugam Madeswaran

    2012-01-01

    New drugs for the inhibition of the enzyme aldose reductase are in development and they have to be screened before being considered for preclinical and clinical evaluation. The current study deals with the evaluation of the cyclooxygenase inhibitory activity of flavonoids using in silico docking studies. In this perspective, flavonoids like Farobin-A, Gericudranin- B, Glaziovianin-A, Rutin, and Xanthotoxin were selected. Epalrestat, a known aldose reductase inhibitor was used as the standard....

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

    OpenAIRE

    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 ...

  12. The role of glutathione reductase and related enzymes on cellular redox homoeostasis network.

    Science.gov (United States)

    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

  13. INHIBITION OF TYPE I 5α-REDUCTASE BY MEDICINAL PLANT EXTRACTS

    Directory of Open Access Journals (Sweden)

    Patil Vijaya

    2011-03-01

    Full Text Available Type I 5α-reductase has been implicated in skin disorders such as acne, hirsutism and male pattern baldness and its inhibition offers a potential treatment for these disorders. The aim of this study was to investigate the inhibition of type I 5α-reductase activity by extracts from Indian medicinal plants. Plant extracts were screened and selected based on their ability to inhibit Propionibacterium acnes and Staphylococcus epidermidis. Since type I 5α-reductase metabolises testosterone to Δ4-androstene-3, 17-dione, the activity of enzyme was determined using RIA for testosterone and Δ4-androstene-3, 17-dione. It was found that methanolic extract of Embelia ribes was a potent inhibitor of type I 5α-reductase (IC50:100μg/mL. Extracts of Vitex negundo, Terminalia chebula, and Terminalia bellerica which also inhibited type I 5α-reductase (IC50: 200-390 μg /mL. Therefore herbal formulation of these plant extracts may be used in the treatment of skin disorders involving type I 5α-reductase.

  14. Nitrate reductase activity and its relationship with applied nitrogen in soybean

    International Nuclear Information System (INIS)

    Field experiments were conducted to study the nitrate reductase activity and its relationship to nitrogen by using frame tests (pot without bottom), sand culture and 15N-urea at transplanting in soybean variety Suinong 14. Results showed that the activity of nitrate reductase in leaf changed as a signal peak curve with the soybean growth, lower in vegetative growth phase, higher in reproductive growth period and reached the peak in blooming period, then decreased gradually. Nitrogen application showed obvious effect on the nitrate reductase activity. The activities of nitrate reductase in leaves followed the order of N135 > N90 > N45 > N0 in vegetative growth stage, no clear regularity was found during the whole reproductive growth period. The activities of nitrate reductase in leaves were accorded with the order of upper leaves > mid leaves > lower leaves, and it was very significant differences (P15N labeling method during beginning seed stage and full seed stage shown that 15N abundance in various organs at different node position also followed the same order, suggesting that high level of nitrate reductase activity at upper leaves of soybean promoted the assimilation of NO3-. (authors)

  15. Isobutyraldehyde production from Escherichia coli by removing aldehyde reductase activity

    Directory of Open Access Journals (Sweden)

    Rodriguez Gabriel M

    2012-06-01

    Full Text Available Abstract Background Increasing global demand and reliance on petroleum-derived chemicals will necessitate alternative sources for chemical feedstocks. Currently, 99% of chemical feedstocks are derived from petroleum and natural gas. Renewable methods for producing important chemical feedstocks largely remain unaddressed. Synthetic biology enables the renewable production of various chemicals from microorganisms by constructing unique metabolic pathways. Here, we engineer Escherichia coli for the production of isobutyraldehyde, which can be readily converted to various hydrocarbons currently derived from petroleum such as isobutyric acid, acetal, oxime and imine using existing chemical catalysis. Isobutyraldehyde can be readily stripped from cultures during production, which reduces toxic effects of isobutyraldehyde. Results We adopted the isobutanol pathway previously constructed in E. coli, neglecting the last step in the pathway where isobutyraldehyde is converted to isobutanol. However, this strain still overwhelmingly produced isobutanol (1.5 g/L/OD600 (isobutanol vs 0.14 g/L/OD600 (isobutyraldehyde. Next, we deleted yqhD which encodes a broad-substrate range aldehyde reductase known to be active toward isobutyraldehyde. This strain produced isobutanol and isobutyraldehyde at a near 1:1 ratio, indicating further native isobutyraldehyde reductase (IBR activity in E. coli. To further eliminate isobutanol formation, we set out to identify and remove the remaining IBRs from the E. coli genome. We identified 7 annotated genes coding for IBRs that could be active toward isobutyraldehyde: adhP, eutG, yiaY, yjgB, betA, fucO, eutE. Individual deletions of the genes yielded only marginal improvements. Therefore, we sequentially deleted all seven of the genes and assessed production. The combined deletions greatly increased isobutyraldehyde production (1.5 g/L/OD600 and decreased isobutanol production (0.4 g/L/OD600. By assessing production by

  16. Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis

    Institute of Scientific and Technical Information of China (English)

    Mee-Kyung; Cha; Yoo-Jeen; Bae; Kyu-Jeong; Kim; Byung-Joon; Park; Il-Han; Kim

    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, C47 S, C166 S, C37/47 S, C37/166 S, C47/166 S, and C37/47/166 S for AhpC_H1; C52 S, C169 S, and C52/169 S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahp C 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

  17. Binding of Natural and Synthetic Polyphenols to Human Dihydrofolate Reductase

    Directory of Open Access Journals (Sweden)

    José Neptuno Rodríguez-López

    2009-12-01

    Full Text Available Dihydrofolate reductase (DHFR is the subject of intensive investigation since it appears to be the primary target enzyme for antifolate drugs. Fluorescence quenching experiments show that the ester bond-containing tea polyphenols (--epigallocatechin gallate (EGCG and (--epicatechin gallate (ECG are potent inhibitors of DHFR with dissociation constants (KD of 0.9 and 1.8 μM, respectively, while polyphenols lacking the ester bound gallate moiety [e.g., (--epigallocatechin (EGC and (--epicatechin (EC] did not bind to this enzyme. To avoid stability and bioavailability problems associated with tea catechins we synthesized a methylated derivative of ECG (3-O-(3,4,5-trimethoxybenzoyl-(--epicatechin; TMECG, which effectively binds to DHFR (KD = 2.1 μM. In alkaline solution, TMECG generates a stable quinone methide product that strongly binds to the enzyme with a KD of 8.2 nM. Quercetin glucuronides also bind to DHFR but its effective binding was highly dependent of the sugar residue, with quercetin-3-xyloside being the stronger inhibitor of the enzyme with a KD of 0.6 μM. The finding that natural polyphenols are good inhibitors of human DHFR could explain the epidemiological data on their prophylactic effects for certain forms of cancer and open a possibility for the use of natural and synthetic polyphenols in cancer chemotherapy.

  18. Rapid identification of aldose reductase inhibitory compounds from Perilla frutescens.

    Science.gov (United States)

    Paek, Ji Hun; Shin, Kuk Hyun; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

    2013-01-01

    The ethyl acetate (EtOAc) soluble fraction of methanol extracts of Perilla frutescens (P. frutescens) inhibits aldose reductase (AR), the key enzyme in the polyol pathway. Our investigation of inhibitory compounds from the EtOAc soluble fraction of P. frutescens was followed by identification of the inhibitory compounds by a combination of HPLC microfractionation and a 96-well enzyme assay. This allowed the biological activities to be efficiently matched with selected HPLC peaks. Structural analyses of the active compounds were performed by LC-MS(n). The main AR inhibiting compounds were tentatively identified as chlorogenic acid and rosmarinic acid by LC-MS(n). A two-step high speed counter current chromatography (HSCCC) isolation method was developed with a solvent system of n-hexane-ethyl acetate-methanol-water at 1.5:5:1:5, v/v and 3:7:5:5, v/v. The chemical structures of the isolated compounds were determined by (1)H- and (13)C-nuclear magnetic resonance spectrometry (NMR). The main compounds inhibiting AR in the EtOAc fraction of methanol extracts of P. frutescens were identified as chlorogenic acid (2) (IC50 = 3.16 μ M), rosmarinic acid (4) (IC50 = 2.77 μ M), luteolin (5) (IC50 = 6.34 μ M), and methyl rosmarinic acid (6) (IC50 = 4.03 μ M). PMID:24308003

  19. Expression analysis of dihydroflavonol 4-reductase genes in Petunia hybrida.

    Science.gov (United States)

    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

  20. 5alpha-reductase: history and clinical importance.

    Science.gov (United States)

    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

  1. Regulation and degradation of HMGCo-A reductase.

    Science.gov (United States)

    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

  2. Methylenetetrahydrofolate Reductase Genotypes, Dietary Habits and Susceptibility to Stomach Cancer

    Institute of Scientific and Technical Information of China (English)

    ChangmingGao; TakezakiToshiro; JianzhongWu; JianhuoDing; YantingLiu; SupingLi; PingSu; XuHu; TianliongXu; HamajimaNobuyuki; TajimaKazuo

    2004-01-01

    OBJECTIVE To study the relation among methylenetetrahydrofolate reductase (MTHFR) C677T genotypes, dietary habits and the risk of stomach cancer (SC).METHODS A case-control study was conducted with 107 cases of SC and 200 population-based controls in Chuzhou district, Huaian, Jiangsu province, China. The epidemiological data were collected, and DNA of peripheral blood leukocytes was obtained from all of the subjects..MTHFR genotypes were detected by PCR-RFLP. RESULTS (1) The prevalence of the MTHFR C/T or T/T genotypes was found to be significantly different between controls (68.5%) and SC cases (79.4%,P=0.0416), the increased risk had an adjusted OR of 1.79 (95%C1:1.01-3.19). (2) Among subjects who had a low intake of garlic or Chinese onion, MTHFR C/T or T/T genotypes significantly increased the risk of developing SC. Among non-tea drinkers or among subjects who had a frequent intakeof meat, the carriers of the MTHFR C/T or T/T genotypes had a higher risk of SC than individuals with the C/C type MTHFR. CONCLUSION The polymorphism of MTHFR C677T was associated with increased risk of developing SC, and that individuals with differing genotypes may have different susceptibilities to SC, based on their exposure level to environmental factors.

  3. Alkyl hydroperoxide reductase: a candidate Helicobacter pylori vaccine.

    Science.gov (United States)

    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

  4. Loss of quinone reductase 2 function selectively facilitates learning behaviors.

    Science.gov (United States)

    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

  5. Determination of Nitrate Reductase Assay Depending on the Microbial Growth

    International Nuclear Information System (INIS)

    A rapid micro-dilution assay for determination of the antimicrobial susceptibility of different bacterial isolates was developed. This assay is based on the ability of the most of viable organisms to reduce nitrates. The MIC or MBC could be determined by nitrate reductase (NR) only after 30 to 90 min of incubation depending on the behaviour of microbial growth. Bacterial viability is detected by a positive nitrite reduction rather than visible turbidity. The nitrate reduction assay was compared with standard micro-assay using 250 isolates of different taxa against 10 antibiotics belonging to different classes. An excellent agreement of 82.5 % was found between the two methods and only 17.5 % of 1794 trials showed difference in the determined MIC by tow-dilution interval above or below the MIC determined by the turbidimetric method under the same test conditions. However, the nitrate reduction assay was more rapid and sensitive in detecting viable bacteria and so, established an accurate estimate of the minimal inhibitory concentration (MIC) or the minimal bacterial concentration (MBC). The nitrate reduction assay offers the additional advantage that it could be used to determine the MBC without having to subculture the broth. 232 cases of resistance were detected by NR and 4 different media were tested for susceptibility test. The bacterial isolates were exposed to ultra violet (UV) light for different period

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

    Science.gov (United States)

    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

  7. Glyoxalase 1 and glutathione reductase 1 regulate anxiety in mice.

    Science.gov (United States)

    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

  8. Feed-forward control of prostate growth: dihydrotestosterone induces expression of its own biosynthetic enzyme, steroid 5 alpha-reductase.

    OpenAIRE

    George, F W; Russell, D.W.; Wilson, J. D.

    1991-01-01

    Dihydrotestosterone, the primary mediator of prostate growth, is synthesized in target tissues from the circulating androgen testosterone through the action of steroid 5 alpha-reductase (EC 1.3.99.5). The expression of 5 alpha-reductase and the level of 5 alpha-reductase messenger RNA in rat ventral prostate are regulated by androgens. To determine whether this control is mediated by dihydrotestosterone or testosterone, we investigated the effect of finasteride, a potent inhibitor of steroid ...

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

    OpenAIRE

    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 b...

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

    International Nuclear Information System (INIS)

    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 C2221 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å

  11. Methylenetetrahydrofolate reductase gene polymorphism in Indian stroke patients

    Directory of Open Access Journals (Sweden)

    Kalita J

    2006-01-01

    Full Text Available Background and Aims: In view of the prevailing controversy about the role of Methylenetetrahydrofolate reductase (MTHFR C677T mutation in stroke and paucity of studies from India, this study has been undertaken to evaluate MTHFR C677T gene polymorphism in consecutive ischemic stroke patients and correlate these with folic acid, homocysteine (Hcy and conventional risk factors. Settings and Design: Ischemic stroke patients prospectively evaluated in a tertiary care teaching hospital. Materials and Methods: Computerized tomography proven ischemic stroke patients were prospectively evaluated including clinical, family history of stroke, dietary habits and addictions. Their fasting and postprandial blood sugar, lipid profile, vitamin B12, folic acid and MTHFR gene analysis were done. Statistical Analysis: MTHFR gene polymorphism was correlated with serum folic acid, Vitamin B12 and Hcy levels; family history of stroke in first-degree relatives; and dietary habits; employing Chi-square test. Results: There were 58 patients with ischemic stroke, whose mean age was 50 (4-79 years; among them, 10 were females. MTHFR gene polymorphism was present in 19 (32.8% patients, 3 were homozygous and 16 were heterozygous. Both serum folate and B12 levels were low in 29 (50% patients and Hcy in 48 (83%. Hypertension was present in 28 (48% patients, diabetes in 12 (21%, hyperlipidemia in 52 (90%, smoking in 17 (29%, obesity in 1 (1.7% and family history of stroke in first-degree relatives in 13 (22.4%. There was no significant relationship of MTHFR gene polymorphism with folic acid, B12, Hcy levels, dietary habits and number of risk factors. Vitamin B12 level was low in vegetarians ( P Conclusion: MTHFR gene polymorphism was found in one-third of patients with ischemic stroke and was insignificantly associated with higher frequency of elevated Hcy.

  12. Metabolism of bupropion by carbonyl reductases in liver and intestine.

    Science.gov (United States)

    Connarn, Jamie N; Zhang, Xinyuan; Babiskin, Andrew; Sun, Duxin

    2015-07-01

    Bupropion's metabolism and the formation of hydroxybupropion in the liver by cytochrome P450 2B6 (CYP2B6) has been extensively studied; however, the metabolism and formation of erythro/threohydrobupropion in the liver and intestine by carbonyl reductases (CR) has not been well characterized. The purpose of this investigation was to compare the relative contribution of the two metabolism pathways of bupropion (by CYP2B6 and CR) in the subcellular fractions of liver and intestine and to identify the CRs responsible for erythro/threohydrobupropion formation in the liver and the intestine. The results showed that the liver microsome generated the highest amount of hydroxybupropion (Vmax = 131 pmol/min per milligram, Km = 87 μM). In addition, liver microsome and S9 fractions formed similar levels of threohydrobupropion by CR (Vmax = 98-99 pmol/min per milligram and Km = 186-265 μM). Interestingly, the liver has similar capability to form hydroxybupropion (by CYP2B6) and threohydrobupropion (by CR). In contrast, none of the intestinal fractions generate hydroxybupropion, suggesting that the intestine does not have CYP2B6 available for metabolism of bupropion. However, intestinal S9 fraction formed threohydrobupropion to the extent of 25% of the amount of threohydrobupropion formed by liver S9 fraction. Enzyme inhibition and Western blots identified that 11β-dehydrogenase isozyme 1 in the liver microsome fraction is mainly responsible for the formation of threohydrobupropion, and in the intestine AKR7 may be responsible for the same metabolite formation. These quantitative comparisons of bupropion metabolism by CR in the liver and intestine may provide new insight into its efficacy and side effects with respect to these metabolites. PMID:25904761

  13. Short-chain dehydrogenases/reductases in cyanobacteria.

    Science.gov (United States)

    Kramm, Anneke; Kisiela, Michael; Schulz, Rüdiger; Maser, Edmund

    2012-03-01

    The short-chain dehydrogenases/reductases (SDRs) represent a large superfamily of enzymes, most of which are NAD(H)-dependent or NADP(H)-dependent oxidoreductases. They display a wide substrate spectrum, including steroids, alcohols, sugars, aromatic compounds, and xenobiotics. On the basis of characteristic sequence motifs, the SDRs are subdivided into two main (classical and extended) and three smaller (divergent, intermediate, and complex) families. Despite low residue identities in pairwise comparisons, the three-dimensional structure among the SDRs is conserved and shows a typical Rossmann fold. Here, we used a bioinformatics approach to determine whether and which SDRs are present in cyanobacteria, microorganisms that played an important role in our ecosystem as the first oxygen producers. Cyanobacterial SDRs could indeed be identified, and were clustered according to the SDR classification system. Furthermore, because of the early availability of its genome sequence and the easy application of transformation methods, Synechocystis sp. PCC 6803, one of the most important cyanobacterial strains, was chosen as the model organism for this phylum. Synechocystis sp. SDRs were further analysed with bioinformatics tools, such as hidden Markov models (HMMs). It became evident that several cyanobacterial SDRs show remarkable sequence identities with SDRs in other organisms. These so-called 'homologous' proteins exist in plants, model organisms such as Drosophila melanogaster and Caenorhabditis  elegans, and even in humans. As sequence identities of up to 60% were found between Synechocystis and humans, it was concluded that SDRs seemed to have been well conserved during evolution, even after dramatic terrestrial changes such as the conversion of the early reducing atmosphere to an oxidizing one by cyanobacteria. PMID:22251568

  14. Aldo-keto reductase (AKR) superfamily: genomics and annotation.

    Science.gov (United States)

    Mindnich, Rebekka D; Penning, Trevor M

    2009-07-01

    Aldo-keto reductases (AKRs) are phase I metabolising enzymes that catalyse the reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)-dependent reduction of carbonyl groups to yield primary and secondary alcohols on a wide range of substrates, including aliphatic and aromatic aldehydes and ketones, ketoprostaglandins, ketosteroids and xenobiotics. In so doing they functionalise the carbonyl group for conjugation (phase II enzyme reactions). Although functionally diverse, AKRs form a protein superfamily based on their high sequence identity and common protein fold, the (alpha/beta) 8 -barrel structure. Well over 150 AKR enzymes, from diverse organisms, have been annotated so far and given systematic names according to a nomenclature that is based on multiple protein sequence alignment and degree of identity. Annotation of non-vertebrate AKRs at the National Center for Biotechnology Information or Vertebrate Genome Annotation (vega) database does not often include the systematic nomenclature name, so the most comprehensive overview of all annotated AKRs is found on the AKR website (http://www.med.upenn.edu/akr/). This site also hosts links to more detailed and specialised information (eg on crystal structures, gene expression and single nucleotide polymorphisms [SNPs]). The protein-based AKR nomenclature allows unambiguous identification of a given enzyme but does not reflect the wealth of genomic and transcriptomic variation that exists in the various databases. In this context, identification of putative new AKRs and their distinction from pseudogenes are challenging. This review provides a short summary of the characteristic features of AKR biochemistry and structure that have been reviewed in great detail elsewhere, and focuses mainly on nomenclature and database entries of human AKRs that so far have not been subject to systematic annotation. Recent developments in the annotation of SNP and transcript variance in AKRs are also summarised. PMID:19706366

  15. Aldo-keto reductase (AKR superfamily: Genomics and annotation

    Directory of Open Access Journals (Sweden)

    Mindnich Rebekka D

    2009-07-01

    Full Text Available Abstract Aldo-keto reductases (AKRs are phase I metabolising enzymes that catalyse the reduced nicotinamide adenine dinucleotide (phosphate (NAD(PH-dependent reduction of carbonyl groups to yield primary and secondary alcohols on a wide range of substrates, including aliphatic and aromatic aldehydes and ketones, ketoprostaglan-dins, ketosteroids and xenobiotics. In so doing they functionalise the carbonyl group for conjugation (phase II enzyme reactions. Although functionally diverse, AKRs form a protein superfamily based on their high sequence identity and common protein fold, the (α/(β8-barrel structure. Well over 150 AKR enzymes, from diverse organisms, have been annotated so far and given systematic names according to a nomenclature that is based on multiple protein sequence alignment and degree of identity. Annotation of non-vertebrate AKRs at the National Center for Biotechnology Information or Vertebrate Genome Annotation (vega database does not often include the systematic nomenclature name, so the most comprehensive overview of all annotated AKRs is found on the AKR website (http://www.med.upenn.edu/akr/. This site also hosts links to more detailed and specialised information (eg on crystal structures, gene expression and single nucleotide polymorphisms [SNPs]. The protein-based AKR nomenclature allows unambiguous identification of a given enzyme but does not reflect the wealth of genomic and transcriptomic variation that exists in the various databases. In this context, identification of putative new AKRs and their distinction from pseudogenes are challenging. This review provides a short summary of the characteristic features of AKR biochemistry and structure that have been reviewed in great detail elsewhere, and focuses mainly on nomenclature and database entries of human AKRs that so far have not been subject to systematic annotation. Recent developments in the annotation of SNP and transcript variance in AKRs are also summarised.

  16. Expression of 5α-Reductase Type 2 Gene in Human Testis, Epididymis and Vas Deferens

    Institute of Scientific and Technical Information of China (English)

    刘德瑜; 吴燕婉; 罗宏志; 张桂元

    2002-01-01

    Objectives To study the expression pattern of 5α-reductase type 2 gene in human malereproductive organsMethods The expression level of 5α-reductase type 2 gene inhuman testis, epididymisand vas deferens tissues was determined by in situ hybridization using Digoxin labeled5α-reductase type 2 cRNA probe.Results The brown granules of hybridizing signals distributed in the cytoplasm ofSertoli and Leydig cells of the testis, the principle cells of epididymis and the epithe-lial cells of vas deferens, but there was no positive signal in the nuclei of above-men-tioned cells. No positive signal was observed in germ cells, basement of the testis,interstium of epididymis and basement, as well as smooth muscle of vas deferens.Conclusion This study confirmed that the 5α-reductase type 2 gene expressed in Ser-toli, Leydig cells of the testis, and the principle cells of epididymis. The expressionpattern of the gene in these cells in human was similar to that of rat and monkey. Thepresence of 5a-reductase type 2 gene in epithelial cells of the vas deferens suggested itmight possess an important physiological role in human reproduction.

  17. Pseudomonas stutzeri N2O reductase contains CuA-type sites

    International Nuclear Information System (INIS)

    N2O reductase (N2O → N2) is the terminal enzyme in the energy-conserving denitrification pathway of soil and marine denitrifying bacteria. The protein is composed of two identical subunits and contains eight copper ions per enzyme molecule. The magnetic circular dichroism spectrum of resting (oxidized) N2O reductase is strikingly similar to the magnetic circular dichroism spectrum of the CuA site in mammalian cytochrome c oxidase and is unlike the magnetic circular dichroism spectra of all other biological copper chromophores obtained to data. Sulfur (or chlorine) scatterers are required to fit the copper extended x-ray absorption fine structure data of both the oxidized and reduced forms of N2O reductase. Satisfactory fits require a Cu-N or Cu-O interaction at 2.0 angstrom, a Cu-(S, Cl) interaction at 2.3 angstrom and an additional Cu-(S, Cl) interaction at ∼ 2.6 angstrom (oxidized) or ∼ 2.7 angstrom (reduced). Comparison of the N2O reductase sequence, determined by translating the structural NosZ gene, with cytochrome c oxidase subunit II sequences from several sources indicates that a Gly-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Ser-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-His stretch if high conserved. This sequence contains three of the probable ligands in a CuA-type site Collectively these data establish that Pseudomonas stutzeri N2O reductase contains CuA-type sites

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

    Directory of Open Access Journals (Sweden)

    M. T. Goodarzi

    2006-05-01

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

  19. Current status of 5α-reductase inhibitors in the treatment of benign hyperplasia of prostate

    Directory of Open Access Journals (Sweden)

    Kumar Vijay

    2008-04-01

    Full Text Available Benign prostatic hyperplasia (BPH is a common problem in aging men, which is associated with lower urinary tract symptoms. This condition is dependent on the presence of androgens for its progression, and medical therapy is the first-line treatment for BPH patients with moderate-to-severe symptoms and includes the use of either alpha 1-adrenergic blockers or 5α-reductase inhibitors. Adrenergic blocking drugs reduce the dynamic component while the 5α-reductase inhibitors reduce the static component of bladder outlet obstruction in BPH. By inhibiting the generation of active form of testosterone, viz., dihydrotestosterone, the 5α-reductase inhibitors not only reduce the symptoms of BPH but also decrease the need for surgery and further progression of BPH. Besides, prolonged use of combination of 5α-reductase inhibitors and alpha 1-adrenergic blockers has been found to be more beneficial than either of the two drugs given alone. This review gives a brief account of rationale and efficacy of treatment by 5α-reductase inhibitors in the management of BPH.

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

    International Nuclear Information System (INIS)

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

  1. Discovery and characterization of a xylose reductase from Zymomonas mobilis ZM4.

    Science.gov (United States)

    Agrawal, Manoj; Chen, Rachel Ruizhen

    2011-11-01

    Formation of xylitol, a byproduct from xylose fermentation, is a major limiting factor in ethanol production from xylose in engineered Zymomonas strains, yet the postulated xylose reductase remains elusive. We report here the discovery of xylose reductase in Zymomonas mobilis and, for the first time, to associate the enzyme function with its gene. Besides xylose and xylulose, the enzyme was active towards benzaldehyde, furfural, 5-hydroxymethyl furfural, and acetaldehyde, exhibiting nearly 150-times higher affinity with benzaldehyde than xylose. The discovery of xylose reductase paves the way for further improvement of xylose fermentation in Z. mobilis. The enzyme may also be used to mitigate toxicity of furfural and other inhibitors from plant biomass. PMID:21720846

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

    Energy Technology Data Exchange (ETDEWEB)

    Kiyota, Eduardo [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil); Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas-SP (Brazil); Sousa, Sylvia Morais de [Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas-SP (Brazil); Santos, Marcelo Leite dos; Costa Lima, Aline da [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil); Menossi, Marcelo [Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas, Campinas-SP (Brazil); Yunes, José Andrés [Laboratório de Biologia Molecular, Centro Infantil Boldrini, Campinas-SP (Brazil); Aparicio, Ricardo, E-mail: aparicio@iqm.unicamp.br [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil)

    2007-11-01

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

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

    Science.gov (United States)

    Reutov, V P

    1999-05-01

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

  4. Crystallization and preliminary X-ray crystallographic studies of pig heart carbonyl reductase

    International Nuclear Information System (INIS)

    Pig heart carbonyl reductase has been crystallized in the presence of NADPH. Diffraction data have been collected using synchrotron radiation. Pig heart carbonyl reductase (PHCR), which belongs to the short-chain dehydrogenase/reductase (SDR) family, has been crystallized by the hanging-drop vapour-diffusion method. Two crystal forms (I and II) have been obtained in the presence of NADPH. Form I crystals belong to the tetragonal space group P42, with unit-cell parameters a = b = 109.61, c = 94.31 Å, and diffract to 1.5 Å resolution. Form II crystals belong to the tetragonal space group P41212, with unit-cell parameters a = b = 120.10, c = 147.00 Å, and diffract to 2.2 Å resolution. Both crystal forms are suitable for X-ray structure analysis at high resolution

  5. Synthetic and Crystallographic Studies of a New Inhibitor Series Targeting Bacillus anthracis Dihydrofolate Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Beierlein, J.; Frey, K; Bolstad, D; Pelphrey, P; Joska, T; Smith, A; Priestley, N; Wright, D; Anderson, A

    2008-01-01

    Bacillus anthracis, the causative agent of anthrax, poses a significant biodefense danger. Serious limitations in approved therapeutics and the generation of resistance have produced a compelling need for new therapeutic agents against this organism. Bacillus anthracis is known to be insensitive to the clinically used antifolate, trimethoprim, because of a lack of potency against the dihydrofolate reductase enzyme. Herein, we describe a novel lead series of B. anthracis dihydrofolate reductase inhibitors characterized by an extended trimethoprim-like scaffold. The best lead compound adds only 22 Da to the molecular weight and is 82-fold more potent than trimethoprim. An X-ray crystal structure of this lead compound bound to B. anthracis dihydrofolate reductase in the presence of NADPH was determined to 2.25 A resolution. The structure reveals several features that can be exploited for further development of this lead series.

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

    International Nuclear Information System (INIS)

    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 P212121, 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

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

    DEFF Research Database (Denmark)

    Andersen, J T

    1995-01-01

    During recent years, pharmacological treatment of symptomatic benign prostatic hyperplasia (BPH) has become the primary treatment choice for an increasing number of patients. The 2 principal drug classes employed are alpha 1-blockers and 5 alpha-reductase inhibitors. Current information from...... patients who will respond well to alpha 1-blockers have yet to be identified, and data concerning the long term effects of these drugs are not yet available. 5 alpha-Reductase inhibitors have a slow onset of effect, but treatment leads to improvement in symptoms, reduction of the size of the prostate gland...... and improvement in objective parameters for bladder outflow obstruction. Approximately 30 to 50% of patients will respond to treatment with 5 alpha-reductase inhibitors. The definitive role of pharmacological treatment in symptomatic BPH remains to be established, although it seems that patients unfit...

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

    Directory of Open Access Journals (Sweden)

    F. Xavier eRuiz

    2012-04-01

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

  9. Putative Role of the Aldo-Keto Reductase from Trypanosoma cruzi in Benznidazole Metabolism.

    Science.gov (United States)

    Garavaglia, Patricia Andrea; Laverrière, Marc; Cannata, Joaquín J B; García, Gabriela Andrea

    2016-05-01

    Benznidazole (Bz), the drug used for treatment of Chagas' disease (caused by the protozoan Trypanosoma cruzi), is activated by a parasitic NADH-dependent type I nitroreductase (NTR I). However, several studies have shown that other enzymes are involved. The aim of this study was to evaluate whether the aldo-keto reductase from T. cruzi (TcAKR), a NADPH-dependent oxido-reductase previously described by our group, uses Bz as the substrate. We demonstrated that both recombinant and native TcAKR enzymes reduce Bz by using NADPH, but not NADH, as a cofactor. TcAKR-overexpressing epimastigotes showed higher NADPH-dependent Bz reductase activity and a 50% inhibitory concentration (IC50) value for Bz 1.8-fold higher than that of the controls, suggesting that TcAKR is involved in Bz detoxification instead of activation. To understand the role of TcAKR in Bz metabolism, we studied TcAKR expression and NADPH/NADH-dependent Bz reductase activities in two T. cruzi strains with differential susceptibility to Bz: CL Brener and Nicaragua. Taking into account the results obtained with TcAKR-overexpressing epimastigotes, we expected the more resistant strain, Nicaragua, to have higher TcAKR levels than CL Brener. However, the results were the opposite. CL Brener showed 2-fold higher TcAKR expression and 5.7-fold higher NADPH-Bz reduction than the Nicaragua strain. In addition, NADH-dependent Bz reductase activity, characteristic of NTR I, was also higher in CL Brener than in Nicaragua. We conclude that although TcAKR uses Bz as the substrate, TcAKR activity is not a determinant of Bz resistance in wild-type strains and may be overcome by other enzymes involved in Bz activation, such as NADPH- and NADH-dependent reductases. PMID:26856844

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

    Directory of Open Access Journals (Sweden)

    Leonora F Ciufo

    Full Text Available Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C(29 and C(28 yielding cholesterol (C(27. The final step of this dealkylation pathway involves desmosterol reductase (DHCR24-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735. Following PCR-based cloning of the cDNA (1.6 kb and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD-dependent reaction.Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250 kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.

  11. Effect of the Concentration Difference between Magnesium Ions and Total Ribonucleotide Triphosphates in Governing the Specificity of T7 RNA Polymerase-Based Rolling Circle Transcription for Quantitative Detection.

    Science.gov (United States)

    Li, Zhiyan; Lau, Choiwan; Lu, Jianzhong

    2016-06-01

    T7 RNA polymerase-based rolling circle transcription (RCT) is a more powerful tool than universal runoff transcription and traditional DNA polymerase-based rolling circle amplification (RCA). However, RCT is rarely employed in quantitative detection due to its poor specificity for small single-stranded DNA (ssDNA), which can be transcribed efficiently by T7 RNA polymerase even without a promoter. Herein we show that the concentration difference between Mg(2+) and total ribonucleotide triphosphates (rNTPs) radically governs the specificity of T7 RNA polymerase. Only when the total rNTP concentration is 9 mM greater than the Mg(2+) concentration can T7 RNA polymerase transcribe ssDNA specifically and efficiently. This knowledge improves our traditional understanding of T7 RNA polymerase and makes convenient application of RCT in quantitative detection possible. Subsequently, an RCT-based label-free chemiluminescence method for microRNA detection was designed to test the capability of this sensing platform. Using this simple method, microRNA as low as 20 amol could be quantitatively detected. The results reveal that the developed sensing platform holds great potential for further applications in the quantitative detection of a variety of targets. PMID:27167591

  12. Aldose reductase inhibition prevents metaplasia of airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Umesh C S Yadav

    Full Text Available BACKGROUND: Goblet cell metaplasia that causes mucus hypersecretion and obstruction in the airway lumen could be life threatening in asthma and chronic obstructive pulmonary disease patients. Inflammatory cytokines such as IL-13 mediate the transformation of airway ciliary epithelial cells to mucin-secreting goblet cells in acute as well as chronic airway inflammatory diseases. However, no effective and specific pharmacologic treatment is currently available. Here, we investigated the mechanisms by which aldose reductase (AR regulates the mucus cell metaplasia in vitro and in vivo. METHODOLOGY/FINDINGS: Metaplasia in primary human small airway epithelial cells (SAEC was induced by a Th2 cytokine, IL-13, without or with AR inhibitor, fidarestat. After 48 h of incubation with IL-13 a large number of SAEC were transformed into goblet cells as determined by periodic acid-schiff (PAS-staining and immunohistochemistry using antibodies against Mucin5AC. Further, IL-13 significantly increased the expression of Mucin5AC at mRNA and protein levels. These changes were significantly prevented by treatment of the SAEC with AR inhibitor. AR inhibition also decreased IL-13-induced expression of Muc5AC, Muc5B, and SPDEF, and phosphorylation of JAK-1, ERK1/2 and STAT-6. In a mouse model of ragweed pollen extract (RWE-induced allergic asthma treatment with fidarestat prevented the expression of IL-13, phosphorylation of STAT-6 and transformation of epithelial cells to goblet cells in the lung. Additionally, while the AR-null mice were resistant, wild-type mice showed goblet cell metaplasia after challenge with RWE. CONCLUSIONS: The results show that exposure of SAEC to IL-13 caused goblet cell metaplasia, which was significantly prevented by AR inhibition. Administration of fidarestat to mice prevented RWE-induced goblet cell metaplasia and AR null mice were largely resistant to allergen induced changes in the lung. Thus our results indicate that AR inhibitors

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

    Science.gov (United States)

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

  14. Feedback regulation of cholesterol synthesis:sterol-accelerated ubiquitination and degradation of HMG CoA reductase

    Institute of Scientific and Technical Information of China (English)

    Russell A DeBose-Boyd

    2008-01-01

    3Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate,an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids.The reductase is subject to an exorbitant amount of feedback control through multiple mechanisms that are mediated by sterol and nonsterol end-products of mevalonate metabolism.Here,Ⅰwill discuss recent advances that shed light on one mechanism for control of reductase,which involves rapid degradation of the enzyme.Accumulation of certain sterols triggers binding of reductase to endoplasmic reticulum (ER) membrane proteins called Insig-1 and Insig-2.Reductase-Insig binding results in recruitment of a membrane-associated ubiquitin ligase called gp78,which initiates ubiquitination of reductase.This ubiquitination is an obligatory reaction for recognition and degradation of reductase from ER membranes by cytosolic 26S proteasomes.Thus,sterol-accelerated degradation of reductase represents an example of how a general cellular process (ER-associated degradation) is used to control an important metabolic pathway (cholesterol synthesis).

  15. Multiple Types of 8-Vinyl Reductases for (Bacterio)Chlorophyll Biosynthesis Occur in Many Green Sulfur Bacteria ▿

    OpenAIRE

    Liu, Zhenfeng; Bryant, Donald A.

    2011-01-01

    Two 8-vinyl reductases, BciA and BciB, have been identified in chlorophototrophs. The bciA gene of Chlorobaculum tepidum was replaced with genes similar to bciB from other green sulfur bacteria. Pigment analyses of the complemented strains showed that the bciB homologs encode 8-vinyl reductases similar to those of cyanobacteria.

  16. Studies on some characteristics of nitrate reductase from sugar beet (Beta vulgaris L.)leaves

    Institute of Scientific and Technical Information of China (English)

    LiWenhua; YanGuiping; 等

    1994-01-01

    Some characteristics of nitrate reductase from sugar beet leaves shown in this paper were as follows:The nitrate reductase from sugar beet leaves required NADH as an electron donor.Accordingly,the nitrate reductase was classified as NADH-dependent(E.C.1.6.61).The Km value of the nitrate reductase for NADH and NO3- were 0.86m mol and 0.18μ mol respectively.The optimum pH in reaction mixture solution for nitrate reduction activity was 7.5.The effect of variable concentrations of inorganic phosphorus in the reaction buffer on nitrate reductase activity was investigated.When the inorganic phosphorus concentration was below 35m mol,the nitrate reductase activity was increased with increase of inorganic phosphorus concentration.Conversely,when the inorganic phosphorus concentration was over 35m mol,the nitrate reductase activity was inhibited.The nitrate reductase activity assayed in vitro was 3.2 and 5.6times of that assayed in vivo under the condition of exogenous and endogenous ground substance respectively.

  17. The role of 5'-adenylylsulfate reductase in the sulfur assimilation pathway of soybean: molecular cloning, kinetic characterization, and gene expression

    Science.gov (United States)

    Soybean seeds are a major source of protein, but contain low levels of sulfur-containing amino acids. With the objective of studying the sulfur assimilation pathway of soybean, a full-length cDNA clone for 5’-adenylylsulfate reductase (APS reductase) was isolated and characterized. The cDNA clone ...

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

    DEFF Research Database (Denmark)

    Nilsson, A; Engberg, G; Henneberg, S; Danielson, K; De Verdier, C H

    1990-01-01

    concentrations of local anaesthetics were low (maximum values: prilocaine 78 ng ml-1, lignocaine 412 ng ml-1). The activity of erythrocyte MetHb reductase (cytochrome b5 reductase) was analysed. Data from a previously studied group of infants aged 3-12 months were included also. Enzyme activity did not reach...

  19. Identification of the bchP gene, encoding geranylgeranyl reductase in Chlorobaculum tepidum

    DEFF Research Database (Denmark)

    Gomez Maqueo Chew, Aline; Frigaard, Niels-Ulrik; Bryant, Donald A

    2008-01-01

    The Chlorobaculum tepidum genome contains two paralogous genes, CT2256 and CT1232, whose products are members of the FixC dehydrogenase superfamily and have sequence similarity to geranylgeranyl reductases. Each gene was insertionally inactivated, and the resulting mutants were characterized. CT2......2256 encodes geranylgeranyl reductase (BchP); CT1232 is not involved in bacteriochlorophyll or chlorophyll biosynthesis....

  20. Synthesis and degradation of nitrate reductase during the cell cycle of Chlorella sorokiniana

    Science.gov (United States)

    Velasco, P. J.; Tischner, R.; Huffaker, R. C.; Whitaker, J. R.

    1989-01-01

    Studies on the diurnal variations of nitrate reductase (NR) activity during the life cycle of synchronized Chlorella sorokiniana cells grown with a 7:5 light-dark cycle showed that the NADH:NR activity, as well as the NR partial activities NADH:cytochrome c reductase and reduced methyl viologen:NR, closely paralleled the appearance and disappearance of NR protein as shown by sodium dodecyl sulfate gel electrophoresis and immunoblots. Results of pulse-labeling experiments with [35S]methionine further confirmed that diurnal variations of the enzyme activities can be entirely accounted for by the concomitant synthesis and degradation of the NR protein.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. Glyphosate inhibition of ferric reductase activity in iron deficient sunflower roots

    OpenAIRE

    Öztürk, Levent; Ozturk, Levent; Yazıcı, Mustafa Atilla; Yazici, Mustafa Atilla; Eker, Selim; Gökmen, Özay Özgür; Gokmen, Ozay Ozgur; Römheld, Volker; Romheld, Volker; ÇAKMAK, İsmail; Cakmak, Ismail

    2007-01-01

    Iron (Fe) deficiency is increasingly being observed in cropping systems with frequent glyphosate applications. A likely reason for this is that glyphosate interferes with root uptake of Fe by inhibiting ferric reductase in roots required for Fe acquisition by dicot and nongrass species. This study investigated the role of drift rates of glyphosate (0.32, 0.95 or 1.89 mM glyphosate corresponding to 1, 3 and 6% of the recommended herbicidal dose, respectively) on ferric reductase activity of...

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

    Science.gov (United States)

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

    1996-12-15

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

  4. Properties of a Thermostable Nitrate Reductase from the Hyperthermophilic Archaeon Pyrobaculum aerophilum

    OpenAIRE

    Afshar, Sepideh; Johnson, Eric; de Vries, Simon; Schröder, Imke

    2001-01-01

    The nitrate reductase of the hyperthermophilic archaeon Pyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49-mol/mol complex) as cofactors. The P. aerophilum nitrate reductase disti...

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, Alessandro S.; Ferrarezi, Thiago [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil); Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A. [Facultad de Ciencias Bioquímicas y Farmacéuticas, Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario (Argentina); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil)

    2006-07-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP{sup +} reductase. Ferredoxin-NADP{sup +} reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source.

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

    International Nuclear Information System (INIS)

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP+ reductase. Ferredoxin-NADP+ reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source

  8. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase

    OpenAIRE

    Trigoso, Yvonne D.; Russell C Evans; Karsten, William E.; Lilian Chooback

    2016-01-01

    The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to i...

  9. Pinpointing a Mechanistic Switch Between Ketoreduction and “Ene” Reduction in Short‐Chain Dehydrogenases/Reductases

    Science.gov (United States)

    Lygidakis, Antonios; Karuppiah, Vijaykumar; Hoeven, Robin; Ní Cheallaigh, Aisling; Leys, David; Gardiner, John M.; Toogood, Helen S.

    2016-01-01

    Abstract 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

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

    Science.gov (United States)

    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

  11. Protective role of glutathione reductase in paraquat induced neurotoxicity.

    Science.gov (United States)

    Djukic, Mirjana M; Jovanovic, Marina D; Ninkovic, Milica; Stevanovic, Ivana; Ilic, Katarina; Curcic, Marijana; Vekic, Jelena

    2012-08-30

    Paraquat (PQ), a widely used herbicide is a well-known free radical producing agent. The mechanistic pathways of PQ neurotoxicity were examined by assessing oxidative/nitrosative stress markers. Focus was on the role of glutathione (GSH) cycle and to examine whether the pre-treatment with enzyme glutathione reductase (GR) could protect the vulnerable brain regions (VBRs) against harmful oxidative effect of PQ. The study was conducted on Wistar rats, randomly divided in five groups: intact-control group, (n = 8) and four experimental groups (n = 24). All tested compounds were administered intrastriatally (i.s.) in one single dose. The following parameters of oxidative status were measured in the striatum, hippocampus and cortex, at 30 min, 24 h and 7 days post treatment: superoxide anion radical (O₂·⁻), nitrate (NO₃⁻), malondialdehyde (MDA), superoxide dismutase (SOD), total GSH (tGSH) and its oxidized, disulfide form (GSSG) and glutathione peroxidase (GPx). Results obtained from the intact and the sham operated groups were not statistically different, confirming that invasive i.s. route of administration would not influence the reliability of results. Also, similar pattern of changes were observed between ipsi- and contra- lateral side of examined VBRs, indicating rapid spatial spreading of oxidative stress. Mortality of the animals (10%), within 24h, along with symptoms of Parkinsonism, after awakening from anesthesia for 2-3 h, were observed in the PQ group, only. Increased levels of O₂·⁻, NO₃⁻ and MDA, increased ratio of GSSG/GSH and considerably high activity of GPx were measured at 30 min after the treatment. Cytotoxic effect of PQ was documented by drastic drop of all measured parameters and extremely high peak of the ratio GSSG/GSH at 24th hrs after the PQ i.s. injection. In the GR+PQ group, markedly low activity of GPx and low content of NO₃⁻ (in striatum and cortex) were measured during whole experiment, while increase value was

  12. Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency

    NARCIS (Netherlands)

    Banka, S.; Blom, H.J.; Walter, J.; Aziz, M.; Urquhart, J.; Clouthier, C.M.; Rice, G.I.; Brouwer, A.P.M. de; Hilton, E.; Vassallo, G.; Will, A.; Smith, D.E.; Smulders, Y.M.; Wevers, R.A.; Steinfeld, R.; Heales, S.; Crow, Y.J.; Pelletier, J.N.; Jones, S.; Newman, W.G.

    2011-01-01

    Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism and an important target of antineoplastic, antimicrobial, and antiinflammatory drugs. We describe three individuals from two families with a recessive inborn error of metabolism, characterized by megaloblastic anemia and/or pan

  13. Direct demonstration of genetic alterations at the dihydrofolate reductase locus after gamma irradiation.

    OpenAIRE

    Graf, L. H.; Chasin, L A

    1982-01-01

    Gamma ray-induced mutants of Chinese hamster ovary cells lacking dihydrofolate reductase activity were screened for DNA sequence changes at the locus specifying this activity by using a cloned cDNA probe. Two of nine mutants screened displayed an altered restriction fragment pattern suggesting the occurrence of DNA deletions or rearrangements.

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

    DEFF Research Database (Denmark)

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

    1998-01-01

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

  15. A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi

    DEFF Research Database (Denmark)

    Pena Diaz, Javier; Montalvetti, A; Camacho, A;

    1997-01-01

    We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest of the...

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial sp...... SOR gene in bioleaching reactors and Acidithiobacillus species....

  17. A Rational Approach to Identify Inhibitors of Mycobacterium tuberculosis Enoyl Acyl Carrier Protein Reductase

    Czech Academy of Sciences Publication Activity Database

    Chhabria, M. T.; Parmar, K. B.; Brahmkshatriya, Pathik

    2013-01-01

    Roč. 19, č. 21 (2013), s. 3878-3883. ISSN 1381-6128 Institutional support: RVO:61388963 Keywords : mycobacterium tuberculosis * enoyl acyl carrier protein reductase * pharmacophore modeling * molecular docking * binding interactions Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.288, year: 2013

  18. Relationships between nitrate uptake and nitrate reductase activity in Cucumis sativus L.

    Directory of Open Access Journals (Sweden)

    Grażyna Kłobus

    2014-02-01

    Full Text Available Anti-NR IgG fragments obtained after papain digestion of polyclonal antibodies gave the positive immunological reaction with both, a soluble and plasma membrane-bound nitrate reductase. Anti-NR antibody as well as IgG fragments almost totally inhibited the nitrate reductase activity in cytosol proving a crossreaction of antibody with the catalytic site of a soluble NR. Anti-NR IgG fragments, but not undigested polyclonal antibodies affected the activity of the nitrate reductase associated with plasma membranes. Discrepancy in the action of intact antibodies and fragments obtained after they digestion were interpreted as a consequence of same differences in the ability of those molecules to the penetration through the membrane. Undigested anti-NR antibody have no effect on the nitrate uptake by intact plants, as well as by the right-side plasma membrane vesicles. On the other hand, IgG fragments of polyclonal antibodies abolished almost totally the nitrate uptake in the case of intact seedlings, but have only slight effect on the N03 uptake in plasma membranes. On the basis of above findings, some relations between nitrate uptake and its assimilation inside the cell are suggested. Since IgG fragments only slightly changed the N03 absorption in vesicles whereas the activity of plasmalemma associated nitrate reductase was strongly repressed, we concluded that the PM-NR is not structurally involved in the nitrate transport through the membrane.

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

    International Nuclear Information System (INIS)

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

  20. Purification and characterization of a novel carbonyl reductase isolated from Rhodococcus erythropolis.

    Science.gov (United States)

    Zelinski, T; Peters, J; Kula, M R

    1994-04-15

    During growth on n-tetradecane a novel NADH-dependent carbonyl reductase is induced in the Gram-positive bacterium Rhodococcus erythropolis (Peters, P., Zelinski, T. and Kula, M.R. (1992) Appl. Microbiol. Biotechnol. 38, 334-340). The enzyme has been purified to homogeneity using fractional pH precipitation, anion exchange chromatography and affinity chromatography. The isoelectric point of the oxidoreductase is 4.4. The apparent molecular mass of the native enzyme is 161 kDa, that of the subunits 40 kDa as determined by SDS gel electrophoresis. A tetrameric structure of the carbonyl reductase is consistent with these results. Important biochemical data concerning the application of the reductase are: a broad pH-optimum, temperature optimum at 40 degrees C and stability at room temperature for more than 5 days. The oxidoreductase accepted as substrate aliphatic and aromatic ketones, keto esters (esters of keto carboxylic acids) and halogenated carbonyl compounds and reduced them to the corresponding hydroxyl compounds with (S)-configuration with more than 98% enantiomeric excess. The NAD(+)-dependent oxidation of primary alcohols was not catalyzed by the carbonyl reductase, whereas secondary alcohols and hydroxy acid esters were oxidized to the corresponding carbonyl compounds at about 10-fold slower reaction rates compared to the reduction. PMID:7764739

  1. Design and synthesis of novel, conformationally restricted HMG-CoA reductase inhibitors.

    Science.gov (United States)

    Pfefferkorn, Jeffrey A; Choi, Chulho; Song, Yuntao; Trivedi, Bharat K; Larsen, Scott D; Askew, Valerie; Dillon, Lisa; Hanselman, Jeffrey C; Lin, Zhiwu; Lu, Gina; Robertson, Andrew; Sekerke, Catherine; Auerbach, Bruce; Pavlovsky, Alexander; Harris, Melissa S; Bainbridge, Graeme; Caspers, Nicole

    2007-08-15

    Using structure-based design, a novel series of conformationally restricted, pyrrole-based inhibitors of HMG-CoA reductase were discovered. Leading analogs demonstrated potent inhibition of cholesterol synthesis in both in vitro and in vivo models and may be useful for the treatment of hypercholesterolemia and related lipid disorders. PMID:17574411

  2. Cloning and expression of Candida guilliermondii xylose reductase gene (xyl1) in Pichia pastoris.

    Science.gov (United States)

    Handumrongkul, C; Ma, D P; Silva, J L

    1998-04-01

    A xylose reductase gene (xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized. The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa. The derived amino acid sequence of C. guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis. The gene was placed under the control of an alcohol oxidase promoter (AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris. Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems. The expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo. The different cofactor specificity between P. pastoris and C. guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two proteins. The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the organism was grown under aerobic conditions. PMID:9615481

  3. HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight

    DEFF Research Database (Denmark)

    Swerdlow, Daniel I; Preiss, David; Kuchenbaecker, Karoline B;

    2015-01-01

    BACKGROUND: Statins increase the risk of new-onset type 2 diabetes mellitus. We aimed to assess whether this increase in risk is a consequence of inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the intended drug target. METHODS: We used single nucleotide polymorphisms in the HMGCR...

  4. Thioredoxin reductase is a key factor in the oxidative stress response of Lactobacillus plantarum WCFS1

    NARCIS (Netherlands)

    Mariela Serrano, L.; Molenaar, D.; Wels, M.; Teusink, B.; Bron, P.A.; Vos, W.M. de; Smid, E.J.

    2007-01-01

    ABSTRACT: BACKGROUND: Thioredoxin (TRX) is a powerful disulfide oxido-reductase that catalyzes a wide spectrum of redox reactions in the cell. The aim of this study is to elucidate the role of the TRX system in the oxidative stress response in Lactobacillus plantarum WCFS1. RESULTS: We have identifi

  5. The 5,10-methylenetetrahydrofolate reductase C677T polymorphism interacts with smoking to increase homocysteine.

    NARCIS (Netherlands)

    Brown, K.S.; Kluijtmans, L.A.J.; Young, I.S.; Murray, L.; McMaster, D.; Woodside, J.; Yarnell, J.W.; Boreham, C.A.; McNulty, H.; Strain, J.J.; McPartlin, J.; Scott, J.M.; Mitchell, L.E.; Whitehead, A.S.

    2004-01-01

    Elevated homocysteine is a risk marker for several human pathologies. Risk factors for elevated homocysteine include low folate and homozygosity for the T allele of the 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism. Because nitric oxide may inhibit folate catabolism and endothe

  6. REGULATION OF NITRATE REDUCTASE ACTIVITY IN RICE (ORYZA SATIVA L. BY GROWTH REGULATORS

    Directory of Open Access Journals (Sweden)

    S HEMALATHA

    2002-12-01

    Full Text Available The effect of three growth regulators, namely kinetin, 6 benzyl adenine, 2 chloro ethyl trimethyl ammonium chloride at three concentrations (10-6 M, 5 x 10-5 M 10-4 M was studied on the catalytic activity of nitrate reductase in green and etiolated seedlings. A concentration of 5 x 10-5 M was optimal for all the growth regulators treatments. All the growth regulators stimulated nitrate reductase activity effectively at 5 x 10-5M concentration in both etiolated and green seedlings and had an additive effect when supplemented by NO-3 up to 140% to 160%. The 99.2% and 93.4% inhibition of nitrate reductase activity resulted in development of etiolated and green seedlings, respectively when treated with eukaryotic 80S ribosome protein synthesis inhibitor cycloheximide. Prokaryotic 70S inhibitor chloromphenicol did not have any effect on measured parameters. Actinomycin D, a RNA synthesis inhibitor also inhibited the enzyme activity as 80s inhibitors (Green 80%, etiolated 98%. One may suggest from this that both DNA and protein synthesis are involved in the induction of nitrate reductase activity. The differential effect of aminoacids was observed on enzyme activity in combination with growth regulators.

  7. NADPH-dependent D-aldose reductases and xylose fermentation in Fusarium oxysporum

    DEFF Research Database (Denmark)

    Panagiotou, Gianni; Christakopoulos, P.

    2004-01-01

    Two aldose (xylose) reductases (ARI and ARII) from Fusarium oxysporum were purified and characterized. The native ARI was a monomer with M-r 41000, pI 5.2 and showed a 52-fold preference for NADPH over NADH, while ARII was homodimeric with a subunit of M-r 37000, pI 3.6 and a 60-fold preference...

  8. Sensing nitrite through a pseudoazurin-nitrite reductase electron transfer relay

    NARCIS (Netherlands)

    Astier, Y; Canters, GW; Davis, JJ; Hill, HAO; Verbeet, MP; Wijma, HJ

    2005-01-01

    Nitrite is converted to nitric oxide by haem or copper-containing enzymes in denitrifying bacteria during the process of denitrification. In designing an efficient biosensor, this enzymic turnover must be quantitatively assessed. The enzyme nitrite reductase from Alcaligenes faecalis contains a redo

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    Cytochrome cd1 nitrite reductase is a bifunctional multiheme enzyme catalyzing the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of dioxygen to water. Kinetics and thermodynamics of the internal electron transfer process in the Pseudomonas stutzeri enzyme have...

  10. Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase

    DEFF Research Database (Denmark)

    Hurtado-Guerrrero, Ramón; Pena Diaz, Javier; Montalvetti, Andrea; Ruiz-Pérez, Luis M; González-Pacanowska, Dolores

    2002-01-01

    A detailed kinetic analysis of the recombinant soluble enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower propo...

  11. In Silico Docking studies of Aldose Reductase Inhibitory activity of selected Flavonoids

    Directory of Open Access Journals (Sweden)

    Muthuswamy Umamaheswari

    2012-09-01

    Full Text Available New drugs for the inhibition of the enzyme aldose reductase are in development and they have to be screened before being considered for preclinical and clinical evaluation. The current study deals with the evaluation of the cyclooxygenase inhibitory activity of flavonoids using in silico docking studies. In this perspective, flavonoids like Farobin-A, Gericudranin- B, Glaziovianin-A, Rutin, and Xanthotoxin were selected. Epalrestat, a known aldose reductase inhibitor was used as the standard. Docking results showed that all the selected flavonoids showed binding energy ranging between -7.91 kcal/mol to - 5.08 kcal/mol when compared with that of the standard (-5.59 kcal/mol. Intermolecular energy (- 9.11 kcal/mol to -8.66 kcal/mol and inhibition constant (1.58 μM to 187.37 μM of the ligands also coincide with the binding energy. Xanthotoxin contributed better aldose reductase inhibitory activity because of its structural parameters. Further studies are required to develop potent aldose reductase inhibitors for the treatment of diabetes.

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

    Science.gov (United States)

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

  13. Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase

    NARCIS (Netherlands)

    Stirpe, A; Guzzi, R; Wijma, H; Verbeet, MP; Canters, GW; Sportelli, L

    2005-01-01

    Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type I is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The tem

  14. Structural and transcriptional analysis of plant genes encoding the bifunctional lysine ketoglutarate reductase saccharopine dehydrogenase enzyme

    Science.gov (United States)

    The analysis of a wheat lysine ketoglutarate reductase – saccharopine dehydrogenase (LKR/SDH) gene and comparative structural and functional analyses among available plant genes provides new information on this important gene. Both the structure of the LKR/SDH gene and the immediately adjacent genes...

  15. Biliverdin Reductase-A correlates with inducible nitric oxide synthasein in atorvastatin treated aged canine brain

    Institute of Scientific and Technical Information of China (English)

    Fabio Di Domenico; Marzia Perluigi; Eugenio Barone

    2013-01-01

    Alzheimer’s disease is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Recent preclinical and epidemiological studies proposed statins as a possible therapeutic drug for Alzheimer’s disease, but the exact mechanisms of action are stil unknown. Biliverdin reductase-A is a pleiotropic enzyme involved in cel ular stress responses. It not only transforms biliverdin-IX alpha into the antioxidant bilirubin-IX alpha but its serine/threonine/tyrosine kinase activity is able to modulate cel signaling networks. We previously reported the beneficial effects of atorvastatin treatment on biliverdin reductase-A and heme oxygenase-1 in the brains of a well characterized pre-clinical model of Alzheimer’s disease, aged beagles, together with observed improvement in cognition. Here we extend our knowledge of the effects of atorvastatin on inducible nitric oxide synthase in parietal cortex, cerebel um and liver of the same animals. We demonstrated that atorvastatin treatment (80 mg/day for 14.5 months) to aged beagles selectively increased inducible nitric oxide synthase in the parietal cortex but not in the cerebel um. In contrast, inducible nitric oxide synthase protein levels were significantly decreased in the liver. Significant positive correlations were found between biliverdin reductase-A and inducible nitric oxide synthase as wel as heme oxygenase-1 protein levels in the parietal cortex. The opposite was observed in the liver. Inducible nitric oxide synthase up-regulation in the parietal cortex was positively associated with improved biliverdin reductase-A functions, whereas the oxidative-induced impairment of biliverdin reductase-A in the liver negatively affected inducible nitric oxide synthase expression, thus suggesting a role for biliverdin reductase-A in atorvastatin-dependent inducible nitric oxide synthase changes. Interestingly, increased inducible nitric oxide synthase levels in the parietal cortex were not

  16. Respiratory detoxification of nitric oxide by the cytochrome c nitrite reductase of Escherichia coli.

    Science.gov (United States)

    Poock, Susannah R; Leach, Emily R; Moir, James W B; Cole, Jeffrey A; Richardson, David J

    2002-06-28

    Nitric oxide is a key element in host defense against invasive pathogens. The periplasmic cytochrome c nitrite reductase (NrfA) of Escherichia coli catalyzes the respiratory reduction of nitrite, but in vitro studies have shown that it can also reduce nitric oxide. The physiological significance of the latter reaction in vivo has never been assessed. In this study the reduction of nitric oxide by Escherichia coli was measured in strains active or deficient in periplasmic nitrite reduction. Nrf(+) cells, harvested from cultures grown anaerobically, possessed a nitric-oxide reductase activity with physiological electron donation of 60 nmol min(-1) x mg dry wt(-1), and an in vivo turnover number of NrfA of 390 NO* s(-1) was calculated. Nitric-oxide reductase activity could not be detected in Nrf(-) strains. Comparison of the anaerobic growth of Nrf(+) and Nrf(-) strains revealed a higher sensitivity to nitric oxide in the NrfA(-) strains. A higher sensitivity to the nitrosating agent S-nitroso-N-acetyl penicillamine (SNAP) was also observed in agar plate disk-diffusion assays. Oxygen respiration by E. coli was also more sensitive to nitric oxide in the Nrf(-) strains compared with the Nrf(+) parent strain. The results demonstrate that active periplasmic cytochrome c nitrite reductase can confer the capacity for nitric oxide reduction and detoxification on E. coli. Genomic analysis of many pathogenic enteric bacteria reveals the presence of nrf genes. The present study raises the possibility that this reflects an important role for the cytochrome c nitrite reductase in nitric oxide management in oxygen-limited environments. PMID:11960983

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

    Science.gov (United States)

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

    1998-01-01

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

  18. Involvement of nitrate reductase and pyoverdine in competitiveness of Pseudomonas fluorescens strain C7R12 in soil.

    Science.gov (United States)

    Mirleau, P; Philippot, L; Corberand, T; Lemanceau, P

    2001-06-01

    Involvement of nitrate reductase and pyoverdine in the competitiveness of the biocontrol strain Pseudomonas fluorescens C7R12 was determined, under gnotobiotic conditions, in two soil compartments (bulk and rhizosphere soil), with the soil being kept at two different values of matric potential (-1 and -10 kPa). Three mutants affected in the synthesis of either the nitrate reductase (Nar(-)), the pyoverdine (Pvd(-)), or both (Nar(-) Pvd(-)) were used. The Nar(-) and Nar(-) Pvd(-) mutants were obtained by site-directed mutagenesis of the wild-type strain and of the Pvd(-) mutant, respectively. The selective advantage given by nitrate reductase and pyoverdine to the wild-type strain was assessed by measuring the dynamic of each mutant-to-total-inoculant (wild-type strain plus mutant) ratio. All three mutants showed a lower competitiveness than the wild-type strain, indicating that both nitrate reductase and pyoverdine are involved in the fitness of P. fluorescens C7R12. The double mutant presented the lowest competitiveness. Overall, the competitive advantages given to C7R12 by nitrate reductase and pyoverdine were similar. However, the selective advantage given by nitrate reductase was more strongly expressed under conditions of lower aeration (-1 kPa). In contrast, the selective advantage given by nitrate reductase and pyoverdine did not differ in bulk and rhizosphere soil, indicating that these bacterial traits are not specifically involved in the rhizosphere competence but rather in the saprophytic ability of C7R12 in soil environments. PMID:11375173

  19. Glutathione Reductase of Vacuole. Comparison of Glutathione Reductase Activity of Vacuole and Tissue Extract of Red Beet Root (Beta vulgaris L.

    Directory of Open Access Journals (Sweden)

    E.V. Pradedova

    2016-02-01

    Full Text Available Glutathione reductase (GR, EC 1.8.1.7 is the enzyme that reduces oxidized glutathione (GSSG and thus regulates the redox state of glutathione (GSH/GSSG. GR has been studied in most plants. This enzyme has been identified in chloroplasts and cytosol, so these cellular compartments are considered to be the main place of the enzyme localization. In the same time, just a little is known about GR vacuoles. There are no conclusive evidences to prove the presence or absence of this enzyme in the vacuoles. GR activity was found in the vacuoles of red beet root cells (Beta vulgaris L.. The level of activity, the optimum pH and isoenzyme composition of GR were compared in the vacuoles and tissue extract of beet root. Vacuolar GR activity was quite high, it was 1.5-2 times higher than the activity of the tissue extract. Enzyme pH optimum of all the objects were identical. pH-optimum depend on the pyridine nucleotide nature: pH 7.0-8.0 was an optimal range with NADPH; pH 5.0 – with NADH. GR activity of the vacuoles and tissue extracts decreased in the presence of a noncompetitive inhibitor 1-chloro-2.4-dinitrobenzene (CDNB, indicating the specificity of this enzymatic reaction. Two bands with glutathione reductase activity have been identified in the vacuoles and tissue extracts using zymography method to determine the enzymatic activity in PAAG after electrophoresis of proteins. Belonging to the GR isoforms of these bands was confirmed by enzyme immunoassay (Western blotting. The electric mobility of isoforms of the study objects did not differ significantly. It is concluded that the biochemical characteristics of vacuolar glutathione reductase were substantially identical to the biochemical characteristics of other localization GR.

  20. Coenzyme A disulfide reductase, the primary low molecular weight disulfide reductase from Staphylococcus aureus. Purification and characterization of the native enzyme.

    Science.gov (United States)

    delCardayre, S B; Stock, K P; Newton, G L; Fahey, R C; Davies, J E

    1998-03-01

    The human pathogen Staphylococcus aureus does not utilize the glutathione thiol/disulfide redox system employed by eukaryotes and many bacteria. Instead, this organism produces CoA as its major low molecular weight thiol. We report the identification and purification of the disulfide reductase component of this thiol/disulfide redox system. Coenzyme A disulfide reductase (CoADR) catalyzes the specific reduction of CoA disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of Mr 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a lambdamax = 452 nm. The liberated flavin from thermally denatured enzyme was identified as flavin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a Km for NADPH of 2 muM and for CoA disulfide of 11 muM. In addition to CoA disulfide CoADR reduces 4,4'-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H2O2. CoADR demonstrates a sequential kinetic mechanism and employs a single active site cysteine residue that forms a stable mixed disulfide with CoA during catalysis. These data suggest that S. aureus employs a thiol/disulfide redox system based on CoA/CoA-disulfide and CoADR, an unorthodox new member of the pyridine nucleotide-disulfide reductase superfamily. PMID:9488707

  1. Glutathione Reductase of Vacuole. Comparison of Glutathione Reductase Activity of Vacuole and Tissue Extract of Red Beet Root (Beta vulgaris L.)

    OpenAIRE

    E.V. Pradedova; O.D. Nimaeva; Salyaev, R. K.

    2016-01-01

    Glutathione reductase (GR, EC 1.8.1.7) is the enzyme that reduces oxidized glutathione (GSSG) and thus regulates the redox state of glutathione (GSH/GSSG). GR has been studied in most plants. This enzyme has been identified in chloroplasts and cytosol, so these cellular compartments are considered to be the main place of the enzyme localization. In the same time, just a little is known about GR vacuoles. There are no conclusive evidences to prove the presence or absence of this enzyme in the ...

  2. Regulation of Escherichia coli fumarate reductase (frdABCD) operon expression by respiratory electron acceptors and the fnr gene product.

    OpenAIRE

    Jones, H. M.; Gunsalus, R P

    1987-01-01

    The fumarate reductase enzyme complex, encoded by the frdABCD operon, allows Escherichia coli to utilize fumarate as a terminal electron acceptor for anaerobic oxidative phosphorylation. To analyze the expression of fumarate reductase, protein and operon fusions were constructed between the frdA and the lacZ genes and introduced onto the E. coli chromosome at the lambda attachment site. Expression of beta-galactosidase from either fusion was increased 10-fold during anaerobic versus aerobic c...

  3. Involvement of Nitrate Reductase and Pyoverdine in Competitiveness of Pseudomonas fluorescens Strain C7R12 in Soil

    OpenAIRE

    Mirleau, Pascal; Philippot, Laurent; Corberand, Thérèse; Lemanceau, Philippe

    2001-01-01

    Involvement of nitrate reductase and pyoverdine in the competitiveness of the biocontrol strain Pseudomonas fluorescens C7R12 was determined, under gnotobiotic conditions, in two soil compartments (bulk and rhizosphere soil), with the soil being kept at two different values of matric potential (−1 and −10 kPa). Three mutants affected in the synthesis of either the nitrate reductase (Nar−), the pyoverdine (Pvd−), or both (Nar− Pvd−) were used. The Nar− and Nar− Pvd− mutants were obtained by si...

  4. Cancer metabolism and oxidative stress: Insights into carcinogenesis and chemotherapy via the non-dihydrofolate reductase effects of methotrexate

    OpenAIRE

    Joshua A. Hess; Mohamad K. Khasawneh

    2015-01-01

    Methotrexate has been in use as an anti-cancer agent for over 60 years. Though inhibition of dihydrofolate reductase is its best known mechanisms of action, its non-dihydrofolate reductase dependent mechanisms disrupt metabolic pathways resulting in a depletion of NAD(P)H and increasing oxidative stress. These mechanisms highlight a novel dependence of cancer cells on their metabolic abnormalities to buffer oxidative stress and chemotherapeutic agents interfere with these cellular abilities. ...

  5. A novel twist on molecular interactions between thioredoxin and nicotinamide adenine dinucleotide phosphate-dependent thioredoxin reductase

    DEFF Research Database (Denmark)

    Kirkensgaard, Kristine Groth; Hägglund, Per; Shahpiri, Azar;

    2013-01-01

    The ubiquitous disulfide reductase thioredoxin (Trx) regulates several important biological processes such as seed germination in plants. Oxidized cytosolic Trx is regenerated by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (NTR) in a multistep transfer of r....... Overall, the findings suggest that NTR:Trx interactions in different biological systems are fine-tuned by multiple intermolecular contacts. Proteins 2014; 82:607-619. (c) 2013 Wiley Periodicals, Inc....

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

    OpenAIRE

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

    2015-01-01

    Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum...

  7. Positive pleiotropic effects of HMG-CoA reductase inhibitor on vitiligo

    Directory of Open Access Journals (Sweden)

    Jobin Jean

    2004-05-01

    Full Text Available Abstract Background HMG-CoA reductase inhibitors (statins are commonly used in medicine to control blood lipid disorder. Large clinical trials have demonstrated that statins greatly reduces cardiovascular-related morbidity and mortality in patients with and without coronary artery disease. Also, the use of HMG-CoA reductase inhibitors has been reported to have immunosuppressive effects. Case presentation We describe an unusual case of regression of vitiligo in a patient treated with high dose simvastatin. The relation between simvastatin and regression of vitiligo in this case report may be related to the autoimmune pathophysiology of the disease. Conclusion This unexpected beneficial impact provides another scientific credence to the hypothesis that immune mechanisms play a role in the development of vitiligo and that the use of statins as immuno-modulator could be of use not only for treatment relative to organ transplant but in other pathologies such as vitiligo.

  8. Purification, crystallization and preliminary X-ray crystallographic analysis of xylose reductase from Candida tropicalis

    International Nuclear Information System (INIS)

    The crystallization of xylose reductase from C. tropicalis is reported. Xylose reductase (XR), which requires NADPH as a co-substrate, catalyzes the reduction of d-xylose to xylitol, which is the first step in the metabolism of d-xylose. The detailed three-dimensional structure of XR will provide a better understanding of the biological significance of XR in the efficient production of xylitol from biomass. XR of molecular mass 36.6 kDa from Candida tropicalis was crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction data from C. tropicalis XR crystals at 2.91 Å resolution, the unit cell belongs to space group P31 or P32. Preliminary analysis indicated the presence of four XR molecules in the asymmetric unit, with 68.0% solvent content

  9. Expression Analysis of Dihydroflavonol 4-Reductase Genes Involved in Anthocyanin Biosynthesis in Purple Grains of Wheat

    Institute of Scientific and Technical Information of China (English)

    Mao-Sen LIU; Fang WANG; Yu-Xiu DONG; Xian-Sheng ZHANG

    2005-01-01

    The grain color of wheat (Triticum aestivum L.) is an important characteristic in crop production.Dihydroflavonol 4-reductase genes (DFR) encode the key enzyme dihydroflavonol 4-reductase, which is involved in the pigmentation of plant tissues. To investigate the molecular mechanism of anthocyanin deposition in grains of wheat, we determined the expression of the wheat DFR gene in purple grains of cultivar Heimai 76. The results showed that DFR transcripts were localized in the seed coat of purple grains rather than in the pericarp, whereas anthocyanins were accumulated in both tissues of purple grains,suggesting that anthocyanin deposition was mainly regulated at the transcriptional level. Overexpression of the TaDFR-A gene in Arabidopsis showed that TaDFR-A was responsible for the pigmentation of Arabidopsis plant tissues, indicating TaDFR-A gene has the same role in Arabidopsis.

  10. Discovery of pyrrole-based hepatoselective ligands as potent inhibitors of HMG-CoA reductase.

    Science.gov (United States)

    Bratton, Larry D; Auerbach, Bruce; Choi, Chulho; Dillon, Lisa; Hanselman, Jeffrey C; Larsen, Scott D; Lu, Gina; Olsen, Karl; Pfefferkorn, Jeffrey A; Robertson, Andrew; Sekerke, Catherine; Trivedi, Bharat K; Unangst, Paul C

    2007-08-15

    In an effort to identify hepatoselective inhibitors of HMG-CoA reductase, two series of pyrroles were synthesized and evaluated. Efforts were made to modify (3R,5R)-7-[3-(4-fluorophenyl)-1-isopropyl-4-phenyl-5-phenylcarbamoyl-1H-pyrrol-2-yl]-3,5-dihydroxy-heptanoic acid sodium salt 30 in order to reduce its lipophilicity and therefore increase hepatoselectivity. Two strategies that were explored were replacement of the lipophilic 3-phenyl substituent with either a polar function (pyridyl series) or with lower alkyl substituents (lower alkyl series) and attachment of additional polar moieties at the 2-position of the pyrrole ring. One compound was identified to be both highly hepatoselective and active in vivo. We report the discovery, synthesis, and optimization of substituted pyrrole-based hepatoselective ligands as potent inhibitors of HMG-CoA reductase for reducing low density lipoprotein cholesterol (LDL-c) in the treatment of hypercholesterolemia. PMID:17560788

  11. The properties of monoclonal antibody against sepiapterin reductase from fat body of the silkworm, Bombyx mori.

    Science.gov (United States)

    Iino, T; Sawada, H; Gyure, W L; Tsusué, M

    1992-12-01

    A specific monoclonal antibody prepared for the 29-kDa a subunit of silkworm fat body sepiapterin reductase (SPR) was able to recognize the subunit in crude extract of fat body after SDS treatment. Although SPR from the silkworm fat body has biochemical properties similar to those reported for SPR from mammalian sources, especially rat erythrocytes, the antibody failed to recognize the 28-kDa subunit of rat erythrocyte SPR. This result indicates that SPR from silkworm fat body has a different amino-acid sequence from that of the rat erythrocyte enzyme. Sepiapterin reductase activity has not been found in crude extract of fat body from the silkworm mutant lemon. Although the antibody recognized only 29-kDa protein in the crude extract of silkworm fat body from normal strain after SDS-treatment, the antibody recognized only an approximately 80-kDa protein in the crude extract of the lemon mutant after SDS-treatment. PMID:1292509

  12. 5alpha-Reductase inhibitor treatment of prostatic diseases: background and practical implications.

    Science.gov (United States)

    Dörsam, J; Altwein, J

    2009-01-01

    This literature review discusses the theoretical background of 5alpha-reductase inhibitor (5ARI) treatment and the resulting clinical implications. A Medline-based search for peer-reviewed articles addressing 5ARIs, benign prostatic hyperplasia and prostate cancer was performed. The 5ARIs Finasteride and Dutasteride, which specifically inhibit the production of dihydrotestosterone by acting as competitive inhibitors of 5alpha-reductase, are clinically well tolerated and represent an effective treatment option for benign prostatic obstruction. Finasteride is the first compound which has a proven efficacy in chemoprevention of prostate cancer. The aim of this review was to elucidate, if there are sufficient data available to point out clinically relevant differences between the drugs. Both compounds achieve a significant reduction of prostate volume, an improvement of symptoms and a lower risk of acute urinary retention. Whether the different pharmacokinetic and pharmacodynamic properties of Finasteride and Dutasteride are of clinical importance cannot be judged at this time. PMID:19030020

  13. Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

    Science.gov (United States)

    Bardon, Clément; Poly, Franck; Piola, Florence; Pancton, Muriel; Comte, Gilles; Meiffren, Guillaume; Haichar, Feth El Zahar

    2016-05-01

    Recently, it has been shown that procyanidins fromFallopiaspp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strainPseudomonas brassicacearumNFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability ofP. brassicacearumwere determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the genenarG) and NO2-reductase (encoded by the genenirS) under NO3or NO2were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearumresponded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the genenarG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and thatP. brassicacearumresponds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI. PMID:26906096

  14. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.

    Science.gov (United States)

    Schumacher, Marc M; Elsabrouty, Rania; Seemann, Joachim; Jo, Youngah; DeBose-Boyd, Russell A

    2015-01-01

    Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD. PMID:25742604

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2014-09-12

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

  17. Theoretical Calculations of the Catalytic Triad in Short-Chain Alcohol Dehydrogenases/Reductases

    OpenAIRE

    Gani, Osman A B S M; Adekoya, Olayiwola A; Giurato, Laura; Spyrakis, Francesca; Cozzini, Pietro; Guccione, Salvatore; Winberg, Jan-Olof; Sylte, Ingebrigt

    2007-01-01

    Three highly conserved active site residues (Ser, Tyr, and Lys) of the family of short-chain alcohol dehydrogenases/reductases (SDRs) were demonstrated to be essential for catalytic activity and have been denoted the catalytic triad of SDRs. In this study computational methods were adopted to study the ionization properties of these amino acids in SDRs from Drosophila melanogaster and Drosophila lebanonensis. Three enzyme models, with different ionization scenarios of the catalytic triad that...

  18. Sulfate reduction in higher plants: Molecular evidence for a novel 5′-adenylylsulfate reductase

    OpenAIRE

    SETYA, AMIT; Murillo, Michael; Leustek, Thomas

    1996-01-01

    Sulfate-assimilating organisms reduce inorganic sulfate for Cys biosynthesis. There are two leading hypotheses for the mechanism of sulfate reduction in higher plants. In one, adenosine 5′-phosphosulfate (APS) (5′-adenylylsulfate) sulfotransferase carries out reductive transfer of sulfate from APS to reduced glutathione. Alternatively, the mechanism may be similar to that in bacteria in which the enzyme, 3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase, catalyzes...

  19. Methylenetetrahydrofolate reductase homozygous mutation in a young boy with cerebellar infarction

    OpenAIRE

    Alberto Spalice; Francesca Del Balzo; Francesco Massimo Perla; Enrico Properzi; Carla Carducci; Italo Antonozzi; Paola Iannetti

    2009-01-01

    Posterior circulation vascular occlusive disease in children is a rare and uncommonly reported event. Among the numerous risk factors, the methylenetetrahydrofolate reductase (MTHFR) mutation is considered to be a common genetic cause of thrombosis in adults and children. Recently, a link between the MTHFR mutation and cerebrovascular disorders was reported in children. Diffusion tensor imaging (DTI) is a great improvement on magnetic resonance imaging (MRI), making the in vivo anatomical and...

  20. Lemierre's syndrome with double heterozygote status in the methylenetetrahydrofolate reductase gene

    Institute of Scientific and Technical Information of China (English)

    Mostafa Behpour-Oskooee; Abdollah Karimi; Shirin Sayyahfar

    2014-01-01

    Background: There are some risk factors being more vulnerable to Lemierre's syndrome such as a hypercoagulable state. Methods: We report a rare case of Lemierre's syndrome with ethmoid and maxillary sinusitis, bilateral mastoiditis, and sigmoid sinus thrombosis. Results: Genetic study revealed a double heterozygote status in the methylenetetrahydrofolate reductase gene including C677T and A1298C. Conclusion: It is suggested to screen patients with Lemierre's syndrome for a hypercoagulable state to consider anticoagulant therapy.

  1. Purine salvage pathways of Bacillus subtilis and effect of guanine on growth of GMP reductase mutants.

    OpenAIRE

    Endo, T.; Uratani, B; Freese, E.

    1983-01-01

    We have isolated numerous mutants containing mutations in the salvage pathways of purine synthesis. The mutations cause deficiencies in adenine phosphoribosyltransferase (adeF), in hypoxanthine-guanine phosphoribosyltransferase (guaF), in adenine deaminase (adeC), in inosine-guanosine phosphorylase, (guaP), and in GMP reductase (guaC). The physiological properties of mutants containing one or more of these mutations and corresponding enzyme measurements have been used to derive a metabolic ch...

  2. The stability of the three transmembrane and the four transmembrane human vitamin K epoxide reductase models

    Science.gov (United States)

    Wu, Sangwook

    2016-04-01

    The three transmembrane and the four transmembrane helix models are suggested for human vitamin K epoxide reductase (VKOR). In this study, we investigate the stability of the human three transmembrane/four transmembrane VKOR models by employing a coarse-grained normal mode analysis and molecular dynamics simulation. Based on the analysis of the mobility of each transmembrane domain, we suggest that the three transmembrane human VKOR model is more stable than the four transmembrane human VKOR model.

  3. Dimethyl sulfoxide reductase activity by anaerobically grown Escherichia coli HB101.

    OpenAIRE

    Bilous, P T; Weiner, J H

    1985-01-01

    Escherichia coli grew anaerobically on a minimal medium with glycerol as the carbon and energy source and dimethyl sulfoxide (DMSO) as the terminal electron acceptor. DMSO reductase activity, measured with an artificial electron donor (reduced benzyl viologen), was preferentially associated with the membrane fraction (77 +/- 10% total cellular activity). A Km for DMSO reduction of 170 +/- 60 microM was determined for the membrane-bound activity. Methyl viologen, reduced flavin mononucleotide,...

  4. Nitrate reductase and acid phosphatase activities as affected by inorganic phosphate in corn roots

    OpenAIRE

    Marie Kummerova; Józef Buczek

    2014-01-01

    The deficieny of inorganic phosphate in nutrient solution reduces by about 50 per cent NO3- absorption in corn seedlings, it decreases both in vitro and in vivo nitrate reductase (NR) activity, as well the potential and actual NR level and has a very weak effect on NR induction. Acid phosphatases activities increase in corn roots when the plants are grown in nutrient solution without phosphorus. We suggest that inorganic phosphate is required mainly for maintenance of NR activity rather, than...

  5. Induction of Glutathione Synthesis and Glutathione Reductase Activity by Abiotic Stresses in Maize and Wheat

    OpenAIRE

    Gabor Kocsy; Gabriella Szalai; Gabor Galiba

    2002-01-01

    The effect of different abiotic stresses (extreme temperatures and osmotic stress) on the synthesis of glutathione and hydroxymethylglutathione, on the ratio of the reduced to oxidised forms of these thiols (GSH/GSSG, hmGSH/hmGSSG), and on the glutathione reductase (GR) activity was studied in maize and wheat genotypes having different sensitivity to low temperature stress. Cold treatment induced a greater increase in total glutathione (TG) content and in GR activity in tolerant genotypes of ...

  6. Thioredoxin reductase-1 (TxnRd1) mediates curcumin-induced radiosensitization of squamous carcinoma cells

    OpenAIRE

    Javvadi, Prashanthi; Hertan, Lauren; Kosoff, Rachelle; Datta, Tatini; Kolev, Johann; Mick, Rosemarie; Tuttle, Stephen W; Koumenis, Constantinos

    2010-01-01

    Curcumin, a plant polyphenol, is a widely studied chemopreventive agent with demonstrated antitumor activities in preclinical studies and low toxicity profiles in multiple clinical trials against human malignancies. We previously demonstrated that curcumin radiosensitizes cervical tumor cells without increasing the cytotoxic effects of radiation on normal human fibroblasts. Here we report that an inhibitory activity of curcumin on the anti-oxidant enzyme Thioredoxin Reductase-1 (TxnRd1) is re...

  7. Tissue distribution and ontogeny of steroid 5 alpha-reductase isozyme expression.

    OpenAIRE

    Thigpen, A E; Silver, R I; Guileyardo, J M; Casey, M L; McConnell, J D; Russell, D W

    1993-01-01

    The synthesis of dihydrotestosterone is catalyzed by steroid 5 alpha-reductase isozymes, designated types 1 and 2. Mutation of type 2 results in male pseudohermaphroditism, in which the external genitalia are phenotypically female at birth. Two striking and unexplained features of this disorder are that external genitalia of affected males undergo virilization during puberty and that these individuals have less temporal hair regression. The tissue-specific and developmental expression pattern...

  8. Dicarbonyl L-Xylulose Reductase (DCXR), a “Moonlighting Protein” in the Bovine Epididymis

    OpenAIRE

    Akintayo, Ayodélé; Légaré, Christine; Sullivan, Robert

    2015-01-01

    During maturation and the acquisition of their fertilization potential, male germ cells are subjected to various sequential modifications that occur in the epididymis. Protein addition, reorganization or withdrawal, comprise some of these modifications. Dicarbonyl L-xylulose reductase (DCXR), a multifunctional protein involved in various enzymatic and protein interaction processes in different physiological systems, is one of the proteins added to spermatozoa in the epididymis. DCXR is a well...

  9. Methionine synthase reductase deficiency results in adverse reproductive outcomes and congenital heart defects in mice

    OpenAIRE

    Deng, Liyuan; Elmore, C. Lee; Lawrance, Andrea K.; Matthews, Rowena G.; Rozen, Rima

    2008-01-01

    Low dietary folate and polymorphisms in genes of folate metabolism can influence risk for pregnancy complications and birth defects. Methionine synthase reductase (MTRR) is required for activation of methionine synthase, a folate- and vitamin B12-dependent enzyme. A polymorphism in MTRR (p.I22M), present in the homozygous state in 25% of many populations, may increase risk for neural tube defects. To examine the impact of MTRR deficiency on early development and congenital heart defects, we u...

  10. Developmental expression of Xenopus short-chain dehydrogenase/reductase 3

    OpenAIRE

    Kam, Richard Kin Ting; Chen, Yonglong; Chan, Sun On; Chan, Wood Yee; Dawid, Igor B.; Hui ZHAO

    2010-01-01

    During early embryonic development, the retinoic acid signaling pathway coordinates with other signaling pathways to regulate body axis patterning and organogenesis. The production of retinoic acid requires two enzymatic reactions, the first of which is the oxidization of vitamin A (all-trans-retinol) to all-trans-retinal, mediated in part by the short-chain dehydrogenase/reductase. Through DNA microarrays, we have identified a gene in Xenopus laevis, which shares a high sequence similarity t...

  11. Differential expression of 5-alpha reductase isozymes in the prostate and its clinical implications

    OpenAIRE

    Kai Wang; Dong-Dong Fan; Song Jin; Nian-Zeng Xing; Yi-Nong Niu

    2014-01-01

    The development of human benign or malignant prostatic diseases is closely associated with androgens, primarily testosterone (T) and dihydrotestosterone (DHT). T is converted to DHT by 5-alpha reductase (5-AR) isozymes. Differential expression of 5-AR isozymes is observed in both human benign and malignant prostatic tissues. 5-AR inhibitors (5-ARI) are commonly used for the treatment of benign prostatic hyperplasia (BPH) and were once promoted as chemopreventive agents for prostate cancer (PC...

  12. Battling Prostate Cancer with 5-Alpha-Reductase Inhibitors: a Pyrrhic Victory?

    OpenAIRE

    Hoffman, Richard M.; Richard G. Roberts; Barry, Michael J.

    2011-01-01

    Given the relatively small impact of prostate cancer screening on cancer mortality, experts are now suggesting that chemoprevention with 5-alpha-reductase inhibitors (5-ARI) may be a more effective strategy for cancer control. Two large placebo-controlled randomized trials found that men receiving 5-ARI were about 25% less likely than controls to be detected with cancer. However, most cancers were detected on routine biopsies required by study protocols. The benefit from receiving 5-ARI was m...

  13. Clinical Effects of Discontinuing 5-Alpha Reductase Inhibitor in Patients With Benign Prostatic Hyperplasia

    OpenAIRE

    Kim, Won; Jung, Jae Hung; Kang, Tae Wook; Song, Jae Mann; Chung, Hyun Chul

    2014-01-01

    Purpose To assess changes in lower urinary tract symptoms (LUTS), prostate volume, and serum prostate-specific antigen (PSA) after discontinuation of 5-alpha reductase inhibitor (5ARI) combination therapy in patients with benign prostatic hyperplasia (BPH). Materials and Methods From December 2003 to December 2012, data were collected retrospectively from 81 men more than 40 years of age with moderate to severe BPH symptoms (International Prostate Symptom Score [IPSS]≥8). The men were classif...

  14. Targeting 5α-reductase for prostate cancer prevention and treatment

    OpenAIRE

    Nacusi, Lucas P.; Tindall, Donald J.

    2011-01-01

    Testosterone is the most abundant circulating androgen, and can be converted to dihydrotestosterone (DHT), a more potent androgen, by the 5α-reductase enzymes in target tissues. Current treatments for prostate cancer consist of reducing androgen levels by chemical or surgical castration or pure antiandrogen therapy that directly targets the androgen receptor (AR). Although these therapies reduce tumor burden and AR activity, the cancer inevitably recurs within 18–30 months. An approach target...

  15. 5-α reductase inhibitors and prostate cancer prevention: where do we turn now?

    OpenAIRE

    Hamilton Robert J; Freedland Stephen J

    2011-01-01

    Abstract With the lifetime risk of being diagnosed with prostate cancer so great, an effective chemopreventive agent could have a profound impact on the lives of men. Despite decades of searching for such an agent, physicians still do not have an approved drug to offer their patients. In this article, we outline current strategies for preventing prostate cancer in general, with a focus on the 5-α-reductase inhibitors (5-ARIs) finasteride and dutasteride. We discuss the two landmark randomized...

  16. Purification and Characterization of (Per)Chlorate Reductase from the Chlorate-Respiring Strain GR-1

    OpenAIRE

    Kengen, Servé W. M.; Rikken, Geoffrey B.; Hagen, Wilfred R.; van Ginkel, Cees G.; Stams, Alfons J. M.

    1999-01-01

    Strain GR-1 is one of several recently isolated bacterial species that are able to respire by using chlorate or perchlorate as the terminal electron acceptor. The organism performs a complete reduction of chlorate or perchlorate to chloride and oxygen, with the intermediate formation of chlorite. This study describes the purification and characterization of the key enzyme of the reductive pathway, the chlorate and perchlorate reductase. A single enzyme was found to catalyze both the chlorate-...

  17. Characterization of the Arsenate Respiratory Reductase from Shewanella sp. Strain ANA-3

    OpenAIRE

    Malasarn, Davin; Keeffe, Jennifer R.; Newman, Dianne K.

    2008-01-01

    Microbial arsenate respiration contributes to the mobilization of arsenic from the solid to the soluble phase in various locales worldwide. To begin to predict the extent to which As(V) respiration impacts arsenic geochemical cycling, we characterized the expression and activity of the Shewanella sp. strain ANA-3 arsenate respiratory reductase (ARR), the key enzyme involved in this metabolism. ARR is expressed at the beginning of the exponential phase and persists throughout the stationary ph...

  18. Induction and inhibition of NAD(P)H: quinone reductase in murine and human skin.

    Science.gov (United States)

    Merk, H; Jugert, F; Bonnekoh, B; Mahrle, G

    1991-01-01

    The purpose of this study was to characterize the human cutaneous NAD(P)H: quinone reductase (NQR) activity by known inhibitors of different reductases and to compare it with the murine skin and liver NQR activity. This enzyme plays a major role in the defence of cells against oxygen stress because it inhibits the 1-electron reduction of quinones to semiquinones and their subsequent oxidation to quinones termed as quinone redox cycle. It belongs to the aromatic hydrocarbon-responsive (Ah) battery. This gene battery includes Cyp1a1 (cytochrome P-450 IA1), Cyp1a2 (cytochrome P-450 IA2) and Nmo-1 [NAD(P)H: quinone reductase]. In the skin cytochrome P-450 IA1-dependent activity is about 1-5% compared to the corresponding activity in the liver, whereas NQR has the same activity in skin and liver. NQR was determined in the cytoplasm of murine skin, liver, and human keratinocytes using 2,6-dichlorophenolindophenol as the substrate. The Ah-receptor binding compounds, such as coal tar constituents, or 3-methylcholanthrene induce cytochrome P-450-dependent activities such as aryl hydrocarbon hydroxylase or 7-ethoxyresorufin-O-de-ethylase and NQR, whereas butyl hydroxytoluol, which does not bind to the Ah receptor, induces only NQR. For inhibition studies several known inhibitors of dihydrodiol dehydrogenase, aldo-keto and carbonyl reductase activities were used. There was a similar pattern of inhibition of the basal and induced activity in all tissues investigated. Pyrazole, progesterone and phenobarbital did not inhibit, whereas dicoumarol, rutin and indomethacin inhibited NQR activity in murine skin and liver as well as in human keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1768430

  19. Novel octaheme cytochrome c tetrathionate reductase (OTR) from Shewanella oneidensis MR-1

    OpenAIRE

    Wu, Fei

    2010-01-01

    Octa-heme cytochrome c tetrathionate reductase (OTR) from Shewanella oneidensis MR-1 is a periplasmic protein and shows several extraordinary structural features around its active-site heme. OTR has been found able to catalyse the in vitro reduction of tetrathionate, nitrite, hydroxylamine and hydrogen peroxide. However the physiological function of this novel protein remains unknown. The subject of this thesis is the in vitro catalytic mechanism and the in vivo function of OTR...

  20. The Effects of Acrolein on Peroxiredoxins, Thioredoxins, and Thioredoxin Reductase in Human Bronchial Epithelial Cells

    OpenAIRE

    Myers, Charles R.; Myers, Judith M.

    2008-01-01

    Inhalation is a common form of exposure to acrolein, a toxic reactive volatile aldehyde that is a ubiquitous environmental pollutant. Bronchial epithelial cells would be directly exposed to inhaled acrolein. The thioredoxin (Trx) system is essential for the maintenance of cellular thiol redox balance, and is critical for cell survival. Normally, thioredoxin reductase (TrxR) maintains the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins in the reduced state, and the thioredoxins keep the...

  1. Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells

    OpenAIRE

    Yadav, Umesh CS; Ramana, KV; Srivastava, SK

    2013-01-01

    Aldose reductase (AR), a glucose metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30μM) than glucose. Acrolein, a major endogenous lipid peroxidation product as well as component of environmental pollutant and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders but the mechanism of acrolein-induced cytotoxicity is not clea...

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

    OpenAIRE

    McCarty, G.W.; Bremner, J. M.

    1992-01-01

    It is well established that assimilatory nitrate reductase (ANR) activity in soil is inhibited by ammonium (NH4+). To elucidate the mechanism of this inhibition, we studied the effect of L-methionine sulfoximine (MSX), an inhibitor of NH4+ assimilation by microorganisms, on assimilatory reduction of nitrate (NO3-) in aerated soil slurries treated with NH4+. We found that NH4+ strongly inhibited ANR activity in these slurries and that MSX eliminated this inhibition. We also found that MSX indu...

  3. Identification and Characterization of Inhibitors of Bacterial Enoyl-Acyl Carrier Protein Reductase

    OpenAIRE

    Ling, Losee L.; Xian, Jun; Ali, Syed; Geng, Bolin; Fan, Jun; Mills, Debra M.; Arvanites, Anthony C.; Orgueira, Hernan; Ashwell, Mark A.; Carmel, Gilles; Xiang, Yibin; Moir, Donald T.

    2004-01-01

    Bacterial enoyl-acyl carrier protein reductase (ENR) catalyzes an essential step in fatty acid biosynthesis. ENR is an attractive target for narrow-spectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species. In addition, the bacterial ENR sequence and structural organization are distinctly different from those of mammalian fatty acid biosynthesis enzymes. High-throughput screening to identify inhibitors of Esch...

  4. Dihydropteroate Synthase and Novel Dihydrofolate Reductase Gene Mutations in Strains of Pneumocystis jirovecii from South Africa

    OpenAIRE

    Robberts, F. J. L.; Chalkley, L J; Weyer, K.; Goussard, P.; Liebowitz, L. D.

    2005-01-01

    Dihydropteroate synthase (DHPS) gene mutations have raised concerns about emerging sulfonamide resistance in Pneumocystis jirovecii. DHPS and dihydrofolate reductase (DHFR) gene products were amplified in clinical specimens from South African patients. One of 53 DHPS genes sequenced contained the double mutation Thr55Ala Pro57Ser. DHFR gene mutations detected were Ala67Val and the new mutations Arg59Gly and C278T.

  5. Dihydrofolate reductase amplification and sensitization to methotrexate of methotrexate-resistant colon cancer cells

    DEFF Research Database (Denmark)

    Morales Torres, Christina; García, Maria J; Ribas, Maria;

    2009-01-01

    have analyzed the structure and dynamics of dihydrofolate reductase (DHFR) gene amplification in HT29 cells treated with methotrexate (MTX). Analysis of the DHFR gene amplification process shows that the amplicon exhibits a complex structure that is consistently reproduced in independent treatments...... responsive to a second round of treatment if left untreated during a sufficient period of time. [Mol Cancer Ther 2009;8(2):424-32]....

  6. Role of 5 alpha-reductase inhibitors in the management of prostate cancer

    OpenAIRE

    Hudak, Steven J.; Hernandez, Javier; Thompson, Ian M

    2006-01-01

    Prostate cancer is one of the most complex and enigmatic oncologic problems in medicine. It is highly prevalent, particularly in elderly males. Unfortunately, its generally protracted and variable clinical course and high association with treatment-related morbidity raise serious questions about the ideal treatment strategy for the individual patient. 5 alpha-reductase (5AR) inhibitors have a dramatic effect on benign prostatic disease with low toxicity. Thus, there is much interest in the po...

  7. Current Status of 5α-Reductase Inhibitors in Prostate Disease Management

    OpenAIRE

    Kang, Dong Il; Chung, Jae Il

    2013-01-01

    The key enzyme in the androgen synthesis and androgen receptor pathways is 5α-reductase (5-AR), which occurs as three isoenzymes. Types I and II 5-ARs the most important clinically, and two different 5-AR inhibitors (5-ARIs), finasteride and dutasteride, have been developed. Several urology associations have recommended and upgraded the use of 5-ARIs for an enlarged prostate with lower urinary tract symptoms. In the Prostate Cancer Prevention Trial and the Reduction by Dutasteride of Prostate...

  8. Epalrestat, an aldose reductase inhibitor, in diabetic neuropathy: An Indian perspective

    OpenAIRE

    Sharma S; Sharma Nalini

    2008-01-01

    Background: A number of diabetic patients with diabetic neuropathy, in India, were treated with epalrestat, an aldose reductase inhibitor. In this study, more than 2000 patients with diabetic neuropathy, who were treated with epalrestat for 3-12 months, were analyzed to assess the efficacy and the adverse reactions of the drug. Method: We analyzed the subjective symptoms (spontaneous pain, numbness, coldness and hypoesthesia) and the nerve function tests (motor nerve conduction velocity, s...

  9. Association of Blood Lead Levels with Methylenetetrahydrofolate Reductase Polymorphisms among Chinese Pregnant Women in Wuhan City

    OpenAIRE

    Shen, Wei; Zhang, Bin; Liu, Shuyun; Wu, Hongling; Gu, Xue; Qin, Lingzhi; Tian, Ping; Zeng, Yun; Ye, Linxiang; Ni, Zemin; Wang, Qi

    2015-01-01

    Background Pregnancy is an important stimulus of bone lead release. Elevated blood lead levels (BLLs) may cause adverse pregnancy outcomes for mothers and harmful lead effects on fetuses. However, the reports about maternal BLL changes during pregnancy are conflicting to some extent. This article is to explore the variations in BLLs among pregnant women. The relationships of BLLs with methylenetetrahydrofolate reductase (MTHFR) gene C677T, A1298C, and G1793A polymorphisms, which are associate...

  10. Altered expression of thioredoxin reductase-1 in dysplastic bile ducts and cholangiocarcinoma in a hamster model

    OpenAIRE

    Yoon, Byung-Il; Kim, Dae-Yong; Jang, Ja-June; Han, Jeong-Hee

    2006-01-01

    Thioredoxin reductase 1 (TrxR) is a homodimeric selenoenzyme catalyzing thioredoxin (Trx) in an NADPH-dependent manner. With regard to carcinogenesis, these redox proteins have been implicated in cell proliferation, transformation and anti-apoptosis. In the present study, using a hamster cholangiocarcinoma (ChC) model, we evaluated the immunohistochemical expression pattern of TrxR in precancerous lesions and ChCs as well as in normal bile ducts. The goal of this study was to determine the po...

  11. Anxiety and Methylenetetrahydrofolate Reductase Mutation Treated With S-Adenosyl Methionine and Methylated B Vitamins.

    Science.gov (United States)

    Anderson, Shanna; Panka, Jacob; Rakobitsch, Robin; Tyre, Kaitlin; Pulliam, Kerry

    2016-04-01

    This case report highlights challenges faced in the clinical management of patients with methylenetetrahydrofolate reductase (MTHFR) gene mutations and the importance of precise dosage when recommending methylated B vitamins to compensate for deficiencies caused by the polymorphism or symptoms related to the polymorphism. It also underscores the importance of obtaining ongoing objective assessments of anxiety (eg, Patient Reported Outcomes Measurement Information System, or PROMIS) to help gauge patient response. PMID:27330489

  12. Influence of methylene tetrahydrofolate reductase polymorphisms and coadministration of antimetabolites on toxicity after high dose methotrexate

    DEFF Research Database (Denmark)

    Niekerk, P.B. van Kooten; Schmiegelow, K.; Schroeder, H.

    2008-01-01

    BACKGROUND AND OBJECTIVE: Through interruption of maintenance treatment with 6-mercaptopurine (6MP), toxicity after high-dose methotrexate (HDMTX) may compromise the efficiency of the treatment of children with acute lymphocytic leukaemia (ALL). We investigated the influence of polymorphisms in the...... methylene tetrahydrofolate reductase (MTHFR) gene and coadministration of antimetabolites on post-HDMTX toxicity. METHODS: Toxicity was retrospectively analysed after 656 HDMTX courses administered to 88 paediatric ALL patients at a single treatment centre. RESULTS: High-dose methotrexate with high...

  13. Astaxanthin prevents changes in the activities of thioredoxin reductase and paraoxonase in hypercholesterolemic rabbits

    OpenAIRE

    Paula R. Augusti; Quatrin, Andréia; Somacal, Sabrina; Conterato, Greicy MM; Sobieski, Rocheli; Ruviaro, Amanda R; Maurer, Luana H; Duarte, Marta MF; Roehrs, Miguel; Emanuelli, Tatiana

    2012-01-01

    This study explored the effects of the antioxidant astaxanthin on paraoxonase and thioredoxin reductase activities as well as on other oxidative stress parameters and on the lipid profile in hypercholesterolemic rabbits. Rabbits were fed a standard or a hypercholesterolemic diet alone or supplemented with 50, 100 and 500 mg/100 g of astaxanthin for 60 days. Antioxidant enzymes activities, lipid profile and oxidative stress markers were evaluated in the serum. The hypercholesterolemic diet inc...

  14. The Silkworm Mutant lemon (lemon lethal) Is a Potential Insect Model for Human Sepiapterin Reductase Deficiency*

    OpenAIRE

    Meng, Yan; Katsuma, Susumu; Daimon, Takaaki; Banno, Yutaka; Uchino, Keiro; Sezutsu, Hideki; Tamura, Toshiki; Mita, Kazuei; Shimada, Toru

    2009-01-01

    Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases, which control the levels of monoamine neurotransmitters. BH4 deficiency has been associated with many neuropsychological disorders. An inherited defect in BH4 biosynthesis is caused by the deficiency of sepiapterin reductase (SPR), which catalyzes the biosynthesis of BH4 from guanosine triphosphate at the terminal step. The human SPR gene has been mapped at the PARK3 locus, which is rela...

  15. Diversity, abundance and expression of nitrite reductase (nirK)-like genes in marine thaumarchaea

    OpenAIRE

    Lund, Marie B; Smith, Jason M.; Francis, Christopher A.

    2012-01-01

    Ammonia-oxidizing archaea (AOA) are widespread and abundant in aquatic and terrestrial habitats and appear to have a significant impact on the global nitrogen cycle. Like the ammonia-oxidizing bacteria, AOA encode a gene homologous to copper-containing nitrite reductases (nirK), which has been studied very little to date. In this study, the diversity, abundance and expression of thaumarchaeal nirK genes from coastal and marine environments were investigated using two mutually excluding primer...

  16. Serum total sialic acid, lipid peroxidation, and glutathione reductase levels in patients with rheumatoid arthritis

    OpenAIRE

    Mohan, Surapaneni Krishna; PRIYAV, Vishnu

    2010-01-01

    The changes in the serum sialic acid levels, total lipid peroxidation products (MDA), and glutathione reductase activity were estimated in patients with rheumatoid arthritis. Serum Sialic acid is known as a parameter of inflammation. This work was undertaken to assess the potential role of sialic acid as well as oxidative stress and antioxidant status in patients with rheumatoid arthritis. Materials and methods: The levels of these parameters in serum were studied in 52 subjects with rheuma...

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

    OpenAIRE

    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 w...

  18. Menthofuran regulates essential oil biosynthesis in peppermint by controlling a downstream monoterpene reductase

    OpenAIRE

    Mahmoud, Soheil S.; Croteau, Rodney B.

    2003-01-01

    (+)-Pulegone is a central intermediate in the biosynthesis of (-)-menthol, the most significant component of peppermint essential oil. Depending on environmental conditions, this branch point metabolite may be reduced to (-)-menthone en route to menthol, by pulegone reductase (PR), or oxidized to (+)-menthofuran, by menthofuran synthase (MFS). To elucidate regulation of pulegone metabolism, we modified the expression of mfs under control of the CaMV 35S promoter in transformed peppermint plan...

  19. Disruption of the plr1+ gene encoding pyridoxal reductase of Schizosaccharomyces pombe.

    Science.gov (United States)

    Morita, Tomotake; Takegawa, Kaoru; Yagi, Toshiharu

    2004-02-01

    Pyridoxal (PL) reductase encoded by the plr1(+) gene practically catalyzes the irreversible reduction of PL by NADPH to form pyridoxine (PN). The enzyme has been suggested to be involved in the salvage synthesis of pyridoxal 5'-phosphate (PLP), a coenzyme form of vitamin B(6), or the excretion of PL as PN from yeast cells. In this study, a PL reductase-disrupted (plr1 Delta) strain was constructed and its phenotype was examined. The plr1 Delta cells showed almost the same growth curve as that of wild-type cells in YNB and EMM media. In EMM, the plr1 Delta strain became flocculent at the late stationary phase for an unknown reason. The plr1 Delta cells showed low but measurable PL reductase activity catalyzed by some other protein(s) than the enzyme encoded by the plr1(+) gene, which maintained the flow of "PL --> PN --> PNP --> PLP" in the salvage synthesis of PLP. The total vitamin B(6) and pyridoxamine 5'-phosphate contents in the plr1 Delta cells were significantly lower than those in the wild-type ones. The percentages of the PLP amount as to the other vitamin B(6) compounds were similar in the two cell types. The amount of PL in the culture medium of the disruptant was significantly higher than that in the wild-type. In contrast, PN was much higher in the latter than the former. The plr1 Delta cells accumulated a 6.1-fold higher amount of PL than the wild-type ones when they were incubated with PL. The results showed that PL reductase encoded by the plr1(+ )gene is involved in the excretion of PL after reducing it to PN, and may not participate in the salvage pathway for PLP synthesis. PMID:15047724

  20. Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis

    Directory of Open Access Journals (Sweden)

    Maíra Bidart Macedo

    2012-09-01

    Full Text Available We validated the nitrate reductase assay (NRA for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB using sodium nitrate (NaNO3 in replacement of potassium nitrate (KNO3 as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.

  1. Mutational analysis of the triclosan-binding region of enoyl-ACP reductase from Plasmodium falciparum

    OpenAIRE

    Kapoor, Mili; Gopalakrishnapai, Jayashree; Surolia, Namita; Surolia, Avadhesha

    2004-01-01

    Triclosan, a known antibacterial, acts by inhibiting enoyl-ACP (acyl-carrier protein) reductase (ENR), a key enzyme of the type II fatty acid synthesis (FAS) system. Plasmodium falciparum, the human malaria-causing parasite, harbours the type II FAS; in contrast. its human host utilizes type I FAS. Due to this striking difference, ENR has emerged as an important target for the development of new antimalarials. Modelling studies, and the crystal structure of P.falciparum ENR, have highlighted ...

  2. Sources of reducing equivalents for nitrite reductase in Pisum arvense roots

    OpenAIRE

    Grażyna Kłobus

    2014-01-01

    Glucose-6-phosphate and NADP+ as well as malic acid and NADP+ present in the incubation mixture enhanced nitrite reductase (EC 1.6.6.4) activity in Pisum arvense roots. This was manifested by a depression of the nitrite level in the tissues and an increased reduction of nitrites by plastids isolated from P. arvense roots. A marked stimulation of plastid malate dehydrogenase was also observed under the influence of nitrates present in the medium. These results suggest that pyridin nucleotides ...

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Rosana Aparecida Manólio Soares

    2015-02-01

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

  5. A preliminary study on estimating extra-cellular nitrate reductase activities in estuarine systems

    Directory of Open Access Journals (Sweden)

    Pant H. K.

    2009-07-01

    Full Text Available Enzymes catalyzing ammonium (NH4+/nitrate (NO3– into nitrous oxide (N2O/molecular nitrogen (N2, play critical roles in water quality management. The objective of this paper was to investigate the role of extra-cellular enzymes in cycling of nitrogen (N in aquatic systems. It appears that N in estuaries, salt marshes, etc., does not stay long enough to be available for uptake, thus, creating N limited conditions. This study showed that indigenous extra-cellular nitrate reductase along with others involved in N transformations in the waters/sediments of estuarine systems can cause complete removal of NH4+ and NO3– from the waters and available NH4+ and NO3– from the sediments. These results indicate that due to high extra-cellular nitrate reductase and other enzymes associated with N transformations in sediments/waters, substantial amounts of NH4+ and NO3– can be quickly lost from the systems as N2O and/or nitric oxide (NO, in turn, creating N limited conditions in estuarine systems. Such high activities of indigenous nitrate reductase and others are useful in removing readily bioavailable N from the systems, thereby avoidance of eutrophic conditions. However, they might contribute in increasing the N2O, a potent greenhouse gas with global warming potential (GWP of 296, in the atmosphere.

  6. 5alpha-reductase inhibitors in benign prostatic hyperplasia and prostate cancer risk reduction.

    Science.gov (United States)

    Rittmaster, Roger S

    2008-04-01

    Androgens play an essential role in prostatic development and function, but are also involved in prostate disease pathogenesis. The primary prostatic androgen, dihydrotestosterone (DHT), is synthesized from testosterone by 5alpha-reductase types 1 and 2. Inhibition of the 5alpha-reductase isoenzymes therefore has potential therapeutic benefit in prostate disease. The two currently approved 5alpha-reductase inhibitors (5ARIs), finasteride and dutasteride, have demonstrated long-term efficacy and safety in the treatment of benign prostatic hyperplasia. Finasteride, a type-2 5ARI, has also been studied for its ability to reduce the incidence of biopsy-detectable prostate cancer in the Prostate Cancer Prevention Trial. Treatment with dutasteride, a dual 5ARI, has been shown to result in a greater degree and consistency of DHT suppression compared with finasteride. Two large-scale studies of dutasteride are currently investigating the role of near-maximal DHT suppression in the settings of prostate cancer risk reduction and expectant management of localized prostate cancer. PMID:18471794

  7. Inhibition of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase (Ex Vivo by Morus indica (Mulberry

    Directory of Open Access Journals (Sweden)

    Vanitha Reddy Palvai

    2014-01-01

    Full Text Available Phytochemicals are the bioactive components that contribute to the prevention of cardiovascular and other degenerative diseases. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA reductase would be an effective means of lowering plasma cholesterol in humans. The present study explores the HMG CoA reductase inhibitory effect of extracts from leaves of Morus indica varieties, M5, V1, and S36, compared with the statin, using an ex vivo method. The assay is based on the stoichiometric formation of coenzyme A during the reduction of microsomal HMG CoA to mevalonate. Dechlorophyllised extract of three varieties was studied at 300 µg. The coenzyme A released at the end of assay in control (100.31 nmoles and statins (94.46 nm was higher than the dechlorphyllised extracts of the samples. The coenzyme A released during the reduction of HMG CoA to mevalonate in dechlorophyllised extracts of the samples was as follows: S36 < M5 < V1. The results indicated that the samples were highly effective in inhibiting the enzyme compared to statins (standard drug. The results indicate the role of Morus varieties extracts in modulating the cholesterol metabolism by inhibiting the activity of HMG CoA reductase. These results provide scope for designing in vivo animal studies to confirm their effect.

  8. Glutathione reductase gsr-1 is an essential gene required for Caenorhabditis elegans early embryonic development.

    Science.gov (United States)

    Mora-Lorca, José Antonio; Sáenz-Narciso, Beatriz; Gaffney, Christopher J; Naranjo-Galindo, Francisco José; Pedrajas, José Rafael; Guerrero-Gómez, David; Dobrzynska, Agnieszka; Askjaer, Peter; Szewczyk, Nathaniel J; Cabello, Juan; Miranda-Vizuete, Antonio

    2016-07-01

    Glutathione is the most abundant thiol in the vast majority of organisms and is maintained in its reduced form by the flavoenzyme glutathione reductase. In this work, we describe the genetic and functional analysis of the Caenorhabditis elegans gsr-1 gene that encodes the only glutathione reductase protein in this model organism. By using green fluorescent protein reporters we demonstrate that gsr-1 produces two GSR-1 isoforms, one located in the cytoplasm and one in the mitochondria. gsr-1 loss of function mutants display a fully penetrant embryonic lethal phenotype characterized by a progressive and robust cell division delay accompanied by an aberrant distribution of interphasic chromatin in the periphery of the cell nucleus. Maternally expressed GSR-1 is sufficient to support embryonic development but these animals are short-lived, sensitized to chemical stress, have increased mitochondrial fragmentation and lower mitochondrial DNA content. Furthermore, the embryonic lethality of gsr-1 worms is prevented by restoring GSR-1 activity in the cytoplasm but not in mitochondria. Given the fact that the thioredoxin redox systems are dispensable in C. elegans, our data support a prominent role of the glutathione reductase/glutathione pathway in maintaining redox homeostasis in the nematode. PMID:27117030

  9. Influence of nitrogen supply on spring barley productivity and nitrate reductase activity

    Directory of Open Access Journals (Sweden)

    U. Wojcieska

    2013-12-01

    Full Text Available The aim of the present study was to obtain some informations on the productivity of four chosen barley varieties growing at low and high nitrogen level. Some parameters of the yield structure and nitrate reductase activity were taken into consideration. It was found that there exist some differences in the yield between the compared varieties and some differences in their reaction to a high N level in the soil. The grain yield increase of the plants treated with high nitrogen doses was above all the result of the increase in dry matter of the lateral shoots and in leaf area. Distinct increase in the number of grains per ear and 1000-grains weight was also observed. The amount of reduced nitrogen collected during the growth season depended, in part, on the nitrate reductase activity and in part on the amount of the enzyme present in the plant. A rise of the nitrogen level caused an increase in nitrate reductase activity, in all varieties. The different influence of nitrogen on the growth of green organs in the compared varieties caused differences in the amount of the enzyme present in the plants and in protein yields.

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

    Science.gov (United States)

    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

  11. Soil acidity as affecting micronutrients concentration, nitrato reductase enzyme activity and yield in upland rice plants

    Directory of Open Access Journals (Sweden)

    Edemar Moro

    2013-12-01

    Full Text Available The lowest grain yield of rice under no-tillage system (NTS in relation to the conventional system may be due to the predominance nitrate in the soil and the low nitrate reductase activity. Another reason may be caused by micronutrient deficiency because of superficially soil acidity corrections. Therefore, the objective of this study was to evaluate the changes caused by soil pH in the N forms in the soil, micronutrients concentration in rice plants, nitrate reductase activity, yield of rice and its components. The experiment was performed in a greenhouse conditions. The experimental design was a completely randomized in a factorial three (levels of soil acidity x five (micronutrients sources with four replications. The addition of micronutrients does not affect levels of nitrate and ammonium in the soil; soil acidity significantly affects levels of nitrate and ammonium in the soil, concentration of micronutrients in rice plants and crop yield and its components; medium soil acidity (pH 5.5 result in medium to high levels of Cu and Fe, medium level of Zn and Mn, high nitrate reductase activity, resulting in higher dry matter, tillers, panicles, spikelets, weight of 100 grains and hence grain yield.

  12. Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean.

    Science.gov (United States)

    Cheng, Qun; Li, Ninghui; Dong, Lidong; Zhang, Dayong; Fan, Sujie; Jiang, Liangyu; Wang, Xin; Xu, Pengfei; Zhang, Shuzhen

    2015-01-01

    Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity. PMID:26635848

  13. Targeting 5α-reductase for prostate cancer prevention and treatment.

    Science.gov (United States)

    Nacusi, Lucas P; Tindall, Donald J

    2011-07-01

    Testosterone is the most abundant circulating androgen, and can be converted to dihydrotestosterone (DHT), a more potent androgen, by the 5α-reductase enzymes in target tissues. Current treatments for prostate cancer consist of reducing androgen levels by chemical or surgical castration or pure antiandrogen therapy that directly targets the androgen receptor (AR). Although these therapies reduce tumor burden and AR activity, the cancer inevitably recurs within 18-30 months. An approach targeting the androgen-AR axis at different levels could, therefore, improve the efficacy of prostate cancer therapy. Inhibition of 5α-reductase is one such approach; however, the two largest trials to investigate the use of the 5α-reductase inhibitors (5ARIs) finasteride and dutasteride in patients with prostate cancer have shown that, although the incidence of cancer was reduced by 5ARI treatment, those cancers that were detected were more aggressive than in patients treated with placebo. Thus, the best practice for using these drugs to prevent and treat prostate cancer remains unclear. PMID:21629218

  14. 5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes.

    Science.gov (United States)

    Nasiri, Maryam; Nikolaou, Nikolaos; Parajes, Silvia; Krone, Nils P; Valsamakis, George; Mastorakos, George; Hughes, Beverly; Taylor, Angela; Bujalska, Iwona J; Gathercole, Laura L; Tomlinson, Jeremy W

    2015-08-01

    Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux. PMID:25974403

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

    Directory of Open Access Journals (Sweden)

    Tae Hyeon Kim

    2013-01-01

    Full Text Available 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.

  16. Evaluation of the Activities of Pyrimethamine Analogs against Plasmodium vivax and Plasmodium falciparum Dihydrofolate Reductase-Thymidylate Synthase Using In Vitro Enzyme Inhibition and Bacterial Complementation Assays▿

    OpenAIRE

    Bunyarataphan, Sasinee ; Leartsakulpanich, Ubolsree; Taweechai, Supannee; Tarnchompoo, Bongkoch; Kamchonwongpaisan, Sumalee; Yuthavong, Yongyuth

    2006-01-01

    Pyrimethamine analogs were examined as potential agents against vivax malaria using a bacterial surrogate system carrying Plasmodium vivax dihydrofolate reductase-thymidylate synthase (PvDHFR-TS), in which the PvDHFR complemented chemically knocked out host dihydrofolate reductase. The system was initially tested with P. falciparum dihydrofolate reductase-thymidylate synthase and was found to have good correlation with the parasite-based system. The 50% inhibitory concentrations derived from ...

  17. Triclosan Resistance of Pseudomonas aeruginosa PAO1 Is Due to FabV, a Triclosan-Resistant Enoyl-Acyl Carrier Protein Reductase

    OpenAIRE

    Zhu, Lei; Lin, Jinshui; Ma, Jincheng; Cronan, John E.; Wang, Haihong

    2009-01-01

    Triclosan, a very widely used biocide, specifically inhibits fatty acid synthesis by inhibition of enoyl-acyl carrier protein (ACP) reductase. Escherichia coli FabI is the prototypical triclosan-sensitive enoyl-ACP reductase, and E. coli is extremely sensitive to the biocide. However, other bacteria are resistant to triclosan, because they encode triclosan-resistant enoyl-ACP reductase isozymes. In contrast, the triclosan resistance of Pseudomonas aeruginosa PAO1 has been attributed to active...

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

    Science.gov (United States)

    Bettiga, Maurizio; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

    2008-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Hahn-Hägerdal Bärbel

    2008-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Baskaran G

    2015-01-01

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

  1. 5α—reductase type 2 gene expression in human testis,epididymis and vas deferens

    Institute of Scientific and Technical Information of China (English)

    LiuDY; WuYW

    2002-01-01

    Objective:To study the expression patterm of 5-α-reductase type 2 gene in human male reproductive organs.Methods:The expression level of 5α-reductase type 2 gene in human testis,epididymis and vas deferens tissues was determined by in situ hybridization using a digoxin-labeled 5α-reductase type 2 cRNA probe.Results:The brown granules of hybridizing signals distributed in the cytoplasm of the Sertoli and Leydig cells of the testis,the principle cells of epididymis and the epithelial cells of vas deferens,but there was no positive signal in the nuclei of these cellsNo positive signal was observed in the germ cells,basement of the testis, interstium of the epididymis and basement and the smooth muscle cells of vas deferens.conclusion:This study confirmed that the 5α-reductase type 2 gene expressed in the Sertoli and Leydig cells of the testis and the principle cells of the epididymis.The expression pattern of the gene in these cells in the human was similar to that in the rat and monkey.The presence of 5α-reductase type 2 gene in the epithelial cells of the vas deferens suggests that it may play a physiological role in human reproduction.

  2. Downregulation of thioredoxin reductase 1 expression in the substantia nigra pars compacta of Parkinson’s disease mice

    Institute of Scientific and Technical Information of China (English)

    Zihua Liu; Yuhong Jing; Jie Yin; Jiying Mu; Tingting Yao; Liping Gao

    2013-01-01

    Because neurons are susceptible to oxidative damage and thioredoxin reductase 1 is extensively distributed in the central nervous system and has antioxidant properties, we speculated that the enzyme may be involved in the pathogenesis of Parkinson’s disease. A Parkinson’s disease model was produced by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine into C57BL/6 mice. Real-time reverse transcription-PCR, western blot analysis and colorimetric assay showed that the levels of thioredoxin reductase 1 mRNA and protein were decreased, along with a significant reduction in thioredoxin reductase activity, in the midbrain of Parkinson’s disease mice compared with normal mice. Immunohistochemical staining revealed that the number of thioredoxin reductase 1-positive neurons in the substantia nigra pars compacta of Parkinson’s disease mice was significantly decreased compared with normal mice. These experimental findings suggest that the expression of thioredoxin reductase 1 in the substantia nigra pars compacta of Parkinson’s disease mice is significantly decreased, and that the enzyme may be associated with disease onset.

  3. ATP-induced temperature independence of hemoglobin-O2 affinity in heterothermic billfish

    DEFF Research Database (Denmark)

    Weber, Roy E.; Campbell, Kevin L.; Fago, Angela;

    2010-01-01

    heterotherms, where it may hamper unloading (e.g. in cold extremities of arctic mammals) or increase the diffusive arterio-venous short-circuiting of O2 (e.g. in counter-current heat exchangers of warm swimming muscles of tuna). We hypothesized analogous blood specializations in heterothermic billfish, whose...... addition to allosterically modulating hemoglobin-O2 affinity, ATP diminishes its temperature sensitivity, reducing deleterious arterio-venous short-circuiting of oxygen in the cranial billfish heat exchangers. The mechanism underlying this reduction in oxygenation enthalpy differs fundamentally from that...

  4. ATP-induced lipid membrane reordering in the myelinated nerve fiber identified using Raman spectroscopy

    International Nuclear Information System (INIS)

    We demonstrate a successful application of Raman spectroscopy to the problem of lipid ordering with microscopic resolution in different regions of the myelinated nerve fiber. Simultaneous collection of Raman spectra of lipids and carotenoids has enabled us to characterize membrane fluidity and the degree of lipid ordering based on intensity ratios for the 1527/1160 and 2940/2885 cm−1 bands. We show that the intensity profiles of the major Raman bands vary significantly between the three major regions of myelinated nerve fiber: internode, paranode and the node of Ranvier. Mapping Raman peak intensities over these areas suggested that the carotenoid molecules are localized in the myelin membranes of nerve cells. Paranodal membranes were sensitive to extracellular ATP. ATP solutions (7 mM) influenced the 1527/1160 and 2940/2885 cm−1 intensity ratios. Changes in both carotenoid and lipid Raman spectra were in accord and indicated an increase in lipid ordering degree and decrease in membrane fluidity under ATP administration. The collected data provide evidence for the existence of a regulatory purinergic signaling pathway in the peripheral nervous system. (letter)

  5. ATP-induced temperature independence of hemoglobin-O2 affinity in heterothermic billfish

    DEFF Research Database (Denmark)

    Weber, Roy E.; Campbell, Kevin L.; Fago, Angela; Malte, Hans; Jensen, Frank Bo

    2010-01-01

      The inverse relationship between temperature and hemoglobin-O2 affinity resulting from the exothermic nature of heme oxygenation favors O2 unloading from blood to warm, metabolically active tissues. However, this temperature sensitivity is maladaptive, and commonly countered in regional heterot...

  6. Phosphocholine-Modified Macromolecules and Canonical Nicotinic Agonists Inhibit ATP-Induced IL-1β Release.

    Science.gov (United States)

    Hecker, Andreas; Küllmar, Mira; Wilker, Sigrid; Richter, Katrin; Zakrzewicz, Anna; Atanasova, Srebrena; Mathes, Verena; Timm, Thomas; Lerner, Sabrina; Klein, Jochen; Kaufmann, Andreas; Bauer, Stefan; Padberg, Winfried; Kummer, Wolfgang; Janciauskiene, Sabina; Fronius, Martin; Schweda, Elke K H; Lochnit, Günter; Grau, Veronika

    2015-09-01

    IL-1β is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1β plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1β release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro-IL-1β synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro-IL-1β by caspase-1, and release of mature IL-1β. Mechanisms controlling IL-1β release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1β release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1β and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host. PMID:26202987

  7. ATP-induced electron transfer by redox-selective partner recognition

    Science.gov (United States)

    Hennig, Sandra E.; Goetzl, Sebastian; Jeoung, Jae-Hun; Bommer, Martin; Lendzian, Friedhelm; Hildebrandt, Peter; Dobbek, Holger

    2014-08-01

    Thermodynamically unfavourable electron transfers are enabled by coupling to an energy-supplying reaction. How the energy is transduced from the exergonic to the endergonic process is largely unknown. Here we provide the structural basis for an energy transduction process in the reductive activation of B12-dependent methyltransferases. The transfer of one electron from an activating enzyme to the cobalamin cofactor is energetically uphill and relies on coupling to an ATPase reaction. Our results demonstrate that the key to coupling is, besides the oxidation state-dependent complex formation, the conformational gating of the electron transfer. Complex formation induces a substitution of the ligand at the electron-accepting Co ion. Addition of ATP initiates electron transfer by provoking conformational changes that destabilize the complex. We show how remodelling of the electron-accepting Co2+ promotes ATP-dependent electron transfer; an efficient strategy not seen in other electron-transferring ATPases.

  8. Extracellular ATP induces a large nonselective conductance in macrophage plasma membranes.

    OpenAIRE

    Buisman, H P; Steinberg, T. H.; FISCHBARG, J.; Silverstein, S C; Vogelzang, S A; Ince, C.; Ypey, D.L.; Leijh, P C

    1988-01-01

    Extracellular ATP in its tetra-anionic form (ATP4-) induces ion fluxes and membrane depolarization in the mouse macrophage-like cell line J774.2 and in resident mouse macrophages. We analyzed the effects of extracellular ATP4- by both patch-clamp and intracellular microelectrode techniques. Whole-cell patch-configuration membrane potential measurements on J774.2 cells revealed that ATP4- -induced depolarization occurred within 40 ms of pulsed application of ATP and was completely reversible. ...

  9. Cdna cloning and expression analyses of the isoflavone reductase-like gene of dendrobium officinale

    International Nuclear Information System (INIS)

    The full length of the isoflavone reductase-like gene (IRL) cDNA of Dendrobium officinale was cloned by using reverse transcription (RT) PCR combined with cDNA library, the IRL function was identified by Bioinformatics and prokaryotic expression analyses, and the IRL expression levels in the organs and tissues of D. officinale plants with different ages were determined by using real-time quantitative PCR (RT-qPCR). The results indicated that the full length of the cDNA of D. officinale IRL, DoIRL, was 1238 bp (accession no. KJ661023). Its open reading frame (ORF) was 930 bp which encoded 309 amino acids with a predicted molecular mass of 34 kDa, the 5 untranslated region (UTR) was 61 bp and the 3 UTR containing a poly (A) tail was 247 bp. The deduced amino acid sequence of DoIRL, DoIRL, was forecast to contain a NAD(P)H-binding motif (GGTGYIG) in the N-terminal region, two conserved N-glycosylation sites, a conserved nitrogen metabolite repression regulator (NmrA) domain and a phenylcoumaran benzylic ether reductase (PCBER) domain, to hold the nearest phylogenetic relationship with the PCBER of Striga asiatica, and to share both 73% identity with the isoflavone reductases-like (IRLs) of Cucumis sativus and Striga asiatica. In Escherichia coli 'BL21' cells, the DoIRL cDNA expression produced a protein band holding the predicted molecular mass of 34 kDa. DoIRL expressed in all organs and tissues of D. officinale plants with different ages at comparatively low levels, and the expression level in the leaves of the two-year-old plants was the highest. (author)

  10. Mechanistic studies with solubilized rat liver steroid 5 alpha-reductase: Elucidation of the kinetic mechanism

    International Nuclear Information System (INIS)

    A solubilized preparation of steroid 5 alpha-reductase from rat liver has been used in studies focused toward an understanding of the kinetic mechanism associated with enzyme catalysis. From the results of analyses with product and dead-end inhibitors, a preferentially ordered binding of substrates and release of products from the surface of the enzyme is proposed. The observations from these experiments were identical with those using the steroid 5 alpha-reductase activity associated with rat liver microsomes. The primary isotope effects on steady-state kinetic parameters when [4S-2H]NADPH was used also were consistent with an ordered kinetic mechanism. Normal isotope effects were observed for all three kinetic parameters (Vm/Km for both testosterone and NADPH and Vm) at all substrate concentrations used experimentally. Upon extrapolation to infinite concentration of testosterone, the isotope effect on Vm/Km for NADPH approached unity, indicating that the nicotinamide dinucleotide phosphate is the first substrate binding to and the second product released from the enzyme. The isotope effects on Vm/Km for testosterone at infinite concentration of cofactor and on Vm were 3.8 +/- 0.5 and 3.3 +/- 0.4, respectively. Data from the pH profiles of these three steady-state parameters and the inhibition constants (1/Ki) of competitive inhibitors versus both substrates indicate that the binding of nicotinamide dinucleotide phosphate involves coordination of its anionic 2'-phosphate to a protonated enzyme-associated base with an apparent pK near 8.0. From these results, relative limits have been placed on several of the internal rate constants used to describe the ordered mechanism of the rat liver steroid 5 alpha-reductase

  11. Dicarbonyl L-xylulose reductase (DCXR), a "moonlighting protein" in the bovine epididymis.

    Science.gov (United States)

    Akintayo, Ayodélé; Légaré, Christine; Sullivan, Robert

    2015-01-01

    During maturation and the acquisition of their fertilization potential, male germ cells are subjected to various sequential modifications that occur in the epididymis. Protein addition, reorganization or withdrawal, comprise some of these modifications. Dicarbonyl L-xylulose reductase (DCXR), a multifunctional protein involved in various enzymatic and protein interaction processes in different physiological systems, is one of the proteins added to spermatozoa in the epididymis. DCXR is a well-conserved protein with multiple characteristics including enzymatic activities and mediation of cell-cell interaction. In this study, we characterized the DCXR gene and protein expression in the bovine epididymis. Dicarbonyl L-xylulose reductase mRNA is differentially expressed in the caput, corpus, and cauda epididymide epithelial cells with a higher level observed in the cauda region. Tissue protein expression follows the same pattern as the corresponding mRNA expression with a cytoplasmic and apical distribution in the corpus and cauda epithelial cells, respectively. The protein can also be found with a nuclear localization in cauda epididymidis epithelial cells. Dicarbonyl L-xylulose reductase is secreted in the epididymis luminal compartment in the soluble fraction and is associated with microvesicular elements named epididymosomes. In spermatozoa, the DCXR protein was found in the cytoplasmic and membranous fractions. Expression of the DCXR protein is higher on caput spermatozoa but finally shows a weak detection in semen. These data describe DCXR in the bovine epididymis and reveal that its behavior differs from that found in humans. It seems that, in this model, the DCXR protein might have a questionable involvement in the fertilization process. PMID:25815750

  12. Dicarbonyl L-xylulose reductase (DCXR, a "moonlighting protein" in the bovine epididymis.

    Directory of Open Access Journals (Sweden)

    Ayodélé Akintayo

    Full Text Available During maturation and the acquisition of their fertilization potential, male germ cells are subjected to various sequential modifications that occur in the epididymis. Protein addition, reorganization or withdrawal, comprise some of these modifications. Dicarbonyl L-xylulose reductase (DCXR, a multifunctional protein involved in various enzymatic and protein interaction processes in different physiological systems, is one of the proteins added to spermatozoa in the epididymis. DCXR is a well-conserved protein with multiple characteristics including enzymatic activities and mediation of cell-cell interaction. In this study, we characterized the DCXR gene and protein expression in the bovine epididymis. Dicarbonyl L-xylulose reductase mRNA is differentially expressed in the caput, corpus, and cauda epididymide epithelial cells with a higher level observed in the cauda region. Tissue protein expression follows the same pattern as the corresponding mRNA expression with a cytoplasmic and apical distribution in the corpus and cauda epithelial cells, respectively. The protein can also be found with a nuclear localization in cauda epididymidis epithelial cells. Dicarbonyl L-xylulose reductase is secreted in the epididymis luminal compartment in the soluble fraction and is associated with microvesicular elements named epididymosomes. In spermatozoa, the DCXR protein was found in the cytoplasmic and membranous fractions. Expression of the DCXR protein is higher on caput spermatozoa but finally shows a weak detection in semen. These data describe DCXR in the bovine epididymis and reveal that its behavior differs from that found in humans. It seems that, in this model, the DCXR protein might have a questionable involvement in the fertilization process.

  13. Functional properties and structural characterization of rice δ1-pyrroline-5-carboxylate reductase

    Science.gov (United States)

    Forlani, Giuseppe; Bertazzini, Michele; Zarattini, Marco; Funck, Dietmar; Ruszkowski, Milosz; Nocek, Bogusław

    2015-01-01

    The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L.) for δ1-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP+ were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP+ ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. Based on the structure, it was possible to identify dynamic structural differences among rice, human, and bacterial enzymes. PMID:26284087

  14. Functional properties and structural characterization of rice δ(1)-pyrroline-5-carboxylate reductase.

    Science.gov (United States)

    Forlani, Giuseppe; Bertazzini, Michele; Zarattini, Marco; Funck, Dietmar; Ruszkowski, Milosz; Nocek, Bogusław

    2015-01-01

    The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L.) for δ(1)-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP(+) were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP(+) ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. Based on the structure, it was possible to identify dynamic structural differences among rice, human, and bacterial enzymes. PMID:26284087

  15. X-ray structural studies of quinone reductase 2 nanomolar range inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Pegan, Scott D.; Sturdy, Megan; Ferry, Gilles; Delagrange, Philippe; Boutin, Jean A.; Mesecar, Andrew D. (IdRS); (Purdue); (Colorado); (UIC)

    2011-09-06

    Quinone reductase 2 (QR2) is one of two members comprising the mammalian quinone reductase family of enzymes responsible for performing FAD mediated reductions of quinone substrates. In contrast to quinone reductase 1 (QR1) which uses NAD(P)H as its co-substrate, QR2 utilizes a rare group of hydride donors, N-methyl or N-ribosyl nicotinamide. Several studies have linked QR2 to the generation of quinone free radicals, several neuronal degenerative diseases, and cancer. QR2 has been also identified as the third melatonin receptor (MT3) through in cellulo and in vitro inhibition of QR2 by traditional MT3 ligands, and through recent X-ray structures of human QR2 (hQR2) in complex with melatonin and 2-iodomelatonin. Several MT3 specific ligands have been developed that exhibit both potent in cellulo inhibition of hQR2 nanomolar, affinity for MT3. The potency of these ligands suggest their use as molecular probes for hQR2. However, no definitive correlation between traditionally obtained MT3 ligand affinity and hQR2 inhibition exists limiting our understanding of how these ligands are accommodated in the hQR2 active site. To obtain a clearer relationship between the structures of developed MT3 ligands and their inhibitory properties, in cellulo and in vitro IC{sub 50} values were determined for a representative set of MT3 ligands (MCA-NAT, 2-I-MCANAT, prazosin, S26695, S32797, and S29434). Furthermore, X-ray structures for each of these ligands in complex with hQR2 were determined allowing for a structural evaluation of the binding modes of these ligands in relation to the potency of MT3 ligands.

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

    Science.gov (United States)

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

    1997-01-01

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

  17. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

    Science.gov (United States)

    Trigoso, Yvonne D; Evans, Russell C; Karsten, William E; Chooback, Lilian

    2016-01-01

    The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification. PMID:26815040

  18. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

    Directory of Open Access Journals (Sweden)

    Yvonne D Trigoso

    Full Text Available The enzyme dihydrodipicolinate reductase (DHDPR is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(PH dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3. The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

  19. Evidence that the intra-amoebal Legionella drancourtii acquired a sterol reductase gene from eukaryotes

    Directory of Open Access Journals (Sweden)

    Fournier Pierre-Edouard

    2009-03-01

    Full Text Available Abstract Background Free-living amoebae serve as a natural reservoir for some bacteria that have evolved into «amoeba-resistant» bacteria. Among these, some are strictly intra-amoebal, such as Candidatus "Protochlamydia amoebophila" (Candidatus "P. amoebophila", whose genomic sequence is available. We sequenced the genome of Legionella drancourtii (L. drancourtii, another recently described intra-amoebal bacterium. By comparing these two genomes with those of their closely related species, we were able to study the genetic characteristics specific to their amoebal lifestyle. Findings We identified a sterol delta-7 reductase-encoding gene common to these two bacteria and absent in their relatives. This gene encodes an enzyme which catalyses the last step of cholesterol biosynthesis in eukaryotes, and is probably functional within L. drancourtii since it is transcribed. The phylogenetic analysis of this protein suggests that it was acquired horizontally by a few bacteria from viridiplantae. This gene was also found in the Acanthamoeba polyphaga Mimivirus genome, a virus that grows in amoebae and possesses the largest viral genome known to date. Conclusion L. drancourtii acquired a sterol delta-7 reductase-encoding gene of viridiplantae origin. The most parsimonious hypothesis is that this gene was initially acquired by a Chlamydiales ancestor parasite of plants. Subsequently, its descendents transmitted this gene in amoebae to other intra-amoebal microorganisms, including L. drancourtii and Coxiella burnetii. The role of the sterol delta-7 reductase in prokaryotes is as yet unknown but we speculate that it is involved in host cholesterol parasitism.

  20. Functional and structural aspects of poplar cytosolic and plastidial type A methionine sulfoxide reductases

    OpenAIRE

    Rouhier, Nicolas; Kauffmann, Brice; Tete-Favier, Frédérique; Palladino, Pasquale; Gans, Pierre; Branlant, Guy; Jacquot, Jean-Pierre; Boschi-Muller, Sandrine

    2007-01-01

    The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism i...

  1. Methylenetetrahydrofolate reductase homozygous mutation in a young boy with cerebellar infarction

    Directory of Open Access Journals (Sweden)

    Alberto Spalice

    2009-11-01

    Full Text Available Posterior circulation vascular occlusive disease in children is a rare and uncommonly reported event. Among the numerous risk factors, the methylenetetrahydrofolate reductase (MTHFR mutation is considered to be a common genetic cause of thrombosis in adults and children. Recently, a link between the MTHFR mutation and cerebrovascular disorders was reported in children. Diffusion tensor imaging (DTI is a great improvement on magnetic resonance imaging (MRI, making the in vivo anatomical and pathological study of the brain and its fibers possible. In our patient cerebellar infarction was associated with MTHFR mutation and, in a standard neurological examination, DTI revealed normal white matter tracts.

  2. Methylenetetrahydrofolate reductase homozygous mutation in a young boy with cerebellar infarction.

    Science.gov (United States)

    Spalice, Alberto; Del Balzo, Francesca; Perla, Francesco Massimo; Properzi, Enrico; Carducci, Carla; Antonozzi, Italo; Iannetti, Paola

    2009-01-01

    Posterior circulation vascular occlusive disease in children is a rare and uncommonly reported event. Among the numerous risk factors, the methylenetetrahydrofolate reductase (MTHFR) mutation is considered to be a common genetic cause of thrombosis in adults and children. Recently, a link between the MTHFR mutation and cerebrovascular disorders was reported in children. Diffusion tensor imaging (DTI) is a great improvement on magnetic resonance imaging (MRI), making the in vivo anatomical and pathological study of the brain and its fibers possible. In our patient cerebellar infarction was associated with MTHFR mutation and, in a standard neurological examination, DTI revealed normal white matter tracts. PMID:21589820

  3. Differential expression of 5-alpha reductase isozymes in the prostate and its clinical implications

    Directory of Open Access Journals (Sweden)

    Kai Wang

    2014-04-01

    Full Text Available The development of human benign or malignant prostatic diseases is closely associated with androgens, primarily testosterone (T and dihydrotestosterone (DHT. T is converted to DHT by 5-alpha reductase (5-AR isozymes. Differential expression of 5-AR isozymes is observed in both human benign and malignant prostatic tissues. 5-AR inhibitors (5-ARI are commonly used for the treatment of benign prostatic hyperplasia (BPH and were once promoted as chemopreventive agents for prostate cancer (PCa. This review discusses the role of the differential expression of 5-AR in the normal development of the human prostate and in the pathogenesis and progression of BPH and PCa.

  4. Purine salvage pathways of Bacillus subtilis and effect of guanine on growth of GMP reductase mutants.

    Science.gov (United States)

    Endo, T; Uratani, B; Freese, E

    1983-07-01

    We have isolated numerous mutants containing mutations in the salvage pathways of purine synthesis. The mutations cause deficiencies in adenine phosphoribosyltransferase (adeF), in hypoxanthine-guanine phosphoribosyltransferase (guaF), in adenine deaminase (adeC), in inosine-guanosine phosphorylase, (guaP), and in GMP reductase (guaC). The physiological properties of mutants containing one or more of these mutations and corresponding enzyme measurements have been used to derive a metabolic chart of the purine salvage pathway of Bacillus subtilis. PMID:6408059

  5. Mutational reconstructed ferric chelate reductase confers enhanced tolerance in rice to iron deficiency in calcareous soil

    OpenAIRE

    Ishimaru, Yasuhiro; Kim, Suyeon; Tsukamoto, Takashi; Oki, Hiroyuki; Kobayashi, Takanori; Watanabe, Satoshi; Matsuhashi, Shinpei; Takahashi, Michiko; Nakanishi, Hiromi; Mori, Satoshi; Naoko K. Nishizawa

    2007-01-01

    Iron (Fe) deficiency is a worldwide agricultural problem on calcareous soils with low-Fe availability due to high soil pH. Rice plants use a well documented phytosiderophore-based system (Strategy II) to take up Fe from the soil and also possess a direct Fe2+ transport system. Rice plants are extremely susceptible to low-Fe supply, however, because of low phytosiderophore secretion and low Fe3+ reduction activity. A yeast Fe3+ chelate-reductase gene refre1/372, selected for better performance...

  6. Purification and characterization of a 15-ketoprostaglandin d-reductase from bovine lung

    DEFF Research Database (Denmark)

    Hansen, Harald S.

    1979-01-01

    15-Ketoprostaglandin d-reductase from bovine lung has been purified using affinity chromatography to apparent homogeneity, as judged from polyacrylamide gel electrophoresis with and without sodium dodecyl sulphate. Valine was identified as the N-terminal amino acid, and the isoelectric point was...... estimated at pH 7.8. Molecular weights of 56 000 and 39 500 were found by the use of gel filtration and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme was found to be specific for the 15-keto group, thus 15-ketoprostaglandin E (apparent K = 10 µm) is a substrate, in contrast to...

  7. 3-hydroxy 3-methylglutaryl coenzyme A reductase: a new biomarker of fish exposure to water pollution.

    Science.gov (United States)

    Pallottini, Valentina; Scalici, Massimiliano; Gibertini, Giancarlo; Marino, Maria; Trentalance, Anna

    2010-10-01

    The aim of this study was to identify a new putative biomarker in Salmo trutta exposed to water pollution. Variations in the levels of hepatic 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMG-CoAR), the rate-limiting enzyme of cholesterol biosynthesis, were compared to heat shock protein 70 and hypoxia inducible factor α, biomarkers of pollution exposure and lowered O₂, respectively. The results confirm that HMG-CoAR levels increase in polluted water irrespective of water temperature or O₂ content, indicating that HMG-CoAR could be used as a specific biomarker for water pollution. PMID:20835703

  8. Activation of mitochondrial glycerophosphate cytochrome c reductase in regenerating rat liver by triiodothyronine

    Czech Academy of Sciences Publication Activity Database

    Lotková, H.; Rauchová, Hana; Drahota, Zdeněk

    2001-01-01

    Roč. 50, č. 3 (2001), s. 333-336. ISSN 0862-8408 R&D Projects: GA MŠk OC 918.50; GA ČR GA303/98/0473; GA ČR GA303/00/1658; GA MŠk LN00A069 Institutional research plan: CEZ:AV0Z5011922 Keywords : liver regeneration * triiodothyromine * glycerophosphate cytochrome c reductase Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 1.027, year: 2001

  9. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.

    OpenAIRE

    Bzik, D J; Li, W B; Horii, T; Inselburg, J

    1987-01-01

    Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum were isolated and sequenced. The deduced DHFR-TS protein contained 608 amino acids (71,682 Da). The coding region for DHFR-TS contained no intervening sequences and had a high A + T content (75%). The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction se...

  10. The HMG-CoA reductase inhibitor fluvastatin inhibits insect juvenile hormone biosynthesis.

    Science.gov (United States)

    Debernard, S; Rossignol, F; Couillaud, F

    1994-07-01

    Fluvastatin (Sandoz Compound XU 62-320), a synthetic HMG-CoA reductase inhibitor, was assayed in vitro and in vivo for its ability to suppress juvenile hormone (JH) biosynthesis by corpora allata of Locusta migratoria migratorioides. Fluvastatin inhibited JH biosynthesis by corpora allata in vitro. Exogenous mevalonic acid lactone restored JH biosynthesis in corpora allata inhibited by fluvastatin. Fluvastatin injected into locusts in vivo inhibited JH biosynthesis, but maximal inhibition lasted for only 6 hr. There were no discernible effects on either JH-regulated metamorphosis or oocyte maturation. Lengthening of the fourth larval stadium was observed and increased doses (single or repeated injections) were fatal. PMID:7926659

  11. Multicenter evaluation of the nitrate reductase assay for drug resistance detection of Mycobacterium tuberculosis.

    Science.gov (United States)

    Martin, Anandi; Montoro, Ernesto; Lemus, Dihadenys; Simboli, Norberto; Morcillo, Nora; Velasco, Maritza; Chauca, José; Barrera, Lucía; Ritacco, Viviana; Portaels, Françoise; Palomino, Juan Carlos

    2005-11-01

    The performance of the nitrate reductase assay was evaluated in a multicenter laboratory study to detect resistance of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin, isoniazid, ethambutol and streptomycin using a set of coded isolates. Compared with the gold standard proportion method on Löwenstein-Jensen medium, the assay was highly accurate in detecting resistance to rifampicin, isoniazid and ethambutol with an accuracy of 98%, 96.6% and 97.9%, respectively. For streptomycin, discrepant results were obtained with an overall accuracy of 85.3%. The assay proved easy to be implemented in countries with limited laboratory facilities. PMID:15893391

  12. 5-α reductase inhibitors, benign prostatic hyperplasia, and risk of male breast cancer

    OpenAIRE

    Robinson, David; Garmo, Hans; Holmberg, Lars; Stattin, Pär

    2015-01-01

    Purpose 5-α reductase inhibitors (5-ARI) have been suggested to increase the risk of male breast cancer. The aim of this study was to study the risk of breast cancer in men on 5-ARI, in men with benign prostatic hyperplasia (BPH) not on 5-ARI, and in men without BPH. Methods We performed a population-based cohort study in Sweden with data from The Prescribed Drug Register, The Patient Register, and The Cancer Register. Men on 5-ARI, men on α-blockers, or men who had undergone a transurethral ...

  13. 5-alpha reductase inhibitors, benign prostatic hyperplasia, and risk of male breast cancer

    OpenAIRE

    Robinson, David; Garmo, Hans; Holmberg, Lars; Stattin, Pär

    2015-01-01

    5-alpha reductase inhibitors (5-ARI) have been suggested to increase the risk of male breast cancer. The aim of this study was to study the risk of breast cancer in men on 5-ARI, in men with benign prostatic hyperplasia (BPH) not on 5-ARI, and in men without BPH. We performed a population-based cohort study in Sweden with data from The Prescribed Drug Register, The Patient Register, and The Cancer Register. Men on 5-ARI, men on alpha-blockers, or men who had undergone a transurethral resectio...

  14. Effect of 5-alpha Reductase Inhibitor on Storage Symptoms in Patients with Benign Prostatic Hyperplasia

    OpenAIRE

    Cho, Kang Jun; Kang, Se Hee; Kim, Hyo Sin; Koh, Jun Sung; Kim, Joon Chul

    2011-01-01

    Purpose Many patients with benign prostatic hyperplasia (BPH) have storage symptoms. The aim of this study was to evaluate the effects of treatment with a 5-alpha reductase inhibitor (5ARI) on storage symptoms in patients with BPH. Methods This study was conducted in 738 patients with lower urinary tract symptoms secondary to BPH. Patients with a prostate volume of higher than 30 mL on the transrectal ultrasound were classified into two groups: group A, in which an alpha blocker was solely ad...

  15. Identification of periplasmic nitrate reductase Mo(V) EPR signals in intact cells of Paracoccus denitrificans.

    Science.gov (United States)

    Sears, H J; Bennett, B; Spiro, S; Thomson, A J; Richardson, D J

    1995-08-15

    EPR spectroscopy has been successfully used to detect signals due to molybdenum (V) and ferric iron in intact cells of aerobically grown Paracoccus denitrificans. The signals are ascribed to the catalytic molybdenum centre and to the haem iron of the periplasmic nitrate reductase. These signals are absent from a mutant strain deficient in this enzyme. The Mo(V) signal is due to the High-g Split species which has been well characterized in the purified enzyme. This confirms that the High-g Split is the physiologically relevant signal of a number observed in the previous work on the purified enzyme. PMID:7646461

  16. Studies on the Iron-Sulfur clusters of hydrogenase, sulfite reductase, nitrogenase and the prismane protein.

    OpenAIRE

    Pierik, A.J.

    1993-01-01

    Iron-sulfur clusters are present in a large number of proteins. Sofar structures of four types of protein-bound iron-sulfur clusters have been determined by X-ray diffraction: rubredoxin-like, [2Fe-2S], [3Fe-4S] and [4Fe-4S] centers. The presence of any of these clusters in a protein can be predicted by comparison of spectroscopic properties. However a number of multiple-electron transferring enzymes, like the Fe-only hydrogenase, sulfite reductase and nitrogenase MoFe protein have enigmatic ...

  17. Normoxic and anoxic metabolism of Nicotiana tabacum transformants lacking root nitrate reductase

    OpenAIRE

    Stoimenova, Maria

    2002-01-01

    The aim of this work was to find out whether and how nitrate reduction in roots would facilitate survival of hypoxic and anoxic (flooding)-phases. For that purpose, we compared the response of roots of hydroponically grown tobacco wildtype (Nicotiana tabacum cv. Gatersleben) and of a transformant (LNR-H) with no nitrate reductase (NR) in the roots but almost normal NR in leaves (based on a nia2-double mutant). As an additional control we used occasionally a 35S-transformant of the same nia2-d...

  18. Vitamin K 2,3-epoxide reductase: the basis for stereoselectivity of 4-hydroxycoumarin anticoagulant activity.

    OpenAIRE

    Thijssen, H. H.; Baars, L. G.; Vervoort-Peters, H. T.

    1988-01-01

    1. The administration of S-warfarin (1 mg kg-1 i.v.) to rats that were pre-loaded 48 h before with tracer doses (6 micrograms) of 14C-labelled R- or S-warfarin caused the plasma levels of these compounds to increase. This is due to the substitution of the microsomal (vitamin K 2,3-epoxide (K0) reductase) bound R- or S-[14C]-warfarin by the unlabelled 4-hydroxycoumarin administered. The rate of reappearance was 3-4 fold higher for R- than for S-warfarin; t1/2 of release: 1.2 +/- 0.04 and 3.7 +...

  19. Effect of ammonium nutrition on the nitrate utilization, nitrate reductase actvity and growth of Spirodela polyrrhiza

    Directory of Open Access Journals (Sweden)

    Ewa Tatkowska

    2014-02-01

    Full Text Available The influence of NH4+ ions on nitrate assimilation and growth of sterile Spirodela polyrrhiza cultures was investigated. S. polyrrhiza utilises both the nitrate and the ammonium form of nitrogen, it prefers, however, NH4+. Ammonium ions present in the nitrate medium inhibit the activity of nitrate reductase (NR, but they do not affect enzyme 'induction and only slightly reduce N03- uptake. These results sugest that the inhibitory effect of NH4+ on the NR activity is the main cause of the decrease in N03- assimilation by S. polyrrhiza cultures growing in nitrate-ammonium medium.

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

    OpenAIRE

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

    2013-01-01

    A chromium-resistant fungus isolated from contaminated air with industrial vapors can be used for reducing toxic Cr(VI) to Cr(III). This study analyzes in vitro reduction of hexavalent chromium using cell free extract(s) of the fungus that was characterized based on optimal temperature, pH, use of electron donors, metal ions and initial Cr(VI) concentration in the reaction mixture. This showed the highest activity at 37°C and pH 7.0; there is an increase in Cr(VI) reductase activity with addi...

  1. Thioredoxin Reductase Was Nitrated in the Aging Heart After Myocardial Ischemia/Reperfusion

    OpenAIRE

    Wang, Ke; Zhang, Jie; Wang, Xiaoliang; Liu, Xin; Zuo, Lin; Bai, Kehua; Shang, Jianyu; Ma, Lu; Liu, Teng; Wang, Li; Wang, Wen; Ma, Xinliang; Liu, Huirong

    2013-01-01

    The age-related loss of anti-oxidant defense reduces recovery from myocardial ischemia/reperfusion injury (MI/R) in aged people. Our previous data showed that inactivation of thioredoxin (Trx) was involved in enhanced aging MI/R injury. Thioredoxin reductase (TrxR), the enzyme known to regulate Trx, is less efficient with age. The aim of the current study was to determine why TrxR activity was reduced and whether reduced TrxR activity contributed to enhanced aging MI/R injury. Both Trx and Tr...

  2. Glutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase.

    OpenAIRE

    Lüer, Corinna; Schauer, Stefan; Virus, Simone; Schubert, Wolf-Dieter; Heinz, Dirk W.; Moser, Jürgen; Jahn, Dieter

    2007-01-01

    The initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR. Similarly unexpectedly, the substituti...

  3. Design of Deinococcus radiodurans thioredoxin reductase with altered thioredoxin specificity using computational alanine mutagenesis

    OpenAIRE

    Obiero, Josiah; Sanders, David AR

    2011-01-01

    In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR–Trx interface suggested...

  4. Polymorphism in methylentetra-hydrofolate reductase gene: important role in diseases.

    Science.gov (United States)

    Kiseljaković, Emina; Jadrić, Radivoj; Hasić, Sabaheta; Skenderi, Faruk; Resić, Halima; Winterhalter-Jadrić, Mira

    2008-05-01

    It has been recognized that some people have a genetic variant which leads to elevated levels of homocysteine and impairs ability to process folate. This condition was recognized as independent risk factor of coronary heart disease. Recently, connection between this termolabile mutation of the methylenetetrahydrofolate reductase and numerous conditions and diseases has been established. Aim of this review is to draw attention to this interesting area in medicine. Additionally, well defined study about presence and frequency of gene polymorphism in our region will provide proper diagnosis and achieve possible delay of development of diseases with vitamin supplementation. PMID:18498269

  5. A structural account of substrate and inhibitor specificity differences between two Naphthol reductases

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Thompson, J.E.; Fahnestock, S.; Valent, B.; Jordan, D.B. (DuPont)

    2010-03-08

    Two short chain dehydrogenase/reductases mediate naphthol reduction reactions in fungal melanin biosynthesis. An X-ray structure of 1,3,6,8-tetrahydroxynaphthalene reductase (4HNR) complexed with NADPH and pyroquilon was determined for examining substrate and inhibitor specificities that differ from those of 1,3,8-trihydroxynaphthalene reductase (3HNR). The 1.5 {angstrom} resolution structure allows for comparisons with the 1.7 {angstrom} resolution structure of 3HNR complexed with the same ligands. The sequences of the two proteins are 46% identical, and they have the same fold. The 30-fold lower affinity of the 4HNR-NADPH complex for pyroquilon (a commercial fungicide that targets 3HNR) in comparison to that of the 3HNR-NADPH complex can be explained by unfavorable interactions between the anionic carboxyl group of the C-terminal Ile282 of 4HNR and CH and CH{sub 2} groups of the inhibitor that are countered by favorable inhibitor interactions with 3HNR. 1,3,8-Trihydroxynaphthalene (3HN) and 1,3,6,8-tetrahydroxynaphthalene (4HN) were modeled onto the cyclic structure of pyroquilon in the 4HNR-NADPH-pyroquilon complex to examine the 300-fold preference of the enzyme for 4HN over 3HN. The models suggest that the C-terminal carboxyl group of Ile282 has a favorable hydrogen bonding interaction with the C6 hydroxyl group of 4HN and an unfavorable interaction with the C6 CH group of 3HN. Models of 3HN and 4HN in the 3HNR active site suggest a favorable interaction of the sulfur atom of the C-terminal Met283 with the C6 CH group of 3HN and an unfavorable one with the C6 hydroxyl group of 4HN, accounting for the 4-fold difference in substrate specificities. Thus, the C-terminal residues of the two naphthol reductase are determinants of inhibitor and substrate specificities.

  6. Steroid 5 α-reductase inhibitors targeting BPH and prostate cancer.

    Science.gov (United States)

    Schmidt, Lucy J; Tindall, Donald J

    2011-05-01

    Steroid 5 alpha-reductase inhibitors (5ARIs) have been approved for use clinically in treatment of benign prostate hyperplasia (BPH) and accompanying lower urinary tract symptoms (LUTS) and have also been evaluated in clinical trials for prevention and treatment of prostate cancer. There are currently two steroidal inhibitors in use, finasteride and dutasteride, both with distinct pharmacokinetic properties. This review will examine the evidence presented by various studies supporting the use of these steroidal inhibitors in the prevention and treatment of prostate disease. Article from the Special issue on Targeted Inhibitors. PMID:20883781

  7. Dual-5α-reductase inhibition promotes hepatic lipid accumulation in man.

    OpenAIRE

    Hazlehurst, JM; Oprescu, AI; N. Nikolaou; Di Guida, R; Grinbergs, AEK; Davies, NP; Flintham, RB; Armstrong, MJ; Taylor, AE; Hughes, BA; Yu, J.; Hodson, L.; Dunn, WB; Tomlinson, JW

    2016-01-01

    5α-Reductase 1 and 2 (SRD5A1, SRD5A2) inactivate cortisol to 5α-dihydrocortisol in addition to their role in the generation of DHT. Dutasteride (dual SRD5A1 and SRD5A2 inhibitor) and finasteride (selective SRD5A2 inhibitor) are commonly prescribed, but their potential metabolic effects have only recently been identified.Our objective was to provide a detailed assessment of the metabolic effects of SRD5A inhibition and in particular the impact on hepatic lipid metabolism.We conducted a randomi...

  8. A preliminary study on estimating extra-cellular nitrate reductase activities in estuarine systems

    OpenAIRE

    Pant H. K.

    2009-01-01

    Enzymes catalyzing ammonium (NH4+)/nitrate (NO3–) into nitrous oxide (N2O)/molecular nitrogen (N2), play critical roles in water quality management. The objective of this paper was to investigate the role of extra-cellular enzymes in cycling of nitrogen (N) in aquatic systems. It appears that N in estuaries, salt marshes, etc., does not stay long enough to be available for uptake, thus, creating N limited conditions. This study showed that indigenous extra-cellular nitrate reductase along wit...

  9. Nitrate Uptake, Nitrate Reductase Distribution and their Relation to Proton Release in Five Nodulated Grain Legumes

    OpenAIRE

    Fan, X. H.; Tang, C; RENGEL, Z.

    2002-01-01

    Nitrate uptake, nitrate reductase activity (NRA) and net proton release were compared in five grain legumes grown at 0·2 and 2 mm nitrate in nutrient solution. Nitrate treatments, imposed on 22‐d‐old, fully nodulated plants, lasted for 21 d. Increasing nitrate supply did not significantly influence the growth of any of the species during the treatment, but yellow lupin (Lupinus luteus) had a higher growth rate than the other species examined. At 0·2 mm nitrate supply, nitrate uptake rates ran...

  10. Crystallographic investigation of the cooperative interaction between trimethoprim, reduced cofactor and dihydrofolate reductase

    International Nuclear Information System (INIS)

    The structure of the complex between E. coli form I dihydrofolate reductase, the antibacterial trimethoprim and NADPH has been determined by X-ray crystallography. The inhibitor and cofactor are in mutual contact. A flexible chain segment which includes Met 20 is in contact with the inhibitor in the presence of NADPH, but more distant in its absence. By contrast, the inhibitor conformation is little changed with NADPH present. The authors discuss these observations with regard to the mutually cooperative binding of these ligands to the protein, and to the associated enhancement of inhibitory selectivity shown by trimethoprim for bacterial as opposed to vertebrate enzyme. (Auth.)

  11. In vivo activation of methyl-coenzyme M reductase by carbon monoxide

    OpenAIRE

    Zhou, Yuzhen; Dorchak, Alexandria E.; Ragsdale, Stephen W.

    2013-01-01

    Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the rate-limiting and final step in methane biosynthesis. Using coenzyme B as the two-electron donor, MCR reduces methyl-coenzyme M (CH3-SCoM) to methane and the mixed disulfide, CoBS-SCoM. MCR contains an essential redox-active nickel tetrahydrocorphinoid cofactor, Coenzyme F430, at its active site. The active form of the enzyme (MCRred1) contains Ni(I)-F430. Rapid and efficient conversion of MCR to MCRred1 is important fo...

  12. In vivo activation of methyl-coenzyme M reductase by carbon monoxide

    OpenAIRE

    StephenWileyRagsdale

    2013-01-01

    Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the rate-limiting and final step in methane biosynthesis. Using coenzyme B (CoBSH) as the two-electron donor, MCR reduces methyl-coenzyme M (CH3-SCoM) to methane and the mixed disulfide, CoBS-SCoM. MCR contains an essential redox-active nickel tetrahydro¬corphinoid cofactor, Coenzyme F430, at its active site. The active form of the enzyme (MCRred1) contains Ni(I)-F430. Rapid and efficient conversion of MCR to MCRred1 i...

  13. STUDY OF THE RELATIONSHIP BETWEEN PSORIASIS AND THE POLYMORPHIC SITE C677T OF METHYLENETETRAHYDROFOLATE REDUCTASE

    Institute of Scientific and Technical Information of China (English)

    王柏秋; 傅松滨; 张贵寅; 李严璞

    2000-01-01

    Objective. In order to investigate 5, l0-methylenetetrahydrofolate reductase (MTHFR)'s polymorphic changes in psoriasis vulgaris. Methods. We detected mutation of site C677V of MTHFR in 39 psoriasfics by PCR-RFLP. Results. Genotype frequencies of the psoriasfics were C/C= 20.15%,C/T= 48.72% and T/T= 30.77%; the allelic frequencies were C = 0.4487 and T=0.5513. Homozygous mutant (TT) of the psoriastics was significantly different from the normal control goup by X2 test. Conclusion. C677V mutant of MTHFR might be related with psoriasis.

  14. STUDY OF THE RELATIONSHIP BETWEEN PSORIASIS AND THE POLYMORPHIC SITE C677T OF METHYLENETETRAHYDROFOLATE REDUCTASE

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective. In order to investigate 5,10-methylenetetrahydrofolate reductase (MTHFR)s polymorphic changes in psoriasis vulgaris.Methods.We detected mutation of site C677V of MTHFR in 39 psoriastics by PCR-RFLP.Results.Genotype frequencies of the psoriastics were C/C=20.15%,C/T=48.72% and T/T=30.77%; the allelic frequencies were C=0.4487 and T=0.5513. Homozygous mutant (TT) of the psoriastics was significantly different from the normal control group by X2 test.Conclusion.C677V mutant of MTHFR might be related with psoriasis.

  15. Cloning, expression and antigenicity of the L. donovani reductase

    DEFF Research Database (Denmark)

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

    2001-01-01

    The protozoan parasite Leishmania undergoes a morphological and biochemical transformation from the promastigote to the amastigote form during its life cycle, which is reflected in the expression of stage-specific proteins. One of these proteins shows homology to a superfamily of reductase proteins......, or not at all. The results indicate the presence of cross-reacting CD45R0 memory T-cells in individuals not exposed to Leishmania. Several previous studies have shown that T-cells from nonexposed individuals often respond to crude Leishmania antigen preparations. The present study suggests that this reactivity...

  16. Genetic variation of Aflatoxin B(1) aldehyde reductase genes (AFAR) in human tumour cells

    DEFF Research Database (Denmark)

    Praml, Christian; Schulz, Wolfgang; Claas, Andreas;

    2008-01-01

    AFAR genes play a key role in the detoxification of the carcinogen Aflatoxin B(1) (AFB(1)). In the rat, Afar1 induction can prevent AFB(1)-induced liver cancer. It has been proposed that AFAR enzymes can metabolise endogenous diketones and dialdehydes that may be cytotoxic and/or genotoxic. Furth...... many aldo-keto reductases. This polarity change may have an effect on the proposed substrate binding amino acids nearby (Met(47), Tyr(48), Asp(50)). Further population analyses and functional studies of the nine variants detected may show if these variants are disease-related....

  17. Cytochrome P450 Reductase: A Harbinger of Diffusible Reduced Oxygen Species

    OpenAIRE

    Manoj, Kelath Murali; Gade, Sudeep Kumar; Mathew, Lazar

    2010-01-01

    The bi-enzymatic system of cytochrome P450 (CYP, a hemoprotein) and cytochrome P450 reductase (CPR, a diflavoenzyme) mediate the redox metabolism of diverse indigenous and xenobiotic molecules in various cellular and organ systems, using oxygen and NADPH. Curiously, when a 1∶1 ratio is seen to be optimal for metabolism, the ubiquitous CYP:CPR distribution ratio is 10 to 100∶1 or higher. Further, the NADPH equivalents consumed in these in vitro or in situ assemblies usually far exceeded the am...

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

    Science.gov (United States)

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

    1989-02-15

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

  19. Expression, purification, crystallization and preliminary X-ray analysis of maleylacetate reductase from Burkholderia sp. strain SJ98

    International Nuclear Information System (INIS)

    Purification and preliminary X-ray crystallographic analysis of maleylacetate reductase encoded by the pnpD gene is reported. Maleylacetate reductase (EC 1.3.1.32) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P21212, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 Å. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 Å resolution, respectively

  20. Crystal structure of the YffB protein from Pseudomonas aeruginosa suggests a glutathione-dependent thiol reductase function

    Directory of Open Access Journals (Sweden)

    Dauter Zbigniew

    2004-03-01

    Full Text Available Abstract Background The yffB (PA3664 gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. Results The structure was determined at 1.0 Å resolution by single-wavelength anomalous diffraction. The fold is very similar to that of arsenate reductase, which is an extension of the thioredoxin fold. Conclusion Given the conservation of the functionally important residues and the ability to bind glutathione, YffB is likely to function as a GSH-dependent thiol reductase.

  1. Effects of some analgesic anaesthetic drugs on human erythrocyte glutathione reductase: an in vitro study.

    Science.gov (United States)

    Senturk, Murat; Irfan Kufrevioglu, O; Ciftci, Mehmet

    2009-04-01

    Inhibitory effects of some analgesic and anaesthetic drugs on human erythrocyte glutathione reductase were investigated. For this purpose, human erythrocyte glutathione reductase was initially purified 2139-fold in a yield of 29% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis confirmed the purity of the enzyme by sharing a single band. A constant temperature (+4 degrees C) was maintained during the purification process. Diclofenac sodium, ketoprofen, lornoxicam, tenoxicam, etomidate, morphine and propofol exhibited inhibitory effects on the enzyme in vitro using the Beutler assay method. K(i) constants and IC(50) values for drugs were determined from Lineweaver-Burk graphs and plotting activity % versus [I] graphs, respectively. The IC(50) values of diclofenac sodium, ketoprofen, lornoxicam, propofol, tenoxicam, etomidate and morphine were 7.265, 6.278, 0.3, 0.242, 0.082, 0.0523 and 0.0128 mM and the K(i) constants were 23.97 +/- 2.1, 22.14 +/- 7.6, 0.42 +/- 0.18, 0.418 +/- 0.056, 0.13 +/- 0.025, 0.0725 +/- 0.0029 and 0.0165 +/- 0.0013 mM, respectively. While diclofenac sodium, ketoprofen, lornoxicam, tenoxicam etomidate and morphine showed competitive inhibition, propofol displayed noncompetitive inhibition. PMID:18608753

  2. Domain motions and electron transfer dynamics in 2Fe-superoxide reductase.

    Science.gov (United States)

    Horch, Marius; Utesch, Tillmann; Hildebrandt, Peter; Mroginski, Maria Andrea; Zebger, Ingo

    2016-08-17

    Superoxide reductases are non-heme iron enzymes that represent valuable model systems for the reductive detoxification of reactive oxygen species. In the present study, we applied different theoretical methods to study the structural dynamics of a prototypical 2Fe-superoxide reductase and its influence on electron transfer towards the active site. Using normal mode and essential dynamics analyses, we could show that enzymes of this type are capable of well-defined, electrostatically triggered domain movements, which may allow conformational proofreading for cellular redox partners involved in intermolecular electron transfer. Moreover, these global modes of motion were found to enable access to molecular configurations with decreased tunnelling distances between the active site and the enzyme's second iron centre. Using all-atom classical molecular dynamics simulations and the tunnelling pathway model, however, we found that electron transfer between the two metal sites is not accelerated under these conditions. This unexpected finding suggests that the unperturbed enzymatic structure is optimized for intramolecular electron transfer, which provides an indirect indication of the biological relevance of such a mechanism. Consistently, efficient electron transfer was found to depend on a distinct route, which is accessible via the equilibrium geometry and characterized by a quasi conserved tyrosine that could enable multistep-tunnelling (hopping). Besides these explicit findings, the present study demonstrates the importance of considering both global and local protein dynamics, and a generalized approach for the functional analysis of these aspects is provided. PMID:27491757

  3. Crystallization and preliminary X-ray crystallographic studies of the alkanesulfonate FMN reductase from Escherichia coli

    International Nuclear Information System (INIS)

    Crystallization of the native and SeMet FMN reductase protein of the E. coli alkanesulfonate monooxygenase two-component enzyme system is reported. The alkanesulfonate FMN reductase (SsuE) from Escherichia coli catalyzes the reduction of FMN by NADPH to provide reduced flavin for the monooxygenase (SsuD) enzyme. The vapor-diffusion technique yielded single crystals that grow as hexagonal rods and diffract to 2.9 Å resolution using synchrotron X-ray radiation. The protein crystallizes in the primitive hexagonal space group P622. The SsuE protein lacks any cysteine or methionine residues owing to the role of the SsuE enzyme in the acquisition of sulfur during sulfate starvation. Therefore, substitution of two leucine residues (Leu114 and Leu165) to methionine was performed to obtain selenomethionine-containing SsuE for MAD phasing. The selenomethionine derivative of SsuE has been expressed and purified and crystals of the protein have been obtained with and without bound FMN. These preliminary studies should lead to the structure solution of SsuE. It is anticipated that this new protein structure will provide detailed structural information on specific active-site regions of the protein and insight into the mechanism of flavin reduction and transfer of reduced flavin

  4. Deletion of thioredoxin reductase and effects of selenite and selenate toxicity in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Christopher J Boehler

    Full Text Available Thioredoxin reductase-1 (TRXR-1 is the sole selenoprotein in C. elegans, and selenite is a substrate for thioredoxin reductase, so TRXR-1 may play a role in metabolism of selenium (Se to toxic forms. To study the role of TRXR in Se toxicity, we cultured C. elegans with deletions of trxr-1, trxr-2, and both in axenic media with increasing concentrations of inorganic Se. Wild-type C. elegans cultured for 12 days in Se-deficient axenic media grow and reproduce equivalent to Se-supplemented media. Supplementation with 0-2 mM Se as selenite results in inverse, sigmoidal response curves with an LC50 of 0.20 mM Se, due to impaired growth rather than reproduction. Deletion of trxr-1, trxr-2 or both does not modulate growth or Se toxicity in C. elegans grown axenically, and (75Se labeling showed that TRXR-1 arises from the trxr-1 gene and not from bacterial genes. Se response curves for selenide (LC50 0.23 mM Se were identical to selenite, but selenate was 1/4(th as toxic (LC50 0.95 mM Se as selenite and not modulated by TRXR deletion. These nutritional and genetic studies in axenic media show that Se and TRXR are not essential for C. elegans, and that TRXR alone is not essential for metabolism of inorganic Se to toxic species.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    Uthus, Eric O

    2010-06-01

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

  7. 15N studies on the in-vivo assay of nitrate reductase in leaves

    International Nuclear Information System (INIS)

    The reduction of nitrate and nitrite in the leaf disks of seven di- and two mono-cotyledonous species under the in-vivo assay conditions of nitrate reductase was studied using N-15 labeled substrates. The significant reduction of both nitrate and nitrite into ammonia and amino acids was detected in the atmosphere of air. In the atmosphere of N2 gas, anaerobic incubation enhanced the accumulation of nitrite, but the subsequent reduction to the basic nitrogen compounds was from 40 to 180 % of the aerobic rate. The present examination indicated that the in-vivo assay of nitrate reductase under aerobic condition may give greatly underestimated results due to nitrite reduction, and that the exclusion of oxygen from the in-vivo assay mixture is desirable. The addition of n- propanol may be desirable for the assay under aerobic condition. Significant difference was not observed in the reduction of nitrate supplied as sodium and potassium salts on the nitrite formation and on the incorporation of nitrate-N into basic fractions. The N-15 experiment on the dark assimilation of nitrate, nitrite and ammonia into amino acids in wheat leaves showed that these three nitrogen sources were assimilated through the same route, and that the glutamine synthetase/glutamate synthetase pathway was the main route. By anaerobic treatment, the incorporation of nitrogen into alanine and serine was relatively high. (Kako, I.)

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

    Science.gov (United States)

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

    2011-01-01

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

  9. Reduction of azo dyes by flavin reductase from Citrobacter freundii A1

    Directory of Open Access Journals (Sweden)

    Mohd Firdaus Abdul-Wahab

    2012-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Carmen Gherasim

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

  11. Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Schormann, Norbert; Velu, Sadanandan E.; Murugesan, Srinivasan; Senkovich, Olga; Walker, Kiera; Chenna, Bala C.; Shinkre, Bidhan; Desai, Amar; Chattopadhyay, Debasish (UAB)

    2010-09-17

    Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas disease. We have undertaken a detailed structure-activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme-ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60-70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.

  12. Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple

    Directory of Open Access Journals (Sweden)

    Yuepeng eHan

    2015-04-01

    Full Text Available Proanthocyanidins (PAs are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR and anthocyanidin reductase (ANR was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis.

  13. The effects of chemical and radioactive properties of Tl-201 on human erythrocyte glutathione reductase activity

    International Nuclear Information System (INIS)

    Aim: The aim of the study was to evaluate the inhibitory effects of thallium-201 (201Tl) solution on human erythrocyte glutathione reductase (GR) activity. Methods: Erythrocyte GR was initially purified by 2′,5′-adenosine diphosphate Sepharose-4B affinity and Sephadex G-200 gel filtration chromatography. The purification was monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, which showed a single band for the final enzyme preparation. The in vitro and in vivo effects of the 201Tl solution including Tl+, Fe+3 and Cu+2 metals and the in vitro effects of the radiation effect of the 201Tl solution and nonradioactive Tl+, Fe+3 and Cu+2 metals on human erythrocyte GR enzyme were studied. Enzyme activity was determined with the Beutler method at 340 nm using a spectrophotometer. All purification procedures were carried out at +4°C. Results: Glutathione reductase was purified 2033-fold at a yield of 28.17%. 201Tl solution and radiation exposure had inhibitory effects on the enzyme activity. Besides, effects of nonradioactive Tl+, Fe+3 and Cu+2 were studied on enzyme activity in vitro. Furthermore, seven human patients were also used for in vivo studies of 201Tl solution. Conclusion: It was detected in in vitro and in vivo studies that the human erythrocyte GR enzyme is inhibited due to the radiation effect of 201Tl solution.

  14. Oxidative stress induced expression of monodehydroascorbate reductase gene in Eleusine coracana.

    Science.gov (United States)

    Sudan, Jebi; Negi, Bhawana; Arora, Sandeep

    2015-10-01

    Abiotic stresses constitute a serious threats to the world food security as they cause significant economic losses in terms of reduction in crop productivity and also greatly limit the geographical locations where crops can be grown. Exposure to abiotic stress causes over-production of reactive oxygen species, leading to oxidative stress in plants. Induction of oxidative stress is primarily responsible for a variety of detrimental changes in the cellular physiology. However, plants have evolved intricate anti-oxidative defence machinery, for their survival under stress. Plant defence strategies for stress tolerance rely on the expression of anti-oxidative genes required for scavenging the toxic reactive oxygen species. Monodehydroascorbate reductase is one of the key anti-oxidant enzyme responsible for scavenging reactive oxygen species. In the present study, efforts have been made to understand the role of monodehydroascorbate reductase in finger millet under different abiotic stresses (drought, salt and UV radiation). The study establishes a differential link between mdar gene expression and enzyme activity under oxidative stress that is validated under different types of imposed stresses. Alteration in correlation between gene expression and enzyme activities under varying magnitude of oxidative stress is elucidated. PMID:26600681

  15. Bee Venom Promotes Hair Growth in Association with Inhibiting 5α-Reductase Expression.

    Science.gov (United States)

    Park, Seeun; Erdogan, Sedef; Hwang, Dahyun; Hwang, Seonwook; Han, Eun Hye; Lim, Young-Hee

    2016-06-01

    Alopecia is an important issue that can occur in people of all ages. Recent studies show that bee venom can be used to treat certain diseases including rheumatoid arthritis, neuralgia, and multiple sclerosis. In this study, we investigated the preventive effect of bee venom on alopecia, which was measured by applying bee venom (0.001, 0.005, 0.01%) or minoxidil (2%) as a positive control to the dorsal skin of female C57BL/6 mice for 19 d. Growth factors responsible for hair growth were analyzed by quantitative real-time PCR and Western blot analysis using mice skins and human dermal papilla cells (hDPCs). Bee venom promoted hair growth and inhibited transition from the anagen to catagen phase. In both anagen phase mice and dexamethasone-induced catagen phase mice, hair growth was increased dose dependently compared with controls. Bee venom inhibited the expression of SRD5A2, which encodes a type II 5α-reductase that plays a major role in the conversion of testosterone into dihydrotestosterone. Moreover, bee venom stimulated proliferation of hDPCs and several growth factors (insulin-like growth factor 1 receptor (IGF-1R), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)2 and 7) in bee venom-treated hDPCs dose dependently compared with the control group. In conclusion, bee venom is a potentially potent 5α-reductase inhibitor and hair growth promoter. PMID:27040904

  16. The effect of 5α-reductase-2 deficiency on human fertility.

    Science.gov (United States)

    Kang, Hey-Joo; Imperato-McGinley, Julianne; Zhu, Yuan-Shan; Rosenwaks, Zev

    2014-02-01

    A most interesting and intriguing male disorder of sexual differentiation is due to 5α-reductase-2 isoenzyme deficiency. These male infants are born with ambiguous external genitalia due to a deficiency in their ability to catalyze the conversion of T to dihydrotestosterone. Dihydrotestosterone is a potent androgen responsible for differentiation of the urogenital sinus and genital tubercle into the external genitalia, urethra, and prostate. Affected males are born with a clitoral-like phallus, bifid scrotum, hypospadias, blind shallow vaginal pouch from incomplete closure of the urogenital sinus, and a rudimentary prostate. At puberty, the surge in mainly T production prompts virilization, causing most boys to choose gender reassignment to male. Fertility is a challenge for affected men for several reasons. Uncorrected cryptorchidism is associated with low sperm production, and there is evidence of defective transformation of spermatogonia into spermatocytes. The underdeveloped prostate and consequent low semen volumes affect sperm transport. In addition, semen may not liquefy due to a lack of prostate-specific antigen. In the present review, we discuss the 5α-reductase-2 deficiency syndrome and its impact on human fertility. PMID:24412121

  17. Selective killing of methotrexate-resistant cells carrying amplified dihydrofolate reductase genes

    International Nuclear Information System (INIS)

    A method for the selective killing of methotrexate (MTX)-resistant cells has been developed. The selection is based on the incorporation of tritiated deoxyuridine into the DNA of MTX-resistant cells but not normal MTX-sensitive cells in the presence of the drug. A Chinese hamster ovary cell mutant that overproduces dihydrofolate reductase was used as an example of a MTX-resistant cell line. In this system, a 10,000-fold enrichment for wild-type MTX-sensitive cells could be achieved after 24 hr of exposure to the drug combination. This selection technique was applied to the isolation of MTX-sensitive segregants from hybrid cells formed between the MTX-resistant mutant and wild-type cells. The loss of MTX resistance and dihydrofolate reductase overproduction was always accompanied by the loss of a homogeneously staining region on chromosome 2 of the resistant parent that contains the amplified genes specifying this enzyme. While this region is always lost, other parts of chromosome 2 are almost always retained, suggesting that deletion rather than chromosome loss underlies marker segregation in this case. When the selection was applied to the resistant mutant itself, no MTX-sensitive revertants were obtained among 10(5) cells screened, attesting to the stability of gene amplification in this clone. It is suggested that this combination of drugs may be useful for the elimination of MTX-resistant tumor cells that develop after MTX chemotherapy

  18. Cloning, expression and enzymatic properties analysis of dihydrofolate reductase gene from the silkworm, Bombyx mori.

    Science.gov (United States)

    Wang, Wenjing; Gao, Junshan; Wang, Jing; Liu, Chaoliang; Meng, Yan

    2012-12-01

    Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases, which control the levels of monoamine neurotransmitters. BH4 deficiency has been associated with many neuropsychological disorders. Dihydrofolate reductase (DHFR) can catalyze 7,8-dihydrobiopterin to 5,6,7,8-tetrahydrobiopterin (BH4) in the salvage pathway of BH4 synthesis from sepiapterin (SP), a major pigment component contained in the integument of silkworm Bombyx mori mutant lemon (lem) in high concentration. In this study, we report the cloning of DHFR gene from the silkworm B. mori (BmDhfr) and identification of enzymatic properties of BmDHFR. BmDhfr is located on scaffold Bm_199 with a predicted gene model BGIBMGA013340, which encodes a 185-aa polypeptide with a predicted molecular mass of about 21 kDa. Biochemical analyses showed that the recombinant BmDHFR protein exhibited high enzymatic activity and suitable parameters to substrate. Together with our previous studies on SP reductase of B. mori (BmSPR) and the lem mutant, it may be an effective way to industrially extract SP from the lem silkworms in large scale to produce BH4 in vitro by co-expressing BmSPR and BmDHFR and using the extracted SP as a substrate in the future. PMID:23065260

  19. Structural Insight into Dihydrodipicolinate Reductase from Corybebacterium glutamicum for Lysine Biosynthesis.

    Science.gov (United States)

    Sagong, Hye-Young; Kim, Kyung-Jin

    2016-02-28

    Dihydrodipicolinate reductase is an enzyme that converts dihydrodipicolinate to tetrahydrodipicolinate using an NAD(P)H cofactor in L-lysine biosynthesis. To increase the understanding of the molecular mechanisms of lysine biosynthesis, we determined the crystal structure of dihydrodipicolinate reductase from Corynebacterium glutamicum (CgDapB). CgDapB functions as a tetramer, and each protomer is composed of two domains, an Nterminal domain and a C-terminal domain. The N-terminal domain mainly contributes to nucleotide binding, whereas the C-terminal domain is involved in substrate binding. We elucidated the mode of cofactor binding to CgDapB by determining the crystal structure of the enzyme in complex with NADP(+) and found that CgDapB utilizes both NADH and NADPH as cofactors. Moreover, we determined the substrate binding mode of the enzyme based on the coordination mode of two sulfate ions in our structure. Compared with Mycobacterium tuberculosis DapB in complex with its cofactor and inhibitor, we propose that the domain movement for active site constitution occurs when both cofactor and substrate bind to the enzyme. PMID:26502738

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

    Science.gov (United States)

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

    2008-10-01

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

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

    Science.gov (United States)

    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

  2. The REDUCE trial: chemoprevention in prostate cancer using a dual 5alpha-reductase inhibitor, dutasteride.

    Science.gov (United States)

    Musquera, Mireia; Fleshner, Neil E; Finelli, Antonio; Zlotta, Alexandre R

    2008-07-01

    Dutasteride, a dual 5alpha-reductase inhibitor, is used in the treatment of benign prostatic hyperplasia (BPH). It reduces serum prostate-specific antigen levels by approximately 50% at 6 months and total prostate volume by 25% after 2 years. Randomized placebo-controlled trials in BPH patients have shown the efficacy of dutasteride in symptomatic relief, improvements in quality of life and peak urinary flow rate. Side effects occurring with dutasteride are decreased libido, erectile dysfunction, ejaculation disorders and gynecomastia. Preliminary data from placebo-controlled BPH trials have shown a decrease in the detection of prostate cancer in patients treated with dutasteride, although these studies were not designed to look at this issue. Dutasteride differs from finasteride in that it inhibits both isoenzymes of 5alpha-reductase, type I and type II. The landmark Prostate Cancer Prevention Trial at the end of the 7-year study demonstrated a 24.8% reduction in the incidence of prostate cancer in the finasteride group compared with placebo. However, a 25.5% increase in the prevalence of high-grade Gleason tumors has been observed, the clinical significance of which has been debated. Preliminary data suggest a decrease in prostate cancer incidence in dutasteride-treated patients and demonstrate type I alphareductase enzyme expression in prostate cancer. As a result, dutasteride is being investigated for prostate cancer prevention in the ongoing Reduction by Dutasteride of Prostate Cancer Events (REDUCE) trial, which is discussed here. PMID:18588452

  3. Nitrofuran drugs as common subversive substrates of Trypanosoma cruzi lipoamide dehydrogenase and trypanothione reductase.

    Science.gov (United States)

    Blumenstiel, K; Schöneck, R; Yardley, V; Croft, S L; Krauth-Siegel, R L

    1999-12-01

    Lipoamide dehydrogenase (LipDH), trypanothione reductase (TR), and glutathione reductase (GR) catalyze the NAD(P)H-dependent reduction of disulfide substrates. TR occurs exclusively in trypanosomatids which lack a GR. Besides their physiological reactions, the flavoenzymes catalyze the single-electron reduction of nitrofurans with the concomitant generation of superoxide anions. Here, we report on the interaction of clinically used antimicrobial nitrofurans with LipDH and TR from Trypanosoma cruzi, the causative agent of Chagas' disease (South American trypanosomiasis), in comparison to mammalian LipDH and GR. The compounds were studied as inhibitors and as subversive substrates of the enzymes. None of the nitrofurans inhibited LipDH, although they did interfere with the disulfide reduction of TR and GR. When the compounds were studied as substrates, T. cruzi LipDH showed a high rate of nitrofuran reduction and was even more efficient than its mammalian counterpart. Several derivatives were also effective subversive substrates of TR, but the respective reaction with human GR was negligible. Nifuroxazide, nifuroxime, and nifurprazine proved to be the most promising derivatives since they were redox-cycled by both T. cruzi LipDH and TR and had pronounced antiparasitic effects in cultures of T. cruzi and Trypanosoma brucei. The results suggest that those nitrofuran derivatives which interact with both parasite flavoenzymes should be revisited as trypanocidal drugs. PMID:10571254

  4. Expression, purification, crystallization and preliminary X-ray crystallographic studies of Deinococcus radiodurans thioredoxin reductase

    International Nuclear Information System (INIS)

    Recombinant D. radiodurans TrxR with a His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. Deinococcus radiodurans, a Gram-positive bacterium capable of withstanding extreme ionizing radiation, contains two thioredoxins (Trx and Trx1) and a single thioredoxin reductase (TrxR) as part of its response to oxidative stress. Thioredoxin reductase is a member of the family of pyridine nucleotide-disulfide oxidoreductase flavoenzymes. Recombinant D. radiodurans TrxR with a His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. X-ray diffraction data were collected on a cryocooled crystal to a resolution of 1.9 Å using a synchrotron-radiation source. The space group was determined to be P3221, with unit-cell parameters a = b = 84.33, c = 159.88 Å. The structure of the enzyme has been solved by molecular-replacement methods and structure refinement is in progress

  5. Molecular dissection of a putative iron reductase from Desulfotomaculum reducens MI-1

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhi; Kim, David D.; Nelson, Ornella D.; Otwell, Annie E.; Richardson, Ruth E.; Callister, Stephen J.; Lin, Hening

    2015-10-08

    Desulfotomaculum reducens MI-1 is a Firmicute strain capable of reducing a variety of heavy metal ions and has a great potential in heavy metal bioremediation.We recently identified Dred_2421 as a potential iron reductase through proteomic study of D. reducens. The current study examines its iron-reduction mechanism. Dred_2421, like its close homolog from Escherichia coli (2, 4-dienoyl-CoA reductase), has an FMN-binding N-terminal domain (NTD), an FAD-binding C-terminal domain (CTD), and a 4Fee4S cluster between the two domains. To understand the mechanism of the iron-reduction activity and the role of each domain, we generated a series of variants for each domain and investigated their iron reduction activity. Our results suggest that CTD is the main contributor of the iron-reduction activity, and that NTD and the 4Fee4S cluster are not directly involved in such activity. This study provides a mechanistic understanding of the ironereductase activity of Dred_2421 and may also help to elucidate other physiological activities this enzyme may have.

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

    Science.gov (United States)

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

    2008-03-01

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

  7. Identification of thioredoxin glutathione reductase inhibitors that kill cestode and trematode parasites.

    Directory of Open Access Journals (Sweden)

    Fabiana Ross

    Full Text Available Parasitic flatworms are responsible for serious infectious diseases that affect humans as well as livestock animals in vast regions of the world. Yet, the drug armamentarium available for treatment of these infections is limited: praziquantel is the single drug currently available for 200 million people infected with Schistosoma spp. and there is justified concern about emergence of drug resistance. Thioredoxin glutathione reductase (TGR is an essential core enzyme for redox homeostasis in flatworm parasites. In this work, we searched for flatworm TGR inhibitors testing compounds belonging to various families known to inhibit thioredoxin reductase or TGR and also additional electrophilic compounds. Several furoxans and one thiadiazole potently inhibited TGRs from both classes of parasitic flatworms: cestoda (tapeworms and trematoda (flukes, while several benzofuroxans and a quinoxaline moderately inhibited TGRs. Remarkably, five active compounds from diverse families possessed a phenylsulfonyl group, strongly suggesting that this moiety is a new pharmacophore. The most active inhibitors were further characterized and displayed slow and nearly irreversible binding to TGR. These compounds efficiently killed Echinococcus granulosus larval worms and Fasciola hepatica newly excysted juveniles in vitro at a 20 µM concentration. Our results support the concept that the redox metabolism of flatworm parasites is precarious and particularly susceptible to destabilization, show that furoxans can be used to target both flukes and tapeworms, and identified phenylsulfonyl as a new drug-hit moiety for both classes of flatworm parasites.

  8. Cloning, expression and crystallization of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus

    International Nuclear Information System (INIS)

    Given the recent rise in antimicrobial resistance, there is an urgent need to identify and characterize new antibiotic drug targets. One such target is dihydrodipicolinate reductase (DHDPR), which is an essential bacterial enzyme that catalyzes the second step in the lysine-biosynthesis pathway. In this paper, the cloning, expression, purification and crystallization of DHDPR from methicillin-resistant S. aureus are presented. Dihydrodipicolinate reductase (DHDPR; EC 1.3.1.26) catalyzes the nucleotide (NADH/NADPH) dependent second step of the lysine-biosynthesis pathway in bacteria and plants. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPR from methicillin-resistant Staphylococcus aureus (MRSA-DHDPR) are presented. The enzyme was crystallized in a number of forms, predominantly with ammonium sulfate as a precipitant, with the best crystal form diffracting to beyond 3.65 Å resolution. Crystal structures of the apo form as well as of cofactor (NADPH) bound and inhibitor (2,6-pyridinedicarboxylate) bound forms of MRSA-DHDPR will provide insight into the structure and function of this essential enzyme and valid drug target

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

    OpenAIRE

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

    2008-01-01

    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.

  10. Quantitative Detection of Perchlorate-Reducing Bacteria by Real-Time PCR Targeting the Perchlorate Reductase Gene▿

    OpenAIRE

    Nozawa-Inoue, Mamie; Jien, Mercy; Hamilton, Nicholas S.; Stewart, Valley; Scow, Kate M.; Hristova, Krassimira R.

    2008-01-01

    A quantitative real-time PCR assay targeting the pcrA gene, encoding the catalytic subunit of perchlorate reductase, detected pcrA genes from perchlorate-reducing bacteria in three different genera and from soil microbial communities. Partial pcrA sequences indicated differences in the composition of perchlorate-reducing bacterial communities following exposure to different electron donors.

  11. Crystallization and preliminary X-ray diffraction analysis of salutaridine reductase from the opium poppy Papaver somniferum

    OpenAIRE

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

    2010-01-01

    Recombinant P. somniferum salutaridine reductase (SalR) was purified and crystallized with NADPH using the hanging-drop vapor-diffusion method. Crystals of the SalR–NADPH complex diffracted X-rays to a resolution of 1.9 Å.

  12. Contribution of thermolabile methylenetetrahydrofolate reductase variant to total plasma homocysteine levels in healthy men and women. Inter99 (2)

    DEFF Research Database (Denmark)

    Husemoen, Lise Lotte N; Thomsen, Troels F; Fenger, Mogens;

    2003-01-01

    Elevation in plasma total homocysteine (tHcy) is believed to be causally related to cardiovascular disease. Like age and sex, the thermolabile variant of methylenetetrahydrofolate reductase (MTHFR(C677T)) is an important nonmodifiable determinant of tHcy, which may be considered when describing...

  13. Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation

    Science.gov (United States)

    Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine forming Met, which is then used for the syn...

  14. Improving xylitol production through recombinant expression of xylose reductase in the white-rot fungus Phanerochaete sordida YK-624.

    Science.gov (United States)

    Hirabayashi, Sho; Wang, Jianqiao; Kawagishi, Hirokazu; Hirai, Hirofumi

    2015-07-01

    We generated an expression construct consisting of the xylose reductase (XR) gene (xr) from Phanerochaete chrysosporium. Transformant X7 exhibited increased xylitol production and markedly higher XR activities than the wild-type strain. RT-PCR analysis demonstrated that the increased XR activity was associated with constant expression of the recombinant xr gene. PMID:25547244

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

    Scoffield, Jessica A; Wu, Hui

    2016-02-01

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

  17. Purification and some properties of sulfur reductase from the iron-oxidizing bacterium Thiobacillus ferrooxidans NASF-1.

    Science.gov (United States)

    Ng, K Y; Sawada, R; Inoue, S; Kamimura, K; Sugio, T

    2000-01-01

    Thiobacillus ferrooxidans strain NASF-1 grown aerobically in an Fe2+ (3%)-medium produces hydrogen sulfide (H2S) from elemental sulfur under anaerobic conditions with argon gas at pH 7.5. Sulfur reductase, which catalyzes the reduction of elemental sulfur (S0) with NAD(P)H as an electron donor to produce hydrogen sulfide (H2S) under anaerobic conditions, was purified 69-fold after 35-65% ammonium sulfate precipitation and Q-Sepharose FF, Phenyl-Toyopearl 650 ML, and Blue Sepharose FF column chromatography, with a specific activity of 57.6 U (mg protein)(-1). The purified enzyme was quite labile under aerobic conditions, but comparatively stable in the presence of sodium hydrosulfite and under anaerobic conditions, especially under hydrogen gas conditions. The purified enzyme showed both sulfur reductase and hydrogenase activities. Both activities had an optimum pH of 9.0. Sulfur reductase has an apparent molecular weight of 120,000 Da, and is composed of three different subunits (M(r) 54,000 Da (alpha), 36,000 Da (beta), and 35,000 Da (gamma)), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is the first report on the purification of sulfur reductase from a mesophilic and obligate chemolithotrophic iron-oxidizing bacterium. PMID:16232842

  18. Mutational Analysis of Pneumocystis jirovecii Dihydropteroate Synthase and Dihydrofolate Reductase Genes in HIV-Infected Patients in China

    OpenAIRE

    Deng, Xilong; Zhuo, Li; Lan, Yun; Dai, Zhaoxia; Chen, Wan-shan; Cai, Weiping; Kovacs, Joseph A.; Ma, Liang; Tang, Xiaoping

    2014-01-01

    We investigated Pneumocystis jirovecii dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) genes for mutations in 25 Chinese HIV-infected patients with P. jirovecii pneumonia. We identified DHPS mutations in 3 (12%) patients and DHFR mutations in 1 (4%) patient. The prevalence of DHPS and DHFR mutations in China remains low, as it does in other developing countries.

  19. Impacts of Elevated CO2 Concentration on Biochemical Composition,Carbonic Anhydrase, and Nitrate Reductase Activity of Freshwater Green Algae

    Institute of Scientific and Technical Information of China (English)

    Jian-Rong XIA; Kun-Shan GAO

    2005-01-01

    To investigate the biochemical response of freshwater green algae to elevated CO2 concentrations,Chlorella pyrenoidosa Chick and Chlamydomonas reinhardtii Dang cells were cultured at different CO2concentrations within the range 3-186 μmol/L and the biochemical composition, carbonic anhydrase (CA),and nitrate reductase activities of the cells were investigated. Chlorophylls (Chl), carotenoids, carbonhydrate,and protein contents were enhanced to varying extents with increasing CO2 concentration from 3-186μmol/L. The CO2 enrichment significantly increased the Chl a/Chl b ratio in Chlorella pyrenoidosa, but not in Chlamydomonas reinhardtii. The CO2 concentration had significant effects on CA and nitrate reductase activity. Elevating CO2 concentration to 186 μmol/L caused a decline in intracellular and extracellullar CA activity. Nitrate reductase activity, under either light or dark conditions, in C. reinhardtii and C. pyrenoidosa was also significantly decreased with CO2 enrichment. From this study, it can be concluded that CO2enrichment can affect biochemical composition, CA, and nitrate reductase activity, and that the biochemical response was species dependent.

  20. Enzymatic reduction of complex redox dyes using NADH-dependent reductase from Bacillus subtilis coupled with cofactor regeneration.

    Science.gov (United States)

    Bozic, Mojca; Pricelius, Sina; Guebitz, Georg M; Kokol, Vanja

    2010-01-01

    Conventional vat dyeing involves chemical reduction of dyes into their water-soluble leuco form generating considerable amounts of toxic chemicals in effluents. In the present study, a new beta-nicotinamide adenine dinucleotide disodium salt (NADH)-dependent reductase isolated from Bacillus subtilis was used to reduce the redox dyes CI Acid Blue 74, CI Natural Orange 6, and CI Vat Blue 1 into their water-soluble leuco form. Enzymatic reduction was optimized in relation to pH and temperature conditions. The reductase was able to reduce Acid Blue 74 and Natural Orange 6 in the presence of the stoichiometrically consumed cofactor NADH; meanwhile, Vat Blue 1 required the presence of mediator 1,8-dihydroxyanthraquinone. Oxygen from air was used to reoxidize the dyes into their initial forms. The enzymatic reduction of the dyes was studied and the kinetic constants determined, and these were compared to the chemically-reduced leuco form. The enzyme responsible for the reduction showed homology to a NADH-dependent reductase from B. subtilis based on results from the MS/MS peptide mass mapping of the tryptically digested protein. Additionally, the reduction of Acid Blue 74 to its leuco form by reductase from B. subtilis was confirmed using NADH regenerated by the oxidation of formic acid with formate dehydrogenase from Candida boidinii in the same solution. PMID:19662398

  1. Quantitative detection of perchlorate-reducing bacteria by real-time PCR targeting the perchlorate reductase gene.

    Science.gov (United States)

    Nozawa-Inoue, Mamie; Jien, Mercy; Hamilton, Nicholas S; Stewart, Valley; Scow, Kate M; Hristova, Krassimira R

    2008-03-01

    A quantitative real-time PCR assay targeting the pcrA gene, encoding the catalytic subunit of perchlorate reductase, detected pcrA genes from perchlorate-reducing bacteria in three different genera and from soil microbial communities. Partial pcrA sequences indicated differences in the composition of perchlorate-reducing bacterial communities following exposure to different electron donors. PMID:18245250

  2. Mechanistic studies on the flavin:NADH reductase (PrnF) from Pseudomonas fluorescens involved in arylamine oxygenation

    DEFF Research Database (Denmark)

    Tiwari, Manish Kumar; Singh, Raushan Kumar; Lee, Jung-Kul;

    2012-01-01

    We report the mechanistic studies of a FAD:NADH reductase (PrnF) involved in arylamine oxygenation. PrnF catalyzes the reduction of FAD via a sequential ordered bi-bi mechanism with NADH as the first substrate to bind and FADH(2) as the first product to be released. The residues Asp145 and His146...

  3. Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural

    Science.gov (United States)

    An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerev...

  4. Cotton Benzoquinon Reductase: Up-Regulation During Early Fiber Development and Heterologous Expresson and Characterization in Pichia Pastoris

    Science.gov (United States)

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

  5. Long-term Use of 5α-Reductase Inhibitors and the Risk of Male Breast Cancer.

    NARCIS (Netherlands)

    Duijnhoven, R.G.; Straus, S.M.J.M.; Souverein, P.C.; de Boer, A.; Bosch, J.L.H.R.; Hoes, A.W.; De Bruin, M.L.; Sub Pharmacoepidemiology; Dep Farmaceutische wetenschappen; Sub Pharmacotherapy, Theoretical

    2014-01-01

    Background The 5α-reductase inhibitors (5-ARI) finasteride and dutasteride are indicated for the treatment of lower urinary tract symptoms caused by benign prostatic hyperplasia. Case reports have suggested that 5-ARIs increase the risk for male breast cancer, with no conclusive evidence. The object

  6. Long-term use of 5α-reductase inhibitors and the risk of male breast cancer

    DEFF Research Database (Denmark)

    Duijnhoven, Ruben G; Straus, Sabine M J M; Souverein, Patrick C;

    2014-01-01

    BACKGROUND: The 5α-reductase inhibitors (5-ARI) finasteride and dutasteride are indicated for the treatment of lower urinary tract symptoms caused by benign prostatic hyperplasia. Case reports have suggested that 5-ARIs increase the risk for male breast cancer, with no conclusive evidence...

  7. Crystallization of mitochondrial rhodoquinol-fumarate reductase from the parasitic nematode Ascaris suum with the specific inhibitor flutolanil

    International Nuclear Information System (INIS)

    Rhodoquinol-fumarate reductase is a key enzyme in the anaerobic respiratory chain of adult A. suum mitochondria. Its crystallization in the presence of a mixture of octaethyleneglycol monododecyl ether and n-dodecyl-β-d-maltopyranoside in a form suitable for X-ray structure analysis is reported. In adult Ascaris suum (roundworm) mitochondrial membrane-bound complex II acts as a rhodoquinol-fumarate reductase, which is the reverse reaction to that of mammalian complex II (succinate-ubiquinone reductase). The adult A. suum rhodoquinol-fumarate reductase was crystallized in the presence of octaethyleneglycol monododecyl ether and n-dodecyl-β-d-maltopyranoside in a 3:2 weight ratio. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 123.75, b = 129.08, c = 221.12 Å, and diffracted to 2.8 Å resolution using synchrotron radiation. The presence of two molecules in the asymmetric unit (120 kDa × 2) gives a crystal volume per protein mass (VM) of 3.6 Å3 Da−1

  8. A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase

    NARCIS (Netherlands)

    Wijma, HJ; Jeuken, LJC; Verbeet, MP; Armstrong, FA; Canters, GW

    2006-01-01

    The homotrimeric copper-containing nitrite reductase ( NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of

  9. Aldo-keto reductase 1B10 and its role in proliferation capacity of drug-resistant cancers

    Directory of Open Access Journals (Sweden)

    Toshiyuki eMatsunaga

    2012-01-01

    Full Text Available 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 towards 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.

  10. Diversity of assimilatory nitrate reductase genes from plankton and epiphytes associated with a seagrass bed.

    Science.gov (United States)

    Adhitya, Anita; Thomas, Florence I M; Ward, Bess B

    2007-11-01

    Assimilatory nitrate reductase gene fragments were isolated from epiphytes and plankton associated with seagrass blades collected from Tampa Bay, Florida, USA. Nitrate reductase genes from diatoms (NR) and heterotrophic bacteria (nasA) were amplified by polymerase chain reaction (PCR) using two sets of degenerate primers. A total of 129 NR and 75 nasA clones from four clone libraries, two from each of epiphytic and planktonic components, were sequenced and aligned. In addition, genomic DNA sequences for the NR fragment were obtained from Skeletonema costatum and Thalassiosira weissflogii diatom cultures. Rarefaction analysis with an operational taxonomic unit cut-off of 6% indicated that diversity of the NR and nasA clone libraries were similar, and that sequencing of the clone libraries was not yet saturated. Phylogenetic analysis indicated that 121 of the 129 NR clones sequenced were similar to diatom sequences. Of the eight non-diatom sequences, four were most closely related to the sequence of Chlorella vulgaris. Introns were found in 8% of the Tampa Bay NR sequences; introns were also observed in S. costatum, but not T. weissflogii. Introns from within the same clone library exhibited close similarity in nucleotide sequence, position and length; the corresponding exon sequences were unique. Introns from within the same component were similar in position and length, but not in nucleotide sequence. These findings raise questions about the function of introns, and mechanisms or time evolution of intron formation. A large cluster of 14 of the 75 nasA sequences was similar to sequences from Vibrio species; other sequences were closely related to sequences from Alteromonas, alpha-proteobacteria and Marinomonas-like species. Biogeographically consistent patterns were observed for the nasA Tampa Bay sequences compared with sequences from other locations: for example, Tampa Bay sequences were similar to those from the South Atlantic Bight, but not the Barents Sea. The

  11. Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina.

    Science.gov (United States)

    Simpson, Philippa J L; Codd, Rachel

    2011-11-01

    The reduction of nitrate to nitrite is catalysed in bacteria by periplasmic nitrate reductase (Nap) which describes a system of variable protein subunits encoded by the nap operon. Nitrate reduction occurs in the NapA subunit, which contains a bis-molybdopterin guanine dinucleotide (Mo-MGD) cofactor and one [4Fe-4S] iron-sulfur cluster. The activity of periplasmic nitrate reductase (Nap) isolated as native protein from the cold-adapted (psychrophilic) Antarctic bacterium Shewanella gelidimarina (Nap(Sgel)) and middle-temperature adapted (mesophilic) Shewanella putrefaciens (Nap(Sput)) was examined at varied temperature. Irreversible deactivation of Nap(Sgel) and Nap(Sput) occurred at 54.5 and 65°C, respectively. When Nap(Sgel) was preincubated at 21-70°C for 30 min, the room-temperature nitrate reductase activity was maximal and invariant between 21 and 54°C, which suggested that Nap(Sgel) was poised for optimal catalysis at modest temperatures and, unlike Nap(Sput), did not benefit from thermally-induced refolding. At 20°C, Nap(Sgel) reduced selenate at 16% of the rate of nitrate reduction. Nap(Sput) did not reduce selenate. Sequence alignment showed 46 amino acid residue substitutions in Nap(Sgel) that were conserved in NapA from mesophilic Shewanella, Rhodobacter and Escherichia species and could be associated with the Nap(Sgel) cold-adapted phenotype. Protein homology modeling of Nap(Sgel) using a mesophilic template with 66% amino acid identity showed the majority of substitutions occurred at the protein surface distal to the Mo-MGD cofactor. Two mesophilic↔psychrophilic substitutions (Asn↔His, Val↔Trp) occurred in a region close to the surface of the NapA substrate funnel resulting in potential interdomain π-π and/or cation-π interactions. Three mesophilic↔psychrophilic substitutions occurred within 4.5Å of the Mo-MGD cofactor (Phe↔Met, Ala↔Ser, Ser↔Thr) resulting in local regions that varied in hydrophobicity and hydrogen bonding

  12. THE IMPACT OF THE ENZYME STEROID 5α-REDUCTASE 2 DEFICIENCY ON UROGENITAL BIRTH DEFECT IN MALES

    Directory of Open Access Journals (Sweden)

    Livia-Irina Olaru

    2014-07-01

    Full Text Available This research work on 5α-reductase 2 is aiming to put into evidence how a mutation of the gene codifying for this enzyme can affect the phenotype of different males, leading at a partial female phenotype.  This type of research focuses on offering a scientific explanation on different health problems affecting the normal lives of some people, even from early stages of their evolution.The experiment is focused on studying the impact of an enzyme called “steroid 5α-reductase” on the development of male urogenital components during the embryological life.  In order to study the steroid 5α-reductase 2 activity, the following procedures were used: isolation of a 5α-reductase 2 gene, genomicDNAisolation, and polymerase chain reactions.Biochemical analysis put into evidence two mutations representing opposite poles of disease manifestation, i.e. feminization of external genitalia versus predominantly male development. It suggested a correlation between clinical expression and severity of the impairment of enzyme function.  As mentioned by Imperato-McGinley, Guerrero, Gautier, & Peterson (1974, one of the mutations is called “G196S mutation.”  This mutation consists of a serine substitution for a glycine at position 196.  In this case, the level of 5α-reductase activity was sufficient to induce partial virilization.  The second type of mutation is called “G34R mutation.”  This mutation consists of an arginine substitution for glycine at position 34.  The G34R enzyme is essentially inactive, thus giving rise to the female phenotype.Mutations in the type 2 gene are responsible for autosomal recessive genetic disease of 5α-reductase deficiency.

  13. 5α-reductase inhibition suppresses testosterone-induced initial regrowth of regressed xenograft prostate tumors in animal models.

    Science.gov (United States)

    Masoodi, Khalid Z; Ramos Garcia, Raquel; Pascal, Laura E; Wang, Yujuan; Ma, Hei M; O'Malley, Katherine; Eisermann, Kurtis; Shevrin, Daniel H; Nguyen, Holly M; Vessella, Robert L; Nelson, Joel B; Parikh, Rahul A; Wang, Zhou

    2013-07-01

    Androgen deprivation therapy (ADT) is the standard treatment for patients with prostate-specific antigen progression after treatment for localized prostate cancer. An alternative to continuous ADT is intermittent ADT (IADT), which allows recovery of testosterone during off-cycles to stimulate regrowth and differentiation of the regressed prostate tumor. IADT offers patients a reduction in side effects associated with ADT, improved quality of life, and reduced cost with no difference in overall survival. Our previous studies showed that IADT coupled with 5α-reductase inhibitor (5ARI), which blocks testosterone conversion to DHT could prolong survival of animals bearing androgen-sensitive prostate tumors when off-cycle duration was fixed. To further investigate this clinically relevant observation, we measured the time course of testosterone-induced regrowth of regressed LuCaP35 and LNCaP xenograft tumors in the presence or absence of a 5ARI. 5α-Reductase inhibitors suppressed the initial regrowth of regressed prostate tumors. However, tumors resumed growth and were no longer responsive to 5α-reductase inhibition several days after testosterone replacement. This finding was substantiated by bromodeoxyuridine and Ki67 staining of LuCaP35 tumors, which showed inhibition of prostate tumor cell proliferation by 5ARI on day 2, but not day 14, after testosterone replacement. 5α-Reductase inhibitors also suppressed testosterone-stimulated proliferation of LNCaP cells precultured in androgen-free media, suggesting that blocking testosterone conversion to DHT can inhibit prostate tumor cell proliferation via an intracrine mechanism. These results suggest that short off-cycle coupled with 5α-reductase inhibition could maximize suppression of prostate tumor growth and, thus, improve potential survival benefit achieved in combination with IADT. PMID:23671262

  14. Characterisation of PduS, the pdu metabolosome corrin reductase, and evidence of substructural organisation within the bacterial microcompartment.

    Directory of Open Access Journals (Sweden)

    Joshua B Parsons

    Full Text Available PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(IIIalamin to cob(IIalamin and cob(IIalamin to cob(Ialamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment.

  15. Intracellular signal transduction of PBAN action in the silkworm, Bombyx mori: involvement of acyl CoA reductase.

    Science.gov (United States)

    Ozawa, R; Matsumoto, S

    1996-03-01

    In the silkworm, Bombyx mori, production of the sex pheromone bombykol is regulated by a neurohormone termed PBAN. We have detected the activity of acyl CoA reductase in the pheromone gland of B. mori by using palmitoyl CoA as a substrate. The acyl CoA reductase requires NADPH, but not NADH, as a proton dono. When the pheromone gland was incubated with the PBAN fragment peptide TKYFSPRLamide, palmitoyl CoA was incorporated and converted into the corresponding C16 alcohols. Radio HPLC analysis revealed that these C16 alcohols were hexadecan-1-ol (81.2%), (Z)-11-hexadecen-1-ol (12.3%), and (E, Z)-10, 12-hexadecadien-1-ol (= bombykol, 6.5%). The production of C16 alcohols in the pheromone gland was inhibited by the known bombykol biosynthesis inhibitors EDTA, LaCl3, W-7, trifluoperazine, p-nitrophenyl phosphate, NaF and compactin. By contrast, when the pheromone gland homogenate was incubated in the presence of palmitoyl CoA and NADPH, production of C16 alcohols was affected by compactin, W-7 and trifluoperazine, but not by EDTA, LaCl3, p-nitrophenyl phosphate and NaF. These results indicate that compactin, W-7 and trifluoperazine directly suppress the step catalyzed by acyl CoA reductase, whereas EDTA, LaCl3, pNPP, and NaF inhibit bombykol production by affecting other biochemical steps in the signal transduction of PBAN action. The present results also imply that PBAN regulates the step catalyzed by acyl CoA reductase and that palmitoyl CoA could be used as a substrate of the acyl CoA reductase that regulates bombykol biosynthesis. PMID:8900596

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    1998-10-01

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

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

    Science.gov (United States)

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

    1991-11-01

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

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

    International Nuclear Information System (INIS)

    An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H2O2. These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reduces peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.

  20. Stress defense mechanisms of NADPH-dependent thioredoxin reductases (NTRs) in plants.

    Science.gov (United States)

    Cha, Joon-Yung; Barman, Dhirendra Nath; Kim, Min Gab; Kim, Woe-Yeon

    2015-01-01

    Plants establish highly and systemically organized stress defense mechanisms against unfavorable living conditions. To interpret these environmental stimuli, plants possess communication tools, referred as secondary messengers, such as Ca(2+) signature and reactive oxygen species (ROS) wave. Maintenance of ROS is an important event for whole lifespan of plants, however, in special cases, toxic ROS molecules are largely accumulated under excess stresses and diverse enzymes played as ROS scavengers. Arabidopsis and rice contain 3 NADPH-dependent thioredoxin reductases (NTRs) which transfer reducing power to Thioredoxin/Peroxiredoxin (Trx/Prx) system for scavenging ROS. However, due to functional redundancy between cytosolic and mitochondrial NTRs (NTRA and NTRB, respectively), their functional involvements under stress conditions have not been well characterized. Recently, we reported that cytosolic NTRA confers the stress tolerance against oxidative and drought stresses via regulation of ROS amounts using NTRA-overexpressing plants. With these findings, mitochondrial NTRB needs to be further elucidated. PMID:26039478