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Sample records for inhibits erythrose reductase

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

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    Ryu Yeon-Woo

    2010-06-01

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

  2. INHIBITION OF RAT LENS ALDOSE REDUCTASE BY QUERCETAGETIN AND PATULETIN

    Institute of Scientific and Technical Information of China (English)

    1991-01-01

    In this paper the results of inhibition of the Aldose reductase(AR) activity on Wistar rat lens by Quercetagetin extracted from Tagetes erects Linn and by Patuletin extracted from Tagetes patula Linn are reported.Quercetagetin inhibited AR of the rat lens by 93.9% at 10~(-4)M, 76.0% at 10~(-5)M and 13.3% at 10~(-6)M. Patuletin inhibited AR of the rat lens by 100% at 10~(-1)M, 80% at 10~(-5)M and 22.7% at 10~(-6)M respectively. The results show that these two flavones are lens AR Inhibitors, but further ...

  3. Inhibition of Aldose Reductase by Gentiana lutea Extracts

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    Chandrasekhar Akileshwari

    2012-01-01

    Full Text Available Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2 activity has been implicated in the development of various secondary complications of diabetes. Thus, ALR2 inhibition could be an effective strategy in the prevention or delay of certain diabetic complications. Gentiana lutea grows naturally in the central and southern areas of Europe. Its roots are commonly consumed as a beverage in some European countries and are also known to have medicinal properties. The water, ethanol, methanol, and ether extracts of the roots of G. lutea were subjected to in vitro bioassay to evaluate their inhibitory activity on the ALR2. While the ether and methanol extracts showed greater inhibitory activities against both rat lens and human ALR2, the water and ethanol extracts showed moderate inhibitory activities. Moreover, the ether and methanol extracts of G. lutea roots significantly and dose-dependently inhibited sorbitol accumulation in human erythrocytes under high glucose conditions. Molecular docking studies with the constituents commonly present in the roots of G. lutea indicate that a secoiridoid glycoside, amarogentin, may be a potential inhibitor of ALR2. This is the first paper that shows G. lutea extracts exhibit inhibitory activity towards ALR2 and these results suggest that Gentiana or its constituents might be useful to prevent or treat diabetic complications.

  4. Inhibition of peroxisomal hydroxypyruvate reductase (HPR1) by tyrosine nitration.

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

    2013-11-01

    Protein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has been reported. We studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and proteomic approaches. Proteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H2O2, NO, GSH and peroxynitrite showed that the ONOO(-) molecule caused the highest inhibition of activity (51% at 5mM SIN-1), with 5mM H2O2 having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite. These findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function. This is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions. © 2013.

  5. A DFT-based QSAR study on inhibition of human dihydrofolate reductase.

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    Karabulut, Sedat; Sizochenko, Natalia; Orhan, Adnan; Leszczynski, Jerzy

    2016-11-01

    Diaminopyrimidine derivatives are frequently used as inhibitors of human dihydrofolate reductase, for example in treatment of patients whose immune system are affected by human immunodeficiency virus. Forty-seven dicyclic and tricyclic potential inhibitors of human dihydrofolate reductase were analyzed using the quantitative structure-activity analysis supported by DFT-based and DRAGON-based descriptors. The developed model yielded an RMSE deviation of 1.1 a correlation coefficient of 0.81. The prediction set was characterized by R(2)=0.60 and RMSE=3.59. Factors responsible for inhibition process were identified and discussed. The resulting model was validated via cross validation and Y-scrambling procedure. From the best model, we found several mass-related descriptors and Sanderson electronegativity-related descriptors that have the best correlations with the investigated inhibitory concentration. These descriptors reflect results from QSAR studies based on characteristics of human dihydrofolate reductase inhibitors.

  6. Biosynthesis of L-Erythrose by Assembly of Two Key Enzymes in Gluconobacter oxydans.

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    Zou, Xingxing; Lin, Jinping; Mao, Xinlei; Zhao, Shengyun; Ren, Yuhong

    2017-09-06

    L-erythrose, a rare aldotetrose, possesses various pharmacological activities. However, efficient L-erythrose production is challenging. Currently, L-erythrose is produced by a two-step fermentation process from erythritol. Here, we describe a novel strategy for the production of L-erythrose in Gluconobacter oxydans (G. oxydans) by localizing the assembly of L-ribose isomerase (L-RI) to membrane-bound sorbitol dehydrogenase (SDH) via the protein-peptide interactions of the PDZ domain and PDZ ligand. To demonstrate this self-assembly, green fluorescent protein (GFP) replaced L-RI and its movement to membrane-bound SDH was observed by fluorescence microscopy. The final L-erythrose production was improved to 23.5 g/L with the stepwise metabolic engineering of G. oxydans, which was 1.4-fold higher than that obtained using coexpression of SDH and L-RI in G. oxydans. This self-assembly strategy shows remarkable potential for further improvement of L-erythrose production.

  7. Laboratory Observation of the Rotational Spectrum of a Potential Interstellar Sugar, Erythrose

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    Pena, I.; Cabezas, C.; Daly, A. M.; Mata, S.; Alonso, J. L.

    2013-06-01

    The rotational spectrum of erythrose has been recorded in the frequency region 6 -- 12 GHz using a chirped-pulse Fourier transform microwave spectrometer (CP-FTMW) combined with a laser ablation (LA) source. The investigation of rotational spectra of erythrose is of astrophysical and biological relevance. However, no gas-phase data were available on erythrose. It is syrup at room temperature and vaporization using conventional methods leads to decomposition. A non convetional laser ablation method has been successfully used to vaporize erythrose and two cyclic forms have been observed using rotational spectroscopy. α-erythrose has been found to have rotational constants are A = 2586.8998 (21) MHz, B = 2353.0837 (41) MHz and C = 1773.2378 (18) MHz and β-erythrose is characterized with rotational constants A = 3109.2005 (46) MHz, B = 1856.1298 (15) MHz, C = 1616.5557 (18) MHz. G. G. Brown, B. C. Dian, K. O. Douglass, S. M. Geyer, S. T. Shipman, B. H. Pate, Rev. Sci. Instrum. 2008, 79, 053103. S. Mata, I. Peña, C. Cabezas, J. C. López, J. L. Alonso, J. Mol. Spectrosc. 2012, 280, 91.

  8. Inhibition of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase (Ex Vivo by Morus indica (Mulberry

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    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.

  9. Aldose Reductase Inhibition Prevents Endotoxin-Induced Inflammatory Responses in Retinal Microglia

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    Chang, Kun-Che; Ponder, Jessica; LaBarbera, Daniel V.; Petrash, J. Mark

    2014-01-01

    Purpose. Retinal microglia become activated in diabetes and produce pro-inflammatory molecules associated with changes in retinal vasculature and increased apoptosis of retinal neurons and glial cells. We sought to determine if the action of aldose reductase (AR), an enzyme linked to the pathogenesis of diabetic retinopathy, contributes to activation of microglial cells. Methods. Involvement of AR in the activation process was studied using primary cultures of retinal microglia (RMG) isolated from wild-type and AR-null mice, or in mouse macrophage cultures treated with either AR inhibitors or small interfering RNA (siRNA) directed to AR. Inflammatory cytokines were measured by ELISA. Cell migration was measured using a transwell assay. Gelatin zymography was used to detect active matrix metalloproteinase (MMP)-9, while RMG-induced apoptosis of adult retinal pigment epithelium (ARPE-19) cells was studied in a cell coculture system. Results. Aldose reductase inhibition or genetic deficiency substantially reduced lipopolysacharide (LPS)-induced cytokine secretion from macrophages and RMG. Aldose reductase inhibition or deficiency also reduced the activation of MMP-9 and attenuated LPS-induced cell migration. Additionally, blockade of AR by sorbinil or through genetic means caused a reduction in the ability of activated RMG to induce apoptosis of ARPE-19 cells. Conclusions. These results demonstrate that the action of AR contributes to the activation of RMG. Inhibition of AR may be a therapeutic strategy to reduce inflammation associated with activation of RMG in disease. PMID:24677107

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

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    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.

  11. Inhibition of aldo-keto reductase family 1 member B10 by unsaturated fatty acids.

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    Hara, Akira; Endo, Satoshi; Matsunaga, Toshiyuki; Soda, Midori; El-Kabbani, Ossama; Yashiro, Koji

    2016-11-01

    A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, is a cytosolic NADPH-dependent reductase toward various carbonyl compounds including reactive aldehydes, and is normally expressed in intestines. The enzyme is overexpressed in several extraintestinal cancers, and suggested as a potential target for cancer treatment. We found that saturated and cis-unsaturated fatty acids inhibit AKR1B10. Among the saturated fatty acids, myristic acid was the most potent, showing the IC50 value of 4.2 μM cis-Unsaturated fatty acids inhibited AKR1B10 more potently, and linoleic, arachidonic, and docosahexaenoic acids showed the lowest IC50 values of 1.1 μM. The inhibition by these fatty acids was reversible and kinetically competitive with respect to the substrate, showing the Ki values of 0.24-1.1 μM. These fatty acids, except for α-linoleic acid, were much less inhibitory to structurally similar aldose reductase. Site-directed mutagenesis study suggested that the fatty acids interact with several active site residues of AKR1B10, of which Gln114, Val301 and Gln303 are responsible for the inhibitory selectivity. Linoleic and arachidonic acids also effectively inhibited AKR1B10-mediated 4-oxo-2-nonenal metabolism in HCT-15 cells. Thus, the cis-unsaturated fatty acids may be used as an adjuvant therapy for treatment of cancers that up-regulate AKR1B10. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Inhibition of aldose reductase by phenylethanoid glycoside isolated from the seeds of Paulownia coreana.

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    Kim, Jin Kyu; Lee, Yeon Sil; Kim, Seon Ha; Bae, Young Soo; Lim, Soon Sung

    2011-01-01

    Aldose reductase (AR) inhibitors have considerable therapeutic potential against diabetic complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of the 70% acetone extract obtained from Paulownia coreana seeds, phenylpropanoid glycosides (compounds 1-4) and 5 phenolic compounds were isolated (compounds 5-9). Their structures were determined on the basis of spectroscopic analysis and comparison with reported data. All the isolates were subjected to in vitro bioassays to evaluate their inhibitory activities against recombinant human aldose reductase (rhAR) and sorbitol formation in human erythrocytes. Phenylethanoid glycosides showed more effective than the phenolic compounds in inhibiting rhAR. Among the compounds, isocampneoside II (3) was found to significantly inhibit rhAR with an IC(50) value of 9.72 µM. In kinetic analyses performed using Lineweaver-Burk plots of 1/velocity and 1/concentration of substrate, isocampneoside II (3) showed uncompetitive inhibition against rhAR. Furthermore, it inhibited sorbitol formation in a rat lens incubated with a high concentration of glucose; this finding indicated that isocampneoside II (3) may effectively prevent osmotic stress in hyperglycemia. Thus, the P. coreana-derived phenylethanoid glycoside isocampneoside II (3) may have a potential therapeutics against diabetic complications.

  13. The molecular basis for inhibition of sulindac and its metabolites towards human aldose reductase.

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    Zheng, Xuehua; Zhang, Liping; Zhai, Jing; Chen, Yunyun; Luo, Haibin; Hu, Xiaopeng

    2012-01-02

    Sulindac (SLD) exhibits both the highest inhibitory activity towards human aldose reductase (AR) among popular non-steroidal anti-inflammatory drugs and clear beneficial clinical effects on Type 2 diabetes. However, the molecular basis for these properties is unclear. Here, we report that SLD and its pharmacologically active/inactive metabolites, SLD sulfide and SLD sulfone, are equally effective as un-competitive inhibitors of AR in vitro. Crystallographic analysis reveals that π-π stacking favored by the distinct scaffold of SLDs is pivotal to their high AR inhibitory activities. These results also suggest that SLD sulfone could be a potent lead compound for AR inhibition in vivo.

  14. Inhibition of methyl-CoM Reductase from Methanobrevibacter ruminantium by 2-bromoethanesulfonate.

    Science.gov (United States)

    Gräwert, Tobias; Hohmann, Hans-Peter; Kindermann, Maik; Duval, Stephane; Bacher, Adelbert; Fischer, Markus

    2014-12-31

    Cattle husbandry is a major contributor to atmospheric methane, which is considered as an important greenhouse gas. Moreover, the generation of methane in the intestine of domestic ruminants by methanogenic bacteria is a drag on feed efficacy. Studies on methanogenesis have typically implied model organisms that are, however, not relevant in the ruminant gut. This paper shows that methyl-CoM reductase catalyzing the final step of methanogenesis in Methanobrevibacter ruminantium, a major participant in methane production by cattle, is inhibited by 2-bromoethanesulfonate, a compound often used as a model in animal agriculture, with an apparent IC50 of 0.4 ± 0.04 μM.

  15. Hypothesis on Serenoa repens (Bartram small extract inhibition of prostatic 5α-reductase through an in silico approach on 5β-reductase x-ray structure

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    Paolo Governa

    2016-11-01

    Full Text Available Benign prostatic hyperplasia is a common disease in men aged over 50 years old, with an incidence increasing to more than 80% over the age of 70, that is increasingly going to attract pharmaceutical interest. Within conventional therapies, such as α-adrenoreceptor antagonists and 5α-reductase inhibitor, there is a large requirement for treatments with less adverse events on, e.g., blood pressure and sexual function: phytotherapy may be the right way to fill this need. Serenoa repens standardized extract has been widely studied and its ability to reduce lower urinary tract symptoms related to benign prostatic hyperplasia is comprehensively described in literature. An innovative investigation on the mechanism of inhibition of 5α-reductase by Serenoa repens extract active principles is proposed in this work through computational methods, performing molecular docking simulations on the crystal structure of human liver 5β-reductase. The results confirm that both sterols and fatty acids can play a role in the inhibition of the enzyme, thus, suggesting a competitive mechanism of inhibition. This work proposes a further confirmation for the rational use of herbal products in the management of benign prostatic hyperplasia, and suggests computational methods as an innovative, low cost, and non-invasive process for the study of phytocomplex activity toward proteic targets.

  16. Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate.

    Science.gov (United States)

    Dawson, Alice; Gibellini, Federica; Sienkiewicz, Natasha; Tulloch, Lindsay B; Fyfe, Paul K; McLuskey, Karen; Fairlamb, Alan H; Hunter, William N

    2006-09-01

    The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 A resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the beta6-alpha6 loop and alpha6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.

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

    Science.gov (United States)

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

    2014-06-01

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

  18. New small-molecule inhibitors of dihydrofolate reductase inhibit Streptococcus mutans.

    Science.gov (United States)

    Zhang, Qiong; Nguyen, Thao; McMichael, Megan; Velu, Sadanandan E; Zou, Jing; Zhou, Xuedong; Wu, Hui

    2015-08-01

    Streptococcus mutans is a major aetiological agent of dental caries. Formation of biofilms is a key virulence factor of S. mutans. Drugs that inhibit S. mutans biofilms may have therapeutic potential. Dihydrofolate reductase (DHFR) plays a critical role in regulating the metabolism of folate. DHFR inhibitors are thus potent drugs and have been explored as anticancer and antimicrobial agents. In this study, a library of analogues based on a DHFR inhibitor, trimetrexate (TMQ), an FDA-approved drug, was screened and three new analogues that selectively inhibited S. mutans were identified. The most potent inhibitor had a 50% inhibitory concentration (IC50) of 454.0±10.2nM for the biofilm and 8.7±1.9nM for DHFR of S. mutans. In contrast, the IC50 of this compound for human DHFR was ca. 1000nM, a >100-fold decrease in its potency, demonstrating the high selectivity of the analogue. An analogue that exhibited the least potency for the S. mutans biofilm also had the lowest activity towards inhibiting S. mutans DHFR, further indicating that inhibition of biofilms is related to reduced DHFR activity. These data, along with docking of the most potent analogue to the modelled DHFR structure, suggested that the TMQ analogues indeed selectively inhibited S. mutans through targeting DHFR. These potent and selective small molecules are thus promising lead compounds to develop new effective therapeutics to prevent and treat dental caries.

  19. Inhibition of thioredoxin reductase but not of glutathione reductase by the major classes of alkylating and platinum-containing anticancer compounds.

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    Witte, Anne-Barbara; Anestål, Karin; Jerremalm, Elin; Ehrsson, Hans; Arnér, Elias S J

    2005-09-01

    Mammalian thioredoxin reductase (TrxR) is important for cell proliferation, antioxidant defense, and redox signaling. Together with glutathione reductase (GR) it is the main enzyme providing reducing equivalents to many cellular processes. GR and TrxR are flavoproteins of the same enzyme family, but only the latter is a selenoprotein. With the active site containing selenocysteine, TrxR may catalyze reduction of a wide range of substrates, but can at the same time easily be targeted by electrophilic compounds due to the extraordinarily high reactivity of a selenolate moiety. Here we addressed the inhibition of the enzyme by major anticancer alkylating agents and platinum-containing compounds and we compared it to that of GR. We confirmed prior studies suggesting that the nitrosourea carmustine can inhibit both GR and TrxR. We next found, however, that nitrogen mustards (chlorambucil and melphalan) and alkyl sulfonates (busulfan) efficiently inhibited TrxR while these compounds, surprisingly, did not inhibit GR. Inhibitions were concentration and time dependent and apparently irreversible. Anticancer anthracyclines (daunorubicin and doxorubicin) were, in contrast to the alkylating agents, not inhibitors but poor substrates of TrxR. We also found that TrxR, but not GR, was efficiently inhibited by both cisplatin, its monohydrated complex, and oxaliplatin. Carboplatin, in contrast, could not inhibit any of the two enzymes. These findings lead us to conclude that representative compounds of the major classes of clinically used anticancer alkylating agents and most platinum compounds may easily target TrxR, but not GR. The TrxR inhibition should thereby be considered as a factor that may contribute to the cytotoxicity seen upon clinical use of these drugs.

  20. Inhibition of aldose reductase and anti-cataract action of trans-anethole isolated from Foeniculum vulgare Mill. fruits.

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    Dongare, Vandana; Kulkarni, Chaitanya; Kondawar, Manish; Magdum, Chandrakant; Haldavnekar, Vivek; Arvindekar, Akalpita

    2012-05-01

    Foeniculum vulgare fruits are routinely consumed for their carminative and mouth freshening effect. The plant was evaluated for aldose reductase inhibition and anti-diabetic action. Bioguided fractionation using silica gel column chromatography, HPLC, and GC-MS analysis revealed trans-anethole as the bioactive constituent possessing potent aldose reductase inhibitory action, with an IC50 value of 3.8μg/ml. Prolonged treatment with the pet ether fraction of the F. vulgare distillate demonstrated improvement in blood glucose, lipid profile, glycated haemoglobin and other parameters in streptozotocin-induced diabetic rats. Trans-anethole could effectively show anti-cataract activity through the increase in soluble lens protein, reduced glutathione, catalase and SOD activity on in vitro incubation of the eye lens with 55mM glucose. Trans-anethole demonstrated noncompetitive to mixed type of inhibition of lens aldose reductase using Lineweaver Burk plot.

  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. HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight : Evidence from genetic analysis and randomised trials

    NARCIS (Netherlands)

    Swerdlow, Daniel I.; Preiss, David; Kuchenbaecker, Karoline B.; Holmes, Michael V.; Engmann, Jorgen E L; Shah, Tina; Sofat, Reecha; Stender, Stefan; Johnson, Paul C D; Scott, Robert A.; Leusink, Maarten; Verweij, Niek; Sharp, Stephen J.; Guo, Yiran; Giambartolomei, Claudia; Chung, Christina; Peasey, Anne; Amuzu, Antoinette; Li, Kawah; Palmen, Jutta; Howard, Philip; Cooper, Jackie A.; Drenos, Fotios; Li, Yun R.; Lowe, Gordon; Gallacher, John; Stewart, Marlene C W; Tzoulaki, Ioanna; Buxbaum, Sarah G.; Van Der A, Daphne L.; Forouhi, Nita G.; Onland-Moret, N. Charlotte; Van Der Schouw, Yvonne T.; Schnabel, Renate B.; Hubacek, Jaroslav A.; Kubinova, Ruzena; Baceviciene, Migle; Tamosiunas, Abdonas; Pajak, Andrzej; Topor-Madry, Romanvan; Stepaniak, Urszula; Malyutina, Sofia; Baldassarre, Damiano; Sennblad, Bengt; Tremoli, Elena; De Faire, Ulf; Veglia, Fabrizio; Ford, Ian; Jukema, J. Wouter; Westendorp, Rudi G J; De Borst, Gert Jan; De Jong, Pim A.; Algra, Ale; Spiering, Wilko; Der Zee, Anke H Maitland Van; Klungel, Olaf H.; De Boer, Anthonius; Doevendans, Pieter A.; Eaton, Charles B.; Robinson, Jennifer G.; Duggan, David; Kjekshus, John; Downs, John R.; Gotto, Antonio M.; Keech, Anthony C.; Marchioli, Roberto; Tognoni, Gianni; Sever, Peter S.; Poulter, Neil R.; Waters, David D.; Pedersen, Terje R.; Amarenco, Pierre; Nakamura, Haruo; McMurray, John J V; Lewsey, James D.; Chasman, Daniel I.; Ridker, Paul M.; Maggioni, Aldo P.; Tavazzi, Luigi; Ray, Kausik K.; Seshasai, Sreenivasa Rao Kondapally; Manson, Joann E.; Price, Jackie F.; Whincup, Peter H.; Morris, Richard W.; Lawlor, Debbie A.; Smith, George Davey; Ben-Shlomo, Yoav; Schreiner, Pamela J.; Fornage, Myriam; Siscovick, David S.; Cushman, Mary; Kumari, Meena; Wareham, Nick J.; Verschuren, W. M Monique; Redline, Susan; Patel, Sanjay R.; Whittaker, John C.; Hamsten, Anders; Delaney, Joseph A.; Dale, Caroline; Gaunt, Tom R.; Wong, Andrew; Kuh, Diana; Hardy, Rebecca; Kathiresan, Sekar; Castillo, Berta A.; Van Der Harst, Pim; Brunner, Eric J.; Tybjaerg-Hansen, Anne; Marmot, Michael G.; Krauss, Ronald M.; Tsai, Michael; Coresh, Josef; Hoogeveen, Ronald C.; Psaty, Bruce M.; Lange, Leslie A.; Hakonarson, Hakon; Dudbridge, Frank; Humphries, Steve E.; Talmud, Philippa J.; Kivimäki, Mika; Timpson, Nicholas J.; Langenberg, Claudia; Asselbergs, Folkert W.; Voevoda, Mikhail; Bobak, Martin; Pikhart, Hynek; Wilson, James G.; Reiner, Alex P.; Keating, Brendan J.; Hingorani, Aroon D.; Sattar, Naveed

    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 gene

  3. HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight : evidence from genetic analysis and randomised trials

    NARCIS (Netherlands)

    Swerdlow, Daniel I.; Preiss, David; Kuchenbaecker, Karoline B.; Holmes, Michael V.; Engmann, Jorgen E. L.; Shah, Tina; Sofat, Reecha; Stender, Stefan; Johnson, Paul C. D.; Scott, Robert A.; Leusink, Maarten; Verweij, Niek; Sharp, Stephen J.; Guo, Yiran; Giambartolomei, Claudia; Chung, Christina; Peasey, Anne; Amuzu, Antoinette; Li, Kawah; Palmen, Jutta; Howard, Philip; Cooper, Jackie A.; Drenos, Fotios; Li, Yun R.; Lowe, Gordon; Gallacher, John; Stewart, Marlene C. W.; Tzoulaki, Ioanna; Buxbaum, Sarah G.; Daphne, L. van der A.; Forouhi, Nita G.; Onland-Moret, N. Charlotte; van der Schouw, Yvonne T.; Schnabel, Renate B.; Hubacek, Jaroslav A.; Kubinova, Ruzena; Baceviciene, Migle; Tamosiunas, Abdonas; Pajak, Andrzej; Topor-Madry, Roman; Stepaniak, Urszula; Malyutina, Sofi A.; Baldassarre, Damiano; Sennblad, Bengt; Tremoli, Elena; de Faire, Ulf; Veglia, Fabrizio; Ford, Ian; Jukema, J. Wouter; Westendorp, Rudi G. J.; de Borst, Gert Jan; de Jong, Pim A.; Algra, Ale; Spiering, Wilko; Maitland-van der Zee, Anke H.; Klungel, Olaf H.; de Boer, Anthonius; Doevendans, Pieter A.; Eaton, Charles B.; Robinson, Jennifer G.; Duggan, David; Kjekshus, John; Downs, John R.; Gotto, Antonio M.; Keech, Anthony C.; Marchioli, Roberto; Tognoni, Gianni; Sever, Peter S.; Poulter, Neil R.; Waters, David D.; Pedersen, Terje R.; Amarenco, Pierre; Nakamura, Haruo; McMurray, John J. V.; Lewsey, James D.; Chasman, Daniel I.; Ridker, Paul M.; Maggioni, Aldo P.; Tavazzi, Luigi; Ray, Kausik K.; Seshasai, Sreenivasa Rao Kondapally; Manson, Joann E.; Price, Jackie F.; Whincup, Peter H.; Morris, Richard W.; Lawlor, Debbie A.; Smith, George Davey; Ben-Shlomo, Yoav; Schreiner, Pamela J.; Fornage, Myriam; Siscovick, David S.; Cushman, Mary; Kumari, Meena; Wareham, Nick J.; Verschuren, W. M. Monique; Redline, Susan; Patel, Sanjay R.; Whittaker, John C.; Hamsten, Anders; Delaney, Joseph A.; Dale, Caroline; Gaunt, Tom R.; Wong, Andrew; Kuh, Diana; Hardy, Rebecca; Kathiresan, Sekar; Castillo, Berta A.; van der Harst, Pim; Brunner, Eric J.; Tybjaerg-Hansen, Anne; Marmot, Michael G.; Krauss, Ronald M.; Tsai, Michael; Coresh, Josef; Hoogeveen, Ronald C.; Psaty, Bruce M.; Lange, Leslie A.; Hakonarson, Hakon; Dudbridge, Frank; Humphries, Steve E.; Talmud, Philippa J.; Kivimaeki, Mika; Timpson, Nicholas J.; Langenberg, Claudia; Asselbergs, Folkert W.; Voevoda, Mikhail; Bobak, Martin; Pikhart, Hynek; Wilson, James G.; Reiner, Alex P.; Keating, Brendan J.; Hingorani, Aroon D.; Sattar, Naveed; Wijmenga, T. N.

    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 ge

  4. HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight : evidence from genetic analysis and randomised trials

    NARCIS (Netherlands)

    Swerdlow, Daniel I; Preiss, David; Kuchenbaecker, Karoline B; Holmes, Michael V; Engmann, Jorgen E L; Shah, Tina; Sofat, Reecha; Stender, Stefan; Johnson, Paul C D; Scott, Robert A; Leusink, Maarten; Verweij, Niek; Sharp, Stephen J; Guo, Yiran; Giambartolomei, Claudia; Chung, Christina; Peasey, Anne; Amuzu, Antoinette; Li, KaWah; Palmen, Jutta; Howard, Philip; Cooper, Jackie A; Drenos, Fotios; Li, Yun R; Lowe, Gordon; Gallacher, John; Stewart, Marlene C W; Tzoulaki, Ioanna; Buxbaum, Sarah G; van der A, Daphne L; Forouhi, Nita G; Onland-Moret, N Charlotte; van der Schouw, Yvonne T; Schnabel, Renate B; Hubacek, Jaroslav A; Kubinova, Ruzena; Baceviciene, Migle; Tamosiunas, Abdonas; Pajak, Andrzej; Topor-Madry, Romanvan; Stepaniak, Urszula; Malyutina, Sofia; Baldassarre, Damiano; Sennblad, Bengt; Tremoli, Elena; de Faire, Ulf; Veglia, Fabrizio; Ford, Ian; Jukema, J Wouter; Westendorp, Rudi G J; de Borst, Gert Jan; de Jong, Pim A; Algra, Ale; Spiering, Wilko; der Zee, Anke H Maitland-van; Klungel, Olaf H; de Boer, Anthonius; Doevendans, Pieter A; Eaton, Charles B; Robinson, Jennifer G; Duggan, David; Kjekshus, John; Downs, John R; Gotto, Antonio M; Keech, Anthony C; Marchioli, Roberto; Tognoni, Gianni; Sever, Peter S; Poulter, Neil R; Waters, David D; Pedersen, Terje R; Amarenco, Pierre; Nakamura, Haruo; McMurray, John J V; Lewsey, James D; Chasman, Daniel I; Ridker, Paul M; Maggioni, Aldo P; Tavazzi, Luigi; Ray, Kausik K; Seshasai, Sreenivasa Rao Kondapally; Manson, JoAnn E; Price, Jackie F; Whincup, Peter H; Morris, Richard W; Lawlor, Debbie A; Smith, George Davey; Ben-Shlomo, Yoav; Schreiner, Pamela J; Fornage, Myriam; Siscovick, David S; Cushman, Mary; Kumari, Meena; Wareham, Nick J; Verschuren, W M Monique; Redline, Susan; Patel, Sanjay R; Whittaker, John C; Hamsten, Anders; Delaney, Joseph A; Dale, Caroline; Gaunt, Tom R; Wong, Andrew; Kuh, Diana; Hardy, Rebecca; Kathiresan, Sekar; Castillo, Berta A; van der Harst, Pim; Brunner, Eric J; Tybjaerg-Hansen, Anne; Marmot, Michael G; Krauss, Ronald M; Tsai, Michael; Coresh, Josef; Hoogeveen, Ronald C; Psaty, Bruce M; Lange, Leslie A; Hakonarson, Hakon; Dudbridge, Frank; Humphries, Steve E; Talmud, Philippa J; Kivimäki, Mika; Timpson, Nicholas J; Langenberg, Claudia; Asselbergs, Folkert W; Voevoda, Mikhail; Bobak, Martin; Pikhart, Hynek; Wilson, James G; Reiner, Alex P; Keating, Brendan J; Hingorani, Aroon D; Sattar, Naveed; DIAGRAM Consortium, MAGIC Consortium, InterAct Consortium

    2014-01-01

    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 ge

  5. Inhibition of HMG-CoA reductase induces the UPR pathway in C. elegans

    DEFF Research Database (Denmark)

    Olsen, Louise Cathrine Braun; Hansen, Nadia Jin Storm; Pilon, Marc

    -requiring enzyme-1 (IRE-1), and activating transcription factor-6 (ATF-6). Using a transgenic GFP reporter strain of the model organism C. elegans, we have recently identified that inhibition of the enzyme HMG-CoA reductase (HMG-CoAR) with Fluvastatin and knock down of HMG-CoAR using RNA interference (RNAi) both...

  6. HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight : evidence from genetic analysis and randomised trials

    NARCIS (Netherlands)

    Swerdlow, Daniel I.; Preiss, David; Kuchenbaecker, Karoline B.; Holmes, Michael V.; Engmann, Jorgen E. L.; Shah, Tina; Sofat, Reecha; Stender, Stefan; Johnson, Paul C. D.; Scott, Robert A.; Leusink, Maarten; Verweij, Niek; Sharp, Stephen J.; Guo, Yiran; Giambartolomei, Claudia; Chung, Christina; Peasey, Anne; Amuzu, Antoinette; Li, Kawah; Palmen, Jutta; Howard, Philip; Cooper, Jackie A.; Drenos, Fotios; Li, Yun R.; Lowe, Gordon; Gallacher, John; Stewart, Marlene C. W.; Tzoulaki, Ioanna; Buxbaum, Sarah G.; Daphne, L. van der A.; Forouhi, Nita G.; Onland-Moret, N. Charlotte; van der Schouw, Yvonne T.; Schnabel, Renate B.; Hubacek, Jaroslav A.; Kubinova, Ruzena; Baceviciene, Migle; Tamosiunas, Abdonas; Pajak, Andrzej; Topor-Madry, Roman; Stepaniak, Urszula; Malyutina, Sofi A.; Baldassarre, Damiano; Sennblad, Bengt; Tremoli, Elena; de Faire, Ulf; Veglia, Fabrizio; Ford, Ian; Jukema, J. Wouter; Westendorp, Rudi G. J.; de Borst, Gert Jan; de Jong, Pim A.; Algra, Ale; Spiering, Wilko; Maitland-van der Zee, Anke H.; Klungel, Olaf H.; de Boer, Anthonius; Doevendans, Pieter A.; Eaton, Charles B.; Robinson, Jennifer G.; Duggan, David; Kjekshus, John; Downs, John R.; Gotto, Antonio M.; Keech, Anthony C.; Marchioli, Roberto; Tognoni, Gianni; Sever, Peter S.; Poulter, Neil R.; Waters, David D.; Pedersen, Terje R.; Amarenco, Pierre; Nakamura, Haruo; McMurray, John J. V.; Lewsey, James D.; Chasman, Daniel I.; Ridker, Paul M.; Maggioni, Aldo P.; Tavazzi, Luigi; Ray, Kausik K.; Seshasai, Sreenivasa Rao Kondapally; Manson, Joann E.; Price, Jackie F.; Whincup, Peter H.; Morris, Richard W.; Lawlor, Debbie A.; Smith, George Davey; Ben-Shlomo, Yoav; Schreiner, Pamela J.; Fornage, Myriam; Siscovick, David S.; Cushman, Mary; Kumari, Meena; Wareham, Nick J.; Verschuren, W. M. Monique; Redline, Susan; Patel, Sanjay R.; Whittaker, John C.; Hamsten, Anders; Delaney, Joseph A.; Dale, Caroline; Gaunt, Tom R.; Wong, Andrew; Kuh, Diana; Hardy, Rebecca; Kathiresan, Sekar; Castillo, Berta A.; van der Harst, Pim; Brunner, Eric J.; Tybjaerg-Hansen, Anne; Marmot, Michael G.; Krauss, Ronald M.; Tsai, Michael; Coresh, Josef; Hoogeveen, Ronald C.; Psaty, Bruce M.; Lange, Leslie A.; Hakonarson, Hakon; Dudbridge, Frank; Humphries, Steve E.; Talmud, Philippa J.; Kivimaeki, Mika; Timpson, Nicholas J.; Langenberg, Claudia; Asselbergs, Folkert W.; Voevoda, Mikhail; Bobak, Martin; Pikhart, Hynek; Wilson, James G.; Reiner, Alex P.; Keating, Brendan J.; Hingorani, Aroon D.; Sattar, Naveed; Wijmenga, T. N.

    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

  7. HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight : evidence from genetic analysis and randomised trials

    NARCIS (Netherlands)

    Swerdlow, Daniel I; Preiss, David; Kuchenbaecker, Karoline B; Holmes, Michael V; Engmann, Jorgen E L; Shah, Tina; Sofat, Reecha; Stender, Stefan; Johnson, Paul C D; Scott, Robert A; Leusink, Maarten|info:eu-repo/dai/nl/357581164; Verweij, Niek; Sharp, Stephen J; Guo, Yiran; Giambartolomei, Claudia; Chung, Christina; Peasey, Anne; Amuzu, Antoinette; Li, KaWah; Palmen, Jutta; Howard, Philip; Cooper, Jackie A; Drenos, Fotios; Li, Yun R; Lowe, Gordon; Gallacher, John; Stewart, Marlene C W; Tzoulaki, Ioanna; Buxbaum, Sarah G; van der A, Daphne L; Forouhi, Nita G; Onland-Moret, N Charlotte; van der Schouw, Yvonne T; Schnabel, Renate B; Hubacek, Jaroslav A; Kubinova, Ruzena; Baceviciene, Migle; Tamosiunas, Abdonas; Pajak, Andrzej; Topor-Madry, Romanvan; Stepaniak, Urszula; Malyutina, Sofia; Baldassarre, Damiano; Sennblad, Bengt; Tremoli, Elena; de Faire, Ulf; Veglia, Fabrizio; Ford, Ian; Jukema, J Wouter; Westendorp, Rudi G J; de Borst, Gert Jan; de Jong, Pim A; Algra, Ale; Spiering, Wilko; der Zee, Anke H Maitland-van|info:eu-repo/dai/nl/255164688; Klungel, Olaf H|info:eu-repo/dai/nl/181447649; de Boer, Anthonius|info:eu-repo/dai/nl/075097346; Doevendans, Pieter A; Eaton, Charles B; Robinson, Jennifer G; Duggan, David; Kjekshus, John; Downs, John R; Gotto, Antonio M; Keech, Anthony C; Marchioli, Roberto; Tognoni, Gianni; Sever, Peter S; Poulter, Neil R; Waters, David D; Pedersen, Terje R; Amarenco, Pierre; Nakamura, Haruo; McMurray, John J V; Lewsey, James D; Chasman, Daniel I; Ridker, Paul M; Maggioni, Aldo P; Tavazzi, Luigi; Ray, Kausik K; Seshasai, Sreenivasa Rao Kondapally; Manson, JoAnn E; Price, Jackie F; Whincup, Peter H; Morris, Richard W; Lawlor, Debbie A; Smith, George Davey; Ben-Shlomo, Yoav; Schreiner, Pamela J; Fornage, Myriam; Siscovick, David S; Cushman, Mary; Kumari, Meena; Wareham, Nick J; Verschuren, W M Monique; Redline, Susan; Patel, Sanjay R; Whittaker, John C; Hamsten, Anders; Delaney, Joseph A; Dale, Caroline; Gaunt, Tom R; Wong, Andrew; Kuh, Diana; Hardy, Rebecca; Kathiresan, Sekar; Castillo, Berta A; van der Harst, Pim; Brunner, Eric J; Tybjaerg-Hansen, Anne; Marmot, Michael G; Krauss, Ronald M; Tsai, Michael; Coresh, Josef; Hoogeveen, Ronald C; Psaty, Bruce M; Lange, Leslie A; Hakonarson, Hakon; Dudbridge, Frank; Humphries, Steve E; Talmud, Philippa J; Kivimäki, Mika; Timpson, Nicholas J; Langenberg, Claudia; Asselbergs, Folkert W; Voevoda, Mikhail; Bobak, Martin; Pikhart, Hynek; Wilson, James G; Reiner, Alex P; Keating, Brendan J; Hingorani, Aroon D; Sattar, Naveed; DIAGRAM Consortium, MAGIC Consortium, InterAct Consortium

    2014-01-01

    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

  8. HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight : Evidence from genetic analysis and randomised trials

    NARCIS (Netherlands)

    Swerdlow, Daniel I.; Preiss, David; Kuchenbaecker, Karoline B.; Holmes, Michael V.; Engmann, Jorgen E L; Shah, Tina; Sofat, Reecha; Stender, Stefan; Johnson, Paul C D; Scott, Robert A.; Leusink, Maarten; Verweij, Niek; Sharp, Stephen J.; Guo, Yiran; Giambartolomei, Claudia; Chung, Christina; Peasey, Anne; Amuzu, Antoinette; Li, Kawah; Palmen, Jutta; Howard, Philip; Cooper, Jackie A.; Drenos, Fotios; Li, Yun R.; Lowe, Gordon; Gallacher, John; Stewart, Marlene C W; Tzoulaki, Ioanna; Buxbaum, Sarah G.; Van Der A, Daphne L.; Forouhi, Nita G.; Onland-Moret, N. Charlotte|info:eu-repo/dai/nl/26504362X; Van Der Schouw, Yvonne T.|info:eu-repo/dai/nl/073449253; Schnabel, Renate B.; Hubacek, Jaroslav A.; Kubinova, Ruzena; Baceviciene, Migle; Tamosiunas, Abdonas; Pajak, Andrzej; Topor-Madry, Romanvan; Stepaniak, Urszula; Malyutina, Sofia; Baldassarre, Damiano; Sennblad, Bengt; Tremoli, Elena; De Faire, Ulf; Veglia, Fabrizio; Ford, Ian; Jukema, J. Wouter; Westendorp, Rudi G J; De Borst, Gert Jan|info:eu-repo/dai/nl/237108151; De Jong, Pim A.|info:eu-repo/dai/nl/287955672; Algra, Ale|info:eu-repo/dai/nl/07483472X; Spiering, Wilko|info:eu-repo/dai/nl/269114173; Der Zee, Anke H Maitland Van; Klungel, Olaf H.; De Boer, Anthonius; Doevendans, Pieter A.|info:eu-repo/dai/nl/164248366; Eaton, Charles B.; Robinson, Jennifer G.; Duggan, David; Kjekshus, John; Downs, John R.; Gotto, Antonio M.; Keech, Anthony C.; Marchioli, Roberto; Tognoni, Gianni; Sever, Peter S.; Poulter, Neil R.; Waters, David D.; Pedersen, Terje R.; Amarenco, Pierre; Nakamura, Haruo; McMurray, John J V; Lewsey, James D.; Chasman, Daniel I.; Ridker, Paul M.; Maggioni, Aldo P.; Tavazzi, Luigi; Ray, Kausik K.; Seshasai, Sreenivasa Rao Kondapally; Manson, Joann E.; Price, Jackie F.; Whincup, Peter H.; Morris, Richard W.; Lawlor, Debbie A.; Smith, George Davey; Ben-Shlomo, Yoav; Schreiner, Pamela J.; Fornage, Myriam; Siscovick, David S.; Cushman, Mary; Kumari, Meena; Wareham, Nick J.; Verschuren, W. M Monique; Redline, Susan; Patel, Sanjay R.; Whittaker, John C.; Hamsten, Anders; Delaney, Joseph A.; Dale, Caroline; Gaunt, Tom R.; Wong, Andrew; Kuh, Diana; Hardy, Rebecca; Kathiresan, Sekar; Castillo, Berta A.; Van Der Harst, Pim; Brunner, Eric J.; Tybjaerg-Hansen, Anne; Marmot, Michael G.; Krauss, Ronald M.; Tsai, Michael; Coresh, Josef; Hoogeveen, Ronald C.; Psaty, Bruce M.; Lange, Leslie A.; Hakonarson, Hakon; Dudbridge, Frank; Humphries, Steve E.; Talmud, Philippa J.; Kivimäki, Mika; Timpson, Nicholas J.; Langenberg, Claudia; Asselbergs, Folkert W.|info:eu-repo/dai/nl/270752137; Voevoda, Mikhail; Bobak, Martin; Pikhart, Hynek; Wilson, James G.; Reiner, Alex P.; Keating, Brendan J.; Hingorani, Aroon D.; Sattar, Naveed

    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

  9. Red wine triggers cell death and thioredoxin reductase inhibition: effects beyond resveratrol and SIRT1.

    Science.gov (United States)

    Wallenborg, Karolina; Vlachos, Pinelopi; Eriksson, Sofi; Huijbregts, Lukas; Arnér, Elias S J; Joseph, Bertrand; Hermanson, Ola

    2009-05-01

    Red wine contains antioxidants and is at moderate amounts believed to exert certain positive health effects. Resveratrol is one of the most studied antioxidants in red wine and has been suggested to activate the longevity- and metabolism-associated histone deacetylase SIRT1. Here we show that relatively low concentrations of resveratrol (0.5-3 microM) specifically inhibited neuronal differentiation of neural stem cells in a SIRT1-dependent manner whereas higher concentrations of resveratrol (> or =10 microM) induced a SIRT1-independent cell death. Surprisingly, using a cell based assay, we found that small amounts of red wine (1-5% v/v)--but not white wine--induced a massive and rapid cell death of various cell types, including neural stem cells and several cancer cell lines. This red wine-induced cell death was ethanol-, SIRT1- and resveratrol-independent but associated with increased oxidative stress and inhibition of thioredoxin reductase (TrxR) activity. The TrxR inhibition correlated with the red color (absorbance at 520 nm) of the wines demonstrating that pigment components of red wine can exert profound cellular effects. Our results unveil important roles for SIRT1 and TrxR in resveratrol and red wine-mediated effects on progenitor and cancer cells, and demonstrate that cellular responses to red wine may be more complex than generally appreciated.

  10. 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.

    2013-01-01

    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

  11. Inhibition of aldose reductase prevents experimental allergic airway inflammation in mice.

    Directory of Open Access Journals (Sweden)

    Umesh C S Yadav

    Full Text Available BACKGROUND: The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR, contributes to the pathogenesis of oxidative stress-induced inflammation by affecting the NF-kappaB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be anti-inflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, ragweed pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma. METHODS AND FINDINGS: Primary Human Small Airway Epithelial Cells (SAEC were used to investigate the in vitro effects of AR inhibition on ragweed pollen extract (RWE-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE -induced apoptotic cell death as measured by annexin-v staining, increase in the activation of NF-kappaB and expression of inflammatory markers such as inducible nitric oxide synthase (iNOS, cycloxygenase (COX-2, Prostaglandin (PG E(2, IL-6 and IL-8. Further, BALB/c mice were sensitized with endotoxin-free RWE in the absence and presence of AR inhibitor and followed by evaluation of perivascular and peribronchial inflammation, mucin production, eosinophils infiltration and

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

    Science.gov (United States)

    Guan, Zhen; Wang, Xiuwei; Dong, Yanting; Xu, Lin; Zhu, Zhiqiang; Wang, Jianhua; Zhang, Ting; Niu, Bo

    2015-02-03

    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.

  13. Inhibition of aldose reductase ameliorates ethanol‑induced steatosis in HepG2 cells.

    Science.gov (United States)

    Qiu, Longxin; Cai, Chengchao; Zhao, Xiangqian; Fang, Yan; Tang, Weibiao; Guo, Chang

    2017-05-01

    Aldose reductase (AR) expression is increased in liver tissue of patients with ethanol‑induced liver disease. However, the exact role of AR in the development of ethanol‑induced liver disease has yet to be elucidated. The present study aimed to determine the effect of an AR inhibitor on ethanol‑induced steatosis in HepG2 cells and to identify possible underlying molecular mechanisms. Steatosis was induced in HepG2 cells by stimulating cells with 100 mM absolute ethanol for 48 h. Oil Red O staining was used to detect the lipid droplet accumulation in cells. Western blot analyses were used to determine protein expression levels and reverse transcription‑quantitative polymerase chain reaction was used to analyze mRNA expression levels. The results showed that AR protein expression was elevated in HepG2 cells stimulated with ethanol. HepG2 cells exhibited marked improvement of ethanol‑induced lipid accumulation following treatment with the AR inhibitor zopolrestat. Phosphorylation levels of 5' adenosine monophosphate‑activated protein kinase (AMPK) were markedly higher, whereas the mRNA expression levels of sterol‑regulatory element‑binding protein (SREBP)‑1c and fatty acid synthase (FAS) were significantly lower in zopolrestat‑treated and ethanol‑stimulated HepG2 cells compared with in untreated ethanol‑stimulated HepG2 cells. In addition, zopolrestat inhibited the ethanol‑induced expression of tumor necrosis factor (TNF)‑α. These results suggested that zopolrestat attenuated ethanol‑induced steatosis by activating AMPK and subsequently inhibiting the expression of SREBP‑1c and FAS, and by suppressing the expression of TNF‑α in HepG2 cells.

  14. Inhibition of fumarate reductase in Leishmania major and L. donovani by chalcones

    DEFF Research Database (Denmark)

    Chen, M; Zhai, L; Christensen, S B

    2001-01-01

    of mitochondrial dehydrogenases of Leishmania parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized Leishmania major....... Since FRD exists in the Leishmania parasite and does not exist in mammalian cells, it could be an excellent target for antiprotozoal drugs....

  15. Substrate pathways and mechanisms of inhibition in the sulfur oxygenase reductase of Acidianus ambivalens

    Directory of Open Access Journals (Sweden)

    Andreas eVeith

    2011-03-01

    Full Text Available Background: The sulfur oxygenase reductase (SOR is the initial enzyme of the sulfur oxidation pathway in the thermoacidophilic Archaeon Acidianus ambivalens. The SOR catalyzes an oxygen-dependent sulfur disproportionation to H2S, sulfite and thiosulfate. The spherical, hollow, cytoplasmic enzyme is composed of 24 identical subunits with an active site pocket each comprising a mononuclear non-heme iron site and a cysteine persulfide. Substrate access and product exit occur via apolar chimney-like protrusions at the four-fold symmetry axes, via narrow polar pores at the three-fold symmetry axes and via narrow apolar pores within in each subunit. In order to investigate the function of the pores we performed site-directed mutagenesis and inhibitor studies. Results: Truncation of the chimney-like protrusions resulted in an up to seven-fold increase in specific enzyme activity compared to the wild type. Replacement of the salt bridge-forming Arg99 residue by Ala at the three-fold symmetry axes doubled the activity and introduced a bias towards reduced reaction products. Replacement of Met296 and Met297, which form the active site pore, lowered the specific activities by 25-55 % with the exception of an M296V mutant. X-ray crystallography of SOR wild type crystals soaked with inhibitors showed that Hg2+ and iodoacetamide bind to cysteines within the active site, whereas Zn2+ binds to a histidine in a side channel of the enzyme. The Zn2+ inhibition was partially alleviated by mutation of the His residue. Conclusions: The expansion of the pores in the outer shell led to an increased enzyme activity while the integrity of the active site pore seems to be important. Hg2+ and iodoacetamide block cysteines in the active site pocket, while Zn2+ interferes over a distance, possibly by restriction of protein flexibility or substrate access or product exit.

  16. Inhibition of Human Steroid 5-Reductase (AKR1D1) by Finasteride and Structure of the Enzyme-Inhibitor Complex

    Energy Technology Data Exchange (ETDEWEB)

    Drury, J.; Di Costanzo, L; Penning, T; Christianson, D

    2009-01-01

    The {Delta}{sup 4}-3-ketosteroid functionality is present in nearly all steroid hormones apart from estrogens. The first step in functionalization of the A-ring is mediated in humans by steroid 5{alpha}- or 5{beta}-reductase. Finasteride is a mechanism-based inactivator of 5{alpha}-reductase type 2 with subnanomolar affinity and is widely used as a therapeutic for the treatment of benign prostatic hyperplasia. It is also used for androgen deprivation in hormone-dependent prostate carcinoma, and it has been examined as a chemopreventive agent in prostate cancer. The effect of finasteride on steroid 5{beta}-reductase (AKR1D1) has not been previously reported. We show that finasteride competitively inhibits AKR1D1 with low micromolar affinity but does not act as a mechanism-based inactivator. The structure of the AKR1D1 {center_dot} NADP{sup +} {center_dot} finasteride complex determined at 1.7 {angstrom} resolution shows that it is not possible for NADPH to reduce the {Delta}{sup 1-2}-ene of finasteride because the cofactor and steroid are not proximal to each other. The C3-ketone of finasteride accepts hydrogen bonds from the catalytic residues Tyr-58 and Glu-120 in the active site of AKR1D1, providing an explanation for the competitive inhibition observed. This is the first reported structure of finasteride bound to an enzyme involved in steroid hormone metabolism.

  17. Deoxynucleoside salvage in fission yeast allows rescue of ribonucleotide reductase deficiency but not Spd1-mediated inhibition of replication

    DEFF Research Database (Denmark)

    Fleck, Oliver; Fahnøe, Ulrik; Løvschal, Katrine Vyff

    2017-01-01

    In fission yeast, the small, intrinsically disordered protein S-phase delaying protein 1 (Spd1) blocks DNA replication and causes checkpoint activation at least in part, by inhibiting the enzyme ribonucleotide reductase, which is responsible for the synthesis of DNA. The CRL4(Cdt2) E3 ubiquitin...... triphosphate (dNTP) pools; yet we find that Spd1 and Spd2 together inhibit DNA replication upon Cdt2 depletion. To directly test whether this block of replication was solely due to insufficient dNTP levels, we established a deoxy-nucleotide salvage pathway in fission yeast by expressing the human nucleoside...... that this inhibition works at the point of the replication clamp proliferating cell nuclear antigen, a co-factor for DNA replication....

  18. 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;

    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...

  19. RNAi silencing of the HaHMG-CoA reductase gene inhibits oviposition in the Helicoverpa armigera cotton bollworm.

    Science.gov (United States)

    Wang, Zhijian; Dong, Yongcheng; Desneux, Nicolas; Niu, Changying

    2013-01-01

    RNA interference (RNAi) has considerable promise for developing novel pest control techniques, especially because of the threat of the development of resistance against current strategies. For this purpose, the key is to select pest control genes with the greatest potential for developing effective pest control treatments. The present study demonstrated that the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase; HMGR) gene is a potential target for insect control using RNAi. HMGR is a key enzyme in the mevalonate pathway in insects. A complete cDNA encoding full length HMGR (encoding an 837-aa protein) was cloned from Helicoverpa armigera (Lepidoptera: Noctuidae). The HaHMGR (H. armigera HMGR) knockdown using systemic RNAi in vivo inhibited the fecundity of the females, effectively inhibited ovipostion, and significantly reduced vitellogenin (Vg) mRNA levels. Moreover, the oviposition rate of the female moths was reduced by 98% by silencing HaHMGR compared to the control groups. One-pair experiments showed that both the proportions of valid mating and fecundity were zero. Furthermore, the HaHMGR-silenced females failed to lay eggs (approximate 99% decrease in oviposition) in the semi-field cage performance. The present study demonstrated the potential implications for developing novel pest management strategies using HaHMGR RNAi in the control of H. armigera and other insect pests.

  20. Enzyme Mechanism and Slow-Onset Inhibition of Plasmodium falciparum Enoyl-Acyl Carrier Protein Reductase by an Inorganic Complex

    Science.gov (United States)

    de Medeiros, Patrícia Soares de Maria; Ducati, Rodrigo Gay; Basso, Luiz Augusto; Santos, Diógenes Santiago; da Silva, Luiz Hildebrando Pereira

    2011-01-01

    Malaria continues to be a major cause of children's morbidity and mortality worldwide, causing nearly one million deaths annually. The human malaria parasite, Plasmodium falciparum, synthesizes fatty acids employing the Type II fatty acid biosynthesis system (FAS II), unlike humans that rely on the Type I (FAS I) pathway. The FAS II system elongates acyl fatty acid precursors of the cell membrane in Plasmodium. Enoyl reductase (ENR) enzyme is a member of the FAS II system. Here we present steady-state kinetics, pre-steady-state kinetics, and equilibrium fluorescence spectroscopy data that allowed proposal of P. falciparum ENR (PfENR) enzyme mechanism. Moreover, building on previous results, the present study also evaluates the PfENR inhibition by the pentacyano(isoniazid)ferrateII compound. This inorganic complex represents a new class of lead compounds for the development of antimalarial agents focused on the inhibition of PfENR. PMID:21603269

  1. Deoxynucleoside Salvage in Fission Yeast Allows Rescue of Ribonucleotide Reductase Deficiency but Not Spd1-Mediated Inhibition of Replication

    Directory of Open Access Journals (Sweden)

    Oliver Fleck

    2017-04-01

    Full Text Available In fission yeast, the small, intrinsically disordered protein S-phase delaying protein 1 (Spd1 blocks DNA replication and causes checkpoint activation at least in part, by inhibiting the enzyme ribonucleotide reductase, which is responsible for the synthesis of DNA. The CRL4Cdt2 E3 ubiquitin ligase mediates degradation of Spd1 and the related protein Spd2 at S phase of the cell cycle. We have generated a conditional allele of CRL4Cdt2, by expressing the highly unstable substrate-recruiting protein Cdt2 from a repressible promoter. Unlike Spd1, Spd2 does not regulate deoxynucleotide triphosphate (dNTP pools; yet we find that Spd1 and Spd2 together inhibit DNA replication upon Cdt2 depletion. To directly test whether this block of replication was solely due to insufficient dNTP levels, we established a deoxy-nucleotide salvage pathway in fission yeast by expressing the human nucleoside transporter human equilibrative nucleoside transporter 1 (hENT1 and the Drosophila deoxynucleoside kinase. We present evidence that this salvage pathway is functional, as 2 µM of deoxynucleosides in the culture medium is able to rescue the growth of two different temperature-sensitive alleles controlling ribonucleotide reductase. However, salvage completely failed to rescue S phase delay, checkpoint activation, and damage sensitivity, which was caused by CRL4Cdt2 inactivation, suggesting that Spd1—in addition to repressing dNTP synthesis—together with Spd2, can inhibit other replication functions. We propose that this inhibition works at the point of the replication clamp proliferating cell nuclear antigen, a co-factor for DNA replication.

  2. Nitrite reductase activity of sulphate-reducing bacteria prevents their inhibition by nitrate-reducing, sulphide-oxidizing bacteria.

    Science.gov (United States)

    Greene, E A; Hubert, C; Nemati, M; Jenneman, G E; Voordouw, G

    2003-07-01

    Sulphate-reducing bacteria (SRB) can be inhibited by nitrate-reducing, sulphide-oxidizing bacteria (NR-SOB), despite the fact that these two groups are interdependent in many anaerobic environments. Practical applications of this inhibition include the reduction of sulphide concentrations in oil fields by nitrate injection. The NR-SOB Thiomicrospira sp. strain CVO was found to oxidize up to 15 mM sulphide, considerably more than three other NR-SOB strains that were tested. Sulphide oxidation increased the environmental redox potential (Eh) from -400 to +100 mV and gave 0.6 nitrite per nitrate reduced. Within the genus Desulfovibrio, strains Lac3 and Lac6 were inhibited by strain CVO and nitrate for the duration of the experiment, whereas inhibition of strains Lac15 and D. vulgaris Hildenborough was transient. The latter had very high nitrite reductase (Nrf) activity. Southern blotting with D. vulgaris nrf genes as a probe indicated the absence of homologous nrf genes from strains Lac3 and Lac6 and their presence in strain Lac15. With respect to SRB from other genera, inhibition of the known nitrite reducer Desulfobulbus propionicus by strain CVO and nitrate was transient, whereas inhibition of Desulfobacterium autotrophicum and Desulfobacter postgatei was long-lasting. The results indicate that inhibition of SRB by NR-SOB is caused by nitrite production. Nrf-containing SRB can overcome this inhibition by further reducing nitrite to ammonia, preventing a stalling of the favourable metabolic interactions between these two bacterial groups. Nrf, which is widely distributed in SRB, can thus be regarded as a resistance factor that prevents the inhibition of dissimilatory sulphate reduction by nitrite.

  3. Quinazolinone-benzimidazole conjugates: Synthesis, characterization, dihydrofolate reductase inhibition, DNA and protein binding properties.

    Science.gov (United States)

    Singla, Prinka; Luxami, Vijay; Paul, Kamaldeep

    2017-03-01

    Quinazolinone and benzimidazole represent as important and abundant classes of fused nitrogen-containing heterocycle. A series of two isomeric quinazolinone-benzimidazole conjugates is synthesized and substitutes with different aromatic rings. These compounds are well characterized by (1)H and (13)C NMR as well as mass spectrometry. Compounds are then evaluated by dihydrofolate reductase inhibitory activity. In vitro assay shows that some compounds are exhibiting significant dihydrofolate reductase inhibitory activities. Compound 14 shows comparable or even superior inhibitory activity with IC50 value of 0.011μM in contrast to methotrexate (IC50=0.02μM). The preliminary interactive investigation of compound 14 is studied with calf thymus DNA by UV-visible and fluorescence spectroscopy. It reveals that compound 14 is effectively intercalated with ct-DNA to form 14.DNA complex which is further supported by ethidium bromide (EB) displacement studies. The compound 14 also shows strong interaction with bovine serum albumin that can helpful in the design, modification and screening of drug molecules. The binding interactions of compound 14 with bovine serum albumin demonstrate that hydrogen bonds and van der Waals forces play important roles in the strong association of compound 14.BSA. These compounds can be considered as useful templates for future development and further derivatization or modification will be helpful to obtain more potent compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Mechanism of inhibition of purified leaping mullet (Liza saliens) NADPH-cytochrome P450 reductase by toxic metals: aluminum and thallium.

    Science.gov (United States)

    Bozcaarmutlu, Azra

    2007-01-01

    Aluminum and thallium may reach life-threatening levels in aquatic systems in the near future because of their extensive use in various industrial fields. It is therefore important to study the mechanism of toxicity of aluminum and thallium on fish enzymes. To this aim, the effects of aluminum and thallium on the activity of purified leaping mullet (Liza saliens) cytochrome P450 reductase, an essential component of the important cytochrome P450 system, have been studied. Results indicated that both metal ions strongly inhibited the NADPH-cytochrome P450 reductase. The IC50 values of AlCl3 and TlCl3 were estimated to be 34 microM and 3 microM, respectively. The Lineweaver-Burk plot and Dixon plot revealed that both metal ions noncompetitively inhibited the purified mullet cytochrome P450 reductase. The K(i) values of Al3+ and Tl3+ were calculated from Dixon plots as 8.9 and 5.6 microM, respectively. The inhibitory effects of Al3+ and Tl3+ on purified cytochrome P450 reductase were partially recovered by 1 mM EDTA. Additionally, tin and magnesium were shown to have no apparent effect on purified mullet cytochrome P450 reductase.

  5. Effects of selenite and chelating agents on mammalian thioredoxin reductase inhibited by mercury: implications for treatment of mercury poisoning.

    Science.gov (United States)

    Carvalho, Cristina M L; Lu, Jun; Zhang, Xu; Arnér, Elias S J; Holmgren, Arne

    2011-01-01

    Mercury toxicity is a highly interesting topic in biomedicine due to the severe endpoints and treatment limitations. Selenite serves as an antagonist of mercury toxicity, but the molecular mechanism of detoxification is not clear. Inhibition of the selenoenzyme thioredoxin reductase (TrxR) is a suggested mechanism of toxicity. Here, we demonstrated enhanced inhibition of activity by inorganic and organic mercury compounds in NADPH-reduced TrxR, consistent with binding of mercury also to the active site selenolthiol. On treatment with 5 μM selenite and NADPH, TrxR inactivated by HgCl(2) displayed almost full recovery of activity. Structural analysis indicated that mercury was complexed with TrxR, but enzyme-generated selenide removed mercury as mercury selenide, regenerating the active site selenocysteine and cysteine residues required for activity. The antagonistic effects on TrxR inhibition were extended to endogenous antioxidants, such as GSH, and clinically used exogenous chelating agents BAL, DMPS, DMSA, and α-lipoic acid. Consistent with the in vitro results, recovery of TrxR activity and cell viability by selenite was observed in HgCl(2)-treated HEK 293 cells. These results stress the role of TrxR as a target of mercurials and provide the mechanism of selenite as a detoxification agent for mercury poisoning.

  6. Inhibition of glutathione synthesis eliminates the adaptive response of ascitic hepatoma 22 cells to nedaplatin that targets thioredoxin reductase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yijun [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China); Lu, Hongjuan [Productivity Center of Jiangsu Province, Nanjing 210042, Jiangsu (China); Wang, Dongxu; Li, Shengrong; Sun, Kang; Wan, Xiaochun [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China); Taylor, Ethan Will [Department of Nanoscience, Joint School of Nanoscience and Nanoengineering, University of North Carolina at Greensboro, Greensboro, NC 27402 (United States); Zhang, Jinsong, E-mail: zjs@ahau.edu.cn [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China)

    2012-12-15

    Thioredoxin reductase (TrxR) is a target for cancer therapy and the anticancer mechanism of cisplatin involves TrxR inhibition. We hypothesize that the anticancer drug nedaplatin (NDP), an analogue of cisplatin and a second-generation platinum complex, also targets TrxR. Furthermore, we investigate whether the therapeutic efficacy of NDP can be enhanced by simultaneous modulation of 1) TrxR, via NDP, and 2) glutathione (GSH), via the GSH synthesis inhibitor buthionine sulfoximine (BSO). Mice bearing ascitic hepatoma 22 (H22) cells were treated with NDP alone or NDP plus BSO. TrxR activity of H22 cells was inhibited by NDP in a dose-dependent manner. A high correlation between the inhibition of TrxR activity at 6 h and the inhibition of ascitic fluid volume at 72 h was established (r = 0.978, p < 0.01). As an adaptive response, the viable ascitic cancer cells after NDP treatment displayed an enlarged cell phenotype, assembled with several-fold more antioxidant enzymes and GSH-predominant non-protein free thiols. This adaptive response was largely eliminated when BSO was co-administered with NDP, leading to the decimation of the H22 cell population without enhancing renal toxicity, since at this dose, NDP did not inhibit renal TrxR activity. In conclusion, the pharmacological effect of NDP involves TrxR inhibition, and the adaptive response of NDP-treated ascitic H22 cells can be efficiently counteracted by BSO. Simultaneous modulation of TrxR and GSH on ascitic H22 cells using NDP plus BSO greatly enhances therapeutic efficacy as compared with the single modulation of TrxR using NDP alone. -- Highlights: ► Nedaplatin at a pharmacological dose inhibits TrxR in cancer cells but not in kidney. ► The nedaplatin-treated cancer cells exhibit adaptive response. ► Buthionine sulfoximine inhibits glutathione in both cancer cells and kidney. ► Buthionine sulfoximine counteracts the adaptive response to the nedaplatin treatment. ► Buthionine sulfoximine does not

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

    Science.gov (United States)

    Aye, Yimon; Stubbe, Joanne

    2011-06-14

    Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of α- and β-subunits that form active α(n)β(m) (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 thought to be associated in part with the triphosphate functioning as an allosteric inhibitor of hRNR. Studies on the mechanism of inhibition of hRNR by ClF di- and triphosphates (ClFDP and ClFTP) are presented. ClFTP is a reversible inhibitor (K(i) = 40 nM) that rapidly inactivates hRNR. However, with time, 50% of the activity is recovered. D57N-α, a mutant with an altered A site, prevents inhibition by ClFTP, suggesting its A site binding. ClFDP is a slow-binding, reversible inhibitor ( K(i)*; t(1/2) = 23 min). CDP protects α from its inhibition. The altered off-rate of ClFDP from E•ClFDP* by ClFTP (A site) or dGTP (S site) and its inhibition of D57N-α together implicate its C site binding. Size exclusion chromatography of hRNR or α alone with ClFDP or ClFTP, ± ATP or dGTP, reveals in each case that α forms a kinetically stable hexameric state. This is the first example of hexamerization of α induced by an NDP analog that reversibly binds at the active site.

  8. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella.

    Science.gov (United States)

    Barbier, Thibault; Collard, François; Zúñiga-Ripa, Amaia; Moriyón, Ignacio; Godard, Thibault; Becker, Judith; Wittmann, Christoph; Van Schaftingen, Emile; Letesson, Jean-Jacques

    2014-12-16

    Erythritol is an important nutrient for several α-2 Proteobacteria, including N2-fixing plant endosymbionts and Brucella, a worldwide pathogen that finds this four-carbon polyol in genital tissues. Erythritol metabolism involves phosphorylation to L-erythritol-4-phosphate by the kinase EryA and oxidation of the latter to L-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted that further steps involve oxidation by the putative dehydrogenase EryC and subsequent decarboxylation to yield triose-phosphates. Accordingly, growth on erythritol as the sole C source should require aldolase and fructose-1,6-bisphosphatase to produce essential hexose-6-monophosphate. However, we observed that a mutant devoid of fructose-1,6-bisphosphatases grew normally on erythritol and that EryC, which was assumed to be a dehydrogenase, actually belongs to the xylose isomerase superfamily. Moreover, we found that TpiA2 and RpiB, distant homologs of triose phosphate isomerase and ribose 5-phosphate isomerase B, were necessary, as previously shown for Rhizobium. By using purified recombinant enzymes, we demonstrated that L-3-tetrulose-4-phosphate was converted to D-erythrose 4-phosphate through three previously unknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (D-3-tetrulose-4-phosphate isomerase; renamed EryH), and RpiB (D-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribution of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on (13)C-labeled erythritol. D-erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the first description to our knowledge of a route feeding carbohydrate metabolism exclusively via D-erythrose 4-phosphate, a pathway that may provide clues to the preferential metabolism of

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

    Directory of Open Access Journals (Sweden)

    Junichi Koseki

    2015-01-01

    Full Text Available Objective. Bokusoku (BK is an extract from the Quercus cortex used in folk medicine for treatment of skin disorders and convergence, and is present in jumihaidokuto, a traditional Japanese medicine that is prescribed for purulent skin diseases like acne vulgaris. The excess of sebum production induced by androgen is involved in the development of acne. Our aim is to examine whether BK and its constituents inhibit testosterone metabolism and testosterone-induced sebum synthesis. Methods. Measurements of 5α-reductase activity and lipogenesis were performed using rat liver microsomes and hamster sebocytes, respectively. Results. BK dose-dependently reduced the conversion of testosterone to a more active androgen, dihydrotestosterone in a 5α-reductase enzymatic reaction. Twenty polyphenols in BK categorized as gallotannin, ellagitannin, and flavonoid were identified by LC-MS/MS. Nine polyphenols with gallate group, tetragalloyl glucose, pentagalloyl glucose, eugeniin, 1-desgalloyl eugeniin, casuarinin, castalagin, stenophyllanin C, (−-epicatechin gallate, and (−-epigallocatechin gallate, inhibited testosterone metabolism. In particular, pentagalloyl glucose showed the strongest activity. BK and pentagalloyl glucose suppressed testosterone-induced lipogenesis, whereas they weakly inhibited the lipogenic action of insulin. Conclusions. BK inhibited androgen-related pathogenesis of acne, testosterone conversion, and sebum synthesis, partially through 5α-reductase inhibition, and has potential to be a useful agent in the therapeutic strategy of acne.

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

    Directory of Open Access Journals (Sweden)

    Izabella Kovacs

    2016-11-01

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

  11. Aldose reductase inhibition suppresses azoxymethane-induced colonic premalignant lesions in C57BL/KsJ-db/db mice.

    Science.gov (United States)

    Saxena, Ashish; Shoeb, Mohammad; Tammali, Ravinder; Ramana, Kota V; Srivastava, Satish K

    2014-12-01

    Type-2 diabetes and obesity-related metabolic abnormalities are major risk factors for the development of colon cancer. In the present study, we examined the effects of polyol pathway enzyme aldose reductase (AR) inhibitor, fidarestat, on the development of azoxymethane (AOM)-induced colonic premalignant lesions in C57BL/KsJ-db/db obese mice. Our results indicate that fidarestat given in the drinking water caused a significant reduction in the total number of colonic premalignant lesions in the AOM treated obese mice. Further, the expression levels of PKC-β2, AKT, COX-2 and iNOS in the colonic mucosa of AOM-treated mice were significantly decreased by fidarestat. The serum levels of IL-1α, IP-10, MIG, TNF-α and VEGF are significantly suppressed in AOM + fidarestat treated obese mice. Fidarestat also decreased the expression of COX-2, iNOS, XIAP, survivin, β-catenin and NF-κB in high glucose-treated HT29 colon cancer cells. In conclusion, our results indicate that fidarestat inhibits the development of colonic premalignant lesions in an obesity-related colon cancer and is chemopreventive to colorectal carcinogenesis in obese individuals. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  12. Inhibition of HMG CoA reductase reveals an unexpected role for cholesterol during PGC migration in the mouse

    Directory of Open Access Journals (Sweden)

    Ewing Andrew G

    2008-12-01

    Full Text Available Abstract Background Primordial germ cells (PGCs are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. Results We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. Conclusion In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.

  13. Scopoletin from the flower buds of Magnolia fargesii inhibits protein glycation, aldose reductase, and cataractogenesis ex vivo.

    Science.gov (United States)

    Lee, Jun; Kim, Nan Hee; Nam, Joo Won; Lee, Yun Mi; Jang, Dae Sik; Kim, Young Sook; Nam, Sang Hae; Seo, Eun-Kyoung; Yang, Min Suk; Kim, Jin Sook

    2010-09-01

    Five compounds previously known structures, scopoletin (1), northalifoline (2), stigmast-4-en-3-one (3), tiliroside (4), and oplopanone (5) were obtained from the flower buds of Magnolia fargesii using chromatographic separation methods. The structures of 1-5 were identified by the interpretation of their spectroscopic data including 1D- and 2D-NMR as well as by comparison with reported values. Three compounds 1-3 were found from M. fargesii for the first time in this study. All the isolates (1-5) were subjected to in vitro bioassays to evaluate the inhibitory activity on advanced glycation end products formation and rat lens aldose reductase (RLAR). Compound 1 showed a remarkable inhibitory activity on advanced glycation end products formation with IC(50) value of 2.93 μM (aminoguanidine: 961 μM), and showed a significant RLAR inhibitory activity with IC(50) value of 22.5 μM (3.3-tetramethyleneglutaric acid: 28.7 μM). Compound 4 exhibited potent inhibitory activity against RLAR (IC(50) = 14.9 μM). In the further experiment ex vivo, cataractogenesis of rat lenses induced with xylose was significantly inhibited by compound 1 treatment.

  14. Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella tularensis: Residence Time and in Vivo Activity

    Energy Technology Data Exchange (ETDEWEB)

    Lu, H.; England, K; Ende, C; Truglio, J; Luckner, S; Reddy, B; Marlenee, N; Knudson, S; Knudson, D; et. al.

    2009-01-01

    Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 ?g mL-1. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.

  15. The selective antifungal activity of Drosophila melanogaster metchnikowin reflects the species-dependent inhibition of succinate-coenzyme Q reductase.

    Science.gov (United States)

    Moghaddam, Mohammad-Reza Bolouri; Gross, Thomas; Becker, Annette; Vilcinskas, Andreas; Rahnamaeian, Mohammad

    2017-08-15

    Insect-derived antifungal peptides have a significant economic potential, particularly for the engineering of pathogen-resistant crops. However, the nonspecific antifungal activity of such peptides could result in detrimental effects against beneficial fungi, whose interactions with plants promote growth or increase resistance against biotic and abiotic stress. The antifungal peptide metchnikowin (Mtk) from Drosophila melanogaster acts selectively against pathogenic Ascomycota, including Fusarium graminearum, without affecting Basidiomycota such as the beneficial symbiont Piriformospora indica. Here we investigated the mechanism responsible for the selective antifungal activity of Mtk by using the peptide to probe a yeast two-hybrid library of F. graminearum cDNAs. We found that Mtk specifically targets the iron-sulfur subunit (SdhB) of succinate-coenzyme Q reductase (SQR). A functional assay based on the succinate dehydrogenase (SDH) activity of mitochondrial complex II clearly demonstrated that Mtk inhibited the SDH activity of F. graminearum mitochondrial SQR by up to 52%, but that the equivalent enzyme in P. indica was unaffected. A phylogenetic analysis of the SdhB family revealed a significant divergence between the Ascomycota and Basidiomycota. SQR is one of the key targets of antifungal agents and we therefore propose Mtk as an environmentally sustainable and more selective alternative to chemical fungicides.

  16. Inhibition of the 3-hydroxy-3-methyl-glutaryl-CoA reductase induces orofacial defects in zebrafish.

    Science.gov (United States)

    Signore, Iskra A; Jerez, Carolina; Figueroa, Diego; Suazo, José; Marcelain, Katherine; Cerda, Oscar; Colombo Flores, Alicia

    2016-10-01

    Orofacial clefts (OFCs) are common birth defects, which include a range of disorders with a complex etiology affecting formation of craniofacial structures. Some forms of syndromic OFCs are produced by defects in the cholesterol pathway. The principal enzyme of the cholesterol pathway is the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Our aim is to study whether defects of HMGCR function would produce orofacial malformation similar to those found in disorders of cholesterol synthesis. We used zebrafish hmgcrb mutants and HMGCR inhibition assay using atorvastatin during early and late stages of orofacial morphogenesis in zebrafish. To describe craniofacial phenotypes, we stained cartilage and bone and performed in situ hybridization using known craniofacial markers. Also, we visualized neural crest cell migration in a transgenic fish. Our results showed that mutants displayed loss of cartilage and diminished orofacial outgrowth, and in some cases palatal cleft. Late treatments with statin show a similar phenotype. Affected-siblings displayed a moderate phenotype, whereas early-treated embryos had a minor cleft. We found reduced expression of the downstream component of Sonic Hedgehog-signaling gli1 in ventral brain, oral ectoderm, and pharyngeal endoderm in mutants and in late atorvastatin-treated embryos. Our results suggest that HMGCR loss-of-function primarily affects postmigratory cranial neural crest cells through abnormal Sonic Hedgehog signaling, probably induced by reduction in metabolites of the cholesterol pathway. Malformation severity correlates with the grade of HMGCR inhibition, developmental stage of its disruption, and probably with availability of maternal lipids. Together, our results might help to understand the spectrum of orofacial phenotypes found in cholesterol synthesis disorders. Birth Defects Research (Part A) 106:814-830, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. HMG-CoA Reductase Inhibition Promotes Neurological Recovery, Peri-Lesional Tissue Remodeling, and Contralesional Pyramidal Tract Plasticity after Focal Cerebral Ischemia

    Science.gov (United States)

    Kilic, Ertugrul; Reitmeir, Raluca; Kilic, Ülkan; Caglayan, Ahmet Burak; Beker, Mustafa Caglar; Kelestemur, Taha; Ethemoglu, Muhsine Sinem; Ozturk, Gurkan; Hermann, Dirk M.

    2014-01-01

    3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors are widely used for secondary stroke prevention. Besides their lipid-lowering activity, pleiotropic effects on neuronal survival, angiogenesis, and neurogenesis have been described. In view of these observations, we were interested whether HMG-CoA reductase inhibition in the post-acute stroke phase promotes neurological recovery, peri-lesional, and contralesional neuronal plasticity. We examined effects of the HMG-CoA reductase inhibitor rosuvastatin (0.2 or 2.0 mg/kg/day i.c.v.), administered starting 3 days after 30 min of middle cerebral artery occlusion for 30 days. Here, we show that rosuvastatin treatment significantly increased the grip strength and motor coordination of animals, promoted exploration behavior, and reduced anxiety. It was associated with structural remodeling of peri-lesional brain tissue, reflected by increased neuronal survival, enhanced capillary density, and reduced striatal and corpus callosum atrophy. Increased sprouting of contralesional pyramidal tract fibers crossing the midline in order to innervate the ipsilesional red nucleus was noticed in rosuvastatin compared with vehicle-treated mice, as shown by anterograde tract tracing experiments. Western blot analysis revealed that the abundance of HMG-CoA reductase was increased in the contralesional hemisphere at 14 and 28 days post-ischemia. Our data support the idea that HMG-CoA reductase inhibition promotes brain remodeling and plasticity far beyond the acute stroke phase, resulting in neurological recovery. PMID:25565957

  18. Aldose reductase inhibition counteracts nitrosative stress and poly(ADP-ribose) polymerase activation in diabetic rat kidney and high-glucose-exposed human mesangial cells

    OpenAIRE

    Drel, Viktor R.; Pacher, Pal; Stevens, Martin J; Obrosova, Irina G.

    2006-01-01

    Both increased aldose reductase (AR) activity and oxidative/nitrosative stress have been implicated in the pathogenesis of diabetic nephropathy, but the relation between the two factors remains a subject of debate. This study evaluated the effects of AR inhibition on nitrosative stress and poly(ADP-ribose) polymerase (PARP) activation in diabetic rat kidney and high-glucose-exposed human mesangial cells. In animal experiments, control (C) and streptozotocin-diabetic (D) rats were treated with...

  19. Inhibition of 5α-Reductase, IL-6 Secretion, and Oxidation Process of Equisetum debile Roxb. ex Vaucher Extract as Functional Food and Nutraceuticals Ingredients

    Directory of Open Access Journals (Sweden)

    Wantida Chaiyana

    2017-10-01

    Full Text Available This study aims to investigate the biological activities related to hair loss of Equisetum debile extracts, including 5α-reductase inhibition, interleukin-6 (IL-6 secretion reduction, and anti-oxidation. E. debile extracts were obtained by maceration in various solvents. Crude extract (CE was obtained by maceration in 95% ethanol. Chlorophyll-free extract (CF was the CE which of the chlorophyll has been removed by electrocoagulation. Hexane extract (HE, ethyl acetate extract (EA, and ethanolic extract (ET were fraction extracts obtained from maceration in hexane, ethyl acetate, and 95% ethanol, respectively. The extracts were investigated for inhibitory activity against 5α-reductase and IL-6 secretion. Total phenolic contents (TPC were investigated and antioxidant activities were determined by means of 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, 2,2′-diphenyl-1-picrylhydrazyl (DPPH, and ferric reducing antioxidant power (FRAP assays. The inhibition of lipid peroxidation was determined by the ferric thiocyanate method. The cytotoxicity of the extracts on dermal papilla cells and irritation test by hen's egg test chorioallantoic membrane assay were also investigated. All extracts could inhibit 5α-reductase and decrease IL-6 secretion in lipopolysaccharide-stimulated macrophage. The antioxidant activity of E. debile extracts was directly related to their TPC. ET which contained the highest TPC (68.8 ± 6.7 mg GA/g showed the highest equivalent concentration (EC1 of 289.1 ± 26.4 mM FeSO4/g, TEAC of 156.6 ± 34.6 mM Trolox/g, and 20.0 ± 6.0% DPPH inhibition. However, EA exhibited the highest inhibition against lipid peroxidation (57.2 ± 0.4%. In addition, EA showed no cytotoxicity on dermal papilla cell line and no irritation on chorioallantoic membrane of hen’s eggs. In conclusion, EA was suggested as the most attractive ingredients for functional food and nutraceuticals because of the high inhibitory activity against 5α-reductase

  20. Antimicrobial effect by extracts of rhizome of Alpinia officinarum Hance may relate to its inhibition of beta-ketoacyl-ACP reductase.

    Science.gov (United States)

    Huang, Hui; Wu, Dan; Tian, Wei-Xi; Ma, Xiao-Feng; Wu, Xiao-Dong

    2008-06-01

    Inhibitory effects on bacterial growth showed that 40% ethanol extract of galangal (rhizome of Alpinia officinarum Hance) can inhibit Staphylococcus aureus, alpha-Hemolytic streptococcus, beta-Hemolytic streptococcus and Streptococcus pneumoniae. beta-ketoacyl-ACP reductase (FabG, EC.1.1.1.100) is a key enzyme in type II fatty acid synthase system in bacteria and catalyzes beta-ketoacyl-ACP reduction. The galangal extracts inhibited FabG with an IC(50) value of only 4.47 +/- 0.10 microg/mL and is more potent than other previously published inhibitors. Kinetics studies showed that the inhibition consisted of both reversible and irreversible inhibition. The extracts of galangal inhibit FabG in a competitive pattern against NADPH. So far, no inhibitor has been reported to exhibit irreversible inhibition of FabG, whereas the galangal ethanol extract can inhibit FabG irreversibly. The irreversible inhibition presented two phases. It is probable that the galangal extract inhibit FabG, thereby displaying antibacterial ability.

  1. Novel synthetic inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity that inhibit tumor cell proliferation and are structurally unrelated to existing statins.

    Science.gov (United States)

    Perchellet, Jean-Pierre H; Perchellet, Elisabeth M; Crow, Kyle R; Buszek, Keith R; Brown, Neil; Ellappan, Sampathkumar; Gao, Ge; Luo, Diheng; Minatoya, Machiko; Lushington, Gerald H

    2009-11-01

    Pilot-scale libraries of eight-membered medium ring lactams (MRLs) and related tricyclic compounds (either seven-membered lactams, thiolactams or amines) were screened for their ability to inhibit the catalytic activity of human recombinant 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in vitro. A dozen of the synthetic compounds mimic the inhibition of purified HMG-CoA reductase activity caused by pravastatin, fluvastatin and sodium salts of lovastatin, mevastatin and simvastatin in this cell-free assay, suggesting direct interaction with the rate-limiting enzyme of cholesterol biosynthesis. Moreover, several MRLs inhibit the metabolic activity of L1210 tumor cells in vitro to a greater degree than fluvastatin, lovastatin, mevastatin and simvastatin, whereas pravastatin is inactive. Although the correlation between the concentration-dependent inhibitions of HMG-CoA reductase activity over 10 min in the cell-free assay and L1210 tumor cell proliferation over 4 days in culture is unclear, some bioactive MRLs elicit interesting combinations of statin-like (IC50: 7.4-8.0 microM) and anti-tumor (IC50: 1.4-2.3 microM) activities. The HMG-CoA reductase-inhibiting activities of pravastatin and an MRL persist in the presence of increasing concentrations of NADPH. But increasing concentrations of HMG-CoA block the HMG-CoA reductase-inhibiting activity of pravastatin without altering that of an MRL, suggesting that MRLs and existing statins may have different mechanisms of enzyme interaction and inhibition. When tested together, suboptimal concentrations of synthetic MRLs and existing statins have additive inhibitory effects on HMG-CoA reductase activity. Preliminary molecular docking studies with MRL-based inhibitors indicate that these ligands fit sterically well into the HMG-CoA reductase statin-binding receptor model and, in contrast to mevastatin, may occupy a narrow channel housing the pyridinium moiety on NADP+.

  2. Phenolics from grapefruit peels inhibit HMG-CoA reductase and angiotensin-I converting enzyme and show antioxidative properties in endothelial EA.Hy 926 cells

    Directory of Open Access Journals (Sweden)

    Ayokunle O. Ademosun

    2015-06-01

    Full Text Available This study sought to investigate the possible mechanisms for the use of phenolic extracts from grapefruit peels in the management/prevention of cardiovascular complications. The effects of the phenolic extracts on key enzymes relevant to cardiovascular diseases [3-hydroxy-methyl-3-glutaryl coenzyme A reductase (HMG-CoA reductase and angiotensin-I converting enzyme (ACE], cellular antioxidant activity in human endothelial cells (EA.Hy 926 and radicals [1,1-diphenyl-2 picrylhydrazyl (DPPH and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS] scavenging abilities were investigated. The phenolic contents of the extracts were investigated using HPLC–DAD. There was no significant (P > 0.05 difference in the HMG-CoA reductase inhibitory ability of the two extracts, while the bound phenolic extracts had a stronger ACE inhibitory ability than the soluble free phenolics. The extracts also showed intracellular antioxidant activity in human endothelial (EA.Hy 926 cells. Furthermore, the bound phenolics had significantly higher radicals (DPPH* and ABTS* scavenging abilities than the free phenolics. The HPLC analysis revealed the presence of flavonoids (quercetin and kaempferol, phenolics acids (resveratrol, gallic acid, ellagic acid and caffeic acid and tannin (catechin. The cellular antioxidative properties and inhibition of enzymes relevant to the management of cardiovascular complications showed that grapefruit peels could be used as nutraceuticals for the management of such conditions.

  3. Evaluation of effect of alcoholic extract of heartwood of Pterocarpus marsupium on in vitro antioxidant, anti-glycation, sorbitol accumulation and inhibition of aldose reductase activity

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    Pankaj Gupta

    2017-07-01

    The alcoholic extract of P. marsupium across varying concentrations showed inhibitory effect as evident by IC50 on advanced glycation-end-products formation (55.39 μg/ml, sorbitol accumulation (151.00 μg/ml and rat kidney aldose reductase (195.88 μg/ml. The phytoextract also exhibited high phenolic and flavonoid contents with promising antioxidant potential against the antioxidant assays evaluated. The present investigation suggests that the phytoextract showed prominent antioxidant, antiglycation property and, inhibited accumulation of sorbitol and ALR enzyme, thus promising a beneficial role in reducing/delaying diabetic complications.

  4. ELEVATED LIPID PEROXIDATION AND DNA OXIDATION IN NERVE FROM DIABETIC RATS: EFFECTS OF ALDOSE REDUCTASE INHIBITION, INSULIN AND NEUROTROPHIC FACTORS

    Science.gov (United States)

    Cunha, Joice M.; Jolivalt, Corinne G.; Ramos, Khara M.; Gregory, Joshua A.; Calcutt, Nigel A.; Mizisin, Andrew P.

    2008-01-01

    We investigated the effect of treatment with an aldose reductase inhibitor, insulin or select neurotrophic factors on the generation of oxidative damage in peripheral nerve. Rats were either treated with streptozotocin (STZ) to induce insulin-deficient diabetes or fed with a diet containing 40% D-galactose to promote hexose metabolism by aldose reductase. Initial time-course studies showed that lipid peroxidation and DNA oxidation were significantly elevated in sciatic nerve after 1 week or 2 weeks of STZ-induced diabetes, respectively, and that both remained elevated after 12 weeks of diabetes. The increase in nerve lipid peroxidation was completely prevented or reversed by treatment with the aldose reductase inhibitor, ICI 222155, or by insulin, but not by the neurotrophic factors, prosaptide TX14(A) or neurotrophin-3. The increase in nerve DNA oxidation was significantly prevented by insulin treatment. In contrast, up to 16 weeks of galactose feeding did not alter nerve lipid peroxidation or protein oxidation, despite evidence of ongoing nerve conduction deficits. These observations demonstrate that nerve oxidative damage develops early after the onset of insulin-deficient diabetes and that it is not induced by increased hexose metabolism by aldose reductase per se, but rather is a downstream consequence of flux through this enzyme. Furthermore, the beneficial effect of prosaptide TX14(A) and neurotrophin-3 on nerve function and structure in diabetic rats are not due to amelioration of increased lipid peroxidation. PMID:18555826

  5. Human carbonyl reductase 1 participating in intestinal first-pass drug metabolism is inhibited by fatty acids and acyl-CoAs.

    Science.gov (United States)

    Hara, Akira; Endo, Satoshi; Matsunaga, Toshiyuki; El-Kabbani, Ossama; Miura, Takeshi; Nishinaka, Toru; Terada, Tomoyuki

    2017-08-15

    Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, reduces a variety of carbonyl compounds including endogenous isatin, prostaglandin E2 and 4-oxo-2-nonenal. It is also a major non-cytochrome P450 enzyme in the phase I metabolism of carbonyl-containing drugs, and is highly expressed in the intestine. In this study, we found that long-chain fatty acids and their CoA ester derivatives inhibit CBR1. Among saturated fatty acids, myristic, palmitic and stearic acids were inhibitory, and stearic acid was the most potent (IC50 9µM). Unsaturated fatty acids (oleic, elaidic, γ-linolenic and docosahexaenoic acids) and acyl-CoAs (palmitoyl-, stearoyl- and oleoyl-CoAs) were more potent inhibitors (IC50 1.0-2.5µM), and showed high inhibitory selectivity to CBR1 over its isozyme CBR3 and other SDR superfamily enzymes (DCXR and DHRS4) with CBR activity. The inhibition by these fatty acids and acyl-CoAs was competitive with respect to the substrate, showing the Ki values of 0.49-1.2µM. Site-directed mutagenesis of the substrate-binding residues of CBR1 suggested that the interactions between the fatty acyl chain and the enzyme's Met141 and Trp229 are important for the inhibitory selectivity. We also examined CBR1 inhibition by oleic acid in cellular levels: The fatty acid effectively inhibited CBR1-mediated 4-oxo-2-nonenal metabolism in colon cancer DLD1 cells and increased sensitivity to doxorubicin in the drug-resistant gastric cancer MKN45 cells that highly express CBR1. The results suggest a possible new food-drug interaction through inhibition of CBR1-mediated intestinal first-pass drug metabolism by dietary fatty acids. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. In vitro and in vivo inhibition of aldose reductase and advanced glycation end products by phloretin, epigallocatechin 3-gallate and [6]-gingerol.

    Science.gov (United States)

    Sampath, Chethan; Sang, Shengmin; Ahmedna, Mohamed

    2016-12-01

    Hyperglycemic stress activates polyol pathway and aldose reductase (AR) key enzyme responsible for generating secondary complications during diabetes. In this study the therapeutic potential of phloretin, epigallocatechin 3-gallate (EGCG) and [6]-gingerol were evaluated for anti-glycating and AR inhibitory activity in vitro and in vivo systems. Human retinal pigment epithelial (HRPE) cells were induced with high glucose supplemented with the phloretin, EGCG and [6]-gingerol. Aldose reductase activity, total advanced glycation end products (AGEs) and enzyme inhibitor kinetics were assessed. Male C57BL/6J mice were randomly assigned to one of the different treatments (bioactive compounds at 2 concentrations each) with either a low fat diet or high fat diet (HFD). After sixteen weeks, AGE accumulation and AR activity was determined in heart, eyes and kidney. High glucose induced toxicity decreased cell viability compared to the untreated cells and AR activity increased to 2-5 folds from 24 to 96h. Pre-treatment of cells with phloretin, EGCG and [6]-gingerol improved cell viability and inhibited AR activity. The enzyme inhibition kinetics followed a non-competitive mode of inhibition for phloretin and EGCG whereas [6]-gingerol indicated uncompetitive type of inhibition against AR. Data from the animal studies showed high plasma glucose levels in HFD group over time, compared to the low fat diet. HFD group developed cataract and AR activity increased to 4 folds compared to the group with low fat diet. Administration of EGCG, phloretin and [6]-gingerol significantly reduced blood sugar levels, AGEs accumulation, and AR activity. These findings could provide a basis to consider using the selected dietary components alone or in combination with other therapeutic approaches to prevent diabetes-related complications in humans. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  7. Caffeoylated phenylpropanoid glycosides from Brandisia hancei inhibit advanced glycation end product formation and aldose reductase in vitro and vessel dilation in larval zebrafish in vivo.

    Science.gov (United States)

    Yu, Song Yi; Lee, Ik-Soo; Jung, Seung-Hyun; Lee, Yun Mi; Lee, Yu-Ri; Kim, Joo-Hwan; Sun, Hang; Kim, Jin Sook

    2013-12-01

    In our continuing efforts to identify effective naturally sourced agents for diabetic complications, five caffeoylated phenylpropanoid glycosides, acteoside (1), isoacteoside (2), poliumoside (3), brandioside (4), and pheliposide (5) were isolated from the 80% EtOH extract of Brandisia hancei stems and leaves. These isolates (1-5) were subjected to an in vitro bioassay evaluating their inhibitory activity on advanced glycation end product formation and rat lens aldose reductase activity. All tested compounds exhibited significant inhibition of advanced glycation end product formation with IC50 values of 4.6-25.7 µM, compared with those of aminoguanidine (IC50=1,056 µM) and quercetin (IC50=28.4 µM) as positive controls. In the rat lens aldose reductase assay, acteoside, isoacteoside, and poliumoside exhibited greater inhibitory effects on rat lens aldose reductase with IC50 values of 0.83, 0.83, and 0.85 µM, respectively, than those of the positive controls, 3,3-tetramethyleneglutaric acid (IC50=4.03 µM) and quercetin (IC50=7.2 µM). In addition, the effect of acteoside on the dilation of hyaloid-retinal vessels induced by high glucose in larval zebrafish was investigated. Acteoside reduced the diameters of high glucose-induced hyaloid-retinal vessels by 69% at 10 µM and 81% at 20 µM, compared to the high glucose-treated control group. These results suggest that B. hancei and its active components might be beneficial in the treatment and prevention of diabetic vascular complications.

  8. NMR evaluation of total statin content and HMG-CoA reductase inhibition in red yeast rice (Monascus spp. food supplements

    Directory of Open Access Journals (Sweden)

    Lachenmeier Dirk W

    2012-03-01

    Full Text Available Abstract Background Red yeast rice (i.e., rice fermented with Monascus spp., as a food supplement, is claimed to be blood cholesterol-lowering. The red yeast rice constituent monacolin K, also known as lovastatin, is an inhibitor of the hydroxymethylglutaryl-CoA (HMG-CoA reductase. This article aims to develop a sensitive nuclear magnetic resonance (NMR method to determine the total statin content of red yeast rice products. Methods The total statin content was determined by a 400 MHz 1H NMR spectroscopic method, based on the integration of the multiplet at δ 5.37-5.32 ppm of a hydrogen at the hexahydronaphthalene moiety in comparison to an external calibration with lovastatin. The activity of HMG-CoA reductase was measured by a commercial spectrophotometric assay kit. Results The NMR detection limit for total statins was 6 mg/L (equivalent to 0.3 mg/capsule, if two capsules are dissolved in 50 mL ethanol. The relative standard deviations were consistently lower than 11%. The total statin concentrations of five red yeast rice supplements were between 1.5 and 25.2 mg per specified daily dose. A dose-dependent inhibition of the HMG-CoA reductase enzyme activity by the red yeast rice products was demonstrated. Conclusion A simple and direct NMR assay was developed to determine the total statin content in red yeast rice. The assay can be applied for the determination of statin content for the regulatory control of red yeast rice products.

  9. Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase.

    Science.gov (United States)

    Wentzinger, Laurent F; Bach, Thomas J; Hartmann, Marie-Andrée

    2002-09-01

    To get some insight into the regulatory mechanisms controlling the sterol branch of the mevalonate pathway, tobacco (Nicotiana tabacum cv Bright Yellow-2) cell suspensions were treated with squalestatin-1 and terbinafine, two specific inhibitors of squalene synthase (SQS) and squalene epoxidase, respectively. These two enzymes catalyze the first two steps involved in sterol biosynthesis. In highly dividing cells, SQS was actively expressed concomitantly with 3-hydroxy-3-methylglutaryl coenzyme A reductase and both sterol methyltransferases. At nanomolar concentrations, squalestatin was found to inhibit efficiently sterol biosynthesis as attested by the rapid decrease in SQS activity and [(14)C]radioactivity from acetate incorporated into sterols. A parallel dose-dependent accumulation of farnesol, the dephosphorylated form of the SQS substrate, was observed without affecting farnesyl diphosphate synthase steady-state mRNA levels. Treatment of tobacco cells with terbinafine is also shown to inhibit sterol synthesis. In addition, this inhibitor induced an impressive accumulation of squalene and a dose-dependent stimulation of the triacylglycerol content and synthesis, suggesting the occurrence of regulatory relationships between sterol and triacylglycerol biosynthetic pathways. We demonstrate that squalene was stored in cytosolic lipid particles, but could be redirected toward sterol synthesis if required. Inhibition of either SQS or squalene epoxidase was found to trigger a severalfold increase in enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, giving first evidence for a positive feedback regulation of this key enzyme in response to a selective depletion of endogenous sterols. At the same time, no compensatory responses mediated by SQS were observed, in sharp contrast to the situation in mammalian cells.

  10. Determination of the potency of a novel saw palmetto supercritical CO2 extract (SPSE for 5α-reductase isoform II inhibition using a cell-free in vitro test system

    Directory of Open Access Journals (Sweden)

    Pais P

    2016-04-01

    Full Text Available Pilar Pais, Agustí Villar, Santiago Rull Euromed, Barcelona, Spain Background: The nicotinamide adenine dinucleotide phosphate-dependent membrane protein 5α-reductase catalyses the conversion of testosterone to the most potent androgen – 5α-dihydrotestosterone. Two 5α-reductase isoenzymes are expressed in humans: type I and type II. The latter is found primarily in prostate tissue. Saw palmetto extract (SPE has been used extensively in the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH. The pharmacological effects of SPE include the inhibition of 5α-reductase, as well as anti-inflammatory and antiproliferative effects. Clinical studies of SPE have been inconclusive – some have shown significant results, and others have not – possibly the result of varying bioactivities of the SPEs used in the studies. Purpose: To determine the in vitro potency in a cell-free test system of a novel SP supercritical CO2 extract (SPSE, an inhibitor of the 5α-reductase isoenzyme type II. Materials and methods: The inhibitory potency of SPSE was compared to that of finasteride, an approved 5α-reductase inhibitor, on the basis of the enzymatic conversion of the substrate androstenedione to the 5α-reduced product 5α-androstanedione. Results: By concentration-dependent inhibition of 5α-reductase type II in vitro (half-maximal inhibitory concentration 3.58±0.05 µg/mL, SPSE demonstrated competitive binding toward the active site of the enzyme. Finasteride, the approved 5α-reductase inhibitor tested as positive control, led to 63%–75% inhibition of 5α-reductase type II. Conclusion: SPSE effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is comparatively low. It can be confirmed from the results of this study that SPSE has bioactivity that promotes prostate health at a level that is superior to that of many other phytotherapeutic extracts. The

  11. Enhancement of sterol synthesis by the monoterpene perillyl alcohol is unaffected by competitive 3-hydroxy-3-methylglutaryl-CoA reductase inhibition.

    Science.gov (United States)

    Cerda, S R; Wilkinson, J; Branch, S K; Broitman, S A

    1999-06-01

    Monoterpenes such as limonene and perillyl alcohol (PA) are currently under investigation for their chemotherapeutic properties which have been tied to their ability to affect protein isoprenylation. Because PA affects the synthesis of isoprenoids, such as ubiquinone, and cholesterol is the end product of the synthetic pathway from which this isoprenoid pathway branches, we investigated the effects of this compound upon cholesterol metabolism in the colonic adenocarcinoma cell line SW480. PA (1 mM) inhibited incorporation of 14C-mevalonate into 21-26 kDa proteins by 25% in SW480 cells. Cholesterol (CH) biosynthesis was assessed by measuring the incorporation of 14C-acetate and 14C-mevalonate into 27-carbon-sterols. Cells treated with PA (1 mM) exhibited a fourfold increase in the incorporation of 14C-acetate but not 14C-mevalonate into cholesterol. Mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase, at 2 microM concentration, inhibited CH synthesis from 14C-acetate by 80%. Surprisingly, concurrent addition of mevinolin and PA did not significantly alter the stimulatory effects of PA. As observed differences in 14C-acetate and 14C-mevalonate precursor labeling could indicate PA affects early pathway events, the effects of this monoterpene on HMG-CoA reductase activity were evaluated. Unexpectedly, 1 mM PA did not stimulate activity of this enzyme. Consistent with its action as a reversibly bound inhibitor, in washed microsomes, 2 microM mevinolin pretreatment increased reductase protein expression causing a 12.7 (+/- 2.4)-fold compensatory HMG-CoA reductase activity increase; concurrent treatment with 1 mM PA attenuated this to a 5.3 (+/- 0.03)-fold increase. Gas chromatographic analysis confirmed CH was the major lipid present in the measured thin-layer chromatography spot. Since 14C-acetate incorporation into free fatty acid and phospholipid pools was not significantly affected by PA treatment, nonspecific changes in whole

  12. 3-Hydroxymethyl coenzyme A reductase inhibition attenuates spontaneous smooth muscle tone via RhoA/ROCK pathway regulated by RhoA prenylation.

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    Rattan, Satish

    2010-06-01

    RhoA prenylation may play an important step in the translocation of RhoA in the basal internal anal sphincter (IAS) smooth muscle tone. Statins inhibit downstream posttranslational RhoA prenylation by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition (HMGCRI). The role of statins in relation to RhoA prenylation in the pathophysiology of the spontaneously tonic smooth muscle has not been investigated. In the present studies, we determined the effect of classical HMGCRI simvastatin on the basal IAS tone and RhoA prenylation and in the levels of RhoA/Rho kinase (ROCK) in the cytosolic vs. membrane fractions of the smooth muscle. Simvastatin produced concentration-dependent decrease in the IAS tone (via direct actions at the smooth muscle cells). The decrease in the IAS tone by simvastatin was associated with the decrease in the prenylation of RhoA, as well as RhoA/ROCK in the membrane fractions of the IAS, in the basal state. The inhibitory effects of the HMGCRI were completely reversible by geranylgeranyltransferase substrate geranylgeranyl pyrophosphate. Relaxation of the IAS smooth muscle via HMGCRI simvastatin is mediated via the downstream decrease in the levels of RhoA prenylation and ROCK activity. Studies support the concept that RhoA prenylation leading to RhoA/ROCK translocation followed by activation is important for the basal tone in the IAS. Data suggest that the role of HMG-CoA reductase may go beyond cholesterol biosynthesis, such as the regulation of the smooth muscle tone. The studies have important implications in the pathophysiological mechanisms and in the novel therapeutic approaches for anorectal motility disorders.

  13. Evaluation of effect of alcoholic extract of heartwood of Pterocarpus marsupium on in vitro antioxidant, anti-glycation, sorbitol accumulation and inhibition of aldose reductase activity.

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    Gupta, Pankaj; Jain, Vivek; Pareek, Ashutosh; Kumari, Preeti; Singh, Randhir; Agarwal, Priyanka; Sharma, Veena

    2017-07-01

    Rising popularity of phytomedicines in various diseased conditions have strengthened the significance of plant-research and evaluation of phytoextracts in clinical manifestations. Pterocarpus marsupium Roxb., a medicinal plant, known for its anti-oxidant and anti-diabetic activity is a rich source of phytochemicals with antihyperglycemic and antihyperlipidemic activities. However, its possible role in diabetic complications is not evaluated yet. The present study explores the possible role of alcoholic extract of heartwood of P. marsupium in the treatment of long-term diabetic complications. The alcoholic extract of P. marsupium was evaluated for advanced glycation-end-products formation, erythrocyte sorbitol accumulation and rat kidney aldose reductase enzyme inhibition at the concentration of 25-400 μg/ml using in-vitro bioassays. Also the phytoextract at the concentration of 10-320 μg/ml was evaluated for its antioxidant potential by in-vitro antioxidant assays which includes, determination of total phenol content; reducing power assay; nitric oxide scavenging activity; superoxide radical scavenging activity; total antioxidant capacity; total flavonoid content; DPPH scavenging activity; and hydrogen peroxide scavenging activity. The alcoholic extract of P. marsupium across varying concentrations showed inhibitory effect as evident by IC50 on advanced glycation-end-products formation (55.39 μg/ml), sorbitol accumulation (151.00 μg/ml) and rat kidney aldose reductase (195.88 μg/ml). The phytoextract also exhibited high phenolic and flavonoid contents with promising antioxidant potential against the antioxidant assays evaluated. The present investigation suggests that the phytoextract showed prominent antioxidant, antiglycation property and, inhibited accumulation of sorbitol and ALR enzyme, thus promising a beneficial role in reducing/delaying diabetic complications.

  14. Application of a simple quantum chemical approach to ligand fragment scoring for Trypanosoma brucei pteridine reductase 1 inhibition

    Science.gov (United States)

    Jedwabny, Wiktoria; Panecka-Hofman, Joanna; Dyguda-Kazimierowicz, Edyta; Wade, Rebecca C.; Sokalski, W. Andrzej

    2017-08-01

    There is a need for improved and generally applicable scoring functions for fragment-based approaches to ligand design. Here, we evaluate the performance of a computationally efficient model for inhibitory activity estimation, which is composed only of multipole electrostatic energy and dispersion energy terms that approximate long-range ab initio quantum mechanical interaction energies. We find that computed energies correlate well with inhibitory activity for a compound series with varying substituents targeting two subpockets of the binding site of Trypanosoma brucei pteridine reductase 1. For one subpocket, we find that the model is more predictive for inhibitory activity than the ab initio interaction energy calculated at the MP2 level. Furthermore, the model is found to outperform a commonly used empirical scoring method. Finally, we show that the results for the two subpockets can be combined, which suggests that this simple nonempirical scoring function could be applied in fragment-based drug design.

  15. Relationship between Aldose reductase and superoxide dismutase inhibition capacities of indole-based analogs of melatonin derivatives

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    Daş-Evcimen N.

    2009-01-01

    Full Text Available Aldose reductase (AR has been implicated in the etiology of diabetic complications. Under diabetic conditions, the elevated vascular glucose level causes an increased flux through the polyol pathway, which induces functional and morphological changes associated with secondary diabetic complications such as cataract, neuropathy, and nephrop­athy. Oxidative stress, antioxidants, and the polyol pathway have recently been found to be linked in pathological states. A large number of structurally different compounds have been studied as potent in vitro AR inhibitors (ARIs. However, with few exceptions, these compounds did not show clinical benefit, and some even produced serious side effects. In view of the ARI activity of certain indole derivative compounds and antioxidant properties of melatonin, we investigated some indole-based analogs of melatonin derivatives. Antioxidant and ARI activity tests were applied to nine indole derivatives that are substituted at the third and fifth positions. Also, the relationship between ARI and antioxidant enzyme activity is discussed.

  16. Opposing Effects of Cyclooxygenase-2 (COX-2) on Estrogen Receptor β (ERβ) Response to 5α-Reductase Inhibition in Prostate Epithelial Cells.

    Science.gov (United States)

    Liu, Teresa T; Grubisha, Melanie J; Frahm, Krystle A; Wendell, Stacy G; Liu, Jiayan; Ricke, William A; Auchus, Richard J; DeFranco, Donald B

    2016-07-08

    Current pharmacotherapies for symptomatic benign prostatic hyperplasia (BPH), an androgen receptor-driven, inflammatory disorder affecting elderly men, include 5α-reductase (5AR) inhibitors (i.e. dutasteride and finasteride) to block the conversion of testosterone to the more potent androgen receptor ligand dihydrotestosterone. Because dihydrotestosterone is the precursor for estrogen receptor β (ERβ) ligands, 5AR inhibitors could potentially limit ERβ activation, which maintains prostate tissue homeostasis. We have uncovered signaling pathways in BPH-derived prostate epithelial cells (BPH-1) that are impacted by 5AR inhibition. The induction of apoptosis and repression of the cell adhesion protein E-cadherin by the 5AR inhibitor dutasteride requires both ERβ and TGFβ. Dutasteride also induces cyclooxygenase type 2 (COX-2), which functions in a negative feedback loop in TGFβ and ERβ signaling pathways as evidenced by the potentiation of apoptosis induced by dutasteride or finasteride upon pharmacological inhibition or shRNA-mediated ablation of COX-2. Concurrently, COX-2 positively impacts ERβ action through its effect on the expression of a number of steroidogenic enzymes in the ERβ ligand metabolic pathway. Therefore, effective combination pharmacotherapies, which have included non-steroidal anti-inflammatory drugs, must take into account biochemical pathways affected by 5AR inhibition and opposing effects of COX-2 on the tissue-protective action of ERβ.

  17. Inhibition of NADPH-cytochrome P450 reductase by tannic acid in rat liver microsomes and primary hepatocytes: methodological artifacts and application to ischemia-reperfusion injury.

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    Pillai, Venkateswaran C; Mehvar, Reza

    2011-08-01

    Tannic acid (TA) inhibits nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) activity, which is measured by reduction of cytochrome c, in rat liver microsomes (RLMs). In the current study, we noticed that TA directly reduces cytochrome c in the absence of microsomes, thus confounding the CPR activity assay. A method is presented that measures CPR activity in the presence of TA by subtracting the cytochrome c reduction in the absence of NADPH (TA effect) from that in the presence of NADPH (TA plus CPR effect). The method was used to determine the inhibitory effect of TA in RLMs, recombinant CPR enzyme, and primary hepatocytes. Additionally, application of TA in a study of role of CPR in a primary rat hepatocyte model of ischemia-reperfusion (IR) was investigated. TA showed concentration-dependent, complete inhibition of CPR with half maximal inhibitory concentration (IC(50) ) values of 58.2 μM in RLMs and 54.6 and 275 μM in primary rat hepatocytes in the absence and presence of serum in the medium, respectively. Additionally, inhibition of CPR by TA was associated with a significant reduction in reactive oxygen species and cell death after IR injury. These data may be useful in future studies using TA as an inhibitor of CPR in microsomes and primary hepatocytes.

  18. Biosynthesis of ribose-5-phosphate and erythrose-4-phosphate in archaea: a phylogenetic analysis of archaeal genomes

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    Tim Soderberg

    2005-01-01

    Full Text Available A phylogenetic analysis of the genes encoding enzymes in the pentose phosphate pathway (PPP, the ribulose monophosphate (RuMP pathway, and the chorismate pathway of aromatic amino acid biosynthesis, employing data from 13 complete archaeal genomes, provides a potential explanation for the enigmatic phylogenetic patterns of the PPP genes in archaea. Genomic and biochemical evidence suggests that three archaeal species (Methanocaldococcus jannaschii, Thermoplasma acidophilum and Thermoplasma volcanium produce ribose-5-phosphate via the nonoxidative PPP (NOPPP, whereas nine species apparently lack an NOPPP but may employ a reverse RuMP pathway for pentose synthesis. One species (Halobacterium sp. NRC-1 lacks both the NOPPP and the RuMP pathway but may possess a modified oxidative PPP (OPPP, the details of which are not yet known. The presence of transketolase in several archaeal species that are missing the other two NOPPP genes can be explained by the existence of differing requirements for erythrose-4-phosphate (E4P among archaea: six species use transketolase to make E4P as a precursor to aromatic amino acids, six species apparently have an alternate biosynthetic pathway and may not require the ability to make E4P, and one species (Pyrococcus horikoshii probably does not synthesize aromatic amino acids at all.

  19. Tactile allodynia and formalin hyperalgesia in streptozotocin-diabetic rats: effects of insulin, aldose reductase inhibition and lidocaine.

    Science.gov (United States)

    Calcutt, N A; Jorge, M C; Yaksh, T L; Chaplan, S R

    1996-12-01

    Rats developed tactile allodynia within days of the onset of diabetes and which persisted for up to 8 weeks. Allodynia was prevented by insulin therapy that maintained normoglycemia while established allodynia was reversed by insulin therapy and normoglycemia of days but not hours duration. Tactile allodynia persisted in diabetic rats that received enough insulin to maintain normal body and foot weights but remained hyperglycemic, whereas this therapy was sufficient to correct other nerve disorders in diabetic rats, including deficits of sensory and motor nerve conduction velocity, nerve blood flow and hyperalgesia during the formalin test. Treating diabetic rats with the aldose reductase inhibitor ICI 222155 did not prevent tactile allodynia. Tactile allodynia was of similar magnitude in diabetic rats and nerve injured control rats and diabetes did not alter the magnitude or time course of nerve injury-induced allodynia. Systemic lidocaine treatment alleviated tactile allodynia in nerve injured control rats and both sham-operated and nerve injured diabetic rats. The streptozotocin-diabetic rat develops tactile allodynia that appears to be related to prolonged periods of insulin deficiency or hyperglycemia and which is amenable to treatment with lidocaine. The model may be of use in investigating the efficacy of other potential therapeutic agents for treating painful diabetic neuropathy.

  20. Self-organizing maps and VolSurf approach to predict aldose reductase inhibition by flavonoid compounds

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    Luciana Scotti

    2011-02-01

    Full Text Available Aldose Reductase (AR is the polyol pathway key enzyme which converts glucose to sorbitol. High glucose availability in insulin resistant tissues in diabetes leads into an accumulation of sorbitol, which has been associated with typical chronic complications of this disease, such as neuropathy, nephropathy and retinopathy. In this study, 71 flavonoids AR inhibitors were subjected to two methods of SAR to verify crucial substituents. The first method used the PCA (Principal Component Analysis to elucidate physical and chemical characteristics in the molecules that would be essential for the activity, employing VolSurf descriptors. The rate obtained explained 53% of the system total variance and revealed that a hydrophobic-hydrophilic balance in the molecules is required, since very polar or nonpolar substituents decrease the activity. Artificial Neural Networks (ANNs was also employed to determine key substituents by evaluating substitution patterns, using NMR data. This study had a high success rate (85% accuracy in the training set and 88% accuracy in the test set and showed polihydroxilations are essential for high activity and methoxylations and glicosilations primarily at positions C7, C3' and C4' decrease the activity.

  1. HMG-CoA reductase inhibition aborts functional differentiation and triggers apoptosis in cultured primary human monocytes: a potential mechanism of statin-mediated vasculoprotection

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    Vamvakopoulos Joannis E

    2003-07-01

    Full Text Available Abstract Background Statins effectively lower blood cholesterol and the risk of cardiovascular death. Immunomodulatory actions, independent of their lipid-lowering effect, have also been ascribed to these compounds. Since macrophages participate in several vascular pathologies, we examined the effect of statin treatment on the survival and differentiation of primary human monocytes. Methods Peripheral blood mononuclear cells (PBMCs from healthy individuals were cultured in the presence or absence of mevastatin. Apoptosis was monitored by annexin V / PI staining and flow cytometry. In parallel experiments, cultures were stimulated with LPS in the presence or absence of mevastatin and the release of IL-1β and IL-1Ra was measured by ELISA. Results Among PBMCs, mevastatin-treated monocytes were particularly susceptible to apoptosis, which occurred at doses >1 microM and was already maximal at 5 microM. However, even at the highest mevastatin dose used (10 microM, apoptosis occurred only after 24 h of culture, possibly reflecting a requirement for cell commitment to differentiation. After 72 h of treatment the vast majority (>50% of monocytes were undergoing apoptosis. Stimulation with LPS revealed that mevastatin-treated monocytes retained the high IL-1β output characteristic of undifferentiated cells; conversely, IL-1Ra release was inhibited. Concurrent treatment with mevalonolactone prevented the induction of apoptosis and suppressed both IL-1β and IL-1Ra release in response to LPS, suggesting a rate-limiting role for HMG-CoA reductase in monocyte differentiation. Conclusions Our findings indicate that statins arrest the functional differentiation of monocytes into macrophages and steer these cells into apoptosis, suggesting a novel mechanism for the vasculoprotective properties of HMG-CoA reductase inhibitors.

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

    Science.gov (United States)

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

    2015-06-01

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

  3. Tissue-specific effects of aldose reductase inhibition on fluorescence and cross-linking of extracellular matrix in chronic galactosemia. Relationship to pentosidine cross-links.

    Science.gov (United States)

    Richard, S; Tamas, C; Sell, D R; Monnier, V M

    1991-08-01

    Chronic experimental hyperglycemia mediated by galactose has been shown to induce browning and cross-linking of rat tail tendon collagen that could be duplicated in vitro by nonenzymatic galactosylation. To investigate the nature of these changes, Sprague-Dawley rats were placed on a 33% galactose diet without and with sorbinil for 6 and 12 mo. Collagen-linked fluorescence and pentosidine cross-links increased with age and galactosemia in tail tendons (P less than 0.001) and skin but were essentially unresponsive to aldose reductase inhibition (ARI). In contrast, tendon breaking time in urea, a likely parameter of cross-linking, was markedly improved (P less than 0.001) by ARI. Fluorescence that was inhibited by sorbinil treatment was increased in pepsin and proteinase K digest of aortic tissue from galactosemic rats (P less than 0.001), but impaired enzymatic digestibility was not observed. Systolic blood pressure as potential consequence of aortic stiffening was not increased in galactosemia. These data suggest that fluorescence in skin and tendon might be in part due to advanced glycosylation and pentosidine formation because these were not decreased by ARI. However, they also suggest that nonfluorescent cross-links may also be forming because, in contrast to fluorescence, tail tendon breaking time was partly corrected by ARI. Thus, it appears that extracellular matrix changes in chronic galactosemia are complex, being partly attributable to advanced glycosylation and partly to polyol-pathway activation.

  4. Methylseleninic acid (MSA) inhibits 17β-estradiol-induced cell growth in breast cancer T47D cells via enhancement of the antioxidative thioredoxin/ thioredoxin reductase system.

    Science.gov (United States)

    Okuno, Tomofumi; Miura, Kiyoshi; Sakazaki, Fumitoshi; Nakamuro, Katsuhiko; Ueno, Hitoshi

    2012-01-01

    The purpose of this study was to clarify the cell growth inhibitory mechanism of human breast cancer cells caused by selenium (Se) compounds. In the presence of 17β-estradiol (E(2)) at physiological concentrations, growth of estrogen receptor α (ERα)-positive T47D cells was markedly inhibited by 1 × 10(-6) mol/L methylseleninic acid (MSA) with no Se related toxicity.Under conditions where cell growth was inhibited, MSA decreased ERα mRNA levels and subsequent protein levels; further decreasing expression of estrogen-responsive finger protein (Efp) which is a target gene product of ERα and promotes G2/M progression of the cell cycle. Therefore, the decline in Efp expression is presumed to be involved in G2 arrest. Coincidentally, the antioxidative thioredoxin/ thioredoxin reductase (Trx/TrxR) system in cells was enhanced by the synergistic action of E(2) and MSA. It has been reported that ROS-induced oxidative stress enhanced ERα expression. E(2) increased production of intracellular ROS in T47D cells. Meanwhile, MSA significantly decreased E(2)-induced ROS accumulation. From these results, activation of the Trx/TrxR system induced by the coexistence of MSA and E(2) suppresses oxidative stress and decreases expression of ERα, and finally induces the growth arrest of T47D cells through disruption of ERα signaling.

  5. An innovative strategy for dual inhibitor design and its application in dual inhibition of human thymidylate synthase and dihydrofolate reductase enzymes.

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    Mahreen Arooj

    Full Text Available Due to the diligence of inherent redundancy and robustness in many biological networks and pathways, multitarget inhibitors present a new prospect in the pharmaceutical industry for treatment of complex diseases. Nevertheless, to design multitarget inhibitors is concurrently a great challenge for medicinal chemists. We have developed a novel computational approach by integrating the affinity predictions from structure-based virtual screening with dual ligand-based pharmacophore to discover potential dual inhibitors of human Thymidylate synthase (hTS and human dihydrofolate reductase (hDHFR. These are the key enzymes in folate metabolic pathway that is necessary for the biosynthesis of RNA, DNA, and protein. Their inhibition has found clinical utility as antitumor, antimicrobial, and antiprotozoal agents. A druglike database was utilized to perform dual-target docking studies. Hits identified through docking experiments were mapped over a dual pharmacophore which was developed from experimentally known dual inhibitors of hTS and hDHFR. Pharmacophore mapping procedure helped us in eliminating the compounds which do not possess basic chemical features necessary for dual inhibition. Finally, three structurally diverse hit compounds that showed key interactions at both active sites, mapped well upon the dual pharmacophore, and exhibited lowest binding energies were regarded as possible dual inhibitors of hTS and hDHFR. Furthermore, optimization studies were performed for final dual hit compound and eight optimized dual hits demonstrating excellent binding features at target systems were also regarded as possible dual inhibitors of hTS and hDHFR. In general, the strategy used in the current study could be a promising computational approach and may be generally applicable to other dual target drug designs.

  6. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and application of statins as a novel effective therapeutic approach against Acanthamoeba infections.

    Science.gov (United States)

    Martín-Navarro, Carmen María; Lorenzo-Morales, Jacob; Machin, Rubén P; López-Arencibia, Atteneri; García-Castellano, José Manuel; de Fuentes, Isabel; Loftus, Brendan; Maciver, Sutherland K; Valladares, Basilio; Piñero, José E

    2013-01-01

    Acanthamoeba is an opportunistic pathogen in humans, whose infections most commonly manifest as Acanthamoeba keratitis or, more rarely, granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba, they are generally lengthy and/or have limited efficacy. Therefore, there is a requirement for the identification, validation, and development of novel therapeutic targets against these pathogens. Recently, RNA interference (RNAi) has been widely used for these validation purposes and has proven to be a powerful tool for Acanthamoeba therapeutics. Ergosterol is one of the major sterols in the membrane of Acanthamoeba. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, one of the precursors for the production of cholesterol in humans and ergosterol in plants, fungi, and protozoa. Statins are compounds which inhibit this enzyme and so are promising as chemotherapeutics. In order to validate whether this enzyme could be an interesting therapeutic target in Acanthamoeba, small interfering RNAs (siRNAs) against HMG-CoA were developed and used to evaluate the effects induced by the inhibition of Acanthamoeba HMG-CoA. It was found that HMG-CoA is a potential drug target in these pathogenic free-living amoebae, and various statins were evaluated in vitro against three clinical strains of Acanthamoeba by using a colorimetric assay, showing important activities against the tested strains. We conclude that the targeting of HMG-CoA and Acanthamoeba treatment using statins is a novel powerful treatment option against Acanthamoeba species in human disease.

  7. Inhibition of HMG-CoA reductase by MFS, a purified extract from the fermentation of marine fungus Fusarium solani FG319, and optimization of MFS production using response surface methodology.

    Science.gov (United States)

    Zhou, Yu; Wu, Wen-Hui; Zhao, Qing-Bo; Wang, Xiao-Yu; Bao, Bin

    2015-05-01

    The present study was designed to isolate and characterize a purified extract from Fusarium solani FG319, termed MFS (Metabolite of Fusarium solani FG319) that showed anti-atherosclerosis activity by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Response surface methodology (RSM) was employed to achieve an improved yield from the fermentation medium. The inhibiting effect of the isolate, MFS, on HMG-CoA reductase was greater than that of the positive control, lovastatin. The average recovery of MFS and the relative standard deviation (RSD) ranged between 99.75% to 101.18%, and 0.31% to 0.74%, respectively. The RSDs intra- and inter-assay of the three samples ranged from 0.288% to 2.438%, and from 0.934% to 2.383%, respectively. From the RSM, the concentration of inducer, cultivation time, and culture temperatures had significant effects on the MFS production, with the effect of inducer concentration being more pronounced that other factors. In conclusion, the optimal conditions for the MFS production were achieved using RSM and that MFS could be explored as an anti-atherosclerosis agent based on its ability to inhibit HMG-CoA reductase. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  8. Gigantol from Dendrobium chrysotoxum Lindl. binds and inhibits aldose reductase gene to exert its anti-cataract activity: An in vitro mechanistic study.

    Science.gov (United States)

    Wu, Jie; Li, Xue; Wan, Wencheng; Yang, Qiaohong; Ma, Weifeng; Chen, Dan; Hu, Jiangmiao; Chen, C-Y Oliver; Wei, Xiaoyong

    2017-02-23

    Dendrobium. chrysotoxum Lindl is a commonly used species of medicinal Dendrobium which belongs to the family of Orchidaceae, locally known as "Shihu" or "Huangcao". D. chrysotoxum Lindl is widely known for medicinal values in traditional Chinese medicine as it possesses anti-inflammatory, anti-hyperglycemic induction, antitumor and antioxidant properties. To characterize the interaction between gigantol extracted from D. chrysotoxum Lindl and the AR gene, and determine gigantol's efficacy against cataractogenesis. Human lens epithelial cells (HLECs) were induced by glucose as the model group. Reverse transcription polymerase chain reaction (RT-PCR) was used to assess AR gene expression. Then, the mode of interaction of gigantol with the AR gene was evaluated by UV-visible spectroscopy, atomic force microscope (AFM) and surface-enhanced Raman spectroscopy (SERS). The binding constant was determined by UV-visible. Gigantol depressed AR gene expression in HLECs. UV-visible spectra preliminarily indicated that interaction between the AR gene and gigantol may follow the groove mode, with a binding constant of 1.85×10(3)L/mol. Atomic force microscope (AFM) data indicated that gigantol possibly bound to insert AR gene base pairs of the double helix. Surface-enhanced Raman spectroscopy (SERS) studies further supported these observations. Gigantol extracted from D. chrysotoxum Lindl not only has inhibitory effects on aldose reductase, but also inhibits AR gene expression. These findings provide a more comprehensive theoretical basis for the use of Dendrobium for the treatment of diabetic cataract. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  9. Inhibiting activities of the secondary metabolites of Phlomis brunneogaleata against parasitic protozoa and plasmodial enoyl-ACP Reductase, a crucial enzyme in fatty acid biosynthesis.

    Science.gov (United States)

    Kirmizibekmez, Hasan; Calis, Ihsan; Perozzo, Remo; Brun, Reto; Dönmez, Ali A; Linden, Anthony; Rüedi, Peter; Tasdemir, Deniz

    2004-08-01

    Anti-plasmodial activity-guided fractionation of Phlomis brunneogaleata (Lamiaceae) led to the isolation of two new metabolites, the iridoid glycoside, brunneogaleatoside and a new pyrrolidinium derivative (2 S,4 R)-2-carboxy-4-( E)- p-coumaroyloxy-1,1-dimethylpyrrolidinium inner salt [(2 S,4 R)-1,1-dimethyl-4-( E)- p-coumaroyloxyproline inner salt]. Moreover, a known iridoid glycoside, ipolamiide, six known phenylethanoid glycosides, verbascoside, isoverbascoside, forsythoside B, echinacoside, glucopyranosyl-(1-->G (i)-6)-martynoside and integrifolioside B, two flavone glycosides, luteolin 7- O-beta- D-glucopyranoside ( 10) and chrysoeriol 7- O-beta- D-glucopyranoside ( 11), a lignan glycoside liriodendrin, an acetophenone glycoside 4-hydroxyacetophenone 4- O-(6'- O-beta- D-apiofuranosyl)-beta- D-glucopyranoside and three caffeic acid esters, chlorogenic acid, 3-O-caffeoylquinic acid methyl ester and 5- O-caffeoylshikimic acid were isolated. The structures of the pure compounds were elucidated by means of spectroscopic methods (UV, IR, MS, 1D and 2D NMR, [alpha] (D)) and X-ray crystallography. Compounds 10 and 11 were determined to be the major anti-malarial principles of the crude extract (IC (50) values of 2.4 and 5.9 micrograms/mL, respectively). They also exhibited significant leishmanicidal activity (IC (50) = 1.1 and 4.1 micrograms/mL, respectively). The inhibitory potential of the pure metabolites against plasmodial enoyl-ACP reductase (FabI), which is the key regulator of type II fatty acid synthases (FAS-II) in P. falciparum, was also assessed. Compound 10 showed promising FabI inhibiting effect (IC (50) = 10 micrograms/mL) and appears to be the first anti-malarial natural product targeting FabI of P. falciparum.

  10. Hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in Acute lung injury to Reduce Pulmonary dysfunction (HARP-2 trial: study protocol for a randomized controlled trial

    Directory of Open Access Journals (Sweden)

    McAuley Daniel F

    2012-09-01

    Full Text Available Abstract Background Acute lung injury (ALI is a common devastating clinical syndrome characterized by life-threatening respiratory failure requiring mechanical ventilation and multiple organ failure. There are in vitro, animal studies and pre-clinical data suggesting that statins may be beneficial in ALI. The Hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in Acute lung injury to Reduce Pulmonary dysfunction (HARP-2 trial is a multicenter, prospective, randomized, allocation concealed, double-blind, placebo-controlled clinical trial which aims to test the hypothesis that treatment with simvastatin will improve clinical outcomes in patients with ALI. Methods/Design Patients fulfilling the American-European Consensus Conference Definition of ALI will be randomized in a 1:1 ratio to receive enteral simvastatin 80 mg or placebo once daily for a maximum of 28 days. Allocation to randomized groups will be stratified with respect to hospital of recruitment and vasopressor requirement. Data will be recorded by participating ICUs until hospital discharge, and surviving patients will be followed up by post at 3, 6 and 12 months post randomization. The primary outcome is number of ventilator-free days to day 28. Secondary outcomes are: change in oxygenation index and sequential organ failure assessment score up to day 28, number of non pulmonary organ failure free days to day 28, critical care unit mortality; hospital mortality; 28 day post randomization mortality and 12 month post randomization mortality; health related quality of life at discharge, 3, 6 and 12 months post randomization; length of critical care unit and hospital stay; health service use up to 12 months post-randomization; and safety. A total of 540 patients will be recruited from approximately 35 ICUs in the UK and Ireland. An economic evaluation will be conducted alongside the trial. Plasma and urine samples will be taken up to day 28 to investigate potential mechanisms

  11. Aldose reductase inhibition counteracts nitrosative stress and poly(ADP-ribose) polymerase activation in diabetic rat kidney and high-glucose-exposed human mesangial cells.

    Science.gov (United States)

    Drel, Viktor R; Pacher, Pal; Stevens, Martin J; Obrosova, Irina G

    2006-04-15

    Both increased aldose reductase (AR) activity and oxidative/nitrosative stress have been implicated in the pathogenesis of diabetic nephropathy, but the relation between the two factors remains a subject of debate. This study evaluated the effects of AR inhibition on nitrosative stress and poly(ADP-ribose) polymerase (PARP) activation in diabetic rat kidney and high-glucose-exposed human mesangial cells. In animal experiments, control (C) and streptozotocin-diabetic (D) rats were treated with/without the AR inhibitor fidarestat (F, 16 mg kg(-1) day(-1)) for 6 weeks starting from induction of diabetes. Glucose, sorbitol, and fructose concentrations were significantly increased in the renal cortex of D vs C (p diabetes-induced increase in kidney weight as well as nitrotyrosine (NT, a marker of peroxynitrite-induced injury and nitrosative stress), and poly(ADP-ribose) (a marker of PARP activation) accumulation, assessed by both immunohistochemistry and Western blot analysis, in glomerular and tubular compartments of the renal cortex. In vitro studies revealed the presence of both AR and PARP-1 in human mesangial cells, and none of these two variables were affected by high glucose or F treatment. Nitrosylated and poly(ADP-ribosyl)ated proteins (Western blot analysis) accumulated in cells cultured in 30 mM D-glucose (vs 5.55 mM glucose, p diabetic renal cortex and high-glucose-exposed human mesangial cells. These findings reveal new beneficial properties of the AR inhibitor F and provide the rationale for detailed studies of F on diabetic nephropathy.

  12. Insights on the mechanism of thioredoxin reductase inhibition by gold N-heterocyclic carbene compounds using the synthetic linear selenocysteine containing C-terminal peptide hTrxR(488-499): an ESI-MS investigation.

    Science.gov (United States)

    Pratesi, Alessandro; Gabbiani, Chiara; Michelucci, Elena; Ginanneschi, Mauro; Papini, Anna Maria; Rubbiani, Riccardo; Ott, Ingo; Messori, Luigi

    2014-07-01

    Gold-based drugs typically behave as strong inhibitors of the enzyme thioredoxin reductase (hTrxR), possibly as the consequence of direct Gold(I) coordination to its active site selenocysteine. To gain a deeper insight into the molecular basis of enzyme inhibition and prove gold-selenocysteine coordination, the reactions of three parent Gold(I) NHC compounds with the synthetic C-terminal dodecapeptide of hTrxR containing Selenocysteine at position 498, were investigated by electrospray ionization mass spectrometry (ESI-MS). Formation of 1:1 Gold-peptide adducts, though in highly different amounts, was demonstrated in all cases. In these adducts the same [Au-NHC](+) moiety is always associated to the intact peptide. Afterward, tandem MS experiments, conducted on a specific Gold-peptide complex, pointed out that Gold is coordinated to the selenolate group. The relatively large strength of the Gold-selenolate coordinative bond well accounts for potent enzyme inhibition typically afforded by these Gold(I) compounds. In a selected case, the time course of enzyme inhibition was explored. Interestingly, enzyme inhibition turned out to show up very quickly and reached its maximum just few minutes after mixing. Overall, the present results offer some clear insight into the process of thioredoxin reductase inhibition by Gold-based compounds. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2016-02-01

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

  14. Sulindac inhibits pancreatic carcinogenesis in LSL-KrasG12D-LSL-Trp53R172H-Pdx-1-Cre mice via suppressing aldo-keto reductase family 1B10 (AKR1B10).

    Science.gov (United States)

    Li, Haonan; Yang, Allison L; Chung, Yeon Tae; Zhang, Wanying; Liao, Jie; Yang, Guang-Yu

    2013-09-01

    Sulindac has been identified as a competitive inhibitor of aldo-keto reductase 1B10 (AKR1B10), an enzyme that plays a key role in carcinogenesis. AKR1B10 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and exhibits lipid substrate specificity, especially for farnesyl and geranylgeranyl. There have been no studies though showing that the inhibition of PDAC by sulindac is via inhibition of AKR1B10, particularly the metabolism of farnesyl/geranylgeranyl and Kras protein prenylation. To determine the chemopreventive effects of sulindac on pancreatic carcinogenesis, 5-week-old LSL-Kras(G12D)-LSL-Trp53(R172H)-Pdx-1-Cre mice (Pan(kras/p53) mice) were fed an AIN93M diet with or without 200 p.p.m. sulindac (n = 20/group). Kaplan-Meier survival analysis showed that average animal survival in Pan(kras/p53) mice was 143.7 ± 8.8 days, and average survival with sulindac was increased to 168.0 ± 8.8 days (P sulindac, the incidence of PDAC was reduced to 56% (P sulindac significantly decreased Ki-67-labeled cell proliferation and markedly reduced the expression of phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Raf and mitogen-activated protein kinase kinase 1 and 2. In in vitro experiments with PDAC cells from Pan(kras/p53) mice, sulindac exhibited dose-dependent inhibition of AKR1B10 activity. By silencing AKR1B10 expression through small interfering RNA or by sulindac treatment, these in vitro models showed a reduction in Kras and human DNA-J homolog 2 protein prenylation, and downregulation of phosphorylated C-raf, ERK1/2 and MEK1/2 expression. Our results demonstrate that sulindac inhibits pancreatic carcinogenesis by the inhibition of Kras protein prenylation by targeting AKR1B10.

  15. Structure-activity studies of the inhibition of FabI, the enoyl reductase from Escherichia coli, by triclosan: kinetic analysis of mutant FabIs.

    Science.gov (United States)

    Sivaraman, Sharada; Zwahlen, Jacque; Bell, Alasdair F; Hedstrom, Lizbeth; Tonge, Peter J

    2003-04-22

    Triclosan, a common antibacterial additive used in consumer products, is an inhibitor of FabI, the enoyl reductase enzyme from type II bacterial fatty acid biosynthesis. In agreement with previous studies [Ward, W. H., Holdgate, G. A., Rowsell, S., McLean, E. G., Pauptit, R. A., Clayton, E., Nichols, W. W., Colls, J. G., Minshull, C. A., Jude, D. A., Mistry, A., Timms, D., Camble, R., Hales, N. J., Britton, C. J., and Taylor, I. W. (1999) Biochemistry 38, 12514-12525], we report here that triclosan is a slow, reversible, tight binding inhibitor of the FabI from Escherichia coli. Triclosan binds preferentially to the E.NAD(+) form of the wild-type enzyme with a K(1) value of 23 pM. In agreement with genetic selection experiments [McMurry, L. M., Oethinger, M., and Levy, S. B. (1998) Nature 394, 531-532], the affinity of triclosan for the FabI mutants G93V, M159T, and F203L is substantially reduced, binding preferentially to the E.NAD(+) forms of G93V, M159T, and F203L with K(1) values of 0.2 microM, 4 nM, and 0.9 nM, respectively. Triclosan binding to the E.NADH form of F203L can also be detected and is defined by a K(2) value of 51 nM. We have also characterized the Y156F and A197M mutants to compare and contrast the binding of triclosan to InhA, the homologous enoyl reductase from Mycobacterium tuberculosis. As observed for InhA, Y156F FabI has a decreased affinity for triclosan and the inhibitor binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3 and 30 nM, respectively. The replacement of A197 with Met has no impact on triclosan affinity, indicating that differences in the sequence of the conserved active site loop cannot explain the 10000-fold difference in affinities of FabI and InhA for triclosan.

  16. Molecular cloning and characterization of Polygalacturonase-Inhibiting Protein and Cinnamoyl-Coa Reductase genes and their association with fruit storage conditions in blueberry (Vaccinium corymbosum)

    KAUST Repository

    Khraiwesh, Basel

    2013-05-13

    Blueberry is a widely grown and easily perishable fruit crop. An efficient post-harvest handling is critical, and for that purpose gene technology methods have been part of ongoing programmes to improve crops with high food values such as blueberry. Here we report the isolation, cloning, characterization and differential expression levels of two cDNAs encoding Polygalacturonase-Inhibitor Protein (PGIP) and Cinnamoyl-Coa Reductase (CCR) from blueberry fruits in relation to various storage conditions. The open reading frame of PGIP and CCR encodes a polypeptide of 329 and 347 amino acids, respectively. To assess changes in the expression of blueberry PGIP and CCR after harvest, a storage trial was initiated. The northern blots hybridization showed a clear differential expression level of PGIP and CCR between freshly harvested and stored fruits as well as between fruits stored under various storage conditions. Although the prospects of exploiting such a strategy for crop improvement are limited, the results provide further insight into the control of the quality over the storage period at the molecular level.

  17. Identification of a novel polyfluorinated compound as a lead to inhibit the human enzymes aldose reductase and AKR1B10: structure determination of both ternary complexes and implications for drug design.

    Science.gov (United States)

    Cousido-Siah, Alexandra; Ruiz, Francesc X; Mitschler, André; Porté, Sergio; de Lera, Ángel R; Martín, María J; Manzanaro, Sonia; de la Fuente, Jesús A; Terwesten, Felix; Betz, Michael; Klebe, Gerhard; Farrés, Jaume; Parés, Xavier; Podjarny, Alberto

    2014-03-01

    Aldo-keto reductases (AKRs) are mostly monomeric enzymes which fold into a highly conserved (α/β)8 barrel, while their substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable external loops. The closely related human enzymes aldose reductase (AR or AKR1B1) and AKR1B10 are of biomedical interest because of their involvement in secondary diabetic complications (AR) and in cancer, e.g. hepatocellular carcinoma and smoking-related lung cancer (AKR1B10). After characterization of the IC50 values of both AKRs with a series of polyhalogenated compounds, 2,2',3,3',5,5',6,6'-octafluoro-4,4'-biphenyldiol (JF0064) was identified as a lead inhibitor of both enzymes with a new scaffold (a 1,1'-biphenyl-4,4'-diol). An ultrahigh-resolution X-ray structure of the AR-NADP(+)-JF0064 complex has been determined at 0.85 Å resolution, allowing it to be observed that JF0064 interacts with the catalytic residue Tyr48 through a negatively charged hydroxyl group (i.e. the acidic phenol). The non-competitive inhibition pattern observed for JF0064 with both enzymes suggests that this acidic hydroxyl group is also present in the case of AKR1B10. Moreover, the combination of surface lysine methylation and the introduction of K125R and V301L mutations enabled the determination of the X-ray crystallographic structure of the corresponding AKR1B10-NADP(+)-JF0064 complex. Comparison of the two structures has unveiled some important hints for subsequent structure-based drug-design efforts.

  18. Aqueous extracts of selenium-fertilized broccoli increase selenoprotein activity and inhibit DNA single-strand breaks, but decrease the activity of quinone reductase in Hepa 1c1c7 cells.

    Science.gov (United States)

    Keck, Anna-Sigrid; Finley, John W

    2006-05-01

    Depending on growth conditions, broccoli may be enriched in the isothiocyanate sulforaphane and/or the mineral selenium (Se); both compounds may play an important role in the reduction of intracellular oxidative stress and chronic disease prevention. Sulforaphane up-regulates transcription of Phase II detoxification proteins (e.g. quinone reductase [QR]), whereas Se is needed for the production of thioredoxin reductase (TR) and glutathione peroxidase-1 (GPx1), both of which exhibit antioxidant activity. The objective of the present study was to determine whether the fertilization of broccoli with Se increases the antioxidant ability of broccoli. Hydrogen peroxide-induced DNA single-strand breaks (measured by single cell electrophoresis, Comet assay) and activity of antioxidant enzymes (GPx, TR and QR) were measured in mouse hepatoma cells (Hepa 1c1c7 cells) treated with purified sulforaphane, sodium selenite or extracts of selenized broccoli. When supplied separately as chemically pure substances, sodium selenite was more effective than sulforaphane for reduction of single-strand breaks. Se-fertilized broccoli extracts were the most effective for reduction of DNA single-strand breaks, and extracts that contained 0.71 microM Se and 0.08 microM sulforaphane inhibited 94% of DNA single-strand breaks. A significant positive association (r = 0.81, p = 0.009) between GPx1 activity and inhibition of DNA single-strand breaks as well as a 24h lag time between addition of Se, sulforaphane or broccoli extract and inhibition of single-strand breaks suggests that some of the antioxidant protection is mediated through selenoproteins. Conversely, fertilization of broccoli with Se decreased the ability of broccoli extract to induce QR activity. These results demonstrate that Se and sulforaphane, alone or as a component of broccoli, may help decrease oxidative stress. They further suggest that Se is the most important for decreasing oxidative stress, but maximizing the Se content

  19. The effect of copper on human erythrocyte glutathione reductase

    NARCIS (Netherlands)

    Flikweert, J.P.; Hoorn, R.K.J.; Staal, Gerard E.J.

    1974-01-01

    1. 1. The influence of copper on purified human erythrocyte glutathione reductase (E.C. 1.6.4.2) was studied. The holoenzyme was inhibited at low oxidized glutathione (GSSG) concentrations. At a glutathione concentration of 1 mM and higher no inhibition at all was found. The inhibition was independe

  20. Quinone Reductase 2 Is a Catechol Quinone Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao (NYMEDCO)

    2008-09-05

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

  1. An inorganic complex that inhibits Mycobacterium tuberculosis enoyl reductase as a prototype of a new class of chemotherapeutic agents to treat tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Basso, Luiz A.; Schneider, Cristopher Z.; Santos, Anderson J.A.B. dos; Souto, Andre A.; Santos, Diogenes S., E-mail: luiz.basso@pucrs.b, E-mail: diogenes@pucrs.b [Pontificia Universidade Catolica do Rio Grande do Sul (PUCRS), Porto Alegre, RS (Brazil). Parque Tecnologico (Tecnopuc). Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF); Santos Junior, Andre A. dos; Campos, Maria M. [Pontificia Universidade Catolica do Rio Grande do Sul (PUCRS), Porto Alegre, RS (Brazil). Inst. Nacional de Ciencia e Tecnologia em Tuberculose (INCT-TB)

    2010-07-01

    Here we describe the inhibitory activity of IQG607, pentacyano(isoniazid)ferrate(II), on isoniazid-sensitive and isoniazid-resistant strains of Mycobacterium tuberculosis, its oral toxicity, and efforts to adapt IQG607 synthesis to large chemical reactors. IQG607 represents a promising chemotherapeutic agent aiming at the inhibition of a validated and druggable molecular target. (author)

  2. Characterization of human platelet glutathione reductase.

    Science.gov (United States)

    Moroff, G; Kosow, D P

    1978-12-08

    Glutathione reductase (NAD(P)h:oxidized glutathione oxidoreductase, EC 1.6.4.2) has been purified 1000-fold from the cytoplasmic fraction of human platelets. Salts, including the heretofore unreported effect of sodium citrate, activate the NADPH-dependent reduction of oxidized glutathione. Sodium citrate and monovalent salt activation appears to involve multiple sites having different binding affinities. At sub-saturating sodium phosphate, non-linear double reciprocal plots indicative of substrate activation by oxidized glutathione were observed. Initial velocity double reciprocal plots at sub-saturating and saturating concentrations of phosphate generate a family of converging lines. NADP+ is a partial inhibitor, indicating that the reduction of oxidized glutathione can proceed by more than one pathway. FMN, FAD, and riboflavin inhibit platelet glutathione reductase by influencing only the V while nitrofurantoin inhibition is associated with an increase Koxidized glutathione and a decreased V.

  3. Ethanol extract of Zhongtian hawthorn lowers serum cholesterol in mice by inhibiting transcription of 3-hydroxy-3-methylglutaryl-CoA reductase via nuclear factor-kappa B signal pathway.

    Science.gov (United States)

    Hu, Hai-Jie; Luo, Xue-Gang; Dong, Qing-Qing; Mu, Ai; Shi, Guo-Long; Wang, Qiu-Tong; Chen, Xiao-Ying; Zhou, Hao; Zhang, Tong-Cun; Pan, Li-Wen

    2016-03-01

    Hawthorn is a berry-like fruit from the species of Crataegus. In China, it has another more famous name, Shan-Zha, which has been used to improve digestion as a traditional Chinese medicine or food for thousands of years. Moreover, during the last decades, hawthorn has received more attention because of its potential to treat cardiovascular diseases. However, currently, only fruits of C. pinnatifida and C. pinnatifida var. major are included as Shan-Zha in the Chinese Pharmacopoeia. In this study, our results showed that the ethanol extract of Zhongtian hawthorn, a novel grafted cultivar of C. cuneata (wild Shan-Zha), could markedly reduce body weight and levels of serum total cholesterol, triglyceride, low-density lipoprotein cholesterol, and liver cholesterol of hyperlipidemia mice. It could suppress the stimulation effect of high-fat diet on the transcription of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and p65, and counteract the downregulation of CYP7A1 and LDLR. In addition, the results of luciferase reporter assay and Western blot showed that the transcriptional activity of HMGCR promoter was inhibited by Zhongtian hawthorn ethanol extract in a dose-dependent manner, while overexpression of p65 could reverse this transcriptional repression effect. These results suggested that Zhongtian hawthorn could provide health benefits by counteracting the high-fat diet-induced hypercholesteolemic and hyperlipidemic effects in vivo, and the mechanism underlying this event was mainly dependent on the suppressive effect of Zhongtian hawthorn ethanol extract on the transcription of HMGCR via nuclear factor-kappa B (NF-κB) signal pathway. Therefore, this novel cultivar of hawthorn cultivar which has much bigger fruits, early bearing, high yield, cold resistance, and drought resistance, might be considered as a good alternative to Shan-Zha and has great value in the food and medicine industry. In addition, to our best knowledge, this is also the first report that the

  4. Imine reductases (IREDs).

    Science.gov (United States)

    Mangas-Sanchez, Juan; France, Scott P; Montgomery, Sarah L; Aleku, Godwin A; Man, Henry; Sharma, Mahima; Ramsden, Jeremy I; Grogan, Gideon; Turner, Nicholas J

    2017-04-01

    Imine reductases (IREDs) have emerged as a valuable new set of biocatalysts for the asymmetric synthesis of optically active amines. The development of bioinformatics tools and searchable databases has led to the identification of a diverse range of new IRED biocatalysts that have been characterised and employed in different synthetic processes. This review describes the latest developments in the structural and mechanistic aspects of IREDs, together with synthetic applications of these enzymes, and identifies ongoing and future challenges in the field. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala)

    Science.gov (United States)

    Herrera-Juárez, Álvaro Miguel; Martínez-González, José de Jesús; del Arenal Mena, Irene Patricia; Flores-Herrera, Óscar

    2017-01-01

    A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life. PMID:28787021

  6. 荞麦芽的抗氧化作用及醛糖还原酶抑制作用的研究%Study on effects of antioxidant and aldose reductase inhibition of buckwheat sprouts

    Institute of Scientific and Technical Information of China (English)

    孙国娟; 李红梅; 李善姬; 林顺成; 崔承弼

    2012-01-01

    以萌发期为4,6,8,9,10 d的甜荞麦芽和苦荞麦芽(黑丰一号,坝上苦荞)为原料,采用高效液相色谱法(HPLC),探明荞麦芽在萌发过程中芦丁的变化趋势,同时观察荞麦芽的ABTS自由基的清除作用以及对大鼠晶状体醛糖还原酶(rAR)的抑制作用.结果表明:无论是甜荞还是苦荞,芦丁的含量都随发芽时间的延长而有所增加,同时黑丰一号苦荞对ABTS自由基的清除作用在萌发的第10天时达最大值(IC50=12.18 μg/mL),作用效果高于芦丁标准品(IC50=21.10 μg/mL), 但是低于对照物Trolox(IC 50 =3.89 μg/mL).坝上苦荞在萌发的第10天时对rAR抑制活性最大(IC 50=2.09 μg/mL),作用效果明显高于槲皮素(IC50=2.57 μg/mL)、芦丁标准品(IC50=4.90 μg/mL).所以萌发的荞麦芽均有一定的抗氧化作用,且对rAR有明显的抑制作用,可以作为醛糖还原酶抑制剂的良好来源.%The contents of rutin in the powders of buckwheat sprouts and tartary buckwheat sprouts ( Heifeng NO. 1, Bashang tartary buckwheat) germinated for 4, 6, 8, 9, 10 days were determined u-sing High-performance liquid chromatography (HPLC). Meanwhile, observed the scavenging effects of ABTS free radical and the inhibi-tive effects of aldose reductase on rat lens (rAR). The results indicated that regardless of buckwheat sprouts and tartary buckwheat sprouts, the contents of rutin were increased with increasing the germination time. When Heifeng NO. 1 germination l0thd reaches the maximum value (IC50 = 12. 18 μg/mL) to the ABTS free radical scavenging action, the function effects is higher than rutin (IC50 = 21. 10 μg/mL) , but less than Trolox ( IC50 = 3. 89 μg/mL). When Bashang tartary buckwheat germination lOthd reaches the maximum value (IC50 = 2.09 μg/mL) on rAR inhibitory activity, the reduction energy is higher than quercetin ( IC50 = 2. 57 μg/mL) and rutin (/C50 = 4. 90 μg/mL). Therefore the germination of buckwheat sprouts has certain antioxidant effects, and

  7. 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

    2014-01-01

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

  8. 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...

  9. Isolated menthone reductase and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L

    2013-04-23

    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  10. Mitochondrial Thioredoxin-Glutathione Reductase from Larval Taenia crassiceps (Cysticerci

    Directory of Open Access Journals (Sweden)

    Alberto Guevara-Flores

    2010-01-01

    Full Text Available Mitochondrial thioredoxin-glutathione reductase was purified from larval Taenia crassiceps (cysticerci. The preparation showed NADPH-dependent reductase activity with either thioredoxin or GSSG, and was able to perform thiol/disulfide exchange reactions. At 25∘C specific activities were 437  ±  27 mU mg-1 and 840  ±  49 mU mg-1 with thioredoxin and GSSG, respectively. Apparent Km values were 0.87  ±  0.04  μM, 41  ±  6  μM and 19  ±  10  μM for thioredoxin, GSSG and NADPH, respectively. Thioredoxin from eukaryotic sources was accepted as substrate. The enzyme reduced H2O2 in a NADPH-dependent manner, although with low catalytic efficiency. In the presence of thioredoxin, mitochondrial TGR showed a thioredoxin peroxidase-like activity. All disulfide reductase activities were inhibited by auranofin, suggesting mTGR is dependent on selenocysteine. The reductase activity with GSSG showed a higher dependence on temperature as compared with the DTNB reductase activity. The variation of the GSSG- and DTNB reductase activities on pH was dependent on the disulfide substrate. Like the cytosolic isoform, mTGR showed a hysteretic kinetic behavior at moderate or high GSSG concentrations, but it was less sensitive to calcium. The enzyme was able to protect glutamine synthetase from oxidative inactivation, suggesting that mTGR is competent to contend with oxidative stress.

  11. Inhibitory Activities of Phenolic Compounds Isolated from Adina rubella Leaves Against 5α-Reductase Associated with Benign Prostatic Hypertrophy

    National Research Council Canada - National Science Library

    Yin, Jun; Heo, Jun Hyeok; Hwang, Yoon Jeong; Le, Thi Tam; Lee, Min Won

    2016-01-01

    .... The current study evaluated the anti-oxidative and anti-inflammatory activities and 5α-reductase inhibition of isolated compounds of AR leaves to find a potential therapeutic agent for benign prostatic hypertrophy (BPH...

  12. Inhibitory Activities of Phenolic Compounds Isolated from Adina rubella Leaves Against 5[alpha]-Reductase Associated with Benign Prostatic Hypertrophy

    National Research Council Canada - National Science Library

    Jun Yin; Jun Hyeok Heo; Yoon Jeong Hwang; Thi Tam Le; Min Won Lee

    2016-01-01

    .... The current study evaluated the anti-oxidative and anti-inflammatory activities and 5[alpha]-reductase inhibition of isolated compounds of AR leaves to find a potential therapeutic agent for benign prostatic hypertrophy...

  13. Trametes versicolor carboxylate reductase uncovered

    OpenAIRE

    Winkler, Margit; Winkler, Christoph K.

    2016-01-01

    Abstract The first carboxylate reductase from Trametes versicolor was identified, cloned, and expressed in Escherichia coli. The enzyme reduces aromatic acids such as benzoic acid and derivatives, cinnamic acid, and 3-phenylpropanoic acid, but also aliphatic acids such as octanoic acid are reduced. Graphical abstract

  14. Inhibition of Prostaglandin Reductase 2, a Putative Oncogene Overexpressed in Human Pancreatic Adenocarcinoma, Induces Oxidative Stress-Mediated Cell Death Involving xCT and CTH Gene Expressions through 15-Keto-PGE2.

    Directory of Open Access Journals (Sweden)

    Emily Yun-Chia Chang

    Full Text Available Prostaglandin reductase 2 (PTGR2 is the enzyme that catalyzes 15-keto-PGE2, an endogenous PPARγ ligand, into 13,14-dihydro-15-keto-PGE2. Previously, we have reported a novel oncogenic role of PTGR2 in gastric cancer, where PTGR2 was discovered to modulate ROS-mediated cell death and tumor transformation. In the present study, we demonstrated the oncogenic potency of PTGR2 in pancreatic cancer. First, we observed that the majority of the human pancreatic ductal adenocarcinoma tissues was stained positive for PTGR2 expression but not in the adjacent normal parts. In vitro analyses showed that silencing of PTGR2 expression enhanced ROS production, suppressed pancreatic cell proliferation, and promoted cell death through increasing 15-keto-PGE2. Mechanistically, silencing of PTGR2 or addition of 15-keto-PGE2 suppressed the expressions of solute carrier family 7 member 11 (xCT and cystathionine gamma-lyase (CTH, two important providers of intracellular cysteine for the generation of glutathione (GSH, which is widely accepted as the first-line antioxidative defense. The oxidative stress-mediated cell death after silencing of PTGR2 or addition of 15-keto-PGE2 was further abolished after restoring intracellular GSH concentrations and cysteine supply by N-acetyl-L-cysteine and 2-Mercaptomethanol. Our data highlight the therapeutic potential of targeting PTGR2/15-keto-PGE2 for pancreatic cancer.

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

    Science.gov (United States)

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

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

  16. Nitrate Reductase: Properties and Regulation

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Nitrate Reductase (NR) is a rating-limit and key enzyme of nitrate assimilation in plants ,so ,NR activity is important for growth,development and the dry matter accumulation of plants. The regulation of NR activity appears to be rather complex and many studies have been devoted to the description of regulation and properties,but in this paper we focus on the properties and regulation of NR in higher plants.

  17. 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.

  18. 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.

  19. Study of HMG-CoA Reductase Inhibition Activity of the Hydrolyzed Product of Snakehead Fish (Channa striata) Skin Collagen with 50 kDa Collagenase from Bacillus licheniformis F11.4.

    Science.gov (United States)

    Virginia, Agnes; Rachmawati, Heni; Riani, Catur; Retnoningrum, Debbie S

    2016-01-01

    Bioactive peptides produced from enzymatic hydrolysis fibrous protein have been proven to have several biological activities. Previous study showed that the hydrolysis product of snakehead fish skin collagen with 26 kDa collagenase from Bacillus licheniformis F11.4 showed HMG-CoA (HMGR) inhibition activity. The aim of this research was to determine the ability of the hydrolysis product produced from snakehead fish skin collagen hydrolysed by 50 kDa collagenase from B. licheniformis F11.4 in inhibiting HMGR activity. Snakehead fish skin collagen was extracted using an acid method and collagenase was produced from B. licheniformis F11.4 using half-strength Luria Bertani (LB) medium containing 5% collagen. Crude collagenase was concentrated and fractionated using the DEAE Sephadex A-25 column eluted with increasing gradient concentrations of NaCl. Collagen, collagenase, and fractions were analyzed using SDS-PAGE and collagenolytic activity was analyzed by the zymography method. Collagenase with 50 kDa molecular weight presented in fraction one was used to hydrolyze the collagen. The reaction was done in 18 hours at 50°C. The hydrolysis product using 3.51 μg collagen and 9 ng collagenase showed 25.8% inhibition activity against pravastatin. This work shows for the first time that the hydrolysis product of snakehead fish skin collagen and 50 kDa collagenase from B. licheniformis F11.4 has potential as an anticholesterol agent.

  20. 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.

  1. Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular trichomes.

    Science.gov (United States)

    Ponnamperuma, K; Croteau, R

    1996-05-01

    Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2', 5'-adenosine diphosphate agarose. SDS-PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. The K(m) values for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 microM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (-)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH-cytochrome P450 reductase.

  2. X-ray structure of trypanothione reductase from Crithidia fasciculata at 2. 4- angstrom resolution

    Energy Technology Data Exchange (ETDEWEB)

    Kuriyan, J.; Xiangpeng Kong; Krishna, T.S.R.; Murgolo, N.J.; Field, H.; Cerami, A.; Henderson, G.B. (Rockefeller Univ., New York, NY (United States)); Sweet, R.M. (Brookhaven National Lab., Upton, NY (United States))

    1991-10-01

    Trypanosomes and related protozoan parasites lack glutathione reductase and possess instead a closely related enzyme that serves as the reductant of a bis(glutathione)-spermidien conjugate, trypanothione. The human and parasite enzymes have mutually exclusive substrate specificities, providing a route for the design of therapeutic agents by specific inhibition of the parasite enzyme. The authors report here the three-dimensional structure of trypanothione reductase from Crithidia fasciculata and show that it closely resembles the structure of human glutathione reductase. In particular, the core structure surrounding the catalytic machinery is almost identical in the two enzymes. However, significant differences are found at the substrate binding sites. A cluster of basic residues in glutathione reductase is replaced by neutral, hydrophobic, or acidic residues in trypanothione reductase, consistent with the nature of the spermidine linkage and the change in overall charge of the substrate from {minus}2 to +1, respectively. The binding site is more open in trypanothione reductase due to rotations of about 4{degree} in the domains that form in site, with relative shifts of as much as 2-3 {angstrom} in residues that can interact with potential inhibitors and complement previous modeling and mutagenesis studies on the two enzymes.

  3. Aldose reductase mediates retinal microglia activation

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-04-29

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

  4. Dimethyl Fumarate Induces Glutathione Recycling by Upregulation of Glutathione Reductase.

    Science.gov (United States)

    Hoffmann, Christina; Dietrich, Michael; Herrmann, Ann-Kathrin; Schacht, Teresa; Albrecht, Philipp; Methner, Axel

    2017-01-01

    Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate (DMF) is an effective oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. DMF activates the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) leading to increased synthesis of the major cellular antioxidant glutathione (GSH) and prominent neuroprotection in vitro. We previously demonstrated that DMF is capable of raising GSH levels even when glutathione synthesis is inhibited, suggesting enhanced GSH recycling. Here, we found that DMF indeed induces glutathione reductase (GSR), a homodimeric flavoprotein that catalyzes GSSG reduction to GSH by using NADPH as a reducing cofactor. Knockdown of GSR using a pool of E. coli RNase III-digested siRNAs or pharmacological inhibition of GSR, however, also induced the antioxidant response rendering it impossible to verify the suspected attenuation of DMF-mediated neuroprotection. However, in cystine-free medium, where GSH synthesis is abolished, pharmacological inhibition of GSR drastically reduced the effect of DMF on glutathione recycling. We conclude that DMF increases glutathione recycling through induction of glutathione reductase.

  5. Dimethyl Fumarate Induces Glutathione Recycling by Upregulation of Glutathione Reductase

    Science.gov (United States)

    Hoffmann, Christina; Dietrich, Michael; Herrmann, Ann-Kathrin; Schacht, Teresa

    2017-01-01

    Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate (DMF) is an effective oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. DMF activates the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) leading to increased synthesis of the major cellular antioxidant glutathione (GSH) and prominent neuroprotection in vitro. We previously demonstrated that DMF is capable of raising GSH levels even when glutathione synthesis is inhibited, suggesting enhanced GSH recycling. Here, we found that DMF indeed induces glutathione reductase (GSR), a homodimeric flavoprotein that catalyzes GSSG reduction to GSH by using NADPH as a reducing cofactor. Knockdown of GSR using a pool of E. coli RNase III-digested siRNAs or pharmacological inhibition of GSR, however, also induced the antioxidant response rendering it impossible to verify the suspected attenuation of DMF-mediated neuroprotection. However, in cystine-free medium, where GSH synthesis is abolished, pharmacological inhibition of GSR drastically reduced the effect of DMF on glutathione recycling. We conclude that DMF increases glutathione recycling through induction of glutathione reductase. PMID:28116039

  6. Dimethyl Fumarate Induces Glutathione Recycling by Upregulation of Glutathione Reductase

    Directory of Open Access Journals (Sweden)

    Christina Hoffmann

    2017-01-01

    Full Text Available Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate (DMF is an effective oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. DMF activates the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2 leading to increased synthesis of the major cellular antioxidant glutathione (GSH and prominent neuroprotection in vitro. We previously demonstrated that DMF is capable of raising GSH levels even when glutathione synthesis is inhibited, suggesting enhanced GSH recycling. Here, we found that DMF indeed induces glutathione reductase (GSR, a homodimeric flavoprotein that catalyzes GSSG reduction to GSH by using NADPH as a reducing cofactor. Knockdown of GSR using a pool of E. coli RNase III-digested siRNAs or pharmacological inhibition of GSR, however, also induced the antioxidant response rendering it impossible to verify the suspected attenuation of DMF-mediated neuroprotection. However, in cystine-free medium, where GSH synthesis is abolished, pharmacological inhibition of GSR drastically reduced the effect of DMF on glutathione recycling. We conclude that DMF increases glutathione recycling through induction of glutathione reductase.

  7. Phellinstatin, a new inhibitor of enoyl-ACP reductase produced by the medicinal fungus Phellinus linteus.

    Science.gov (United States)

    Cho, Jun-Young; Kwon, Yun-Ju; Sohn, Mi-Jin; Seok, Soon-Ja; Kim, Won-Gon

    2011-03-15

    A new trimeric hispidin derivative, phellinstatin, was isolated from a culture broth of the medicinal fungus Phellinus linteus and its structure was established by various spectral analysis. Phellinstatin strongly inhibited Staphylococcus aureus enoyl-ACP reductase with an IC(50) of 6 μM and also showed antibacterial activity against S. aureus and MRSA.

  8. 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

  9. The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain.

    Directory of Open Access Journals (Sweden)

    Hu Peng

    Full Text Available In addition to superoxide (O2.- generation from nitric oxide synthase (NOS oxygenase domain, a new O2.- generation site has been identified in the reductase domain of inducible NOS (iNOS and neuronal NOS (nNOS. Cysteine S-glutathionylation in eNOS reductase domain also induces O2.- generation from eNOS reductase domain. However, the characteristics and regulatory mechanism of the O2.- generation from NOS reductase domain remain unclear. We cloned and purified the wild type bovine eNOS (WT eNOS, a mutant of Serine 1179 replaced with aspartic acid eNOS (S1179D eNOS, which mimics the negative charge caused by phosphorylationand truncated eNOS reductase domain (eNOS RD. Both WT eNOS and S1179D eNOS generated significant amount of O2.- in the absence of BH4 and L-arginine. The capacity of O2.- generation from S1179D eNOS was significantly higher than that of WT eNOS (1.74:1. O2.- generation from both WT eNOS and S1179D eNOS were not completely inhibited by 100nM tetrahydrobiopterin(BH4. This BH4 un-inhibited O2.- generation from eNOS was blocked by 10mM flavoprotein inhibitor, diphenyleneiodonium (DPI. Purified eNOS reductase domain protein confirmed that this BH4 un-inhibited O2.- generation originates at the FMN or FAD/NADPH binding site of eNOS reductase domain. DEPMPO-OOH adduct EPR signals and NADPH consumptions analyses showed that O2.- generation from eNOS reductase domain was regulated by Serine 1179 phosphorylation and DPI, but not by L-arginine, BH4 or calmodulin (CaM. In addition to the heme center of eNOS oxygenase domain, we confirmed another O2.- generation site in the eNOS reductase domain and characterized its regulatory properties.

  10. A second target of benzamide riboside: dihydrofolate reductase.

    Science.gov (United States)

    Roussel, Breton; Johnson-Farley, Nadine; Kerrigan, John E; Scotto, Kathleen W; Banerjee, Debabrata; Felczak, Krzysztof; Pankiewicz, Krzysztof W; Gounder, Murugesan; Lin, HongXia; Abali, Emine Ercikan; Bertino, Joseph R

    2012-11-01

    Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

  11. [Progress in research of aldose reductase inhibitors in traditional medicinal herbs].

    Science.gov (United States)

    Feng, Chang-Gen; Zhang, Lin-Xia; Liu, Xia

    2005-10-01

    The traditional medicinal herbs are natural product, and have no obviously toxic action and side effect, and their resources are extensive. The adverse effects produced by aldose reductase inhibitors in traditional medicinal herbs are less than those from chemical synthesis and micro-organism, they can effectively prevent and delay diabetic complication, such as diabetic nephropathy, vasculopathy, retinopathy, peripheral neuropathy, and so on. They will have a wonderful respect. Flavonoid compounds and their derivates from traditional medicinal herbs are active inhibitors to aldose reductase, such as quercetin, silymarin, puerarin, baicalim, berberine and so on. In addition, some compound preparations show more strongly activity in inhibiting aldose reductase and degrading sorbitol contents, such as Shendan in traditional medicinal herbs being active inhibitors and Jianyi capsule, Jinmaitong composita, Liuwei Di-huang pill, et al. The progresses definite functions of treating diabetes complications have been reviewed.

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

    Science.gov (United States)

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

    2015-09-01

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

  13. Aldose reductases influence prostaglandin F2α levels and adipocyte differentiation in male mouse and human species.

    Science.gov (United States)

    Pastel, Emilie; Pointud, Jean-Christophe; Loubeau, Gaëlle; Dani, Christian; Slim, Karem; Martin, Gwenaëlle; Volat, Fanny; Sahut-Barnola, Isabelle; Val, Pierre; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie

    2015-05-01

    Aldose reductases (AKR1B) are widely expressed oxidoreductases whose physiological function remains elusive. Some isoforms are genuine prostaglandin F2α (PGF2α) synthases, suggesting they might influence adipose homeostasis because PGF2α inhibits adipogenesis. This was shown by Akr1b7 gene ablation in the mouse, which resulted in increased adiposity related to a lower PGF2α content in fat. Yet humans have no ortholog gene for Akr1b7, so the role of aldose reductases in human adipose homeostasis remains to be explored. We analyzed expression of genes encoding human and mouse aldose reductase isoforms in adipose tissues and differentiating adipocytes to assess conserved mechanisms regulating PGF2α synthesis and adipogenesis. The Akr1b3 gene encoded the most abundant isoform in mouse adipose tissue, whereas Akr1b7 encoded the only isoform enriched in the stromal vascular fraction. Most mouse aldose reductase gene expression peaked in early adipogenesis of 3T3-L1 cells and diminished with differentiation. In contrast with its mouse ortholog Akr1b3, AKR1B1 expression increased throughout differentiation of human multipotent adipose-derived stem cells, paralleling PGF2α release, whereas PGF2α receptor (FP) levels collapsed in early differentiation. Pharmacological inhibition of aldose reductase using Statil altered PGF2α production and enhanced human multipotent adipose-derived stem adipocyte differentiation. As expected, the adipogenic effects of Statil were counteracted by an FP agonist (cloprostenol). Thus, in both species aldose reductase-dependent PGF2α production could be important in early differentiation to restrict adipogenesis. PGF2α antiadipogenic signaling could then be toned down through the FP receptor or aldose reductases down-regulation in human and mouse cells, respectively. Our data suggest that aldose reductase inhibitors could have obesogenic potential.

  14. The aldo-keto reductase superfamily homepage.

    Science.gov (United States)

    Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

    2003-02-01

    The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

  15. 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.

  16. Asymmetric Ketone Reduction by Imine Reductases.

    Science.gov (United States)

    Lenz, Maike; Meisner, Jan; Quertinmont, Leann; Lutz, Stefan; Kästner, Johannes; Nestl, Bettina M

    2017-02-01

    The rapidly growing area of asymmetric imine reduction by imine reductases (IREDs) has provided alternative routes to chiral amines. Here we report the expansion of the reaction scope of IREDs by showing the stereoselective reduction of 2,2,2-trifluoroacetophenone. Assisted by an in silico analysis of energy barriers, we evaluated asymmetric hydrogenations of carbonyls and imines while considering the influence of substrate reactivity on the chemoselectivity of this novel class of reductases. We report the asymmetric reduction of C=N as well as C=O bonds catalysed by members of the IRED enzyme family. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Regulation of HMG-CoA reductase in MCF-7 cells by genistein, EPA, and DHA, alone and in combination with mevastatin.

    Science.gov (United States)

    Duncan, Robin E; El-Sohemy, Ahmed; Archer, Michael C

    2005-06-28

    We investigated the regulation of HMG-CoA reductase in MCF-7 human breast cancer cells by genistein, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). All three compounds down-regulated reductase activity, primarily through post-transcriptional effects. In mevastatin-treated cells, only genistein and DHA abrogated the induction of reductase activity caused by this competitive inhibitor. Diets rich in soy isoflavones and fish oils, therefore, may exert anti-cancer effects through the inhibition of mevalonate synthesis in the breast. Genistein and DHA, in particular, may augment the efficacy of statins, increasing the potential for use of these drugs in adjuvant therapy for breast cancer.

  18. Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases.

    Science.gov (United States)

    Gang, D R; Kasahara, H; Xia, Z Q; Vander Mijnsbrugge, K; Bauw, G; Boerjan, W; Van Montagu, M; Davin, L B; Lewis, N G

    1999-03-12

    Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.

  19. Go Green: The Antiinflammatory Effects of Biliverdin Reductase

    Directory of Open Access Journals (Sweden)

    Barbara eWegiel

    2012-03-01

    Full Text Available Biliverdin (BV has emerged as a cytoprotective and important anti-inflammatory molecule. Conversion of BV to bilirubin (BR is catalyzed by biliverdin reductase (BVR and is required for the downstream signaling and nuclear localization of BVR. Recent data by others and us make clear that BVR is a critical regulator of innate immune responses resulting from acute insult and injury and moreover, that a lack of BVR results in an enhanced pro-inflammatory phenotype. In macrophages, BVR is regulated by its substrate BV which leads to activation of the PI3K-Akt-IL10 axis and inhibition of TLR4 expression via direct binding of BVR to the TLR4 promoter. In this review, we will summarize recent findings on the role of BVR and the bile pigments in inflammation in context with its activity as an enzyme, receptor and transcriptional regulator.

  20. Mitochondrial fumarate reductase as a target of chemotherapy: from parasites to cancer cells.

    Science.gov (United States)

    Sakai, Chika; Tomitsuka, Eriko; Esumi, Hiroyasu; Harada, Shigeharu; Kita, Kiyoshi

    2012-05-01

    Recent research on respiratory chain of the parasitic helminth, Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system (fumarate respiration), which is composed of complex I (NADH-rhodoquinone reductase), rhodoquinone and complex II (rhodoquinol-fumarate reductase) plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts. The enzymes in these parasite-specific pathways are potential target for chemotherapy. We isolated a novel compound, nafuredin, from Aspergillus niger, which inhibits NADH-fumarate reductase in helminth mitochondria at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep indicating that mitochondrial complex I is a promising target for chemotherapy. In addition to complex I, complex II is a good target because its catalytic direction is reverse of succinate-ubiquionone reductase in the host complex II. Furthermore, we found atpenin and flutolanil strongly and specifically inhibit mitochondrial complex II. Interestingly, fumarate respiration was found not only in the parasites but also in some types of human cancer cells. Analysis of the mitochondria from the cancer cells identified an anthelminthic as a specific inhibitor of the fumarate respiration. Role of isoforms of human complex II in the hypoxic condition of cancer cells and fetal tissues is a challenge. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. HMG-CoA Reductase Inhibitors from Monascus-Fermented Rice

    Directory of Open Access Journals (Sweden)

    Xuemei Li

    2013-01-01

    Full Text Available Seven compounds were isolated from Monascus-fermented rice by column chromatography with silica gel and semiprep HPLC. Their structures were elucidated by extensive spectroscopic methods. All compounds displayed HMG-CoA reductase inhibitory potential, among them compound 7 exhibited strong inhibition with IC50 value comparable with lovastatin. In this study, two compounds (1 and 2 were obtained from natural source for the first time.

  2. Stereochemistry of Furfural Reduction by a Saccharomyces cerevisiae Aldehyde Reductase That Contributes to In Situ Furfural Detoxification

    Science.gov (United States)

    Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by the yeast, particularly when the carbon source is acid-treated lignocell...

  3. Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci.

    Science.gov (United States)

    Plancarte, Agustin; Nava, Gabriela

    2015-02-01

    Thioredoxin glutathione reductases (TGRs) (EC 1.8.1.9) were purified to homogeneity from the cytosolic (cTsTGR) and mitochondrial (mTsTGR) fractions of Taenia solium, the agent responsible for neurocysticercosis, one of the major central nervous system parasitic diseases in humans. TsTGRs had a relative molecular weight of 132,000, while the corresponding value per subunit obtained under denaturing conditions, was of 62,000. Specific activities for thioredoxin reductase and glutathione reductase substrates for both TGRs explored were in the range or lower than values obtained for other platyhelminths and mammalian TGRs. cTsTGR and mTsTGR also showed hydroperoxide reductase activity using hydroperoxide as substrate. Km(DTNB) and Kcat(DTNB) values for cTsTGR and mTsTGR (88 µM and 1.9 s(-1); 45 µM and 12.6 s(-1), respectively) and Km(GSSG) and Kcat(GSSG) values for cTsTGR and mTsTGR (6.3 µM and 0.96 s(-1); 4 µM and 1.62 s(-1), respectively) were similar to or lower than those reported for mammalian TGRs. Mass spectrometry analysis showed that 12 peptides from cTsTGR and seven from mTsTGR were a match for gi|29825896 thioredoxin glutathione reductase [Echinococcus granulosus], confirming that both enzymes are TGRs. Both T. solium TGRs were inhibited by the gold compound auranofin, a selective inhibitor of thiol-dependent flavoreductases (I₅₀ = 3.25, 2.29 nM for DTNB and GSSG substrates, respectively for cTsTGR; I₅₀ = 5.6, 25.4 nM for mTsTGR toward the same substrates in the described order). Glutathione reductase activity of cTsTGR and mTsTGR exhibited hysteretic behavior with moderate to high concentrations of GSSG; this result was not observed either with thioredoxin, DTNB or NADPH. However, the observed hysteretic kinetics was suppressed with increasing amounts of both parasitic TGRs. These data suggest the existence of an effective substitute which may account for the lack of the detoxification enzymes glutathione reductase

  4. Aldose reductase induced by hyperosmotic stress mediates cardiomyocyte apoptosis: differential effects of sorbitol and mannitol.

    Science.gov (United States)

    Galvez, Anita S; Ulloa, Juan Alberto; Chiong, Mario; Criollo, Alfredo; Eisner, Verónica; Barros, Luis Felipe; Lavandero, Sergio

    2003-10-03

    Cells adapt to hyperosmotic conditions by several mechanisms, including accumulation of sorbitol via induction of the polyol pathway. Failure to adapt to osmotic stress can result in apoptotic cell death. In the present study, we assessed the role of aldose reductase, the key enzyme of the polyol pathway, in cardiac myocyte apoptosis. Hyperosmotic stress, elicited by exposure of cultured rat cardiac myocytes to the nonpermeant solutes sorbitol and mannitol, caused identical cell shrinkage and adaptive hexose uptake stimulation. In contrast, only sorbitol induced the polyol pathway and triggered stress pathways as well as apoptosis-related signaling events. Sorbitol resulted in activation of the extracellular signal-regulated kinase (ERK), p54 c-Jun N-terminal kinase (JNK), and protein kinase B. Furthermore, sorbitol treatment resulting in induction and activation of aldose reductase, decreased expression of the antiapoptotic protein Bcl-xL, increased DNA fragmentation, and glutathione depletion. Apoptosis was attenuated by aldose reductase inhibition with zopolrestat and also by glutathione replenishment with N-acetylcysteine. In conclusion, our data show that hypertonic shrinkage of cardiac myocytes alone is not sufficient to induce cardiac myocyte apoptosis. Hyperosmolarity-induced cell death is sensitive to the nature of the osmolyte and requires induction of aldose reductase as well as a decrease in intracellular glutathione levels.

  5. Purification to Homogeneity and Characterization of a Novel Pseudomonas putida Chromate Reductase

    Science.gov (United States)

    Park, C. H.; Keyhan, M.; Wielinga, B.; Fendorf, S.; Matin, A.

    2000-01-01

    Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80°C and 5, respectively; and the Km was 374 μM, with a Vmax of 1.72 μmol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50°C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction. PMID:10788340

  6. Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Park, C.H.; Keyhan, M.; Wielinga, B.; Fendorf, S.; Matin, A.

    2000-05-01

    Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, the authors purified to homogeneity and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation, anion-exchange chromatography, chromatofocusing, and gel filtration. The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80 C and 5, respectively; and the K{sub m} was 374 {micro}M, with a V{sub max} of 1.72 {micro}mol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50 C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction.

  7. In silico docking studies of aldose reductase inhibitory activity of commercially available flavonoids

    Directory of Open Access Journals (Sweden)

    Arumugam Madeswaran

    2012-12-01

    Full Text Available The primary objective of this study was to investigate the aldose reductase inhibitory activity of flavonoids using in silico docking studies. In this perspective, flavonoids like biochanin, butein, esculatin, fisetin and herbacetin were selected. Epalrestat, a known aldose reductase inhibitor was used as the standard. In silico docking studies were carried out using AutoDock 4.2, based on the Lamarckian genetic algorithm principle. The results showed that all the selected flavonoids showed binding energy ranging between -9.33 kcal/mol to -7.23 kcal/mol when compared with that of the standard (-8.73 kcal/mol. Inhibition constant (144.13 µM to 4.98 µM and intermolecular energy (-11.42 kcal/mol to -7.83 kcal/mol of the flavonoids also coincide with the binding energy. All the selected flavonoids contributed aldose reductase inhibitory activity because of its structural properties. These molecular docking analyses could lead to the further development of potent aldose reductase inhibitors for the treatment of diabetes.

  8. Structural and docking studies of Leucaena leucocephala Cinnamoyl CoA reductase.

    Science.gov (United States)

    Prasad, Nirmal K; Vindal, Vaibhav; Kumar, Vikash; Kabra, Ashish; Phogat, Navneet; Kumar, Manoj

    2011-03-01

    Lignin, a major constituent of plant call wall, is a phenolic heteropolymer. It plays a major role in the development of plants and their defense mechanism against pathogens. Therefore Lignin biosynthesis is one of the critical metabolic pathways. In lignin biosynthesis, the Cinnamoyl CoA reductase is a key enzyme which catalyzes the first step in the pathway. Cinnamoyl CoA reductase provides the substrates which represent the main transitional molecules of lignin biosynthesis pathway, exhibits a high in vitro kinetic preference for feruloyl CoA. In present study, the three-dimensional model of cinnamoyl CoA reductase was constructed based on the crystal structure of Grape Dihydroflavonol 4-Reductase. Furthermore, the docking studies were performed to understand the substrate interactions to the active site of CCR. It showed that residues ARG51, ASN52, ASP54 and ASN58 were involved in substrate binding. We also suggest that residue ARG51 in CCR is the determinant residue in competitive inhibition of other substrates. This structural and docking information have prospective implications to understand the mechanism of CCR enzymatic reaction with feruloyl CoA, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well.

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

    Energy Technology Data Exchange (ETDEWEB)

    Higashi, Yasuhiro; Kutchan, Toni M.; Smith, Thomas J. (Danforth)

    2011-11-18

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

  10. Discovery of pinoresinol reductase genes in sphingomonads.

    Science.gov (United States)

    Fukuhara, Y; Kamimura, N; Nakajima, M; Hishiyama, S; Hara, H; Kasai, D; Tsuji, Y; Narita-Yamada, S; Nakamura, S; Katano, Y; Fujita, N; Katayama, Y; Fukuda, M; Kajita, S; Masai, E

    2013-01-10

    Bacterial genes for the degradation of major dilignols produced in lignifying xylem are expected to be useful tools for the structural modification of lignin in plants. For this purpose, we isolated pinZ involved in the conversion of pinoresinol from Sphingobium sp. strain SYK-6. pinZ showed 43-77% identity at amino acid level with bacterial NmrA-like proteins of unknown function, a subgroup of atypical short chain dehydrogenases/reductases, but revealed only 15-21% identity with plant pinoresinol/lariciresinol reductases. PinZ completely converted racemic pinoresinol to lariciresinol, showing a specific activity of 46±3 U/mg in the presence of NADPH at 30°C. In contrast, the activity for lariciresinol was negligible. This substrate preference is similar to a pinoresinol reductase, AtPrR1, of Arabidopsis thaliana; however, the specific activity of PinZ toward (±)-pinoresinol was significantly higher than that of AtPrR1. The role of pinZ and a pinZ ortholog of Novosphingobium aromaticivorans DSM 12444 were also characterized.

  11. Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules.

    Science.gov (United States)

    Horchani, Faouzi; Prévot, Marianne; Boscari, Alexandre; Evangelisti, Edouard; Meilhoc, Eliane; Bruand, Claude; Raymond, Philippe; Boncompagni, Eric; Aschi-Smiti, Samira; Puppo, Alain; Brouquisse, Renaud

    2011-02-01

    Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.

  12. Cadmium and vanadate oligomers effects on methaemoglobin reductase activity from Lusitanian toadfish: in vivo and in vitro studies.

    Science.gov (United States)

    Soares, S S; Aureliano, M; Joaquim, N; Coucelo, J M

    2003-03-01

    Cadmium and two vanadate solutions as 'metavanadate' (containing ortho and metavanadate species) and 'decavanadate' (containing decameric species) (5 mM) were injected intraperitoneously in Halobatrachus didactylus (Lusitanian toadfish), in order to evaluate the effects of cadmium and oligomeric vanadate species on methaemoglobin reductase activity from fish red blood cells. Following short-term exposure (1 and 7 days), different changes were observed on enzyme activity. After 7 days of exposure, 'metavanadate' increased methaemoglobin reductase activity by 67% (P < 0.05), whereas, minor effects were observed on enzymatic activity upon cadmium and 'decavanadate' administration. However, in vitro studies indicate that decameric vanadate, in concentrations as low as 50 microM, besides strongly inhibiting methaemoglobin reductase activity, promotes haemoglobin oxidation to methaemoglobin. Although decameric vanadate species showed to be unstable in the different media used in this work, the rate of decameric vanadate deoligomerization is in general slow enough, making it possible to study its effects. It is concluded that the increase in H. didactylus methaemoglobin reductase activity is more pronounced upon exposition to 'metavanadate' than to cadmium and decameric species. Moreover, only decameric vanadate species promoted haemoglobin oxidation, suggesting that vanadate speciation is important to evaluate in vivo and in vitro effects on methaemoglobin reductase activity.

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

    Directory of Open Access Journals (Sweden)

    Shamala Salvamani

    2016-01-01

    Full Text Available Inflammation and oxidative stress are believed to contribute to the pathology of several chronic diseases including hypercholesterolemia (elevated levels of cholesterol in blood and atherosclerosis. HMG-CoA reductase inhibitors of plant origin are needed as synthetic drugs, such as statins, which are known to cause adverse effects on the liver and muscles. Amaranthus viridis (A. viridis has been used from ancient times for its supposedly medically beneficial properties. In the current study, different parts of A. viridis (leaf, stem, and seed were evaluated for potential anti-HMG-CoA reductase, antioxidant, and anti-inflammatory activities. The putative HMG-CoA reductase inhibitory activity of A. viridis extracts at different concentrations was determined spectrophotometrically by NADPH oxidation, using HMG-CoA as substrate. A. viridis leaf extract revealed the highest HMG-CoA reductase inhibitory effect at about 71%, with noncompetitive inhibition in Lineweaver-Burk plot analysis. The leaf extract showed good inhibition of hydroperoxides, 2,2-diphenyl-1-picrylhydrazyl (DPPH, nitric oxide (NO, and ferric ion radicals in various concentrations. A. viridis leaf extract was proven to be an effective inhibitor of hyaluronidase, lipoxygenase, and xanthine oxidase enzymes. The experimental data suggest that A. viridis leaf extract is a source of potent antioxidant and anti-inflammatory agent and may modulate cholesterol metabolism by inhibition of HMG-CoA reductase.

  14. Effect of mercury, cadmium, nickel, chromium and zinc on kinetic properties of NADPH-cytochrome P450 reductase purified from leaping mullet (Liza saliens).

    Science.gov (United States)

    Bozcaarmutlu, Azra; Arinç, Emel

    2007-04-01

    Information on the mechanism of metal ion inhibition of NADPH-cytochrome P450 reductase is limited. The purpose of the present paper was to elucidate in vitro effect of Hg(+2), Cd(+2), Ni(+2), Cr(+3) and Zn(+2) ions on the purified mullet NADPH-cytochrome P450 reductase. NADPH-cytochrome P450 reductase was purified from detergent-solubilized liver microsomes from leaping mullet (Liza saliens). All of the metal ions caused inhibition of the enzyme activity except Zn(+2). At 50 microM metal concentration, Hg(+2) inhibited the cytochrome P450 reductase activity completely (100%), while, at the same concentrations, Cd(+2), Cr(+3) and Ni(+2) caused 66%, 65% and 37% inhibition, respectively. At 50 microM metal concentration, Zn(+2) had no apparent effect on cytochrome P450 reductase activity. The IC(50) values of HgCl(2), CrCl(3), CdCl(2) and NiCl(2) were estimated to be 0.07 microM, 24 microM, 33 microM and 143 microM, respectively. Of the metal ions tested, Hg(+2) exhibited much higher inhibitory effect at lower concentrations, so it was evidently a more potent inhibitor than the others. All four metal ions displayed noncompetitive type of inhibition mechanism for the purified reductase as analyzed by Dixon plot. K(i) values of Hg(+2), Cr(+3), Cd(+2), and Ni(+2) were calculated from Dixon plots as 0.048 microM, 18 microM, 73 microM and 329 microM, respectively.

  15. Two mutations of dihydropteridine reductase deficiency.

    Science.gov (United States)

    Ponzone, A; Guardamagna, O; Ferraris, S; Bracco, G; Niederwieser, A; Cotton, R G

    1988-02-01

    Two patients with dihydropteridine reductase (DHPR) deficiency, in one case due to the absence of any enzyme protein (DHPR- cross reactive material (CRM)-) and in the other case due to the production of a mutant type devoid of catalytic activity (DHPR- CRM+) were examined. This latter form of malignant phenylketonuria, whose relative frequency seems to be higher in the Italian population, possibly has a worse prognosis. The earlier onset and the greater severity of clinical symptoms are associated with a more pronounced hydroxylation defect, as shown by higher degree of neonatal hyperphenylalaninaemia, unresponsiveness to an oral tetrahydrobiopterin load, lower concentrations of neurotransmitter metabolites, and reduced tyrosine production after an oral phenylalanine load.

  16. Statins: 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors demonstrate anti-atherosclerotic character due to their antioxidant capacity.

    Science.gov (United States)

    Puttananjaiah, Mohan-Kumari H; Dhale, Mohan Appasaheb; Gaonkar, Vaishali; Keni, Shradha

    2011-01-01

    Atherosclerosis is a chronic inflammatory disease of multiple etiologies. It is associated with the accumulation of oxidized lipids in arterial lesions leading to coronary heart disease. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (commonly known as statins) are widely used in cardiovascular disease prevention to lower the cholesterol. The antioxidant activity of HMG-CoA reductase inhibitors was studied by lipid peroxidation inhibition assay, DPPH, and hydroxyl radical scavenging-activity methods. The lovastatin (93%) and simvastatin (96%) showed significant action of lipid peroxidation inhibition compared to other HMG-CoA reductase inhibitors. The DPPH radical and hydroxyl radical scavenging activity of simvastatin was 38% and 33%, respectively. The oxidative modification of serum lipid due to reactive oxygen species causes atherosclerosis. This study revealed the importance of lovastatin and simvastatin to prevent oxidative stress-related cardiovascular diseases.

  17. Enzyme toolbox: novel enantiocomplementary imine reductases.

    Science.gov (United States)

    Scheller, Philipp N; Fademrecht, Silvia; Hofelzer, Sebastian; Pleiss, Jürgen; Leipold, Friedemann; Turner, Nicholas J; Nestl, Bettina M; Hauer, Bernhard

    2014-10-13

    Reducing reactions are among the most useful transformations for the generation of chiral compounds in the fine-chemical industry. Because of their exquisite selectivities, enzymatic approaches have emerged as the method of choice for the reduction of C=O and activated C=C bonds. However, stereoselective enzymatic reduction of C=N bonds is still in its infancy-it was only recently described after the discovery of enzymes capable of imine reduction. In our work, we increased the spectrum of imine-reducing enzymes by database analysis. By combining the currently available knowledge about the function of imine reductases with the experimentally uncharacterized diversity stored in protein sequence databases, three novel imine reductases with complementary enantiopreference were identified along with amino acids important for catalysis. Furthermore, their reducing capability was demonstrated by the reduction of the pharmaceutically relevant prochiral imine 2-methylpyrroline. These novel enzymes exhibited comparable to higher catalytic efficiencies than previously described enzymes, and their biosynthetic potential is highlighted by the full conversion of 2-methylpyrroline in whole cells with excellent selectivities. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

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

    2014-01-01

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

  19. Iron-mediated effects on nitrate reductase in marine phytoplankton

    NARCIS (Netherlands)

    Timmermans, K.R.; Stolte, W.; Baar, H.J.W. de

    1994-01-01

    The potential activity of nitrate reductase was determined in uni-algal cultures in the laboratory and in natural marine phytoplankton assemblages. In the laboratory bioassays, distinct differences in nitrate reductase activity were observed in iron replete versus depleted cultures for Emiliania hux

  20. 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.

  1. Identification of thioredoxin glutathione reductase inhibitors that kill cestode and trematode parasites.

    Science.gov (United States)

    Ross, Fabiana; Hernández, Paola; Porcal, Williams; López, Gloria V; Cerecetto, Hugo; González, Mercedes; Basika, Tatiana; Carmona, Carlos; Fló, Martín; Maggioli, Gabriela; Bonilla, Mariana; Gladyshev, Vadim N; Boiani, Mariana; Salinas, Gustavo

    2012-01-01

    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.

  2. The Inhibitory Effect of Prunella vulgaris L. on Aldose Reductase and Protein Glycation

    Directory of Open Access Journals (Sweden)

    Hong Mei Li

    2012-01-01

    Full Text Available To evaluate the aldose reductase (AR enzyme inhibitory ability of Prunella vulgaris L. extract, six compounds were isolated and tested for their effects. The components were subjected to in vitro bioassays to investigate their inhibitory assays using rat lens aldose reductase (rAR and human recombinant AR (rhAR. Among them, caffeic acid ethylene ester showed the potent inhibition, with the IC50 values of rAR and rhAR at 3.2±0.55 μM and 12.58±0.32 μM, respectively. In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/concentration of substrate, this compound showed noncompetitive inhibition against rhAR. Furthermore, it inhibited galactitol formation in a rat lens incubated with a high concentration of galactose. Also it has antioxidative as well as advanced glycation end products (AGEs inhibitory effects. As a result, this compound could be offered as a leading compound for further study as a new natural products drug for diabetic complications.

  3. 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).

  4. 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.

  5. Insights into Enzyme Catalysis and Thyroid Hormone Regulation of Cerebral Ketimine Reductase/μ-Crystallin Under Physiological Conditions.

    Science.gov (United States)

    Hallen, André; Cooper, Arthur J L; Jamie, Joanne F; Karuso, Peter

    2015-06-01

    Mammalian ketimine reductase is identical to μ-crystallin (CRYM)-a protein that is also an important thyroid hormone binding protein. This dual functionality implies a role for thyroid hormones in ketimine reductase regulation and also a reciprocal role for enzyme catalysis in thyroid hormone bioavailability. In this research we demonstrate potent sub-nanomolar inhibition of enzyme catalysis at neutral pH by the thyroid hormones L-thyroxine and 3,5,3'-triiodothyronine, whereas other thyroid hormone analogues were shown to be far weaker inhibitors. We also investigated (a) enzyme inhibition by the substrate analogues pyrrole-2-carboxylate, 4,5-dibromopyrrole-2-carboxylate and picolinate, and (b) enzyme catalysis at neutral pH of the cyclic ketimines S-(2-aminoethyl)-L-cysteine ketimine (owing to the complex nomenclature trivial names are used for the sulfur-containing cyclic ketimines as per the original authors' descriptions) (AECK), Δ(1)-piperideine-2-carboxylate (P2C), Δ(1)-pyrroline-2-carboxylate (Pyr2C) and Δ(2)-thiazoline-2-carboxylate. Kinetic data obtained at neutral pH suggests that ketimine reductase/CRYM plays a major role as a P2C/Pyr2C reductase and that AECK is not a major substrate at this pH. Thus, ketimine reductase is a key enzyme in the pipecolate pathway, which is the main lysine degradation pathway in the brain. In silico docking of various ligands into the active site of the X-ray structure of the enzyme suggests an unusual catalytic mechanism involving an arginine residue as a proton donor. Given the critical importance of thyroid hormones in brain function this research further expands on our knowledge of the connection between amino acid metabolism and regulation of thyroid hormone levels.

  6. Disruption of narH, narJ, and moaE Inhibits Heterotrophic Nitrification in Pseudomonas Strain M19

    OpenAIRE

    2002-01-01

    Interruptions in three nitrate reductase-related genes, narH, narJ, and moaE, inhibited heterotrophic nitrification in Pseudomonas strain M19. No nitrate was detected in the medium, and nitrification proceeded in the presence of a nitrate reductase inhibitor. Heterotrophic nitrification was greatly stimulated by the addition of nitrate.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Ke-Wu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Li, Jun [Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029 (China); Dong, Xin; Wang, Ying-Hong; Ma, Zhi-Zhong; Jiang, Yong; Jin, Hong-Wei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Tu, Peng-Fei, E-mail: pengfeitu@vip.163.com [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029 (China)

    2013-11-15

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

  8. YqhD. A broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Jarboe, Laura R. [Iowa State Univ., Ames, IA (United States). Dept. of Chemical and Biological Engineering

    2011-01-15

    The Escherichia coli NADPH-dependent aldehyde reductase YqhD has contributed to a variety of metabolic engineering projects for production of biorenewable fuels and chemicals. As a scavenger of toxic aldehydes produced by lipid peroxidation, YqhD has reductase activity for a broad range of short-chain aldehydes, including butyraldehyde, glyceraldehyde, malondialdehyde, isobutyraldehyde, methylglyoxal, propanealdehyde, acrolein, furfural, glyoxal, 3-hydroxypropionaldehyde, glycolaldehyde, acetaldehyde, and acetol. This reductase activity has proven useful for the production of biorenewable fuels and chemicals, such as isobutanol and 1,3- and 1,2-propanediol; additional capability exists for production of 1-butanol, 1-propanol, and allyl alcohol. A drawback of this reductase activity is the diversion of valuable NADPH away from biosynthesis. This YqhD-mediated NADPH depletion provides sufficient burden to contribute to growth inhibition by furfural and 5-hydroxymethyl furfural, inhibitory contaminants of biomass hydrolysate. The structure of YqhD has been characterized, with identification of a Zn atom in the active site. Directed engineering efforts have improved utilization of 3-hydroxypropionaldehyde and NADPH. Most recently, two independent projects have demonstrated regulation of yqhD by YqhC, where YqhC appears to function as an aldehyde sensor. (orig.)

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

    Directory of Open Access Journals (Sweden)

    Danièle Klett

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

  10. A 5α-reductase inhibitor, finasteride, increases differentiation and proliferation of embryonal carcinoma cell-derived-neural cells.

    Science.gov (United States)

    Shoae-Hassani, Alireza; Sharif, Shiva; Verdi, Javad

    2011-01-01

    Recent advances in stem cell biology have resulted in identifying new agents to differentiate stem cell-derived-neural cells. Different stem cell types have been shown to differentiate into neural cells. It has been shown that P19 line of embryonal carcinoma cells develops into neurons and astroglia after exposure to some hormones such as dehydroepiandrosterone (DHEA). Steroid 5α-reductase is a key enzyme in the conversion of several Δ4-3 keto steroids, such as testosterone into their respective 5α-reductase derivatives. Finasteride is a 5α-reductase inhibitor that inhibits conversion of testosterone to the more potent androgen dihydrotestosterone (DHT). Reduction in DHT and sustaining testosterone levels has an important impact on differentiation and proliferation of embryonal carcinoma cells to neural cells. We hypothesize that finasteride, a 5α-reductase inhibitor, will be differentiate embryonal carcinoma cell to the neural cell and increase their proliferation due to the elevation levels of testosterone, a neuroprotective neurosteroid. Copyright © 2010. Published by Elsevier Ltd.

  11. Trypanosoma cruzi trypanothione reductase inhibitors: phenothiazines and related compounds modify experimental Chagas' disease evolution.

    Science.gov (United States)

    Rivarola, H W; Paglini-Oliva, P A

    2002-06-01

    Chagas' disease affects about 18 million people and 25% of the population of Latin America is at risk of acquiring Chagas' disease. The chemotherapy of Chagas' disease is still an open field and remains as an unsolved problem. Nifurtimox and benznidazole are currently used to treat this disease, however, both drugs have high toxicity and are mutagenic with the result that the patients frequently fail to follow treatment. T. cruzi enzimes such as trypanothione reductase, represent potential drug targets because they play an essential role in the life of this organism. This enzyme has been isolated, purified and studied by X ray crystallography. Phenothiazines and related compounds inhibit trypanothione reductase and a specially favoured fit is a phenothiazine with a 2- substitued with 2- chloro and 2- trifluoromethyl with a remote hydrophobic patch. The essential phenothiazine nucleus can adopt more than one inhibitory orientation in its binding site. Phenothiazines and related compounds are drugs used in psychiatric treatments. These anti-depressants inhibit trypanothione reductase through the peroxidase/ H2O2/ system, and also exert other trypanocidal effects upon epimastigotes and tripomastigotes forms: clomipramine through an anticalmodulin action; trifluopherazine and thioridazine induced disruption of mitochondria and prometazine provoked serious cell membrane disorganization. Clomipramine and thioridazine were also effective in treatment of mice with experimental Chagas' disease, significantly modifying the natural evolution of the infection; cardiac function and survival of infected and treated animals were not different from non infected animals. Phenothiazines and related compounds are promising trypanocidal agents for treatment of Chagas' disease. Other trypanocidal agents as nifurtimox, benznidazol,Allopurinol, cystein protease inhibitors and others, are also discussed.

  12. Biomarkers of adverse response to mercury: histopathology versus thioredoxin reductase activity.

    Science.gov (United States)

    Branco, Vasco; Ramos, Paula; Canário, João; Lu, Jun; Holmgren, Arne; Carvalho, Cristina

    2012-01-01

    Exposure to mercury is normally assessed by measuring its accumulation in hair, blood or urine. Currently, the biomarkers of effect that have been proposed for mercurials, such as coproporphyrines or oxidative stress markers, are not sensitive enough and lack specificity. Selenium and selenoproteins are important targets for mercury and thioredoxin reductase (TrxR) in particular was shown to be very sensitive to mercury compounds both in vitro and in vivo. In this study we looked into the relation between the inhibition of thioredoxin reductase (TrxR) activity and histopathological changes caused by exposure to mercurials. Juvenile zeabra-seabreams were exposed to Hg(2+) or MeHg for 28 days and histopathological changes were analyzed in the liver and kidney as well as TrxR activity. Both mercurials caused histopathological changes in liver and kidney, albeit Hg(2+) caused more extensive and severe lesions. Likewise, both mercurials decreased TrxR activity, being Hg(2+) a stronger inhibitor. Co-exposure to Hg(2+) and Se fully prevented TrxR inhibition in the liver and reduced the severity of lesions in the organ. These results show that upon exposure to mercurials, histopathological alterations correlate with the level of TrxR activity and point to the potential use of this enzyme as a biomarker of mercury toxicity.

  13. Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

    DEFF Research Database (Denmark)

    Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie

    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...

  14. Cyclic Voltammetric Responses of Nitrate Reductase on Chemical Modified Electrodes

    Institute of Scientific and Technical Information of China (English)

    YaRuSONG; HuiBoSHAO; 等

    2002-01-01

    Electrochemistry of nitrate reductases (NR) incorporated into 2-aminoethanethiol self-assembled on the gold electrode and polyacrylamide cast on the pyrolytic graphite electrode was examined. NR on chemical modified electrode showed electrochemical cyclic voltammetric responses in phosphate buffers.

  15. Enantioselective imine reduction catalyzed by imine reductases and artificial metalloenzymes.

    Science.gov (United States)

    Gamenara, Daniela; Domínguez de María, Pablo

    2014-05-21

    Adding value to organic synthesis. Novel imine reductases enable the enantioselective reduction of imines to afford optically active amines. Likewise, novel bioinspired artificial metalloenzymes can perform the same reaction as well. Emerging proof-of-concepts are herein discussed.

  16. The dependence of nitrate reductase activity on the level of soluble sugars in wheat and cucumber roots growing in the presence of simazine, in light or in darkness

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-01-01

    Full Text Available At concentrations of 25 and 5 µM, simazine inhibited nitrate reductase activity in wheat and cucumber roots, respectively. It also lowered the content of soluble sugars and decreased the activities of NADH malate dehydrogenase and NADP+ glucose-6-phosphate dehydrogenase. The inclusion of 50 mM glucose into the medium partially reversed the inhibitory effect of simazine on the activity of nitrate reductase in cucumber roots and slightly increased the activity of this enzyme in wheat roots These results suggest a complex influence of the herbicide on the activity of nitrate reductase: simazine lowers the level of soluble sugars in roots and decreases the activity of the dehydrogenases supplying the reduced nucleotides indispensable for reduction of nitrates.

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

    Science.gov (United States)

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

    1996-07-01

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

  18. Distribution of Prx-linked hydroperoxide reductase activity among microorganisms.

    Science.gov (United States)

    Takeda, Kouji; Nishiyama, Yoshitaka; Yoda, Koji; Watanabe, Toshihiro; Nimura-Matsune, Kaori; Mura, Kiyoshi; Tokue, Chiyoko; Katoh, Tetzuya; Kawasaki, Shinji; Niimura, Youichi

    2004-01-01

    Peroxiredoxin (Prx) constitutes a large family of enzymes found in microorganisms, animals, and plants, but the detection of the activities of Prx-linked hydroperoxide reductases (peroxiredoxin reductases) in cell extracts, and the purification based on peroxide reductase activity, have only been done in bacteria and Trypanosomatidae. A peroxiredoxin reductase (NADH oxidase) from a bacterium, Amphibacillus, displayed only poor activities in the presence of purified Prx from Saccharomyces or Synechocystis, while it is highly active in the presence of bacterial Prx. These results suggested that an enzyme system different from that in bacteria might exist for the reduction of Prx in yeast and cyanobacteria. Prx-linked hydroperoxide reductase activities were detected in cell extracts of Saccharomyces, Synechocystis, and Chlorella, and the enzyme activities of Saccharomyces and Chlorella were induced under vigorously aerated culture conditions and intensive light exposure conditions, respectively. Partial purification of Prx-linked peroxidase from the induced yeast cells indicated that the Prx-linked peroxidase system consists of two protein components, namely, thioredoxin and thioredoxin reductase. This finding is consistent with the previous report on its purification based on its protein protection activity against oxidation [Chae et al., J. Biol. Chem., 269, 27670-27678 (1994)]. In this study we have confirmed that Prx-linked peroxidase activity are widely distributed, not only in bacteria species and Trypanosomatidae, but also in yeast and photosynthetic microorganisms, and showed reconstitution of the activity from partially purified interspecies components.

  19. Dimethoxycurcumin, a metabolically stable analogue of curcumin enhances the radiosensitivity of cancer cells: Possible involvement of ROS and thioredoxin reductase

    Energy Technology Data Exchange (ETDEWEB)

    Jayakumar, Sundarraj; Patwardhan, R.S.; Pal, Debojyoti [Radiation Biology & Health Sciences Division, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Sharma, Deepak [Radiation Biology & Health Sciences Division, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Homi Bhabha National Institute, Anushaktinagar, Mumbai 400094 (India); Sandur, Santosh K., E-mail: sskumar@barc.gov.in [Radiation Biology & Health Sciences Division, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Homi Bhabha National Institute, Anushaktinagar, Mumbai 400094 (India)

    2016-09-09

    Dimethoxycurcumin (DIMC), a structural analogue of curcumin, has been shown to have more stability, bioavailability, and effectiveness than its parent molecule curcumin. In this paper the radiosensitizing effect of DIMC has been investigated in A549 lung cancer cells. As compared to its parent molecule curcumin, DIMC showed a very potent radiosensitizing effect as seen by clonogenic survival assay. DIMC in combination with radiation significantly increased the apoptosis and mitotic death in A549 cells. This combinatorial treatment also lead to effective elimination of cancer stem cells. Further, there was a significant increase in cellular ROS, decrease in GSH to GSSG ratio and also significant slowdown in DNA repair when DIMC was combined with radiation. In silico docking studies and in vitro studies showed inhibition of thioredoxin reductase enzyme by DIMC. Overexpression of thioredoxin lead to the abrogation of radiosensitizing effect of DIMC underscoring the role of thioredoxin reductase in radiosensitization. Our results clearly demonstrate that DIMC can synergistically enhance the cancer cell killing when combined with radiation by targeting thioredoxin system. - Highlights: • DIMC enhances radiosensitivity of cancer cells by inducing cell death. • DIMC with radiation disrupted the cellular redox and targeted cancer stem cells. • DNA repair is hampered when cells are treated with DIMC. • DIMC inhibited thioredoxin reductase in cancer cells.

  20. The Cholesterol-Lowering Effect of Alisol Acetates Based on HMG-CoA Reductase and Its Molecular Mechanism

    Directory of Open Access Journals (Sweden)

    Fei Xu

    2016-01-01

    Full Text Available This study measured the impact of alisol B 23-acetate and alisol A 24-acetate, the main active ingredients of the traditional Chinese medicine Alismatis rhizoma, on total cholesterol (TC, triglyceride (TG, high density lipoprotein-cholesterol (HDL-C, and low density lipoprotein-cholesterol (LDL-C levels of hyperlipidemic mice. The binding of alisol B 23-acetate and alisol A 24-acetate to the key enzyme involved in the metabolism of TC, 3-hydroxy-3-methylglutary-coenzyme A (HMG-CoA reductase, was studied using the reagent kit method and the western blotting technique combined with a molecular simulation technique. According to the results, alisol acetates significantly lower the TC, TG, and LDL-C concentrations of hyperlipidemic mice, while raising HDL-C concentrations. Alisol acetates lower HMG-CoA reductase activity in a dose-dependent fashion, both in vivo and in vitro. Neither of these alisol acetates significantly lower the protein expression of HMG-CoA. This suggests that alisol acetates lower the TC level via inhibiting the activity of HMG-CoA reductase by its prototype drug, which may exhibit an inhibition effect via directly and competitively binding to HMG-CoA. The side chain of the alisol acetate was the steering group via molecular simulation.

  1. 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.

  2. Aldose reductase, oxidative stress and diabetic mellitus

    Directory of Open Access Journals (Sweden)

    Waiho eTang

    2012-05-01

    Full Text Available Diabetes mellitus (DM is a complex metabolic disorder arising from lack of insulin production or insulin resistance 1. DM is a leading cause of morbidity and mortality in the developed world, particularly from vascular complications such as atherothrombosis in the coronary vessels. Aldose reductase (AR [ALR2; EC 1.1.1.21], a key enzyme in the polyol pathway, catalyzes NADPH-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS in various tissues of DM including the heart, vasculature, neurons, eyes and kidneys. As an example, hyperglycemia through such polyol pathway induced oxidative stress, may have dual heart actions, on coronary blood vessel (atherothrombosis and myocardium (heart failure leading to severe morbidity and mortality (reviewed in 2. In cells cultured under high glucose conditions, many studies have demonstrated similar AR-dependent increases in ROS production, confirming AR as an important factor for the pathogenesis of many diabetic complications. Moreover, recent studies have shown that AR inhibitors may be able to prevent or delay the onset of cardiovascular complications such as ischemia/reperfusion injury, atherosclerosis and atherothrombosis. In this review, we will focus on describing pivotal roles of AR in the pathogenesis of cardiovascular diseases as well as other diabetic complications, and the potential use of AR inhibitors as an emerging therapeutic strategy in preventing DM complications.

  3. 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.

  4. Methylenetetrahydrofolate Reductase Activity and Folate Metabolism

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    Nursen Keser

    2014-04-01

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

  5. Synthesis of 3-[(N-carboalkoxy)ethylamino]-indazole-dione derivatives and their biological activities on human liver carbonyl reductase.

    Science.gov (United States)

    Berhe, Solomon; Slupe, Andrew; Luster, Choice; Charlier, Henry A; Warner, Don L; Zalkow, Leon H; Burgess, Edward M; Enwerem, Nkechi M; Bakare, Oladapo

    2010-01-01

    A series of indazole-dione derivatives were synthesized by the 1,3-dipolar cycloaddition reaction of appropriate substituted benzoquinones or naphthoquinones and N-carboalkoxyamino diazopropane derivatives. These compounds were evaluated for their effects on human carbonyl reductase. Several of the analogs were found to serve as substrates for carbonyl reductase with a wide range of catalytic efficiencies, while four analogs display inhibitory activities with IC(50) values ranging from 3-5 microM. Two of the inhibitors were studied in greater detail and were found to be noncompetitive inhibitors against both NADPH and menadione with K(I) values ranging between 2 and 11 microM. Computational studies suggest that conformation of the compounds may determine whether the indazole-diones bind productively to yield product or nonproductively to inhibit the enzyme.

  6. Isolation and characterization of cDNAs encoding leucoanthocyanidin reductase and anthocyanidin reductase from Populus trichocarpa.

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    Lijun Wang

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

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

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    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.

  8. Effect of cytochrome P450 and aldo-keto reductase inhibitors on progesterone inactivation in primary bovine hepatic cell cultures.

    Science.gov (United States)

    Lemley, C O; Wilson, M E

    2010-10-01

    Progesterone is required for maintenance of pregnancy, and peripheral concentrations of progesterone are affected by both production and inactivation. Hepatic cytochrome P450 (EC 1.14.14.1) and aldo-keto reductase (EC 1.1.1.145-151) enzymes play a pivotal role in the first step of steroid inactivation, which involves the addition of hydroxyl groups to various sites of the cyclopentanoperhydrophenanthrene nucleus. The current objective was to discern the proportional involvement of hepatic progesterone inactivating enzymes on progesterone decay using specific enzyme inhibitors. Ticlopidine, diltiazem, curcumin, dicumarol, and naproxen were used because of their selective inhibition of cytochrome P450s, aldo-keto reductases, and glucuronosyltransferases. Liver biopsies were collected from 6 lactating Holstein dairy cows, and cells were dissociated using a nonperfusion technique. Confluent wells were preincubated for 4 h with enzyme inhibitor and then challenged with progesterone for 1 h. Cell viability was unaffected by inhibitor treatment and averaged 84±1%. In control wells, 50% of the progesterone had been inactivated after a 1-h challenge with 5 ng/mL of progesterone. Preincubation with curcumin, ticlopidine, or naproxen caused the greatest reduction in progesterone inactivation compared with controls and averaged 77, 39, or 37%, respectively. Hydroxylation of 4-nitrophenol to 4-nitrocatechol in intact cells was inhibited by approximately 65% after treatment with curcumin or ticlopidine. Glucuronidation of phenol red or 4-nitrocatechol in intact cells was inhibited by treatment with curcumin, dicumarol, or naproxen. In cytoplasmic preparations, aldo-keto reductase 1C activity was inhibited by curcumin, dicumarol, or naproxen treatment. Microsomal cytochrome P450 2C activity was inhibited by treatment with curcumin or ticlopidine, whereas cytochrome P450 3A activity was inhibited by treatment with curcumin or diltiazem. The contribution of cytochrome P450 2C and

  9. Inhibitory Effects of Colocasia esculenta (L. Schott Constituents on Aldose Reductase

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    Hong Mei Li

    2014-08-01

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

  10. Auranofin inactivates Trichomonas vaginalis thioredoxin reductase and is effective against trichomonads in vitro and in vivo.

    Science.gov (United States)

    Hopper, Melissa; Yun, Jeong-Fil; Zhou, Bianhua; Le, Christine; Kehoe, Katelin; Le, Ryan; Hill, Ryan; Jongeward, Gregg; Debnath, Anjan; Zhang, Liangfang; Miyamoto, Yukiko; Eckmann, Lars; Land, Kirkwood M; Wrischnik, Lisa A

    2016-12-01

    Trichomoniasis, caused by the protozoan parasite Trichomonas vaginalis, is the most common, non-viral, sexually transmitted infection in the world, but only two closely related nitro drugs are approved for its treatment. New antimicrobials against trichomoniasis remain an urgent need. Several organic gold compounds were tested for activity against T. vaginalis thioredoxin reductase (TrxR) in cell-free systems as well as for activity against different trichomonads in vitro and in a murine infection model. The organic gold(I) compounds auranofin and chloro(diethylphenylphosphine)gold(I) inhibited TrxR in a concentration-dependent manner in assays with recombinant purified reductase and in cytoplasmic extracts of T. vaginalis transfected with a haemagglutinin epitope-tagged form of the reductase. Auranofin potently suppressed the growth of three independent clinical T. vaginalis isolates as well as several strains of another trichomonad (Tritrichomonas foetus) in a 24 h-assay, with 50% inhibitory concentrations of 0.7-2.5 µM and minimum lethal concentrations of 2-6 µM. The drug also compromised the ability of the parasite to overcome oxidant stress, supporting the notion that auranofin acts, in part, by inactivating TrxR-dependent antioxidant defences. Chloro(diethylphenylphosphine)gold(I) was 10-fold less effective against T. vaginalis in vitro than auranofin. Oral administration of auranofin for 4 days cleared the parasites in a murine model of vaginal T. foetus infection without displaying any apparent adverse effects. The approved human drug auranofin may be a promising agent as an alternative treatment of trichomoniasis in cases when standard nitro drug therapies have failed.

  11. Selective non-steroidal inhibitors of 5 alpha-reductase type 1.

    Science.gov (United States)

    Occhiato, Ernesto G; Guarna, Antonio; Danza, Giovanna; Serio, Mario

    2004-01-01

    The enzyme 5 alpha-reductase (5 alpha R) catalyses the reduction of testosterone (T) into the more potent androgen dihydrotestosterone (DHT). The abnormal production of DHT is associated to pathologies of the main target organs of this hormone: the prostate and the skin. Benign prostatic hyperplasia (BPH), prostate cancer, acne, androgenetic alopecia in men, and hirsutism in women appear related to the DHT production. Two isozymes of 5 alpha-reductase have been cloned, expressed and characterized (5 alpha R-1 and 5 alpha R-2). They share a poor homology, have different chromosomal localization, enzyme kinetic parameters, and tissue expression patterns. Since 5 alpha R-1 and 5 alpha R-2 are differently distributed in the androgen target organs, a different involvement of the two isozymes in the pathogenesis of prostate and skin disorders can be hypothesized. High interest has been paid to the synthesis of inhibitors of 5 alpha-reductase for the treatment of DHT related pathologies, and the selective inhibition of any single isozyme represents a great challenge for medical and pharmaceutical research in order to have more specific drugs. At present, no 5 alpha R-1 inhibitor is marketed for the treatment of 5 alpha R-1 related pathologies but pharmaceutical research is very active in this field. This paper will review the major classes of 5 alpha R inhibitors focusing in particular on non-steroidal inhibitors and on structural features that enhance the selectivity versus the type 1 isozyme. Biological tests to assess the inhibitory activity towards the two 5 alpha R isozymes will be also discussed.

  12. Thioredoxin reductase deficiency potentiates oxidative stress, mitochondrial dysfunction and cell death in dopaminergic cells.

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    Pamela Lopert

    Full Text Available Mitochondria are considered major generators of cellular reactive oxygen species (ROS which are implicated in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD. We have recently shown that isolated mitochondria consume hydrogen peroxide (H₂O₂ in a substrate- and respiration-dependent manner predominantly via the thioredoxin/peroxiredoxin (Trx/Prx system. The goal of this study was to determine the role of Trx/Prx system in dopaminergic cell death. We asked if pharmacological and lentiviral inhibition of the Trx/Prx system sensitized dopaminergic cells to mitochondrial dysfunction, increased steady-state H₂O₂ levels and death in response to toxicants implicated in PD. Incubation of N27 dopaminergic cells or primary rat mesencephalic cultures with the Trx reductase (TrxR inhibitor auranofin in the presence of sub-toxic concentrations of parkinsonian toxicants paraquat; PQ or 6-hydroxydopamine; 6OHDA (for N27 cells resulted in a synergistic increase in H₂O₂ levels and subsequent cell death. shRNA targeting the mitochondrial thioredoxin reductase (TrxR2 in N27 cells confirmed the effects of pharmacological inhibition. A synergistic decrease in maximal and reserve respiratory capacity was observed in auranofin treated cells and TrxR2 deficient cells following incubation with PQ or 6OHDA. Additionally, TrxR2 deficient cells showed decreased basal mitochondrial oxygen consumption rates. These data demonstrate that inhibition of the mitochondrial Trx/Prx system sensitizes dopaminergic cells to mitochondrial dysfunction, increased steady-state H₂O₂, and cell death. Therefore, in addition to their role in the production of cellular H₂O₂ the mitochondrial Trx/Prx system serve as a major sink for cellular H₂O₂ and its disruption may contribute to dopaminergic pathology associated with PD.

  13. Nodule and Leaf Nitrate Reductases and Nitrogen Fixation in Medicago sativa L. under Water Stress.

    Science.gov (United States)

    Aparicio-Tejo, P; Sánchez-Díaz, M

    1982-02-01

    The effect of water stress on patterns of nitrate reductase activity in the leaves and nodules and on nitrogen fixation were investigated in Medicago sativa L. plants watered 1 week before drought with or without NO(3) (-). Nitrogen fixation was decreased by water stress and also inhibited strongly by the presence of NO(3) (-). During drought, leaf nitrate reductase activity (NRA) decreased significantly particularly in plants watered with NO(3) (-), while with rewatering, leaf NRA recovery was quite important especially in the NO(3) (-)-watered plants. As water stress progressed, the nodular NRA increased both in plants watered with NO(3) (-) and in those without NO(3) (-) contrary to the behavior of the leaves. Beyond -15.10(5) pascal, nodular NRA began to decrease in plants watered with NO(3) (-). This phenomenon was not observed in nodules of plants given water only.Upon rewatering, it was observed that in plants watered with NO(3) (-) the nodular NRA increased again, while in plants watered but not given NO(3) (-), such activity began to decrease. Nitrogen fixation increased only in plants without NO(3) (-).

  14. The effects of chemical and radioactive properties of Tl-201 on human erythrocyte glutathione reductase activity.

    Science.gov (United States)

    Sahin, Ali; Senturk, Murat; Akkemik, Ebru; Ciftci, Mehmet

    2012-01-01

    The aim of the study was to evaluate the inhibitory effects of thallium-201 ((201)Tl) solution on human erythrocyte glutathione reductase (GR) activity. 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 (201)Tl solution including Tl(+), Fe(+3) and Cu(+2) metals and the in vitro effects of the radiation effect of the (201)Tl 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. Glutathione reductase was purified 2033-fold at a yield of 28.17%. (201)Tl 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 (201)Tl solution. It was detected in in vitro and in vivo studies that the human erythrocyte GR enzyme is inhibited due to the radiation effect of (201)Tl solution. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. New drug target in protozoan parasites: the role of thioredoxin reductase.

    Science.gov (United States)

    Andrade, Rosa M; Reed, Sharon L

    2015-01-01

    Amebiasis causes approximately 70,000 deaths annually and is the third cause of death due to parasites worldwide. It is treated primarily with metronidazole, which has adverse side effects, is mutagenic and carcinogenic, and emergence of resistance is an increasing concern. Unfortunately, better therapeutic alternatives are lacking. Re-purposing of older FDA approved drugs is advantageous to drug discovery since safety and pharmacokinetic effects in humans are already known. In high throughput screening studies, we recently demonstrated that auranofin, a gold containing compound originally approved to treat rheumatoid arthritis, has activity against trophozoites of E. histolytica, the causative agent of amebiasis. Auranofin's anti-parasitic activity is attributed to its monovalent gold molecule that readily inhibits E. histolytica thioredoxin reductase. This anti-oxidant enzyme is the only thiol-dependent flavo-reductase present in E. histolytica. Auranofin has also shown promising activity against other protozoans of significant public health importance. Altogether, this evidence suggests that auranofin has the potential to become a broad spectrum alternative therapeutic agent for diseases with a large global burden.

  16. New drug target in protozoan parasites: the role of thioredoxin reductase

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    Rosa M. Andrade

    2015-09-01

    Full Text Available Amebiasis causes approximately 70,000 deaths annually and is the third cause of death due to parasites worldwide. It is treated primarily with metronidazole, which has adverse side effects, is mutagenic and carcinogenic, and emergence of resistance is an increasing concern. Unfortunately, better therapeutic alternatives are lacking. Re-purposing of older FDA approved drugs is advantageous to drug discovery since safety and pharmacokinetic effects in humans are already known. In high throughput screening studies, we recently demonstrated that auranofin, a gold containing compound originally approved to treat rheumatoid arthritis, has activity against trophozoites of E. histolytica, the causative agent of amebiasis. Auranofin’s anti-parasitic activity is attributed to its monovalent gold molecule that readily inhibits E.histolytica thioredoxin reductase. This anti-oxidant enzyme is the only thiol-dependent flavo-reductase present in E.histolytica. Auranofin has also shown promising activity against other protozoans of significant public health importance. Altogether, this evidence suggests that auranofin has the potential to become a broad spectrum alternative therapeutic agent for diseases with a large global burden.

  17. The Nox/Ferric reductase/Ferric reductase-like families of Eumycetes.

    Science.gov (United States)

    Grissa, Ibtissem; Bidard, Frédérique; Grognet, Pierre; Grossetete, Sandrine; Silar, Philippe

    2010-09-01

    Reactive Oxygen Species (ROS) are involved in plant biomass degradation by fungi and development of fungal structures. While the ROS-generating NADPH oxidases from filamentous fungi are under strong scrutiny, much less is known about the related integral Membrane (or Ferric) Reductases (IMRs). Here, we present a survey of these enzymes in 29 fungal genomes covering the entire available range of fungal diversity. IMRs are present in all fungal genomes. They can be classified into at least 24 families, underscoring the high diversity of these enzymes. Some are differentially regulated during colony or fruiting body development, as well as by the nature of the carbon source of the growth medium. Importantly, functional characterization of IMRs has been made on proteins belonging to only two families, while nothing or very little is known about the proteins of the other 22 families.

  18. Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase.

    Science.gov (United States)

    Liu, Yi; Shi, Zi; Maximova, Siela; Payne, Mark J; Guiltinan, Mark J

    2013-12-05

    The proanthocyanidins (PAs), a subgroup of flavonoids, accumulate to levels of approximately 10% total dry weight of cacao seeds. PAs have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant. To dissect the genetic basis of PA biosynthetic pathway in cacao (Theobroma cacao), we have isolated three genes encoding key PA synthesis enzymes, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). We measured the expression levels of TcANR, TcANS and TcLAR and PA content in cacao leaves, flowers, pod exocarp and seeds. In all tissues examined, all three genes were abundantly expressed and well correlated with PA accumulation levels, suggesting their active roles in PA synthesis. Overexpression of TcANR in an Arabidopsis ban mutant complemented the PA deficient phenotype in seeds and resulted in reduced anthocyanidin levels in hypocotyls. Overexpression of TcANS in tobacco resulted in increased content of both anthocyanidins and PAs in flower petals. Overexpression of TcANS in an Arabidopsis ldox mutant complemented its PA deficient phenotype in seeds. Recombinant TcLAR protein converted leucoanthocyanidin to catechin in vitro. Transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin. Our results confirm the in vivo function of cacao ANS and ANR predicted based on sequence homology to previously characterized enzymes from other species. In addition, our results provide a clear functional analysis of a LAR gene in vivo.

  19. Selenium in Thioredoxin Reductase: A Mechanistic Perspective†

    Science.gov (United States)

    Lacey, Brian M.; Eckenroth, Brian E.; Flemer, Stevenson; Hondal, Robert J.

    2013-01-01

    Most high Mr thioredoxin reductases (TRs) have the unusual feature of utilizing a vicinal disulfide bond (Cys1-Cys2) which form an eight-membered ring during the catalytic cycle. Many eukaryotic TRs have replaced the Cys2 position of the dyad with the rare amino acid selenocysteine (Sec). Here we demonstrate that Cys- and Sec-containing TRs are distinguished by the importance each class of enzymes places on the 8-membered ring structure in the catalytic cycle. This hypothesis was explored by studying the truncated enzyme missing the C-terminal ring structure in conjunction with oxidized peptide substrates to investigate the reduction and opening of this dyad. The peptide substrates were identical in sequence to the missing part of the enzyme, containing either a disulfide or selenylsulfide linkage, but were differentiated by the presence (cyclic) and absence (acyclic) of the ring structure. The ratio of these turnover rates informs that the ring is only of modest importance for the truncated mouse mitochondrial Sec-TR (ring/no ring = 32), while the ring structure is highly important for the truncated Cys-TRs from D. melanogaster and C. elegans (ring/no ring > 1000). All three enzymes exhibit a similar dependence upon leaving group pKa as shown by the use of the acyclic peptides as substrates. These two factors can be reconciled for Cys-TRs if the ring functions to simultaneously allow for attack by a nearby thiolate while correctly positioning the leaving group sulfur atom to accept a proton from the enzymic general acid. For Sec-TRs the ring is unimportant because the lower pKa of the selenol relative to a thiol obviates its need to be protonated upon S-Se bond scission and permits physical separation of the selenol and the general acid. Further study of the biochemical properties of the truncated Cys and Sec TR enzymes demonstrates that the chemical advantage conferred on the eukaryotic enzyme by a selenol is the ability to function at acidic pH. PMID:18986163

  20. 4-Dimethylaminoazobenzenes: carcinogenicities and reductive cleavage by microsomal azo reductase.

    Science.gov (United States)

    Lambooy, J P; Koffman, B M

    1985-01-01

    Twenty-four 4-dimethylaminoazobenzenes (DABs) in which systematic structural modifications have been made in the prime ring have been studied for substrate specificity for microsomal azo reductase. The DABs were also evaluated for carcinogenicity and it was found that there was no correlation between carcinogenicity and extent of azo bond cleavage by azo reductase. While any substituent in the prime ring reduces the rate of cleavage of the azo bond relative to the unsubstituted dye, there is a correlation between substituent size and susceptibility to the enzyme. Substituent size was also found to be a significant factor in the induction of hepatomas by the dyes. Preliminary studies have shown that there appears to be a positive correlation between microsomal riboflavin content and the activity of the azo reductase.

  1. Stimulatory Effect of Insulin on 5α-Reductase Type 1 (SRD5A1) Expression through an Akt-Dependent Pathway in Ovarian Granulosa Cells

    Science.gov (United States)

    Kayampilly, Pradeep P.; Wanamaker, Brett L.; Stewart, James A.; Wagner, Carrie L.; Menon, K. M. J.

    2010-01-01

    Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P < 0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P < 0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation. PMID:20810561

  2. Effect of dehydroepiandrosterone derivatives on the activity of 5α-reductase isoenzymes and on cancer cell line PC-3.

    Science.gov (United States)

    Bratoeff, Eugene; Garrido, Mariana; Ramírez-Apan, Teresa; Heuze, Yvonne; Sánchez, Araceli; Soriano, Juan; Cabeza, Marisa

    2014-11-01

    It is well known that testosterone (T) under the influence of 5α-reductase enzyme is converted to dihydrotestosterone (DHT), which causes androgen-dependent diseases. The aim of this study was to synthesize new dehydroepiandrosterone derivatives (3a-e, 4a-i, 6 and 7) having potential inhibitory activity against the 5α-reductase enzyme. This paper also reports the in vivo pharmacological effect of these steroidal molecules. The results from this study showed that all compounds exhibited low inhibitory activity for 5α-reductase type 1 and 2 enzymes and they failed to bind to the androgen receptor. Furthermore, in the in vivo experiment, steroids 3b, 4f, and 4 g showed comparable antiandrogenic activity to that of finasteride; only derivatives 4d and 7 produced a considerable decrease in the weight of the prostate gland of gonadectomized hamsters treated with (T). On the other hand, compounds 4a, f and h showed 100% inhibition of the growth of prostate cancer cell line PC-3, with compound 4 g having a 98.2% antiproliferative effect at 50 μM. The overall data indicated that these steroidal molecules, having an aromatic ester moiety at C-3 (4f-h), could have anticancer properties.

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

    Science.gov (United States)

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

    2011-10-01

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

  4. The 5 Alpha-Reductase Isozyme Family: A Review of Basic Biology and Their Role in Human Diseases

    Directory of Open Access Journals (Sweden)

    Faris Azzouni

    2012-01-01

    Full Text Available Despite the discovery of 5 alpha-reduction as an enzymatic step in steroid metabolism in 1951, and the discovery that dihydrotestosterone is more potent than testosterone in 1968, the significance of 5 alpha-reduced steroids in human diseases was not appreciated until the discovery of 5 alpha-reductase type 2 deficiency in 1974. Affected males are born with ambiguous external genitalia, despite normal internal genitalia. The prostate is hypoplastic, nonpalpable on rectal examination and approximately 1/10th the size of age-matched normal glands. Benign prostate hyperplasia or prostate cancer does not develop in these patients. At puberty, the external genitalia virilize partially, however, secondary sexual hair remains sparse and male pattern baldness and acne develop rarely. Several compounds have been developed to inhibit the 5 alpha-reductase isozymes and they play an important role in the prevention and treatment of many common diseases. This review describes the basic biochemical properties, functions, tissue distribution, chromosomal location, and clinical significance of the 5 alpha-reductase isozyme family.

  5. NITRATE REDUCTASE ACTIVITY DURING HEAT SHOCK IN WINTER WHEAT

    Directory of Open Access Journals (Sweden)

    Klimenko S.B.

    2006-03-01

    Full Text Available Nitrates are the basic source of nitrogen for the majority of plants. Absorption and transformation of nitrates in plants are determined by external conditions and, first of all, temperature and light intensity. The influence of the temperature increasing till +40 0С on activity of nitrate reductase was studied. It is shown, that the rise of temperature was accompanied by sharp decrease of activity nitrate reductase in leaves of winter wheat, what, apparently, occurred for the account deactivations of enzyme and due to its dissociation.

  6. Do cytochromes function as oxygen sensors in the regulation of nitrate reductase biosynthesis?

    Science.gov (United States)

    MacGregor, C H; Bishop, C W

    1977-01-01

    The observation that oxygen represses nitrate reductase biosynthesis in a hemA mutant grown aerobically with or without delta-aminolevulinic acid indicates that cytochromes are not responsible for nitrate reductase repression in aerobically grown cells. PMID:326768

  7. Statin Drugs Markedly Inhibit Testosterone Production by Rat Leydig Cells In Vitro: Implications for Men

    Science.gov (United States)

    Statin drugs lower blood cholesterol by inhibiting hepatic 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase. During drug development it was shown that statins inhibit production of cholesterol in the testis. We evaluated testosterone production in vitro, using highly purified rat ...

  8. 8-Methoxy-naphtho[2,3-b]thiophen-4,9-quinone, a non-competitive inhibitor of trypanothione reductase

    Directory of Open Access Journals (Sweden)

    Zani Carlos L

    2003-01-01

    Full Text Available The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione and to the co-factor (NADPH, with Ki-values of 5 and 3.6 µM, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 µM, indicating a good selectivity against the parasite enzyme.

  9. Role of 5α-reductase inhibitors in androgen-stimulated skin disorders.

    Science.gov (United States)

    Azzouni, Faris; Zeitouni, Nathalie; Mohler, James

    2013-02-01

    5α-reductase (5α-R) isozymes are ubiquitously expressed in human tissues. This enzyme family is composed of 3 members that perform several important biologic functions. 5α-R isozymes play an important role in benign prostate hyperplasia, prostate cancer, and androgen-stimulated skin disorders, which include androgenic alopecia, acne, and hirsutism. Discovery of 5α-R type 2 deficiency in 1974 sparked interest in development of pharmaceutical agents to inhibit 5α-R isozymes, and 2 such inhibitors are currently available for clinical use: finasteride and dutasteride. 5α-R inhibitors are US Food and Drug Administration (FDA)-approved for the treatment of benign prostate hyperplasia. Only finasteride is FDA-approved for treatment of male androgenic alopecia. This article reviews the pathophysiology of androgen-stimulated skin disorders and the key clinical trials using 5α-R inhibitors in the treatment of androgen-stimulated skin disorders.

  10. Existence and characteristics of nitrate reductase in plasma membrane of maize roots

    Institute of Scientific and Technical Information of China (English)

    陈珈; 王学臣

    1995-01-01

    The existence and characteristics of nitrate reductase (NR) have been investigated with microsomes and purified plasma membrane vesicles (RV and IV) from the primary root tips of maize (Zea mays L.). An integral membrane protein capable of reducing nitrate is presented in the plasma membrane which is obviously different from the soluble cytoplasmic NR in respect of NO3- induction and Triton X-100 activation Plasma membrane-bound NR did not have direct coupling relationship with the transmembrane H-transport, however, it could inhibit the electron transmission from NADH to K3[Fe(CN)6]. The possible action mode of plasma membrane redox system that the membrane-bound NR is involved in is discussed.

  11. Aldose Reductase as a Drug Target for Treatment of Diabetic Nephropathy: Promises and Challenges.

    Science.gov (United States)

    El Gamal, Heba; Munusamy, Shankar

    2016-11-28

    Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes mellitus and the leading cause of end stage renal disease. One of the key pathways activated in DN is the polyol pathway, in which glucose is converted to sorbitol (a relatively non-metabolizable sugar) by the enzyme aldose reductase (AR). Shunting of glucose into this pathway causes disruption to glucose metabolism and subsequently damages the tissues via increased oxidative stress, protein kinase c activation and production of advanced glycation end products (AGE) in the kidney. This review aims to provide a comprehensive overview of the AR enzyme structure, substrate specificity and topology in normal physiology; to elaborate on the deleterious effects of AR activation in DN; and to summarize the potential therapeutic benefits and major challenges associated with AR inhibition in patients with DN.

  12. Potential use of HMG-CoA reductase inhibitors (statins) as radioprotective agents.

    Science.gov (United States)

    Fritz, Gerhard; Henninger, Christian; Huelsenbeck, Johannes

    2011-01-01

    HMG-CoA reductase inhibitors (statins) are widely used in the therapy of hypercholesterolemia. Apart from their lipid-lowering activity, they have pleiotropic effects that are attributed to the inhibition of regulatory proteins, including Ras-homologous (Rho) GTPases. Here, we discuss the potential usefulness of statins to prevent normal tissue damage provoked by radiotherapy. Statins reduce the mRNA expression of pro-inflammatory and pro-fibrotic cytokines stimulated by ionizing radiation in vitro and alleviate IR-induced inflammation and fibrosis in vivo. The currently available data indicate that statins accelerate the rapid repair of DNA double-strand breaks and, moreover, mitigate the DNA damage response induced by IR. Furthermore, statins increase the mRNA expression of DNA repair factors in vivo. Thus, although the molecular mechanisms involved are still ambiguous, preclinical data concordantly show a promising radioprotective capacity of statins.

  13. Discovery of Potent and Selective Leads against Toxoplasma gondii Dihydrofolate Reductase via Structure-Based Design.

    Science.gov (United States)

    Welsch, Matthew E; Zhou, Jian; Gao, Yueqiang; Yan, Yunqing; Porter, Gene; Agnihotri, Gautam; Li, Yingjie; Lu, Henry; Chen, Zhongguo; Thomas, Stephen B

    2016-12-08

    Current treatment of toxoplasmosis targets the parasite's folate metabolism through inhibition of dihydrofolate reductase (DHFR). The most widely used DHFR antagonist, pyrimethamine, was introduced over 60 years ago and is associated with toxicity that can be largely attributed to a similar affinity for parasite and human DHFR. Computational analysis of biochemical differences between Toxoplasma gondii and human DHFR enabled the design of inhibitors with both improved potency and selectivity. The approach described herein yielded TRC-19, a promising lead with an IC50 of 9 nM and 89-fold selectivity in favor of Toxoplasma gondii DHFR, as well as crystallographic data to substantiate in silico methodology. Overall, 50% of synthesized in silico designs met hit threshold criteria of IC50 2-fold selectivity favoring Toxoplasma gondii, further demonstrating the efficiency of our structure-based drug design approach.

  14. Charaterization of bumarsin, a 3-hydroxy-3-methylglutaryl-coenzyme reductase inhibitor from Mesobuthus martensii Karsch venom.

    Science.gov (United States)

    Chai, S C; Armugam, A; Strong, P N; Jeyaseelan, K

    2012-09-01

    Scorpion venoms are rich sources of bioactive peptides and are widely known for their ion channel inhibiting properties. We have isolated, cloned and characterized a venom protein (Bumarsin) from the Chinese scorpion, Mesobuthus martensii Karsch. Bumarsin cDNA encodes a 8132 Da, 72 amino acid mature protein that most probably exists in its native form as a Cys-bridged homodimer. We have identified this novel protein to be an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. 0.6 μM of Bumarsin inhibits 32% of the HMG-CoA reductase activity, in comparison to 10 μM simvastatin which only inhibits 35% of the activity. RT-PCR and SELDI-TOF mass spectrometric studies demonstrate that bumarsin regulates the expression of both genes and proteins involved in cholesterol homeostasis. Our results suggest that bumarsin may provide a model for the design of novel drugs that can be used to modulate cholesterol homeostasis.

  15. Kinetics of mercury reduction by Serratia marcescens mercuric reductase expressed by pseudomonas putida strains

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, M.; Deckwer, W.D. [GBF-Gesellschaft fuer Biotechnologische Forschung mbH, Abteilung TU-BCE, Mascheroder Weg 1, D-38124 Braunschweig (Germany)

    2005-10-01

    Mercury (Hg) resistance is widespread among microorganisms and is based on the intracellular transformation of Hg(II) to less toxic elemental Hg(0). The use of microbial consortia to demercurize polluted wastewater streams and environments has been demonstrated. To develop efficient and versatile microbial cleanup strategies requires detailed knowledge of transport and reaction rates. This study focuses on the kinetics of the key enzyme of the microbial transformation, e.g., the mercuric reductase (MerA) under conditions closely resembling the cell interior. To this end, previously constructed and characterized Pseudomonas putida strains expressing MerA from Serratia marcescens were applied. Of the P. putida strains considered in this study P. putida KT2442::mer73 constitutively expressing broad spectrum mercury resistance (merTPAB) yielded the highest mercuric reductase (MerA) activity directly after cell disruption. MerA in the raw extract was further purified (about 100 fold). Reduction rates were measured for various substrates (HgCl{sub 2}, Hg{sub 2}SO{sub 4}, Hg(NO{sub 3}){sub 2} and phenyl mercury acetate) up to high concentrations dependent on the purification grade. In all cases, a pronounced substrate inhibition was found. The kinetic constants determined for the cell raw extract are in agreement with those measured for intact cells. However, the rate data exhibit reduced affinity and inhibition with rising purification grade (specific activity). Therefore, the findings seemingly point to reactions preceding the catalytic reduction. Based on simplified assumptions, a kinetic model is suggested which reasonably describes the experimental findings and can advantageously be applied to the bioreactor design. (Abstract Copyright [2005], Wiley Periodicals, Inc.)

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

    Science.gov (United States)

    Yadav, Umesh C S; Ramana, K V; Srivastava, Satish K

    2013-12-01

    Aldose reductase (AR), a glucose-metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30 µM) relative to glucose. Acrolein, a major endogenous lipid peroxidation product as well as a component of environmental pollutants 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 clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells (SAECs). Exposure of SAECs to varying concentrations of acrolein caused cell death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low-dose (5-10 µM) but not the high-dose (>10 µM) acrolein-induced SAEC death. AR inhibition protected SAECs from low-dose (5 µM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail moment, and annexin V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of the proapoptotic proteins Bax and Bad from the cytosol to the mitochondria and that of Bcl2 and BclXL from the mitochondria to the cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases 1 and 2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38MAPK, and c-Jun were transiently activated in airway epithelial cells by acrolein in a concentration- and time-dependent fashion, which was significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells.

  17. HMG-CoA reductase inhibitors decrease angiotensin II-induced vascular fibrosis: role of RhoA/ROCK and MAPK pathways.

    Science.gov (United States)

    Rupérez, Mónica; Rodrigues-Díez, Raquel; Blanco-Colio, Luis Miguel; Sánchez-López, Elsa; Rodríguez-Vita, Juan; Esteban, Vanesa; Carvajal, Gisselle; Plaza, Juan José; Egido, Jesús; Ruiz-Ortega, Marta

    2007-08-01

    3-Hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) present beneficial effects in cardiovascular diseases. Angiotensin II (Ang II) contributes to cardiovascular damage through the production of profibrotic factors, such as connective tissue growth factor (CTGF). Our aim was to investigate whether HMG-CoA reductase inhibitors could modulate Ang II responses, evaluating CTGF expression and the mechanisms underlying this process. In cultured vascular smooth muscle cells (VSMCs) atorvastatin and simvastatin inhibited Ang II-induced CTGF production. The inhibitory effect of statins on CTGF upregulation was reversed by mevalonate and geranylgeranylpyrophosphate, suggesting that RhoA inhibition could be involved in this process. In VSMCs, statins inhibited Ang II-induced Rho membrane localization and activation. In these cells Ang II regulated CTGF via RhoA/Rho kinase activation, as shown by inhibition of Rho with C3 exoenzyme, RhoA dominant-negative overexpression, and Rho kinase inhibition. Furthermore, activation of p38MAPK and JNK, and redox process were also involved in Ang II-mediated CTGF upregulation, and were downregulated by statins. In rats infused with Ang II (100 ng/kg per minute) for 2 weeks, treatment with atorvastatin (5 mg/kg per day) diminished aortic CTGF and Rho activation without blood pressure modification. Rho kinase inhibition decreased CTGF upregulation in rat aorta, mimicking statin effect. CTGF is a vascular fibrosis mediator. Statins diminished extracellular matrix (ECM) overexpression caused by Ang II in vivo and in vitro. In summary, HMG-CoA reductase inhibitors inhibit several intracellular signaling systems activated by Ang II (RhoA/Rho kinase and MAPK pathways and redox process) involved in the regulation of CTGF. Our results may explain, at least in part, some beneficial effects of statins in cardiovascular diseases.

  18. 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...

  19. 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

  20. Plasmid-encoded diacetyl (acetoin) reductase in Leuconostoc pseudomesenteroides

    DEFF Research Database (Denmark)

    Rattray, Fergal P; Myling-Petersen, Dorte; Larsen, Dianna;

    2003-01-01

    ) reductases reported previously. Downstream of the butA gene of L. pseudomesenteroides, but coding in the opposite orientation, a putative DNA recombinase was identified. A two-step PCR approach was used to construct FPR02, a butA mutant of the wild-type strain, CHCC2114. FPR02 had significantly reduced...

  1. Sepiapterin reductase deficiency : A Treatable Mimic of Cerebral Palsy

    NARCIS (Netherlands)

    Friedman, Jennifer; Roze, Emmanuel; Abdenur, Jose E.; Chang, Richard; Gasperini, Serena; Saletti, Veronica; Wali, Gurusidheshwar M.; Eiroa, Hernan; Neville, Brian; Felice, Alex; Parascandalo, Ray; Zafeiriou, Dimitrios I.; Arrabal-Fernandez, Luisa; Dill, Patricia; Eichler, Florian S.; Echenne, Bernard; Gutierrez-Solana, Luis G.; Hoffmann, Georg F.; Hyland, Keith; Kusmierska, Katarzyna; Tijssen, Marina A. J.; Lutz, Thomas; Mazzuca, Michel; Penzien, Johann; Bwee Tien Poll-The, [No Value; Sykut-Cegielska, Jolanta; Szymanska, Krystyna; Thoeny, Beat; Blau, Nenad

    2012-01-01

    Objective: Sepiapterin reductase deficiency (SRD) is an under-recognized levodopa-responsive disorder. We describe clinical, biochemical, and molecular findings in a cohort of patients with this treatable condition. We aim to improve awareness of the phenotype and available diagnostic and therapeuti

  2. Direct Electrochemistry With Nitrate Reductase in Chitosan Films

    Institute of Scientific and Technical Information of China (English)

    Xiao Xia CHEN; Jing Bo HU; Hong WU; Hui Bo SHAO

    2004-01-01

    Stable films made from chitosan(CS)on pyrolytic graphite electrode(PGE)gave direct electrochemistry for incorporated enzyme nitrate reductase(NR).Cyclic voltammetry of CS/NR films showed a pair of well-defined and nearly reversible redox peaks at about-0.430 V vs.SCE at pH 7.0 phosphate buffers.

  3. 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...

  4. 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.

  5. A novel zwitterionic inhibitor of aldose reductase interferes with polyol pathway in ex vivo and in vivo models of diabetic complications.

    Science.gov (United States)

    Karasova, M Juskova; Prnova, M Soltesova; Stefek, M

    2014-10-01

    Recently a zwitterionic principle has been suggested as an alternative to bioisosteric replacement for increasing low bioavailability of aldose reductase inhibitors bearing an acidic function. In the present work we studied the effect of a novel zwitterionic inhibitor of aldose reductase [(2-benzyl-2,3,4,5-tetrahydro-1 H-pyrido[4,3-b]indole-8-yl)-acetic acid, compound 1] on sorbitol accumulation in ex vivo and in vivo models of diabetic complications. The effect of 1 on sorbitol accumulation in isolated rat eye lenses incubated with high glucose and in selected organs of streptozotocin-induced diabetic rats was evaluated. Significantly increased sorbitol levels were recorded in the lenses incubated with 50 mM glucose in comparison with controls. Sorbitol production was inhibited by 1 at concentrations of 25 and 100 μM. Under in vivo conditions in diabetic rats, significant elevation of sorbitol levels in selected organs was recorded. Compound 1 administered i.g. for five consecutive days (twice a day 25 mg/kg) inhibited sorbitol accumulation in erythrocytes and the sciatic nerve, yet it was without effect in eye lenses. A similar picture of inhibition was observed after i.p. administration of 1. To conclude, the results suggest that the zwitterionic principle may represent a practicable way of improving bioavailability of aldose reductase inhibitors bearing an acidic function.

  6. 3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

    Science.gov (United States)

    Sheldon, P S; Kekwick, R G; Smith, C G; Sidebottom, C; Slabas, A R

    1992-04-01

    3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.

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

    Science.gov (United States)

    Zhao, Xianxian; Tang, Juan; Wang, Xu; Yang, Ruoheng; Zhang, Xiaoping; Gu, Yunfu; Li, Xi; Ma, Menggen

    2015-05-01

    Furfural and 5-hydroxymethylfurfural (HMF) are the two main aldehyde compounds derived from pentoses and hexoses, respectively, during lignocellulosic biomass pretreatment. These two compounds inhibit microbial growth and interfere with subsequent alcohol fermentation. Saccharomyces cerevisiae has the in situ ability to detoxify furfural and HMF to the less toxic 2-furanmethanol (FM) and furan-2,5-dimethanol (FDM), respectively. Herein, we report that an uncharacterized gene, YNL134C, was highly up-regulated under furfural or HMF stress and Yap1p and Msn2/4p transcription factors likely controlled its up-regulated expression. Enzyme activity assays showed that YNL134C is an NADH-dependent aldehyde reductase, which plays a role in detoxification of furfural to FM. However, no NADH- or NADPH-dependent enzyme activity was observed for detoxification of HMF to FDM. This enzyme did not catalyse the reverse reaction of FM to furfural or FDM to HMF. Further studies showed that YNL134C is a broad-substrate aldehyde reductase, which can reduce multiple aldehydes to their corresponding alcohols. Although YNL134C is grouped into the quinone oxidoreductase family, no quinone reductase activity was observed using 1,2-naphthoquinone or 9,10-phenanthrenequinone as a substrate, and phylogenetic analysis indicates that it is genetically distant to quinone reductases. Proteins similar to YNL134C in sequence from S. cerevisiae and other microorganisms were phylogenetically analysed.

  8. Phytochemical Analysis of Agrimonia pilosa Ledeb, Its Antioxidant Activity and Aldose Reductase Inhibitory Potential

    Science.gov (United States)

    Kim, Set Byeol; Hwang, Seung Hwan; Suh, Hong-Won; Lim, Soon Sung

    2017-01-01

    The aim of this study was to determine aldose reductase (AR) inhibitory activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of compounds from Agrimonia pilosa Ledeb (AP). We isolated agrimoniin (AM), four flavonoid glucosides and two flavonoid glucuronides from the n-butanol fraction of AP 50% methanol extract. In addition to isolated compounds, the AR-inhibitory activity and the DPPH free radical scavenging activity of catechin, 5-flavonoids, and 4-flavonoid glucosides (known components of AP) against rat lens AR (RLAR) and DPPH assay were measured. AM showed IC50 values of 1.6 and 13.0 μM against RLAR and DPPH scavenging activity, respectively. Additionally, AM, luteolin-7-O-glucuronide (LGN), quercitrin (QU), luteolin (LT) and afzelin (AZ) showed high inhibitory activity against AR and were first observed to decrease sorbitol accumulation in the rat lens under high-sorbitol conditions ex vivo with inhibitory values of 47.6%, 91.8%, 76.9%, 91.8% and 93.2%, respectively. Inhibition of recombinant human AR by AM, LGN and AZ exhibited a noncompetitive inhibition pattern. Based on our results, AP and its constituents may play partial roles in RLAR and oxidative radical inhibition. Our results suggest that AM, LGN, QU, LT and AZ may potentially be used as natural drugs for treating diabetic complications. PMID:28208627

  9. Effects of HMG-CoA reductase inhibitors (statins) on progression of kidney disease.

    Science.gov (United States)

    Fried, Linda F

    2008-09-01

    Chronic kidney disease, especially in the setting of proteinuria, is characterized by hyperlipidemia. In animal models, hyperlipidemia causes glomerular foam cells and glomerulosclerosis. Treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) ameliorates kidney disease in these models. The data of the role of hyperlipidemia in progression of human kidney disease are less clear. Data from small studies in glomerular disease suggest that statins decrease proteinuria. Data mainly from cardiovascular studies suggest that statins decrease the loss of glomerular filtration. The benefit of statins may derive from their lipid lowering effects. More recently, data suggest that the benefit of statins is greater than lipid lowering alone. The pleiotropic effects of statins may derive from inhibition of other downstream targets (isoprenoids) of the mevalonic acid pathway that are separate from cholesterol synthesis. Statins inhibits isoprenylation of Ras and Rho GTPases. These effects may lead to decreased monocyte/macrophage infiltration in the glomerulus, decreased mesangial proliferation and decreased accumulation of extracellular matrix and fibrosis. In addition, inhibition of RhoA and Ras may decrease inflammation and increase eNOS activity. These effects could lead to improvement in the progression of kidney disease.

  10. (+)-Pinoresinol/(+)-lariciresinol reductase from Forsythia intermedia. Protein purification, cDNA cloning, heterologous expression and comparison to isoflavone reductase.

    Science.gov (United States)

    Dinkova-Kostova, A T; Gang, D R; Davin, L B; Bedgar, D L; Chu, A; Lewis, N G

    1996-11-15

    Lignans are a widely distributed class of natural products, whose functions and distribution suggest that they are one of the earliest forms of defense to have evolved in vascular plants; some, such as podophyllotoxin and enterodiol, have important roles in cancer chemotherapy and prevention, respectively. Entry into lignan enzymology has been gained by the approximately 3000-fold purification of two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase, a pivotal branchpoint enzyme in lignan biosynthesis. Both have comparable ( approximately 34.9 kDa) molecular mass and kinetic (Vmax/Km) properties and catalyze sequential, NADPH-dependent, stereospecific, hydride transfers where the incoming hydride takes up the pro-R position. The gene encoding (+)-pinoresinol/(+)-lariciresinol reductase has been cloned and the recombinant protein heterologously expressed as a functional beta-galactosidase fusion protein. Its amino acid sequence reveals a strong homology to isoflavone reductase, a key branchpoint enzyme in isoflavonoid metabolism and primarily found in the Fabaceae (angiosperms). This is of great evolutionary significance since both lignans and isoflavonoids have comparable plant defense properties, as well as similar roles as phytoestrogens. Given that lignans are widespread from primitive plants onwards, whereas the isoflavone reductase-derived isoflavonoids are mainly restricted to the Fabaceae, it is tempting to speculate that this branch of the isoflavonoid pathway arose via evolutionary divergence from that giving the lignans.

  11. 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

    2003-12-12

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

  12. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Norman G. (Pullman, WA); Davin, Laurence B. (Pullman, WA); Dinkova-Kostova, Albena T. (Baltimore, MD); Fujita, Masayuki (Kita-gun, JP), Gang; David R. (Ann Arbor, MI), Sarkanen; Simo (Minneapolis, MN), Ford; Joshua D. (Pullman, WA)

    2003-10-21

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Norman G. (Pullman, WA); Davin, Laurence B. (Pullman, WA); Dinkova-Kostova, Albena T. (Baltimore, MD); Fujita, Masayuki (Kagawa, JP); Gang, David R. (Ann Arbor, MI); Sarkanen, Simo (S. Minneapolis, MN); Ford, Joshua D. (Pullman, WA)

    2001-04-03

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

  15. Comparison of inhibitory effects between acetaminophen-glutathione conjugate and reduced glutathione in human glutathione reductase.

    Science.gov (United States)

    Nýdlová, Erika; Vrbová, Martina; Cesla, Petr; Jankovičová, Barbora; Ventura, Karel; Roušar, Tomáš

    2014-09-01

    Acetaminophen overdose is the most frequent cause of acute liver injury. The main mechanism of acetaminophen toxicity has been attributed to oxidation of acetaminophen. The oxidation product is very reactive and reacts with glutathione generating acetaminophen-glutathione conjugate (APAP-SG). Although this conjugate has been recognized to be generally nontoxic, we have found recently that APAP-SG could produce a toxic effect. Therefore, the aim of our study was to estimate the toxicity of purified APAP-SG by characterizing the inhibitory effect in human glutathione reductase (GR) and comparing that to the inhibitory effect of the natural inhibitor reduced glutathione. We used two types of human GR: recombinant and freshly purified from red blood cells. Our results show that GR was significantly inhibited in the presence of both APAP-SG and reduced glutathione. For example, the enzyme activity of recombinant and purified GR was reduced in the presence of 4 mm APAP-SG (with 0.5 mm glutathione disulfide) by 28% and 22%, respectively. The type of enzyme inhibition was observed to be competitive in the cases of both APAP-SG and glutathione. As glutathione inhibits GR activity in cells under physiological conditions, the rate of enzyme inhibition ought to be weaker in the case of glutathione depletion that is typical of acetaminophen overdose. Notably, however, enzyme activity likely remains inhibited due to the presence of APAP-SG, which might enhance the pro-oxidative status in the cell. We conclude that our finding could reflect some other pathological mechanism that may contribute to the toxicity of acetaminophen.

  16. Peroxynitrite-mediated nitration of the stable free radical tyrosine residue of the ribonucleotide reductase small subunit.

    Science.gov (United States)

    Guittet, O; Decottignies, P; Serani, L; Henry, Y; Le Maréchal, P; Laprévote, O; Lepoivre, M

    2000-04-25

    Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase. The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity. This radical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues. In this report, nitrated residues in the E. coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing. Mass analysis allowed the detection of protein R2 as a native dimer with two iron clusters per subunit. The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da. Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein. A mean of two nitrotyrosines per E. coli protein R2 dimer were obtained at 400 microM peroxynitrite. Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122. Despite its radical state, that should favor nitration, the buried Tyr122 was not nitrated with a high yield, probably owing to its restricted accessibility. Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed. However, protein R2 inactivation was higher than nitration of Tyr122, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for peroxynitrite-mediated inhibition of E. coli protein R2.

  17. The respiratory arsenate reductase from Bacillus selenitireducens strain MLS10

    Science.gov (United States)

    Afkar, E.; Lisak, J.; Saltikov, C.; Basu, P.; Oremland, R.S.; Stolz, J.F.

    2003-01-01

    The respiratory arsenate reductase from the Gram-positive, haloalkaliphile, Bacillus selenitireducens strain MLS10 was purified and characterized. It is a membrane bound heterodimer (150 kDa) composed of two subunits ArrA (110 kDa) and ArrB (34 kDa), with an apparent Km for arsenate of 34 ??M and Vmax of 2.5 ??mol min-1 mg-1. Optimal activity occurred at pH 9.5 and 150 g l-1 of NaCl. Metal analysis (inductively coupled plasma mass spectrometry) of the holoenzyme and sequence analysis of the catalytic subunit (ArrA; the gene for which was cloned and sequenced) indicate it is a member of the DMSO reductase family of molybdoproteins. ?? 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

  18. Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase

    Institute of Scientific and Technical Information of China (English)

    Rajib; Sengupta; Arne; Holmgren

    2014-01-01

    Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.

  19. Molecular modeling, structural analysis and identification of ligand binding sites of trypanothione reductase from Leishmania mexicana

    Directory of Open Access Journals (Sweden)

    Ozal Mutlu

    2013-01-01

    Full Text Available Background & objectives: Trypanothione reductase (TR is a member of FAD-dependent NADPH oxidoreductase protein family and it is a key enzyme which connects the NADPH and the thiol-based redox system. Inhibition studies indicate that TR is an essential enzyme for parasite survival. Therefore, it is an attractive target enzyme for novel drug candidates. There is no structural model for TR of Leishmania mexicana (LmTR in the protein databases. In this work, 3D structure of TR from L. mexicana was identified by template-based in silico homology modeling method, resultant model was validated, structurally analyzed and possible ligand binding pockets were identified. Methods: For computational molecular modeling study, firstly, template was identified by BLAST search against PDB database. Multiple alignments were achieved by ClustalW2. Molecular modeling of LmTR was done and possible drug targeting sites were identified. Refinement of the model was done by performing local energy minimization for backbone, hydrogen and side chains. Model was validated by web-based servers. Results: A reliable 3D model for TR from L. mexicana was modeled by using L. infantum trypanothione reductase (LiTR as a template. RMSD results according to C-alpha, visible atoms and backbone were 0.809 Å, 0.732 Å and 0.728 Å respectively. Ramachandran plot indicates that model shows an acceptable stereochemistry. Conclusion: Modeled structure of LmTR shows high similarity with LiTR based on overall structural features like domains and folding patterns. Predicted structure will provide a source for the further docking studies of various peptide-based inhibitors.

  20. In vitro and in vivo biotransformation of simvastatin, an inhibitor of HMG CoA reductase.

    Science.gov (United States)

    Vickers, S; Duncan, C A; Vyas, K P; Kari, P H; Arison, B; Prakash, S R; Ramjit, H G; Pitzenberger, S M; Stokker, G; Duggan, D E

    1990-01-01

    Simvastatin (SV), an analog of lovastatin, is the lactone form of 1', 2', 6', 7', 8', 8a'-hexahydro-3,5-dihydroxy-2', 6'-dimethyl-8' (2", 2"-dimethyl-1"-oxobutoxy)-1'-naphthalene-heptanoic acid (SVA) which lowers plasma cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase. SV but not its corresponding hydroxy acid form SVA underwent microsomal metabolism. Major in vitro metabolites were 6'-OH-SV (I) and 3"-OH-SV (III) formed by allylic and aliphatic hydroxylation, respectively, and 6'-exomethylene-SV (IV) formed by dehydrogenation. In rats, dogs, and humans, biliary excretion is the major route of elimination. Biliary metabolites (as both hydroxy acids and lactones) also included 6'-CH2OH-SV (V) and 6'-COOH-SV (VI) in both of which the 6'-chiral center had been inverted. High levels of esterase in rodent plasma favored the formation of SVA from SV. The formation of 1', 2', 6', 7', 8', 8a'-hexahydro-2', 6'-dimethyl-8'-(2",2"-dimethyl-1-oxobutoxy)-1'-naphthalene-pentano ic acid (VII) only in rodents represented a species difference in the metabolism of SV. It is proposed that VII is formed by beta-oxidation pathways of fatty acid intermediary metabolism. Several metabolites resulting from microsomal oxidation (after subsequent conversion from lactones to hydroxy acids) are effective inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and may contribute to the cholesterol lowering effect of SV. Qualitatively, the metabolism of SV closely resembles that of lovastatin.

  1. Role of aldo-keto reductase enzymes in mediating the timing of parturition

    Directory of Open Access Journals (Sweden)

    Michael C. Byrns

    2012-01-01

    Full Text Available A better understanding of the mechanisms underlying the initiation of parturition would provide an important step towards the development of more effective therapies for the prevention of pre-term labor and the associated neonatal morbidity and mortality. Expression of aldo-keto reductases (AKR from the 1D, 1C, and 1B subfamilies are likely to play an important role in determining the timing of parturition through their effects on metabolism of progesterone and prostaglandins. Placental expression of AKR1D1 (human 5β reductase likely contributes to the maintenance of pregnancy by inhibiting myometrial contractility through the formation of 5β-dihydroprogesterone (DHP. AKR1C1, AKR1C2, and AKR1C3 all catalyze the 20-ketosteroid reduction of progestins and can also catalyze the 3-ketosteroid reduction of pregnanes. They could therefore be playing an important role in eliminating tocolytic progestins at term. Activation of the F prostanoid receptor by its ligands also plays a critical role in initiation of labor. AKR1C3 also has prostaglandin (PG F synthase activities that would contribute to the initiation of labor. AKR1C3 converts PGH2 to PGF2α and PGD2 to 9α,11β-PGF2. AKR1B1 also contributes to the conversion of PGH2 to PGF2α, but does not form 9α,11β-PGF2. Consistent with the potential role for AKR1C3 in the initiation of parturition, indomethacin, which is a potent and isoform selective inhibitor of AKR1C3, has long been used for tocolysis.

  2. Structural and functional diversity of ferredoxin-NADP(+) reductases.

    Science.gov (United States)

    Aliverti, Alessandro; Pandini, Vittorio; Pennati, Andrea; de Rosa, Matteo; Zanetti, Giuliana

    2008-06-15

    Although all ferredoxin-NADP(+) reductases (FNRs) catalyze the same reaction, i.e. the transfer of reducing equivalents between NADP(H) and ferredoxin, they belong to two unrelated families of proteins: the plant-type and the glutathione reductase-type of FNRs. Aim of this review is to provide a general classification scheme for these enzymes, to be used as a framework for the comparison of their properties. Furthermore, we report on some recent findings, which significantly increased the understanding of the structure-function relationships of FNRs, i.e. the ability of adrenodoxin reductase and its homologs to catalyze the oxidation of NADP(+) to its 4-oxo derivative, and the properties of plant-type FNRs from non-photosynthetic organisms. Plant-type FNRs from bacteria and Apicomplexan parasites provide examples of novel ways of FAD- and NADP(H)-binding. The recent characterization of an FNR from Plasmodium falciparum brings these enzymes into the field of drug design.

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

    Science.gov (United States)

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

    2016-01-01

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

  4. 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.

  5. HMG-CoA reductase inhibitor rosuvastatin improves abnormal brain electrical activity via mechanisms involving eNOS.

    Science.gov (United States)

    Seker, F B; Kilic, U; Caglayan, B; Ethemoglu, M S; Caglayan, A B; Ekimci, N; Demirci, S; Dogan, A; Oztezcan, S; Sahin, F; Yilmaz, B; Kilic, E

    2015-01-22

    Apart from its repressing effect on plasma lipid levels, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors exert neuroprotective functions in animal models of neurodegenerative disorders. In view of these promising observations, we were interested in whether HMG-CoA reductase inhibition would affect epileptiform activity in the brain. To elucidate this issue, atorvastatin, simvastatin and rosuvastatin were administered orally at a dose of 20 mg/kg each for 3 days and their anti-epileptic activities were tested and compared in rats. Epileptiform activity in the brain was induced by an intracortical penicillin G injection. Among HMG-CoA reductase inhibitors, simvastatin-treatment was less effective in terms of spike frequency as compared with atorvastatin- and rosuvastatin-treated animals. Atorvastatin treatment reduced spike frequencies and amplitudes significantly throughout the experiment. However, the most pronounced anti-epileptic effect was observed in rosuvastatin-treated animals, which was associated with improved blood-brain barrier (BBB) integrity, increased expression of endothelial nitric oxide synthase (eNOS) mRNA and decreased expressions of pro-apoptotic p53, Bax and caspase-3 mRNAs. Inhibition of eNOS activity with L-NG-Nitroarginine Methyl Ester (L-NAME) reversed the anti-epileptic effect of rosuvastatin significantly. However, L-NAME did not alter the effect of rosuvastatin on the levels of p53, Bax and caspase-3 mRNA expression. Here, we provide evidence that among HMG-CoA reductase inhibitors, rosuvastatin was the most effective statin on the reduction of epileptiform activity, which was associated with improved BBB permeability, increased expression of eNOS and decreased expressions of pro-apoptotic p53, Bax and caspase-3. Our observation also revealed that the anti-epileptic effect of rosuvastatin was dependent on the increased expression level of eNOS. The robust anti-epileptic effect encourages proof-of-concept studies with

  6. Trypanothione reductase: a target protein for a combined in vitro and in silico screening approach.

    Directory of Open Access Journals (Sweden)

    Mathias Beig

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

  7. Novel dehydroepiandrosterone benzimidazolyl derivatives as 5α-reductase isozymes inhibitors.

    Science.gov (United States)

    Arellano, Yazmín; Bratoeff, Eugene; Segura, Tania; Mendoza, Maria Eugenia; Sánchez-Márquez, Araceli; Medina, Yesica; Heuze, Yvonne; Soriano, Juan; Cabeza, Marisa

    2016-12-01

    5α-R isozymes (types 1 and 2) play an important role in prostate gland development because they are responsible for intraprostatic dihydrotestosterone (DHT) levels when the physiological serum testosterone (T) concentration is low. In this study, we synthesized seven novel dehydroepiandrosterone derivatives with benzimidazol moiety at C-17, and determined their effect on the activity of 5α-reductase types 1 and 2. The derivatives with an aliphatic ester at C-3 of the dehydroepiandrosterone scaffold induced specific inhibition of 5α-R1 activity, whereas those with a cycloaliphatic ester (cyclopropyl, cyclobutyl, or cyclopentyl ring) or an alcohol group at C-3 inhibited the activity of both isozymes. Derivatives with a cyclohexyl or cycloheptyl ester at C-3 showed no inhibitory activity. In pharmacological experiments, derivatives with esters having an alcohol or the aliphatic group or one of the three smaller cycloaliphatic rings at C-3 decreased the diameter of male hamster flank organs, with the cyclobutyl and cyclopentyl esters exhibiting higher effect. With exception of the cyclobutyl and cyclopentyl esters, these compounds reduced the weight of the prostate and seminal vesicles.

  8. Dihydrofolate reductase is required for the development of heart and outflow tract in zebrafish

    Institute of Scientific and Technical Information of China (English)

    Shuna Sun; Yonghao Gui; Qiu Jiang; Houyan Song

    2011-01-01

    Folic acid is very important for embryonic development and folic acid inhibition can cause congenital heart defects in vertebrates.Dihydrofolate reductase (DHFR) is a key enzyme in folate-mediated metabolism.The dysfunction of DHFR disrupts the key biological processes which folic acid participates in.DHFR gene is conserved during vertebrate evolution.It is important to investigate the roles of DHFR in cardiac developments.In this study,we showed that DHFR knockdown resulted in the abnormal developments of zebrafish embryos in the early stages.Obvious malformations in heart and outflow tract (OFT) were also observed in DHFR knockdown embryos.DHFR overexpression rescued the abnormal phenotypes in the DHFR knockdown group.DHFR knockdown had negative impacts on the expressions of NKX2.5 (NK2 transcription factor-related 5),MEF2C (myocyte-specific enhancer factor 2C),TBX20 (T-box 20),and TBX1 (T-box 1) which are important transcriptional factors during cardiac development process,while DHFR overexpression had positive effects.DHFR was required for Hedgehog pathway.DHFR knockdown caused reduced cell proliferation and increased apoptosis,while its overexpression promoted cell proliferation and inhibited apoptosis.Taken together,our study suggested that DHFR plays crucial roles in the development of heart and OFT in zebrafish by regulating gene transcriptions and affecting cell proliferation and apoptosis.

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

    Directory of Open Access Journals (Sweden)

    Marek Burzyński

    2014-02-01

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

  10. Redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase is required for high-light stress tolerance in Arabidopsis.

    Science.gov (United States)

    Noshi, Masahiro; Hatanaka, Risa; Tanabe, Noriaki; Terai, Yusuke; Maruta, Takanori; Shigeoka, Shigeru

    2016-05-01

    Chloroplasts are a significant site for reactive oxygen species production under illumination and, thus, possess a well-organized antioxidant system involving ascorbate. Ascorbate recycling occurs in different manners in this system, including a dehydroascorbate reductase (DHAR) reaction. We herein investigated the physiological significance of DHAR3 in photo-oxidative stress tolerance in Arabidopsis. GFP-fused DHAR3 protein was targeted to chloroplasts in Arabidopsis leaves. A DHAR3 knockout mutant exhibited sensitivity to high light (HL). Under HL, the ascorbate redox states were similar in mutant and wild-type plants, while total ascorbate content was significantly lower in the mutant, suggesting that DHAR3 contributes, at least to some extent, to ascorbate recycling. Activation of monodehydroascorbate reductase occurred in dhar3 mutant, which might compensate for the lack of DHAR3. Interestingly, glutathione oxidation was consistently inhibited in dhar3 mutant. These findings indicate that DHAR3 regulates both ascorbate and glutathione redox states to acclimate to HL.

  11. Trypanothione reductase activity is prominent in metacyclic promastigotes and axenic amastigotes of Leishmania amazonesis. Evaluation of its potential as a therapeutic target.

    Science.gov (United States)

    Castro-Pinto, Denise B; Echevarria, Aurea; Genestra, Marcelo S; Cysne-Finkelstein, Léa; Leon, Leonor L

    2004-02-01

    The activity of trypanothione reductase in Leishmania amazonensis was evaluated and it was demonstrated that TR is expressed in the soluble fractions of infective promastigotes and amastigotes, while non-infective promastigotes expressed the enzyme at basal levels. This data allows an association of enzyme activity and the infective capacity of the parasite. We have also previously demonstrated that amidine compounds (N, N'-diphenyl-4-methoxy-benzamidine and pentamidine) were active against this parasite. Here, experiments concerning the effect of these compounds on TR activity, showed that both compounds significantly inhibited the enzyme. However, against glutathione reductase, only pentamidine showed a significant inhibitory action, suggesting an association with the toxic effects of this drug used in the clinic for the treatment of leishmaniasis.

  12. Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase

    Directory of Open Access Journals (Sweden)

    Justin R. Prigge

    2017-06-01

    Full Text Available Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1 disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1 and glutathione reductase (Gsr, respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

  13. 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

    can replace light in eliciting an increase of nitrate reductase mRNA accumulation in dark-adapted green Arabidopsis plants. We show further that sucrose alone is sufficient for the full expression of nitrate reductase genes in etiolated Arabidopsis plants. Finally, using a reporter gene, we show......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....... Located in the cytosol, nitrate reductase obtains its reductant not from photosynthesis but from carbohydrate catabolism. This relationship prompted us to investigate the indirect role that light might play, via photosynthesis, in the regulation of nitrate reductase gene expression. We show that sucrose...

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

    Directory of Open Access Journals (Sweden)

    Jolanta Marciniak

    2014-01-01

    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.

  15. Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening

    Directory of Open Access Journals (Sweden)

    Lin SH

    2015-06-01

    Full Text Available Shih-Hung Lin,1 Kao-Jean Huang,1,2 Ching-Feng Weng,1 David Shiuan1 1Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, Republic of China; 2Development Center of Biotechnology, Taipei, Taiwan, Republic of China Abstract: Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR. The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening. Keywords: HMG-CoA reductase, virtual screening, curcumin, salvianolic acid C

  16. Comparative studies on the soluble and plasma membrane associated nitrate reductase from Cucumis sativus L.

    Directory of Open Access Journals (Sweden)

    Grażyna Kłobus

    2014-02-01

    Full Text Available The biochemical comparison between two forms of nitrate reductase from cucumber roots: the soluble enzyme and the plasma membrane-associated one was made. Soluble nitrate reductase was purified on the blue-Sepharose 4B. The nitrate reductase bound with plasma membranes was isolated from cucumber roots by partition of microsomes in the 6.5% dextran-PEG two phase system. The molecular weight of native enzyme estimated with HPLC was 240 kDa and 114 kDa for the soluble and membrane bounded enzyme, respectively. Temperature induced phase separation in Triton X-114 indicated a huge difference in hydrophobicity of the plasma membrane associated nitrate reductase and soluble form of enzyme. Small differences were observed in partial activities of plasma membrane nitrate reductase and soluble nitrate reductase. Also experiments with polyclonal antiserum raised against the native nitrate reductase showed some differences in the immunological properties of both forms of the nitrate reductase. The above results indicated that in cucumber roots two different forms of the nitrate reductase are present.

  17. 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.

  18. 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

    allele 1·02, 95% CI 1·00-1·05); the rs12916-T allele association was consistent (1·06, 1·03-1·09). In 129 170 individuals in randomised trials, statins lowered LDL cholesterol by 0·92 mmol/L (95% CI 0·18-1·67) at 1-year of follow-up, increased bodyweight by 0·24 kg (95% CI 0·10-0·38 in all trials; 0...

  19. Enhanced Ascorbate Regeneration Via Dehydroascorbate Reductase Confers Tolerance to Photo-Oxidative Stress in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Lin, Shu-Tseng; Chiou, Chih-Wen; Chu, Yen-Lin; Hsiao, Yu; Tseng, Yu-Fei; Chen, Yi-Chun; Chen, Hsien-Jung; Chang, Hsin-Yang; Lee, Tse-Min

    2016-10-01

    The role of ascorbate (AsA) recycling via dehydroascorbate reductase (DHAR) in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress was examined. The activity of DHAR and the abundance of the CrDHAR1 (Cre10.g456750) transcript increased after moderate light (ML; 750 µmol m(-2) s(-1)) or high light (HL; 1,800 µmol m(-2) s(-1)) illumination, accompanied by dehydroascorbate (DHA) accumulation, decreased AsA redox state, photo-inhibition, lipid peroxidation, H2O2 overaccumulation, growth inhibition and cell death. It suggests that DHAR and AsA recycling is limiting under high-intensity light stress. The CrDHAR1 gene was cloned and its recombinant CrDHAR1 protein was a monomer (25 kDa) detected by Western blot that exhibits an enzymatic activity of 965 µmol min(-1)( )mg(-1) protein. CrDHAR1 was overexpressed driven by a HSP70A:RBCS2 fusion promoter or down-regulated by artificial microRNA (amiRNA) to examine whether DHAR-mediated AsA recycling is critical for the tolerance of C. reinahartii cells to photo-oxidative stress. The overexpression of CrDHAR1 increased DHAR protein abundance and enzyme activity, AsA pool size, AsA:DHA ratio and the tolerance to ML-, HL-, methyl viologen- or H2O2-induced oxidative stress. The CrDHAR1-knockdown amiRNA lines that have lower DHAR expression and AsA recycling ability were sensitive to high-intensity illumination and oxidative stress. The glutathione pool size, glutathione:oxidized glutathione ratio and glutathione reductase and ascorbate peroxidase activities were increased in CrDHAR1-overexpressing cells and showed a further increase after high-intensity illumination but decreased in wild-type cells after light stress. The present results suggest that increasing AsA regeneration via enhanced DHAR activity modulates the ascorbate-glutathione cycle activity in C. reinhardtii against photo-oxidative stress.

  20. Inhibitory effect of fruit juices on the doxorubicin metabolizing activity of carbonyl reductase 1.

    Science.gov (United States)

    Miura, Takeshi; Nagai, Katsutoshi; Kaneshiro, Shingo; Taketomi, Ayako; Nakabayashi, Toshikatsu; Konishi, Hiroki; Nishinaka, Toru; Terada, Tomoyuki

    2017-03-09

    Doxorubicin, an anthracycline anti-cancer drug, is effective for breast cancer and childhood lymphoma. Chronic cardiotoxicity has been known as a critical adverse effect of doxorubicin and is attributed to its metabolite doxorubicinol produced by carbonyl reductase 1 (CBR1, SDR21C1). Some flavonoids have been reported to act as inhibitors for CBR1, therefore, commercially available juices containing flavonoids are likely to be applicable as a prophylactic treatment against doxorubicin-induced cardiotoxicity by suppression doxorubicinol production. In the study, fruit juices containing flavonoids were investigated on inhibitory effects for CBR1. Recombinant CBR1 protein was subjected to in vitro enzymatic assays with/without juices. An apple juice and a grapefruit juice were selected in the present study as candidates capable of inhibiting CBR1. The enzymatic assays revealed that both juices potently inhibit the CBR1-mediated metabolic conversion of doxorubicin to doxorubicinol in concertation-dependent manners. The concentrations required for obtaining 50% inhibition (IC50) were 0.0012% (v/v) and 0.0014% (v/v) for apple and grapefruit juices, respectively. Additionally, it is worth noting that these juices showed inhibitory effects on doxorubicin metabolism by CBR1 even at very low concentrations (0.0001% (v/v)). An apple juice and a grape fruit juice showed strong inhibitory effects on doxorubicin metabolism by CBR1 in vitro. These results suggest that the intake of flavonoid-containing juices can be promising measure for the protection against doxorubicin-induced cardiac toxicity, enabling patients to keep higher adherence with routine use in light of safety, economic performance and stable supply to market. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  1. Binding of DNA-Intercalating Agents to Oxidized and Reduced Quinone Reductase 2.

    Science.gov (United States)

    Leung, Kevin K K; Shilton, Brian H

    2015-12-29

    Quinone reductase 2 (NQO2) is an enzyme that might have intracellular signaling functions. NQO2 can exist in either an oxidized state or a reduced state, and binding of compounds to one or both of these states inhibits enzymatic activity and could also affect intracellular signaling. A wide range of planar aromatic compounds bind NQO2, and we have identified three DNA-intercalating agents [ethidium bromide, acridine orange (AO), and doxorubicin] as novel nanomolar inhibitors of NQO2. Ethidium and AO, which carry a positive charge in their aromatic ring systems, bound reduced NQO2 with an affinity 50-fold higher than that of oxidized NQO2, while doxorubicin bound only oxidized NQO2. Crystallographic analyses of oxidized NQO2 in complex with the inhibitors indicated that the inhibitors were situated deep in the active site. The aromatic faces were sandwiched between the isoalloxazine ring of FAD and the phenyl ring of F178, with their edges making direct contact with residues lining the active site. In reduced NQO2, ethidium and AO occupied a more peripheral position in the active site, allowing several water molecules to interact with the polar end of the negatively charged isoalloxazine ring. We also showed that AO inhibited NQO2 at a nontoxic concentration in cells while ethidium was less effective at inhibiting NQO2 in cells. Together, this study shows that reduced NQO2 has structural and electrostatic properties that yield a preference for binding of planar, aromatic, and positively charged molecules that can also function as DNA-intercalating agents.

  2. Aldo-keto reductases 1B in adrenal cortex physiology

    Directory of Open Access Journals (Sweden)

    Emilie PASTEL

    2016-07-01

    Full Text Available Aldose reductase proteins are cytosolic monomeric enzymes, belonging to the aldo-keto reductase (AKR superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates such as aliphatic and aromatic aldehydes or ketones. The Aldose reductase subgroup (AKR1B is one of the most characterized because of its involvement in human diseases such as diabetic complications resulting from the ability of its human archetype AKR1B1 to reduce glucose into sorbitol. However the issue of AKR1B function in non pathologic condition remains poorly resolved. Adrenal steroidogenesis is strongly associated with high production of endogenous harmful lipid aldehyde by-products including isocaproaldehyde (4-methylpentanal derived from cholesterol side chain cleavage (the first step of steroid synthesis and 4-hydroxynonenal (4- HNE that can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase activity, suggesting that in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, previous studies have established that the adrenal gland is one of the major site for human and murine AKR1B expression suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms.This review presents the molecular mechanisms accounting for the adrenal specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions.

  3. 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.

  4. Soybean nitrate reductase activity influenced by manganese nutrition

    OpenAIRE

    Damien P., Heenan; Lindsay C., Campbell; Department of Agronomy and Horticultural Science, University of Sydney

    1980-01-01

    Nitrate assimilation by soybeans [Glycine max (L.) Merrill cvv. Lee and Bragg] was investigated in plants grown in solution culture at manganese concentrations of 0, 1.8 and 275 μM and at day-night temperatures of 33-28℃ and 22-17℃. Manganese deficiency occurred in plants of both cultivars grown at 0 μM Mn; under these conditions, leaf nitrate concentration increased in both cultivars and nitrate reductase activity in vivo but not in vitro was reduced. High solution Mn (275 μM) produced sympt...

  5. 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.

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

    Science.gov (United States)

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

    2016-03-01

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

  7. Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase-dihydrofolate reductase.

    Science.gov (United States)

    Kumar, Vidya P; Cisneros, Jose A; Frey, Kathleen M; Castellanos-Gonzalez, Alejandro; Wang, Yiqiang; Gangjee, Aleem; White, A Clinton; Jorgensen, William L; Anderson, Karen S

    2014-09-01

    Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS-DHFR specific inhibitors.

  8. Design and synthesis of chiral 2H-chromene-N-imidazolo-amino acid conjugates as aldose reductase inhibitors.

    Science.gov (United States)

    Gopinath, Gudipudi; Sankeshi, Venu; Perugu, Shaym; Alaparthi, Malini D; Bandaru, Srinivas; Pasala, Vijay K; Chittineni, Prasad Rao; Krupadanam, G L David; Sagurthi, Someswar R

    2016-11-29

    Aldose reductase (ALR2) inhibitors provide a viable mode to fight against diabetic complications. ALR2 exhibit plasticity in the active site vicinities and possible shifts in the nearby two supporting alpha helices. Therefore, a novel series of amino acid conjugates of chromene-3-imidazoles (13-15) were designed and synthesized based on natural isoflavonoids. The compounds were identified on the basis of spectral ((1)H NMR, (13)C NMR and MS) data and tested in vitro for ALR2 inhibitory activity with an IC50 value ranges from 0.031 ± 0.082 μM to 4.29 ± 0.55 μM. Our in silico and biochemical studies confirmed that 15e has the best inhibition activity among the synthesized compounds with a high selective index against the Aldehyde reductase (ALR1). Supplementation of 15e to STZ induced rats decreased the blood glucose levels and delayed the progression of cataract in a dose-dependent manner. The present study thus provides novel series of compounds with a promising inhibitor to prevent or delay the cataract progression. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  9. An integrated approach towards the discovery of novel non-nucleoside Leishmania major pteridine reductase 1 inhibitors.

    Science.gov (United States)

    Leite, Franco Henrique Andrade; Froes, Thamires Quadros; da Silva, Suellen Gonçalves; de Souza, Evandro Italo Macêdo; Vital-Fujii, Drielli Gomes; Trossini, Gustavo Henrique Goulart; Pita, Samuel Silva da Rocha; Castilho, Marcelo Santos

    2017-05-26

    Despite the fact that Leishmania ssp are pteridine auxotrophs, Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) inhibitors are ineffective against Leishmania major. On the other hand Pteridine Reductase 1 (PTR1) inhibitors proved to be lethal to the parasite. Aiming at identifying hits that lie outside the chemical space of known PTR1 inhibitors, pharmacophore models that differentiate true-binders from decoys and explain the structure-activity relationships of known inhibitors were employed to virtually screen the lead-like subset of ZINC database. This approach leads to the identification of Z80393 (IC50 = 32.31 ± 1.18 μM), whose inhibition mechanism was investigated by Thermal Shift Assays. This experimental result supports a competitive mechanism and was crucial to establish the docking search space as well as select the best pose, which was then investigated by molecular dynamics studies that corroborate the hit putative binding profile towards LmPTR1. The information gathered from such studies shall be useful to design more potent non-nucleoside LmPTR1 inhibitors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  10. Dimethoxycurcumin, a metabolically stable analogue of curcumin enhances the radiosensitivity of cancer cells: Possible involvement of ROS and thioredoxin reductase.

    Science.gov (United States)

    Jayakumar, Sundarraj; Patwardhan, R S; Pal, Debojyoti; Sharma, Deepak; Sandur, Santosh K

    2016-09-09

    Dimethoxycurcumin (DIMC), a structural analogue of curcumin, has been shown to have more stability, bioavailability, and effectiveness than its parent molecule curcumin. In this paper the radiosensitizing effect of DIMC has been investigated in A549 lung cancer cells. As compared to its parent molecule curcumin, DIMC showed a very potent radiosensitizing effect as seen by clonogenic survival assay. DIMC in combination with radiation significantly increased the apoptosis and mitotic death in A549 cells. This combinatorial treatment also lead to effective elimination of cancer stem cells. Further, there was a significant increase in cellular ROS, decrease in GSH to GSSG ratio and also significant slowdown in DNA repair when DIMC was combined with radiation. In silico docking studies and in vitro studies showed inhibition of thioredoxin reductase enzyme by DIMC. Overexpression of thioredoxin lead to the abrogation of radiosensitizing effect of DIMC underscoring the role of thioredoxin reductase in radiosensitization. Our results clearly demonstrate that DIMC can synergistically enhance the cancer cell killing when combined with radiation by targeting thioredoxin system.

  11. Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

    Science.gov (United States)

    Ericson, Megan E.; Frank, Matthew W.

    2016-01-01

    Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. PMID:27736774

  12. Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes.

    Science.gov (United States)

    Yao, Jiangwei; Ericson, Megan E; Frank, Matthew W; Rock, Charles O

    2016-12-01

    Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. Inhibitory Activities of Phenolic Compounds Isolated from Adina rubella Leaves Against 5α-Reductase Associated with Benign Prostatic Hypertrophy.

    Science.gov (United States)

    Yin, Jun; Heo, Jun Hyeok; Hwang, Yoon Jeong; Le, Thi Tam; Lee, Min Won

    2016-07-07

    Adina rubella Hance (AR), a plant native to Korea, has been used as traditional medicine for dysentery, eczema, intoxication, and external hemorrhages. Previous phytochemical studies of AR have reported several components, including terpenoids, phenolics, and alkaloids. The current study evaluated the anti-oxidative and anti-inflammatory activities and 5α-reductase inhibition of isolated compounds of AR leaves to find a potential therapeutic agent for benign prostatic hypertrophy (BPH). Repeated chromatographic isolation of an 80% acetone extract of AR leaves yielded seven phenolic compounds: caffeic acid (1), chlorogenic acid (2), methyl chlorogenate (3), quercetin-3-rutinoside (4), kaempferol-3-O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside (5), hyperoside (6), and grandifloroside (7). Compound 7 is a novel compound in AR. Caffeoyl derivatives 1-3 and 7 showed good anti-oxidative activities. In particular, caffeic acid (1) and grandifloroside (7) showed potent anti-inflammatory activities, and 7 also exhibited potent inhibitory activity against TNF-α and 5α-reductase. Our results show that the extract and grandifloroside (7) from leaves of AR might be developed as a source of potent anti-oxidative and anti-inflammatory agents and therapeutic agent for BPH.

  14. Inhibitory Activities of Phenolic Compounds Isolated from Adina rubella Leaves Against 5α-Reductase Associated with Benign Prostatic Hypertrophy

    Directory of Open Access Journals (Sweden)

    Jun Yin

    2016-07-01

    Full Text Available Adina rubella Hance (AR, a plant native to Korea, has been used as traditional medicine for dysentery, eczema, intoxication, and external hemorrhages. Previous phytochemical studies of AR have reported several components, including terpenoids, phenolics, and alkaloids. The current study evaluated the anti-oxidative and anti-inflammatory activities and 5α-reductase inhibition of isolated compounds of AR leaves to find a potential therapeutic agent for benign prostatic hypertrophy (BPH. Repeated chromatographic isolation of an 80% acetone extract of AR leaves yielded seven phenolic compounds: caffeic acid (1, chlorogenic acid (2, methyl chlorogenate (3, quercetin-3-rutinoside (4, kaempferol-3-O-α-l-rhamnopyranosyl-(1→6-β-d-glucopyranoside (5, hyperoside (6, and grandifloroside (7. Compound 7 is a novel compound in AR. Caffeoyl derivatives 1–3 and 7 showed good anti-oxidative activities. In particular, caffeic acid (1 and grandifloroside (7 showed potent anti-inflammatory activities, and 7 also exhibited potent inhibitory activity against TNF-α and 5α-reductase. Our results show that the extract and grandifloroside (7 from leaves of AR might be developed as a source of potent anti-oxidative and anti-inflammatory agents and therapeutic agent for BPH.

  15. A novel NADPH thioredoxin reductase, localized in the chloroplast, which deficiency causes hypersensitivity to abiotic stress in Arabidopsis thaliana.

    Science.gov (United States)

    Serrato, Antonio Jesús; Pérez-Ruiz, Juan Manuel; Spínola, María Cristina; Cejudo, Francisco Javier

    2004-10-15

    Plants contain three thioredoxin systems. Chloroplast thioredoxins are reduced by ferredoxin-thioredoxin reductase, whereas the cytosolic and mitochondrial thioredoxins are reduced by NADPH thioredoxin reductase (NTR). There is high similarity among NTRs from plants, lower eukaryotes, and bacteria, which are different from mammal NTR. Here we describe the OsNTRC gene from rice encoding a novel NTR with a thioredoxin-like domain at the C terminus, hence, a putative NTR/thioredoxin system in a single polypeptide. Orthologous genes were found in other plants and cyanobacteria, but not in bacteria, yeast, or mammals. Full-length OsNTRC and constructs with truncated NTR and thioredoxin domains were expressed in Escherichia coli as His-tagged polypeptides, and a polyclonal antibody specifically cross-reacting with the OsNTRC enzyme was raised. An in vitro activity assay showed that OsNTRC is a bifunctional enzyme with both NTR and thioredoxin activity but is not an NTR/thioredoxin system. Although the OsNTRC gene was expressed in roots and shoots of rice seedlings, the protein was exclusively found in shoots and mature leaves. Moreover, fractionation experiments showed that OsNTRC is localized to the chloroplast. An Arabidopsis NTRC knock-out mutant showed growth inhibition and hypersensitivity to methyl viologen, drought, and salt stress. These results suggest that the NTRC gene is involved in plant protection against oxidative stress.

  16. Spectroscopic and kinetic properties of a recombinant form of the flavin domain of spinach NADH: nitrate reductase.

    Science.gov (United States)

    Quinn, G B; Trimboli, A J; Prosser, I M; Barber, M J

    1996-03-01

    The C-terminal 268 residues of the spinach assimilatory NADH:nitrate reductase amino acid sequence that correspond to the flavin-containing domain of the enzyme have been selectively amplified and expressed as a recombinant protein in Escherichia coli. The recombinant protein, which was produced in both soluble and insoluble forms, was purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose and FPLC gel filtration. The purified domain exhibited a molecular weight of approximately 30 kDa, estimated by polyacrylamide gel electrophoresis, and a molecular mass of 30,169 for the apoprotein determined by mass spectrometry, which also confirmed the presence of FAD. The UV/visible spectrum was typical of a flavoprotein, with maxima at 272, 386, and 461 nm in the oxidized form while CD spectroscopy yielded both positive and negative maxima at 313 and 382 nm and 461 and 484 nm, respectively. The purified domain showed immunological cross-reactivity with anti-spinach nitrate reductase polyclonal antibodies while both N-terminal and internal amino acid sequencing of isolated peptides confirmed the fidelity of the domain's primary sequence. The protein retained NADH-ferricyanide reductase activity (Vmax=84 micromol NADH consumer/min/nmol FAD) with Km's of 17 and 34 microM for NADH and ferricyanide, respectively, with a pH optimum of approximately 6.5 A variety of NADH-analogs could also function as electron donors, though with decreased efficiency, the most effective being reduced nicotinamide hypoxanthine dinucleotide (V(max) = 35 micromol NHDH consumer/min/nmol FAD) and Km = 22 microM). NAD+ was demonstrated to be a competitive inhibitor (Ki = 1.9 mM) while analysis of inhibition by a variety of NAD+-analogs indicated the most efficient inhibitor to be ADP (Ki = 0.2 mM), with analogs devoid of either the phosphate, ribose, or adenine moieties proving to be markedly less-efficient inhibitors. The isolated domain

  17. 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...

  18. Stereospecificity of (+)-pinoresinol and (+)-lariciresinol reductases from Forsythia intermedia.

    Science.gov (United States)

    Chu, A; Dinkova, A; Davin, L B; Bedgar, D L; Lewis, N G

    1993-12-25

    Pinoresinol/lariciresinol reductase catalyzes the first known example of a highly unusual benzylic ether reduction in plants; its mechanism of hydride transfer is described. The enzyme was found in Forsythia intermedia and catalyzes the presumed regulatory branch-points in the pathway leading to benzylaryltetrahydrofuran, dibenzylbutane, dibenzylbutyrolactone, and aryltetrahydronaphthalene lignans. Using [7,7'-2H2]-pinoresinol and [7,7'-2H3]lariciresinol as substrates, the hydride transfers of the highly unusual reductase were demonstrated to be completely stereospecific (> 99%). The incoming hydrides were found to take up the pro-R position at C-7' (and/or C-7) in lariciresinol and secoisolariciresinol, thereby eliminating the possibility of random hydride delivery to a planar quinone methide intermediate. As might be expected, the mode of hydride abstraction from NADPH was also stereospecific: using [4R-3H] and [4S-3H]NADPH, it was found that only the 4 pro-R hydrogen was abstracted for enzymatic hydride transfer.

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

    DEFF Research Database (Denmark)

    Farver, Ole; Brunori, Maurizio; Cutruzzolà, Francesca

    2009-01-01

    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......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......-controlled bimolecular process, followed by unimolecular electron equilibration between the c and d(1) hemes (k(ET) = 4.3 s(-1) and K = 1.4 at 298 K, pH 7.0). In the case of the mutant, the latter ET rate was faster by almost one order of magnitude. Moreover, the internal ET rate dropped (by approximately 30-fold...

  20. Properties of the arsenate reductase of plasmid R773.

    Science.gov (United States)

    Gladysheva, T B; Oden, K L; Rosen, B P

    1994-06-14

    Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resistance to arsenate is conferred by the product of the arsC gene. In this report, purified ArsC protein was shown to catalyze reduction of arsenate to arsenite. The enzymatic activity of the ArsC protein required glutaredoxin as a source of reducing equivalents. Other reductants, including glutathione and thioredoxin, were not effective electron donors. A spectrophotometric assay was devised in which arsenate reduction was coupled to NADPH oxidation. The results obtained with the coupled assay corresponded to those found by direct reduction of radioactive arsenate to arsenite. The only substrate of the reaction was arsenate (Km = 8 mM); other oxyanions including phosphate, sulfate, and antimonate were not reduced. Phosphate and sulfate were weak inhibitors, while the product, arsenite, was a stronger inhibitor (Ki = 0.1 mM). Arsenate reductase activity exhibited a pH optimum of 6.3-6.8. These results indicate that the ArsC protein is a novel reductase, and elucidation of its enzymatic mechanism should be of interest.

  1. VC对土壤亚硝酸还原酶动力学参数的影响%Effect of VC on Soil Nitrite Reductase Kinetic Parameters

    Institute of Scientific and Technical Information of China (English)

    许明惠; 李昌满

    2012-01-01

    采用rpиcc试剂比色法,研究了VC对灰棕紫泥土亚硝酸还原酶动力学参数的影响.结果表明,VC的加入降低了V0、Km和Vmax值,表明VC对土壤亚硝酸还原酶的作用类型表现为反竞争性抑制作用,但低浓度VC会减弱亚硝酸还原酶与底物亲和力,而加快产物与酶的分离.由于VC具有抗氧化作用,对土壤具有良好作用,作为安全、有效的反硝化抑制剂使用.%The article researched the effect of Vc treatment on the nitrite reductase kinetic parameter of gray-brown purple clay soil by rpHCC reagent colorimetric method. The result showed that Vc treatment reduced the values of V0, Km and Vmax, the effect type of Vc on soil nitrite reductase was uncompetitive inhibition. Low concentration Vc reduced the affinity of nitrite reductase with substrate, and accelerated the separation of products and enzyme. Vc has antioxidant effect, and is good for to soil, it could be the safe and functional nitrification inhibitors.

  2. ADP-ribosylation of dinitrogenase reductase in Azospirillum brasilense is regulated by AmtB-dependent membrane sequestration of DraG.

    Science.gov (United States)

    Huergo, Luciano F; Souza, Emanuel M; Araujo, Mariana S; Pedrosa, Fábio O; Chubatsu, Leda S; Steffens, Maria B R; Merrick, Mike

    2006-01-01

    Nitrogen fixation in some diazotrophic bacteria is regulated by mono-ADP-ribosylation of dinitrogenase reductase (NifH) that occurs in response to addition of ammonium to the extracellular medium. This process is mediated by dinitrogenase reductase ADP-ribosyltransferase (DraT) and reversed by dinitrogenase reductase glycohydrolase (DraG), but the means by which the activities of these enzymes are regulated are unknown. We have investigated the role of the P(II) proteins (GlnB and GlnZ), the ammonia channel protein AmtB and the cellular localization of DraG in the regulation of the NifH-modification process in Azospirillum brasilense. GlnB, GlnZ and DraG were all membrane-associated after an ammonium shock, and both this membrane sequestration and ADP-ribosylation of NifH were defective in an amtB mutant. We now propose a model in which membrane association of DraG after an ammonium shock creates a physical separation from its cytoplasmic substrate NifH thereby inhibiting ADP-ribosyl-removal. Our observations identify a novel role for an ammonia channel (Amt) protein in the regulation of bacterial nitrogen metabolism by mediating membrane sequestration of a protein other than a P(II) family member. They also suggest a model for control of ADP-ribosylation that is likely to be applicable to all diazotrophs that exhibit such post-translational regulation of nitrogenase.

  3. THE EFFECTS OF AN ALDOSE REDUCTASE INHIBITOR ON THE PROGRESSION OF DIABETIC-RETINOPATHY

    NARCIS (Netherlands)

    TROMP, A; HOOYMANS, JMM; BARENDSEN, BC; VONDOORMAAL, JJ

    1991-01-01

    The polyol pathway has long been associated with diabetic retinopathy. Glucose is converted to sorbitol with the aid of the enzyme aldose reductase. Aldose reductase inhibitors can prevent changes induced by diabetes. A total of 30 patients with minimal background retinopathy were randomly divided i

  4. Determination of potential N2O-reductase activity in soil

    NARCIS (Netherlands)

    Qin, S.P.; Yuan, H.J.; Hu, C.S.; Oenema, O.; Zhang, Y.M.; Li, X.X.

    2014-01-01

    Determination of N2O-reductase activity in soil is important for understanding the microbial regulation of nitrous oxide (N2O) concentrations in soil. Unfortunately, there are no easily applicable and accurate methods for determining N2O-reductase activity, which frustrates the understanding of the

  5. Separation and distribution of thiosulfate-oxidizing enzyme, tetrathionate reductase, and thiosulfate reductase in extracts of marine heterotroph strain 16B.

    OpenAIRE

    Whited, G M; Tuttle, J.H.

    1983-01-01

    Thiosulfate-oxidizing enzyme (TSO), tetrathionate reductase (TTR), and thiosulfate reductase (TSR) were demonstrated in cell-free extracts of the marine heterotrophic thiosulfate-oxidizing bacterium strain 16B. Extracts prepared from cells cultured aerobically in the absence of thiosulfate or tetrathionate exhibited constitutive TSO and TTR activity which resided in the soluble fraction of ultracentrifuged crude extracts. Constitutive TSO and TTR cochromatographed on DEAE-Sephadex A-50, Celle...

  6. Extraction and identification of three major aldose reductase inhibitors from Artemisia montana.

    Science.gov (United States)

    Jung, Hyun Ah; Islam, M D Nurul; Kwon, Yong Soo; Jin, Seong Eun; Son, You Kyung; Park, Jin Ju; Sohn, Hee Sook; Choi, Jae Sue

    2011-02-01

    Aldose reductase inhibitors (ARIs) provide an important therapeutic and preventive opportunity against hyperglycemia associated diabetic complications. The methanolic extracts of 12 species from the genus Artemisia exhibited significant in vitro rat lens AR (RLAR) inhibitory activities with IC(50) values ranging from 0.51 to 13.45 μg/mL (quercetin, 0.64 μg/mL). Since the whole plant of Artemisia montana showed the highest RLAR inhibitory activity, bioassay-guided fractionation was performed to obtain ethyl acetate and n-butanol fractions. Repeated column chromatography of two active fractions, yielded fifteen compounds, including four chlorogenic acids (3,5-di-O-caffeoylquinic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid), six flavonoids (apigenin, luteolin, quercetin, isoquercitrin, hyperoside, luteolin 7-rutinoside), and five coumarins (umbelliferone, scoparone, scopoletin, esculetin, and scopolin); their structures were confirmed by spectroscopic methods. 3,5-Di-O-caffeoylquinic acid and chlorogenic acid, as well as test flavonoids, displayed the most potent RLAR inhibitory activities with IC(50) values ranging from 0.19 to 5.37 μM. Furthermore, the HPLC profiles of the ethyl acetate and n-butanol fractions indicated that 3,5-di-O-caffeoylquinic acid, chlorogenic acid, and hyperoside, as major compounds, might play crucial roles in RLAR inhibition. The results suggest that A. montana and three key AR inhibitors therein would clearly be potential candidates as therapeutic or preventive agents for diabetic complications. Copyright © 2010 Elsevier Ltd. All rights reserved.

  7. Aldo-keto reductase enzymes detoxify glyphosate and improve herbicide resistance in plants.

    Science.gov (United States)

    Vemanna, Ramu S; Vennapusa, Amaranatha Reddy; Easwaran, Murugesh; Chandrashekar, Babitha K; Rao, Hanumantha; Ghanti, Kirankumar; Sudhakar, Chinta; Mysore, Kirankumar S; Makarla, Udayakumar

    2017-07-01

    In recent years, concerns about the use of glyphosate-resistant crops have increased because of glyphosate residual levels in plants and development of herbicide-resistant weeds. In spite of identifying glyphosate-detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an aldo-keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologues in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate-mediated cucumber seedling growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1- or OsAKRI-expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    2015-07-01

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

  9. Synthesis of xanthohumol analogues and discovery of potent thioredoxin reductase inhibitor as potential anticancer agent.

    Science.gov (United States)

    Zhang, Baoxin; Duan, Dongzhu; Ge, Chunpo; Yao, Juan; Liu, Yaping; Li, Xinming; Fang, Jianguo

    2015-02-26

    The selenoprotein thioredoxin reductases (TrxRs) are attractive targets for anticancer drugs development. Xanthohumol (Xn), a naturally occurring polyphenol chalcone from hops, has received increasing attention because of its multiple pharmacological activities. We synthesized Xn and its 43 analogues and discovered that compound 13n displayed the highest cytotoxicity toward HeLa cells (IC50 = 1.4 μM). Structure-activity relationship study indicates that the prenyl group is not necessary for cytotoxicity, and introducing electron-withdrawing group, especially on the meta-position, is favored. In addition, methylation of the phenoxyl groups generally improves the potency. Mechanistic study revealed that 13n selectively inhibits TrxR and induces reactive oxygen species and apoptosis in HeLa cells. Cells overexpressing TrxR are resistant to 13n insult, while knockdown of TrxR sensitizes cells to 13n treatment, highlighting the physiological significance of targeting TrxR by 13n. The clarification of the structural determinants for the potency would guide the design of novel potent molecules for future development.

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

    Energy Technology Data Exchange (ETDEWEB)

    J Beierlein; N Karri; A Anderson

    2011-12-31

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

  11. Nitrosylation of c heme in cd(1)-nitrite reductase is enhanced during catalysis.

    Science.gov (United States)

    Rinaldo, Serena; Giardina, Giorgio; Cutruzzolà, Francesca

    2014-08-29

    The reduction of nitrite into nitric oxide (NO) in denitrifying bacteria is catalyzed by nitrite reductase. In several species, this enzyme is a heme-containing protein with one c heme and one d1 heme per monomer (cd1NiR), encoded by the nirS gene. For many years, the evidence of a link between NO and this hemeprotein represented a paradox, given that NO was known to tightly bind and, possibly, inhibit hemeproteins, including cd1NiRs. It is now established that, during catalysis, cd1NiRs diverge from "canonical" hemeproteins, since the product NO rapidly dissociates from the ferrous d1 heme, which, in turn, displays a peculiar "low" affinity for NO (KD=0.11 μM at pH 7.0). It has been also previously shown that the c heme reacts with NO at acidic pH but c heme nitrosylation was not extensively investigated, given that in cd1NiR it was considered a side reaction, rather than a genuine process controlling catalysis. The spectroscopic study of the reaction of cd1NiR and its semi-apo derivative (containing the sole c heme) with NO reported here shows that c heme nitrosylation is enhanced during catalysis; this evidence has been discussed in order to assess the potential of c heme nitrosylation as a regulatory process, as observed for cytochrome c nitrosylation in mammalian mitochondria.

  12. Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase

    Science.gov (United States)

    Bachmann, M.; Shiraishi, N.; Campbell, W. H.; Yoo, B. C.; Harmon, A. C.; Huber, S. C.; Davies, E. (Principal Investigator)

    1996-01-01

    Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

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

    Science.gov (United States)

    Zarnowski, Robert; Woods, Jon P.

    2009-01-01

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

  14. Lepidopteran HMG-CoA reductase is a potential selective target for pest control

    Directory of Open Access Journals (Sweden)

    Yuan-mei Li

    2017-01-01

    Full Text Available As a consequence of the negative impacts on the environment of some insecticides, discovery of eco-friendly insecticides and target has received global attention in recent years. Sequence alignment and structural comparison of the rate-limiting enzyme HMG-CoA reductase (HMGR revealed differences between lepidopteran pests and other organisms, which suggested insect HMGR could be a selective insecticide target candidate. Inhibition of JH biosynthesis in vitro confirmed that HMGR inhibitors showed a potent lethal effect on the lepidopteran pest Manduca sexta, whereas there was little effect on JH biosynthesis in Apis mellifera and Diploptera punctata. The pest control application of these inhibitors demonstrated that they can be insecticide candidates with potent ovicidal activity, larvicidal activity and insect growth regulatory effects. The present study has validated that Lepidopteran HMGR can be a potent selective insecticide target, and the HMGR inhibitors (especially type II statins could be selective insecticide candidates and lead compounds. Furthermore, we demonstrated that sequence alignment, homology modeling and structural comparison may be useful for determining potential enzymes or receptors which can be eco-friendly pesticide  targets.

  15. Lepidopteran HMG-CoA reductase is a potential selective target for pest control

    Science.gov (United States)

    Li, Yuan-mei; Huang, Juan; Tobe, Stephen S.

    2017-01-01

    As a consequence of the negative impacts on the environment of some insecticides, discovery of eco-friendly insecticides and target has received global attention in recent years. Sequence alignment and structural comparison of the rate-limiting enzyme HMG-CoA reductase (HMGR) revealed differences between lepidopteran pests and other organisms, which suggested insect HMGR could be a selective insecticide target candidate. Inhibition of JH biosynthesis in vitro confirmed that HMGR inhibitors showed a potent lethal effect on the lepidopteran pest Manduca sexta, whereas there was little effect on JH biosynthesis in Apis mellifera and Diploptera punctata. The pest control application of these inhibitors demonstrated that they can be insecticide candidates with potent ovicidal activity, larvicidal activity and insect growth regulatory effects. The present study has validated that Lepidopteran HMGR can be a potent selective insecticide target, and the HMGR inhibitors (especially type II statins) could be selective insecticide candidates and lead compounds. Furthermore, we demonstrated that sequence alignment, homology modeling and structural comparison may be useful for determining potential enzymes or receptors which can be eco-friendly pesticide  targets. PMID:28133568

  16. STRUCTURAL BASIS FOR ALLOSTERIC REGULATION OF HUMAN RIBONUCLEOTIDE REDUCTASE BY NUCLEOTIDE-INDUCED OLIGOMERIZATION

    Science.gov (United States)

    Fairman, James Wesley; Wijerathna, Sanath Ranjan; Ahmad, Md. Faiz; Xu, Hai; Nakano, Ryo; Jha, Shalini; Prendergast, Jay; Welin, Martin; Flodin, Susanne; Roos, Annette; Nordlund, Pär; Li, Zongli; Walz, Thomas; Dealwis, Chris Godfrey

    2011-01-01

    Ribonucleotide reductase (RR) is an αnβn (RR1●RR2) complex that maintains balanced dNTP pools by reducing ribonucleoside diphosphates to deoxyribonucleoside diphosphates. 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-only, dATP-only, TTP●GDP, TTP●ATP, and TTP●dATP. These structures provide insights into ATP/dATP regulation of RR. 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 α6●ββ’ 1●dATP holo complex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data provide an elegant mechanism for regulating RR activity by dATP-induced oligomerization. PMID:21336276

  17. The role of S-nitrosoglutathione reductase (GSNOR) in human disease and therapy.

    Science.gov (United States)

    Barnett, Scott D; Buxton, Iain L O

    2017-04-10

    S-nitrosoglutathione reductase (GSNOR), or ADH5, is an enzyme in the alcohol dehydrogenase (ADH) family. It is unique when compared to other ADH enzymes in that primary short-chain alcohols are not its principle substrate. GSNOR metabolizes S-nitrosoglutathione (GSNO), S-hydroxymethylglutathione (the spontaneous adduct of formaldehyde and glutathione), and some alcohols. GSNOR modulates reactive nitric oxide (•NO) availability in the cell by catalyzing the breakdown of GSNO, and indirectly regulates S-nitrosothiols (RSNOs) through GSNO-mediated protein S-nitrosation. The dysregulation of GSNOR can significantly alter cellular homeostasis, leading to disease. GSNOR plays an important regulatory role in smooth muscle relaxation, immune function, inflammation, neuronal development and cancer progression, among many other processes. In recent years, the therapeutic inhibition of GSNOR has been investigated to treat asthma, cystic fibrosis and interstitial lung disease (ILD). The direct action of •NO on cellular pathways, as well as the important regulatory role of protein S-nitrosation, is closely tied to GSNOR regulation and defines this enzyme as an important therapeutic target.

  18. Design and Synthesis of Aryl Ether Inhibitors of the Bacillus Anthracis Enoyl–ACP Reductase

    Science.gov (United States)

    Tipparaju, Suresh K.; Mulhearn, Debbie C.; Klein, Gary M.; Chen, Yufeng; Tapadar, Subhasish; Bishop, Molly H.; Yang, Shuo; Chen, Juan; Ghassemi, Mahmood; Santarsiero, Bernard D.; Cook, James L.; Johlfs, Mary; Mesecar, Andrew D.; Johnson, Michael E.; Kozikowski, Alan P.

    2009-01-01

    The problem of increasing bacterial resistance to the current generation of antibiotics is well documented. This includes such pathogens as methicillin–resistant Staphylococcus aureus and the potential for developing drug–resistant pathogens for use as bioweapons, such as Bacillus anthracis. The biphenyl ether, antibacterial triclosan exhibits broad–spectrum activity and provides a potential scaffold for the development of new, broad–spectrum antibiotics targeting the fatty acid biosynthetic pathway, via inhibition of enoyl–acyl carrier protein reductase (ENR). We have utilized a structure–based approach to develop novel aryl ether analogs of triclosan that target ENR, the product of the FabI gene, from Bacillus anthracis (BaENR). Structure–based design methods were used for the expansion of the compound series including X-ray crystal structure determination, molecular docking, and QSAR methods. Structural modifications were made to both phenyl rings of the 2-phenoxyphenyl core. A number of compounds were derived that exhibited improved potency against BaENR and increased efficacy against both the Sterne strain of B. anthracis and the methicillin–resistant strain of S. aureus. X-ray crystal structures of BaENR in complex with triclosan and two other compounds help explain the improved efficacy of the new compounds and suggest future rounds of optimisation that might be used to improve their potency. PMID:18663709

  19. Design and synthesis of aryl ether inhibitors of the Bacillus anthracis enoyl-ACP reductase.

    Science.gov (United States)

    Tipparaju, Suresh K; Mulhearn, Debbie C; Klein, Gary M; Chen, Yufeng; Tapadar, Subhasish; Bishop, Molly H; Yang, Shuo; Chen, Juan; Ghassemi, Mahmood; Santarsiero, Bernard D; Cook, James L; Johlfs, Mary; Mesecar, Andrew D; Johnson, Michael E; Kozikowski, Alan P

    2008-08-01

    The problem of increasing bacterial resistance to the current generation of antibiotics is well documented. Known resistant pathogens such as methicillin-resistant Staphylococcus aureus are becoming more prevalent, while the potential exists for developing drug-resistant pathogens for use as bioweapons, such as Bacillus anthracis. The biphenyl ether antibacterial agent, triclosan, exhibits broad-spectrum activity by targeting the fatty acid biosynthetic pathway through inhibition of enoyl-acyl carrier protein reductase (ENR) and provides a potential scaffold for the development of new, broad-spectrum antibiotics. We used a structure-based approach to develop novel aryl ether analogues of triclosan that target ENR, the product of the fabI gene, from B. anthracis (BaENR). Structure-based design methods were used for the expansion of the compound series including X-ray crystal structure determination, molecular docking, and QSAR methods. Structural modifications were made to both phenyl rings of the 2-phenoxyphenyl core. A number of compounds exhibited improved potency against BaENR and increased efficacy against both the Sterne strain of B. anthracis and the methicillin-resistant strain of S. aureus. X-ray crystal structures of BaENR in complex with triclosan and two other compounds help explain the improved efficacy of the new compounds and suggest future rounds of optimization that might be used to improve their potency.

  20. Enoyl acyl carrier protein reductase inhibitors: an updated patent review (2011 - 2015).

    Science.gov (United States)

    Zitko, Jan; Doležal, Martin

    2016-09-01

    Enoyl-(acyl-carrier-protein) reductase (ENR) is a limiting step enzyme in the Fatty Acid Synthase II system. In mammals, there is no homologue to ENR, which makes it an optimal candidate target for selective anti-infective drugs. Up-to-date, only two ENR inhibitors are used in clinical practice. This review is a survey on important patents on low molecular weight compounds with ENR inhibiting activity published in 2011-2015. Common patent databases (SciFinder, esp@cenet, WIPO) were used to locate patent applications on the proposed topic and in the timespan of 2011-2015. In 2011-2015, we have observed patents in previously known structural groups of diphenyl ethers and acrylamides as well as new structural classes, often identified by high-throughput screening campaigns. The spectrum of activity of applied derivatives covers significant bacteria, mycobacteria, and apicomplexan parasites (Plasmodia and Toxoplasma). Good news from research of ENR inhibitors: a) four selective anti-staphylococcal compounds applied in 2011-2015 or earlier were pushed to Phase I or Phase II clinical trials and some of them proved safety and tolerability after peroral and/or intravenous administration; b) big pharma companies have renewed their interest in the development of new anti-infective compounds against resistant strains of clinical relevance.

  1. Selenium and the selenoprotein thioredoxin reductase in the prevention, treatment and diagnostics of cancer.

    Science.gov (United States)

    Selenius, Markus; Rundlöf, Anna-Klara; Olm, Eric; Fernandes, Aristi P; Björnstedt, Mikael

    2010-04-01

    Selenium is an essential element that is specifically incorporated as selenocystein into selenoproteins. It is a potent modulator of eukaryotic cell growth with strictly concentration-dependant effects. Lower concentrations are necessary for cell survival and growth, whereas higher concentrations inhibit growth and induce cell death. It is well established that selenium has cancer preventive effects, and several studies also have shown that it has strong anticancer effects with a selective cytotoxicity on malignant drug-resistant cells while only exerting marginal effects on normal and benign cells. This cancer-specific cytotoxicity is likely explained by high affinity selenium uptake dependent on proteins connected to multidrug resistance. One of the most studied selenoproteins in cancer is thioredoxin reductase (TrxR) that has important functions in neoplastic growth and is an important component of the resistant phenotype. Several reports have shown that TrxR is induced in tumor cells and pre-neoplastic cells, and several commonly used drugs interact with the protein. In this review, we summarize the current knowledge of selenium as a potent preventive and tumor selective anticancer drug, and we also discuss the potential of using the expression and modulation of the selenoprotein TrxR in the diagnostics and treatment of cancer.

  2. Fitness trade-offs in the evolution of dihydrofolate reductase and drug resistance in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Marna S Costanzo

    Full Text Available BACKGROUND: Patterns of emerging drug resistance reflect the underlying adaptive landscapes for specific drugs. In Plasmodium falciparum, the parasite that causes the most serious form of malaria, antifolate drugs inhibit the function of essential enzymes in the folate pathway. However, a handful of mutations in the gene coding for one such enzyme, dihydrofolate reductase, confer drug resistance. Understanding how evolution proceeds from drug susceptibility to drug resistance is critical if new antifolate treatments are to have sustained usefulness. METHODOLOGY/PRINCIPAL FINDINGS: We use a transgenic yeast expression system to build on previous studies that described the adaptive landscape for the antifolate drug pyrimethamine, and we describe the most likely evolutionary trajectories for the evolution of drug resistance to the antifolate chlorcycloguanil. We find that the adaptive landscape for chlorcycloguanil is multi-peaked, not all highly resistant alleles are equally accessible by evolution, and there are both commonalities and differences in adaptive landscapes for chlorcycloguanil and pyrimethamine. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that cross-resistance between drugs targeting the same enzyme reflect the fitness landscapes associated with each particular drug and the position of the genotype on both landscapes. The possible public health implications of these findings are discussed.

  3. Cardiomyocyte aldose reductase causes heart failure and impairs recovery from ischemia.

    Directory of Open Access Journals (Sweden)

    Ni-Huiping Son

    Full Text Available Aldose reductase (AR, an enzyme mediating the first step in the polyol pathway of glucose metabolism, is associated with complications of diabetes mellitus and increased cardiac ischemic injury. We investigated whether deleterious effects of AR are due to its actions specifically in cardiomyocytes. We created mice with cardiac specific expression of human AR (hAR using the α-myosin heavy chain (MHC promoter and studied these animals during aging and with reduced fatty acid (FA oxidation. hAR transgenic expression did not alter cardiac function or glucose and FA oxidation gene expression in young mice. However, cardiac overexpression of hAR caused cardiac dysfunction in older mice. We then assessed whether hAR altered heart function during ischemia reperfusion. hAR transgenic mice had greater infarct area and reduced functional recovery than non-transgenic littermates. When the hAR transgene was crossed onto the PPAR alpha knockout background, another example of greater heart glucose oxidation, hAR expressing mice had increased heart fructose content, cardiac fibrosis, ROS, and apoptosis. In conclusion, overexpression of hAR in cardiomyocytes leads to cardiac dysfunction with aging and in the setting of reduced FA and increased glucose metabolism. These results suggest that pharmacological inhibition of AR will be beneficial during ischemia and in some forms of heart failure.

  4. Design and Synthesis of Chalcone Derivatives as Inhibitors of the Ferredoxin — Ferredoxin-NADP+ Reductase Interaction of Plasmodium falciparum: Pursuing New Antimalarial Agents

    Directory of Open Access Journals (Sweden)

    Hery Suwito

    2014-12-01

    Full Text Available Some chalcones have been designed and synthesized using Claisen-Schmidt reactions as inhibitors of the ferredoxin and ferredoxin-NADP+ reductase interaction to pursue a new selective antimalaria agent. The synthesized compounds exhibited inhibition interactions between PfFd-PfFNR in the range of 10.94%–50%. The three strongest inhibition activities were shown by (E-1-(4-aminophenyl-3-(4-methoxyphenylprop-2-en-1-one (50%, (E-1-(4-aminophenyl-3-(2,4-dimethoxyphenylprop-2-en-1-one (38.16%, and (E-1-(4-aminophenyl-3-(2,3-dimethoxyphenylprop-2-en-1-one (31.58%. From the docking experiments we established that the amino group of the methoxyamino chlacone derivatives plays an important role in the inhibition activity by electrostatic interaction through salt bridges and that it forms more stable and better affinity complexes with FNR than with Fd.

  5. Human biliverdin reductase suppresses Goodpasture antigen-binding protein (GPBP) kinase activity: the reductase regulates tumor necrosis factor-alpha-NF-kappaB-dependent GPBP expression.

    Science.gov (United States)

    Miralem, Tihomir; Gibbs, Peter E M; Revert, Fernando; Saus, Juan; Maines, Mahin D

    2010-04-23

    The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-alpha). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-kappaB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-alpha-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-kappaB show the hBVR role in the initial stimulation of GPBP expression by TNF-alpha-activated NF-kappaB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the (281)CX(10)C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC(281), corresponding to the core of the consensus D(delta)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-alpha dependent NF-kappaB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-alpha-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis.

  6. Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

    DEFF Research Database (Denmark)

    Chen, Z. W.; Jiang, C. Y.; She, Qunxin;

    2005-01-01

    Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system......). The thio-modifying reagent N-ethylmaleimide and Zn2+ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant...... proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have...

  7. Adverse reactions of HMG—CoA reductase inhibitors as a consequence of drug—drug interaction

    Institute of Scientific and Technical Information of China (English)

    IkedaT

    2002-01-01

    Use of HMG-CoA reductase inhibitors in treating hypercholesterolemia is a well-established therapy.Presently,atorvastatin,fluvastatin,lovastatin,simvastatin and pravastatin are used clinically.Cerivastatin was pulled from the market in 2001 due to its higher risk of inducing rhabdomyolysis than all other drugs.Hepatotoxicity and rhabdomyolysis are the known adverse reactions by these drugs.However,the hepatotoxicity has been regarded to be mild,and is now referred to as transaminitis.Rhadomyolysis occurs in rare instances but is sometimes life threatening as a result of renal failure caused by myoglobinemia.The mechanism leading to rhabdomyolysis is unknown but in many of the reported cases,increased plasma concentratinos of thes drugs have been observed,most likely as a consequence of drug interaction.Inhibition of CYP 3A4 and UGT is believed to be the reason for this interaction.

  8. Drug interactions with HMG-CoA reductase inhibitors (statins): the importance of CYP enzymes, transporters and pharmacogenetics.

    Science.gov (United States)

    Neuvonen, Pertti J

    2010-03-01

    HMG-CoA reductase inhibitors (statins) can cause skeletal muscle toxicity; the risk of toxicity is elevated by drug interactions and pharmacogenetic factors that increase the concentration of statins in the plasma. Statins are substrates for several membrane transporters that may mediate drug interactions. Inhibitors of the organic anion transporting polypeptide 1B1 can decrease the hepatic uptake of many statins, as well as the therapeutic index of these agents. Potent inhibitors of cytochrome P450 (CYP)3A4 can significantly increase the plasma concentrations of the active forms of simvastatin, lovastatin and atorvastatin. Fluvastatin, which is metabolized by CYP2C9, is less prone to pharmacokinetic interactions, while pravastatin, rosuvastatin and pitavastatin are not susceptible to any CYP inhibition. An understanding of the mechanisms of statin interactions will help to minimize drug interactions and to develop statins that are less prone to adverse interactions.

  9. A human fatty acid synthase inhibitor binds β-ketoacyl reductase in the keto-substrate site.

    Science.gov (United States)

    Hardwicke, Mary Ann; Rendina, Alan R; Williams, Shawn P; Moore, Michael L; Wang, Liping; Krueger, Julie A; Plant, Ramona N; Totoritis, Rachel D; Zhang, Guofeng; Briand, Jacques; Burkhart, William A; Brown, Kristin K; Parrish, Cynthia A

    2014-09-01

    Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the β-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.

  10. Crystal Structure of Human Liver delta {4}-3-Ketosteroid 5 beta-Reductase (AKR1D1) and Implications for Substrate Binding and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Di Costanzo,L.; Drury, J.; Penning, T.; Christianson, D.

    2008-01-01

    AKR1D1 (steroid 5{beta}-reductase) reduces all 4-3-ketosteroids to form 5{beta}-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an a,{beta}-unsaturated ketone by 5{beta}-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the {beta}-face of a 4-3-ketosteroid yields a cis-A/B-ring configuration with an {approx}90 bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human 4-3-ketosteroid 5{beta}-reductase (AKR1D1), and its complexes with intact substrates. We have determined the structures of AKR1D1 complexes with NADP+ at 1.79- and 1.35- Angstroms resolution (HEPES bound in the active site), NADP+ and cortisone at 1.90- Angstroms resolution, NADP+ and progesterone at 2.03- Angstroms resolution, and NADP+ and testosterone at 1.62- Angstroms resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP+. This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr58 and Glu120. The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.

  11. HMG-CoA reductase inhibitors prevent migration of human coronary smooth muscle cells through suppression of increase in oxidative stress.

    Science.gov (United States)

    Yasunari, K; Maeda, K; Minami, M; Yoshikawa, J

    2001-06-01

    In vitro and in vivo evidence of a decrease in vascular smooth muscle cell (SMC) migration induced by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has been reported. When added to SMC cultures for 6 hours, the HMG-CoA reductase inhibitors fluvastatin, simvastatin, and pravastatin at 1 micromol/L resulted in a 48%, 50%, and 16% suppression, respectively, of human coronary SMC migration; these reductions mirrored the suppression in oxidative stress induced by 1 micromol/L lysophosphatidylcholine (lyso-PC) of 50%, 53% and 19%, respectively. The hydroxylated metabolites of fluvastatin, M(2) and M(3), at 1 micromol/L also suppressed the enhancement of SMC migration by 58% and 45% and the increase in oxidative stress induced by lyso-PC of 58% and 49%, respectively. Lyso-PC activated phospholipase D and protein kinase C (PKC), and this activation was also suppressed by HMG-CoA reductase inhibitors. The inhibition of phospholipase D and PKC was reversed by 100 micromol/L mevalonate, its isoprenoid derivative, farnesol, and geranylgeraniol but not by 10 micromol/L squalene. Antisense oligodeoxynucleotides at 5 micromol/L to PKC-alpha, but not those to the PKC-beta isoform, suppressed the lyso-PC-mediated increases in SMC migration and oxidative stress. These findings suggest that HMG-CoA reductase inhibitors have direct antimigratory effects on the vascular wall beyond their effects on plasma lipids and that they might exert such antimigratory effects via suppression of the phospholipase D- and PKC (possibly PKC-alpha)-induced increase in oxidative stress, which might in turn prevent significant coronary artery disease.

  12. Purification of NADPH-cytochrome c reductase from swine testis microsomes by chromatofocusing and characterization of the purified reductase.

    Science.gov (United States)

    Kuwada, M; Ohsawa, Y; Horie, S

    1985-07-18

    A purified NADPH-cytochrome c reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was prepared from swine testis microsomes by detergent solubilization followed by a procedure including chromatofocusing. The reductase was eluted at an isoelectric point of 4.8 from the chromatofocusing column. 730-fold purification was achieved with an overall yield of 1.2%. The preparation was found to be homogeneous upon polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (SDS). Upon SDS-polyacrylamide gel electrophoresis, however, the purified preparation resolved into one major band (Mr 78 000) and two minor bands (Mr 60 000 and 15 000). The enzyme contained about 1 mol each of FMN and FAD, which were both extractable with trichloroacetic acid and also boiling water. The oxidized form of the enzyme showed the absorption spectrum of a typical flavoprotein. Aerobic reduction with NADPH resulted in conversion of the spectrum into one of an air-stable semiquinone form. The activity of the purified preparation was 26 mumol cytochrome c reduced/min per mg protein under the standard assay conditions at 22 degrees C. The enzyme catalyzed the reaction through a ping-pong mechanism.

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

    Directory of Open Access Journals (Sweden)

    Hui eZhou

    2015-10-01

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

  14. Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems.

    Science.gov (United States)

    Ondarza, Raúl N; Hurtado, Gerardo; Tamayo, Elsa; Iturbe, Angélica; Hernández, Eva

    2006-11-01

    This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.

  15. Proximal FAD histidine residue influences interflavin electron transfer in cytochrome P450 reductase and methionine synthase reductase.

    Science.gov (United States)

    Meints, Carla E; Parke, Sarah M; Wolthers, Kirsten R

    2014-04-01

    Cytochrome P450 reductase (CPR) and methionine synthase reductase (MSR) transfer reducing equivalents from NADPH to FAD to FMN. In CPR, hydride transfer and interflavin electron transfer are kinetically coupled steps, but in MSR the two catalytic steps are represented by two distinct kinetic phases leading to transient formation of the FAD hydroquinone. In human CPR, His(322) forms a hydrogen-bond with the highly conserved Asp(677), a member of the catalytic triad. The catalytic triad is present in MSR, but Ala(312) replaces the histidine residue. To examine if this structural variation accounts for differences in their kinetic behavior, reciprocal substitutions were created. Substitution of His(322) for Ala in CPR does not affect the rate of NADPH hydride transfer or the FAD redox potentials, but does impede interflavin electron transfer. For MSR, swapping Ala(312) for a histidine residue resulted in the kinetic coupling of hydride and interflavin electron transfer, and eliminated the formation of the FAD hydroquinone intermediate. For both enzymes, placement of the His residue in the active site weakens coenzyme binding affinity. The data suggest that the proximal FAD histidine residue accelerates proton-coupled electron transfer from FADH2 to the higher potential FMN; a mechanism for this catalytic role is discussed.

  16. Structural analysis of sulindac as an inhibitor of aldose reductase and AKR1B10.

    Science.gov (United States)

    Cousido-Siah, Alexandra; Ruiz, Francesc X; Crespo, Isidro; Porté, Sergio; Mitschler, André; Parés, Xavier; Podjarny, Alberto; Farrés, Jaume

    2015-06-05

    Aldose reductase (AR, AKR1B1) and AKR1B10 are enzymes implicated in important pathologies (diabetes and cancer) and therefore they have been proposed as suitable targets for drug development. Sulindac is the metabolic precursor of the potent non-steroidal anti-inflammatory drug (NSAID) sulindac sulfide, which suppresses prostaglandin production by inhibition of cyclooxygenases (COX). In addition, sulindac has been found to be one of the NSAIDs with higher antitumoral activity, presumably through COX inhibition. However, sulindac anticancer activity could be partially mediated through COX-independent mechanisms, including the participation of AR and AKR1B10. Previously, it had been shown that sulindac and sulindac sulfone were good AR inhibitors and the structure of the ternary complex with NADP(+) and sulindac was described (PDB ID 3U2C). In this work, we determined the three-dimensional structure of AKR1B10 with sulindac and established structure-activity relationships (SAR) of sulindac and their derivatives with AR and AKR1B10. The difference in the IC50 values for sulindac between AR (0.36 μM) and AKR1B10 (2.7 μM) might be explained by the different positioning and stacking interaction given by Phe122/Phe123, and by the presence of two buried and ordered water molecules in AKR1B10 but not in AR. Moreover, SAR analysis shows that the substitution of the sulfinyl group is structurally allowed in sulindac derivatives. Hence, sulindac and its derivatives emerge as lead compounds for the design of more potent and selective AR and AKR1B10 inhibitors.

  17. Aldose reductase inhibitory potential of different fractions ofHouttuynia cordata Thunb

    Institute of Scientific and Technical Information of China (English)

    Manish Kumar; Damiki Laloo; Satyendra K. Prasad; Siva Hemalatha

    2014-01-01

    Objective:To evaluate the aldose reductase(AR) inhibitory activity of different fractions from Houttuynia cordata(H. cordata) which used as a medicinal salad for lowering of blood sugar level. Methods:AR inhibitory activity along with protein content was evaluatedin vitro in rat lens. Total phenol and flavonoid contents were also determined in all the fractions.Results:All the four fractions were found to inhibit lensAR activity, but to differentextent.From dose response curve(DRC), aqueous fraction(AQ) was found to be the most effectiveAR inhibitor followed by ethyl acetate(EA), chloroform(CL) and hexane fraction(HEX).TheIC50 values ofAQ,EA,CL and HEX were calculated to be(64.62±3.90),(90.69±7.50),(134.59±4.90) and(151.58±3.30) μg/mL respectively.Quercetin was taken as positive control which exhibitedAR inhibition withanIC50 value of(3.21±0.60) μg/mL in a non-competitive manner.Conclusion:These findings indicated that,AQ fraction ofH. cordata exhibited significant inhibitory effect onAR in a non-competitive manner, which may be attributed to the presence of high phenolic and flavonoid contents.Thus, the plantH. cordata may act as a promising source in the treatment of secondary complications like cataract associated with diabetes.

  18. The impact of HMG-CoA reductase therapy on serum PSA.

    Science.gov (United States)

    Mener, David J; Cambio, Angelo; Stoddard, David G; Martin, Brad A; Palapattu, Ganesh S

    2010-05-01

    3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, otherwise known as statins, inhibit the enzyme that controls the conversion of HMG-CoA to mevalonate, a precursor for cholesterol. Statins may be important to prostate cancer biology by inhibiting cell growth, inflammation, and oxidative stress. The purpose of this study was to assess the influence of statin therapy on serum prostate-specific antigen (PSA) levels. The computerized medical records at the University of Rochester Medical Center were used to identify men who filled statin prescriptions between May 31st, 2008 and September 30th, 2008. Men with at least one PSA assay performed within 2 years before and at least one PSA assay performed within 1 year after starting a statin medication were included. The primary endpoint was the change in PSA concentration computed as the difference between PSA levels before and after starting a statin medication. Paired t-tests were used to analyze the mean differences in PSA values. A total of 962 patients were identified. The mean difference in serum PSA level after statin administration was -0.29 ng/ml (-8.04%). Subgroup analyses for mean PSA concentration change before and after statin administration by age group revealed: 50-59 years old (-0.1609, 95% CI: -0.2444, -0.0775, P 70 years old (-0.351, 95% CI: -0.490, -0.212, P < 0.0001). These observations suggest a statistically significant reduction in serum PSA level that is associated with the onset of statin therapy.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    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....... 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...... 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...

  20. Dynamic Changes of Nitrate Reductase Activity within 24 Hours

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    [Objective] The research aimed to study the circadian rhythm of nitrate re- ductase activity (NRA) in plant. [Method] The wheat plants at heading stage were used as the materials for the measurement of dynamic changes of nitrate reductase activity (NRA) within 24 h under the conditions of constant high temperature. [Resulti The fluctuation of NRA in wheat changed greatly from 20:00 pm to 11:00 am. The enzyme activity remained constant, but at 14:00 the enzyme activity was the high- est, higher than all the other time points except the enzyme activity measured at11:00. The enzyme activity was the lowest of 17:00, which was lower than all the other time points except the enzyme activity measured at 2:00. [Conclusion] There were autonomous rhythm changes of NRA in wheat in a certain degree.

  1. 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.

  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......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. Aspects of ribonucleotide reductase regulation and genome stability

    DEFF Research Database (Denmark)

    Nielsen, Helena Berner Nedergaard

    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......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...... the human RNR enzyme. In this study regulation of human RNR was investigated using a fission yeast strain that depended solely on the human genes of R1 and R2 for dNTP synthesis. Even though this strain could grow like wild-type fission yeast it was hypersensitive to hydroxyurea (HU) and depended...

  4. 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.

  5. 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.

  6. 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 GLGxMGx(5 )[ATS]x(4) Gx(4) [VIL]WNR[TS]x(2) [KR] and the active site motif Gx[DE]x[GDA]x[APS]x(3){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. © 2016 Wiley Periodicals, Inc.

  7. 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.

  8. In vivo and in vitro effect of androstene derivatives as 5α-reductase type 1 enzyme inhibitors.

    Science.gov (United States)

    Bratoeff, Eugene; Sánchez, Araceli; Arellano, Yazmín; Heuze, Yvonne; Soriano, Juan; Cabeza, Marisa

    2013-12-01

    The aim of these studies was to synthesize twelve ester derivatives of dehydroepiandrosterone with therapeutic potential. The effect of 1-12 was demonstrated in the flank organs of gonadectomized hamsters treated with testosterone and the synthesized steroids. In vitro studies were carried out determining the IC50 values for the inhibition of the activity of 5α-reductase type 1 and 2, which are present in rat liver and human prostate respectively. The binding of 1-12 to the androgen receptors (AR) was determined using rat's prostate cytosol. Steroids 1-12 containing different substituents in the phenyl group of the ester moiety in C-3 reduced the flank organs and inhibited the activity of 5α-R type 1; however only steroids 1 and 2 inhibited 5α-R type 2. 1-12 did not bind to the AR. The modification of one atom of the substituents in the phenyl group of the ester moiety in C-3 changed their biological potency (IC50).

  9. Steroid 5β-Reductase from Leaves of Vitis vinifera: Molecular Cloning, Expression, and Modeling.

    Science.gov (United States)

    Ernst, Mona; Munkert, Jennifer; Campa, Manuela; Malnoy, Mickael; Martens, Stefan; Müller-Uri, Frieder

    2015-11-25

    A steroid 5β-reductase gene corresponding to the hypothetical protein LOC100247199 from leaves of Vitis vinifera (var. 'Chardonnay') was cloned and overexpressed in Escherichia coli. The recombinant protein showed 5β-reductase activity when progesterone was used as a substrate. The reaction was stereoselective, producing only 5β-products such as 5β-pregnane-3,20-dione. Other small substrates (terpenoids and enones) were also accepted as substrates, indicating the highly promiscuous character of the enzyme class. Our results show that the steroid 5β-reductase gene, encoding an orthologous enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in leaves of the cardenolide-free plant V. vinifera. We emphasize the fact that, on some occasions, different reductases (e.g., progesterone 5β-reductase and monoterpenoid reductase) can also use molecules that are similar to the final products as a substrate. Therefore, in planta, the different reductases may contribute to the immense number of diverse small natural products finally leading to the flavor of wine.

  10. Aldose reductase inhibitor improves insulin-mediated glucose uptake and prevents migration of human coronary artery smooth muscle cells induced by high glucose.

    Science.gov (United States)

    Yasunari, K; Kohno, M; Kano, H; Minami, M; Yoshikawa, J

    2000-05-01

    We examined involvement of the polyol pathway in high glucose-induced human coronary artery smooth muscle cell (SMC) migration using Boyden's chamber method. Chronic glucose treatment for 72 hours potentiated, in a concentration-dependent manner (5.6 to 22.2 mol/L), platelet-derived growth factor (PDGF) BB-mediated SMC migration. This potentiation was accompanied by an increase in PDGF BB binding, because of an increased number of PDGF-beta receptors, and this potentiation was blocked by the aldose reductase inhibitor epalrestat. Epalrestat at concentrations of 10 and 100 nmol/L inhibited high glucose-potentiated (22.2 mmol/L), PDGF BB-mediated migration. Epalrestat at 100 nmol/L inhibited a high glucose-induced increase in the reduced/oxidized nicotinamide adenine dinucleotide ratio and membrane-bound protein kinase C (PKC) activity in SMCs. PKC inhibitors calphostin C (100 nmol/L) and chelerythrine (1 micromol/L) each inhibited high glucose-induced, PDGF BB-mediated SMC migration. High glucose-induced suppression of insulin-mediated [(3)H]-deoxyglucose uptake, which was blocked by both calphostin C (100 nmol/L) and chelerythrine (1 micromol/L), was decreased by epalrestat (100 nmol/L). Chronic high glucose treatment for 72 hours increased intracellular oxidative stress, which was directly measured by flow cytometry using carboxydichlorofluorescein diacetate bis-acetoxymethyl ester, and this increase was significantly suppressed by epalrestat (100 nmol/L). Antisense oligonucleotide to PKC-beta isoform inhibited high glucose-mediated changes in SMC migration, insulin-mediated [(3)H]-deoxyglucose uptake, and oxidative stress. These findings suggest that high glucose concentrations potentiate SMC migration in coronary artery and that the aldose reductase inhibitor epalrestat inhibits high glucose-potentiated, PDGF BB-induced SMC migration, possibly through suppression of PKC (PKC-beta), impaired insulin-mediated glucose uptake, and oxidative stress.

  11. Methionine sulfoxide reductase A protects hepatocytes against acetaminophen-induced toxicity via regulation of thioredoxin reductase 1 expression.

    Science.gov (United States)

    Singh, Mahendra Pratap; Kwak, Geun-Hee; Kim, Ki Young; Kim, Hwa-Young

    2017-06-03

    Thioredoxin reductase 1 (TXNRD1) is associated with susceptibility to acetaminophen (APAP)-induced liver damage. Methionine sulfoxide reductase A (MsrA) is an antioxidant and protein repair enzyme that specifically catalyzes the reduction of methionine S-sulfoxide residues. We have previously shown that MsrA deficiency exacerbates acute liver injury induced by APAP. In this study, we used primary hepatocytes to investigate the underlying mechanism of the protective effect of MsrA against APAP-induced hepatotoxicity. MsrA gene-deleted (MsrA(-/-)) hepatocytes showed higher susceptibility to APAP-induced cytotoxicity than wild-type (MsrA(+/+)) cells, consistent with our previous in vivo results. MsrA deficiency increased APAP-induced glutathione depletion and reactive oxygen species production. APAP treatment increased Nrf2 activation more profoundly in MsrA(-/-) than in MsrA(+/+) hepatocytes. Basal TXNRD1 levels were significantly higher in MsrA(-/-) than in MsrA(+/+) hepatocytes, while TXNRD1 depletion in both MsrA(-/-) and MsrA(+/+) cells resulted in increased resistance to APAP-induced cytotoxicity. In addition, APAP treatment significantly increased TXNRD1 expression in MsrA(-/-) hepatocytes, while no significant change was observed in MsrA(+/+) cells. Overexpression of MsrA reduced APAP-induced cytotoxicity and TXNRD1 expression levels in APAP-treated MsrA(-/-) hepatocytes. Collectively, our results suggest that MsrA protects hepatocytes from APAP-induced cytotoxicity through the modulation of TXNRD1 expression. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Emmanuel O. AJANI

    2016-09-01

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

  13. In vivo assay for conversion of testosterone to dihydrotestosterone by rat prostatic steroid 5 alpha-reductase and comparison of two inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Toomey, R.E.; Goode, R.L.; Petrow, V.; Neubauer, B.L. (Endocrine Research, Lilly Research Labs, Indianapolis, IN (USA))

    1991-01-01

    An in vivo assay for steroid 5 alpha-reductase in rat ventral prostate has been developed and used to compare the inhibitory activity of N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and 6-methylene-4-pregnene-3,20-dione (LY207320). Immature rats (70-80 g) received test compounds 30 min prior to s.c. injection of (3H)-T. The rats were sacrificed 30 min later and the ventral prostates were analyzed for (3H)-T metabolites. Intraprostatic (3H)-T and (3H)-DHT reached peak levels within 5 min after injection of (3H)-T and declined to about 25% of peak levels after 2 hr. 4-MA was a very potent inhibitor of (3H)-DHT formation with an estimated IC50 of 0.2 mg/kg. LY207320, an inhibitor of 5 alpha-reductase in vitro, was weakly active in vivo and did not achieve greater than 45% inhibition at high doses (greater than 200 mg/kg, s.c.). Tissue uptake of (3H)-T was also inhibited by LY207320, which may contribute to its inhibitory activity on accessory sex organ growth in the rat.

  14. Molecular effects of bioactive fraction of Curcuma mangga (DLBS4847 as a downregulator of 5α-reductase activity pathways in prostatic epithelial cells

    Directory of Open Access Journals (Sweden)

    Karsono AH

    2014-06-01

    Full Text Available Agung Heru Karsono, Olivia Mayasari Tandrasasmita, Raymond R TjandrawinataSection of Molecular Pharmacology, Research Innovation and Invention, Dexa Laboratories of Biomolecular Sciences, Dexa Medica, Cikarang, IndonesiaAbstract: DLBS4847 is a standardized bioactive fraction of Curcuma mangga. In this study, we used prostate cancer (PC-3 as the cell line to study the effects of DLBS4847 on prostatic cell viability, as well as related molecular changes associated with the decreased cell number. The observation revealed that DLBS4847 inhibited the growth of PC3 cells through downregulation of the 5α-reductase (5AR pathway. At the transcription level, 5AR1 and androgen-receptor gene expressions were downregulated in a dose-dependent manner. Furthermore, 5AR-1 and dihydrotestosterone expression were also downregulated at the protein level. A microarray study was also performed to see the effects of DLBS4847 on differential gene expressions in prostate cancer 3 cells. Among others, DLBS4847 downregulated genes related to prostate growth and hypertrophy. Our results suggested that DLBS4847 could potentially become an alternative treatment for prostate disorders, such as benign prostatic hyperplasia. In this regard, DLBS4847 exerts its growth inhibition partially through downregulation of the 5AR pathway.Keywords: DLBS4847, Curcuma mangga, 5α-reductase inhibitor, benign prostatic hyperplasia (BPH, prostate cancer

  15. Effect of inhibition of cholesterol synthetic pathway on the activation of Ras and MAP kinase in mesangial cells

    National Research Council Canada - National Science Library

    Bassa, Babu V; Roh, Daeyoung D; Vaziri, Nosratola D; Kirschenbaum, Michael A; Kamanna, Vaijinath S

    1999-01-01

    ... of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthetic pathway, to animals with experimental renal disease significantly inhibited mesangial hypercellularity, ECM expansion and prevented the progression of glomerular disease [10–12] . In vitro studies have also shown that the inhibiti...

  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. Survival and psychomotor development with early betaine treatment in patients with severe methylenetetrahydrofolate reductase deficiency

    NARCIS (Netherlands)

    Diekman, E.F.; Koning, T.J. de; Verhoeven-Duif, N.M.; Rovers, M.M.; Hasselt, P.M. van

    2014-01-01

    IMPORTANCE The impact of betaine treatment on outcome in patients with severe methylenetetrahydrofolate reductase (MTHFR) deficiency is presently unclear. OBJECTIVE To investigate the effect of betaine treatment on development and survival in patients with severe MTHFR deficiency. DATA SOURCES MEDLI

  18. Survival and Psychomotor Development With Early Betaine Treatment in Patients With Severe Methylenetetrahydrofolate Reductase Deficiency

    NARCIS (Netherlands)

    Diekman, Eugene F.; de Koning, Tom J.; Verhoeven-Duif, Nanda M.; Rovers, Maroeska M.; van Hasselt, Peter M.

    2014-01-01

    IMPORTANCE The impact of betaine treatment on outcome in patients with severe methylenetetrahydrofolate reductase (MTHFR) deficiency is presently unclear. OBJECTIVE To investigate the effect of betaine treatment on development and survival in patients with severe MTHFR deficiency. DATA SOURCES MEDLI

  19. 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

  20. Localization and Solubilization of the Iron(III) Reductase of Geobacter sulfurreducens

    OpenAIRE

    1998-01-01

    The iron(III) reductase activity of Geobacter sulfurreducens was determined with the electron donor NADH and the artificial electron donor horse heart cytochrome c. The highest reduction rates were obtained with Fe(III) complexed by nitrilotriacetic acid as an electron acceptor. Fractionation experiments indicated that no iron(III) reductase activity was present in the cytoplasm, that approximately one-third was found in the periplasmic fraction, and that two-thirds were associated with the m...

  1. Measurement of nitrite reductase in leaf tissue of Vigna mungo : A new method.

    Science.gov (United States)

    Srivastava, R C; Bose, B; Mukerji, D; Mathur, S N; Srivastava, H S

    1979-12-01

    The enzyme nitrite reductase (EC 1.6.6.4) is generally assayed in terms of disappearance of nitrite from the assay medium. We describe a technique which allowed estimation of the enzyme level in leaf tissues of Vigna mungo (L). Hepper in terms of the release of the product (NH3) of the enzyme reaction. The technique is offered as an alternative, possibly more convenient method for assay of nitrite reductase in plant tissue in vivo.

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

    OpenAIRE

    2004-01-01

    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 dos...

  3. On the inhibitor effects of bergamot juice flavonoids binding to the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme.

    Science.gov (United States)

    Leopoldini, Monica; Malaj, Naim; Toscano, Marirosa; Sindona, Giovanni; Russo, Nino

    2010-10-13

    Density functional theory was applied to study the binding mode of new flavonoids as possible inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), an enzyme that catalyzes the four-electron reduction of HMGCoA to mevalonate, the committed step in the biosynthesis of sterols. The investigated flavonoid conjugates brutieridin and melitidin were recently quantified in the bergamot fruit extracts and identified to be structural analogues of statins, lipids concentration lowering drugs that inhibit HMGR. Computations allowed us to perform a detailed analysis of the geometrical and electronic features affecting the binding of these compounds, as well as that of the excellent simvastatin drug, to the active site of the enzyme and to give better insight into the inhibition process.

  4. Staying green postharvest: how three mutations in the Arabidopsis chlorophyll b reductase gene NYC1 delay degreening by distinct mechanisms.

    Science.gov (United States)

    Jibran, Rubina; Sullivan, Kerry L; Crowhurst, Ross; Erridge, Zoe A; Chagné, David; McLachlan, Andrew R G; Brummell, David A; Dijkwel, Paul P; Hunter, Donald A

    2015-11-01

    Stresses such as energy deprivation, wounding and water-supply disruption often contribute to rapid deterioration of harvested tissues. To uncover the genetic regulation behind such stresses, a simple assessment system was used to detect senescence mutants in conjunction with two rapid mapping techniques to identify the causal mutations. To demonstrate the power of this approach, immature inflorescences of Arabidopsis plants that contained ethyl methanesulfonate-induced lesions were detached and screened for altered timing of dark-induced senescence. Numerous mutant lines displaying accelerated or delayed timing of senescence relative to wild type were discovered. The underlying mutations in three of these were identified using High Resolution Melting analysis to map to a chromosomal arm followed by a whole-genome sequencing-based mapping method, termed 'Needle in the K-Stack', to identify the causal lesions. All three mutations were single base pair changes and occurred in the same gene, NON-YELLOW COLORING1 (NYC1), a chlorophyll b reductase of the short-chain dehydrogenase/reductase (SDR) superfamily. This was consistent with the mutants preferentially retaining chlorophyll b, although substantial amounts of chlorophyll b were still lost. The single base pair mutations disrupted NYC1 function by three distinct mechanisms, one by producing a termination codon, the second by interfering with correct intron splicing and the third by replacing a highly conserved proline with a non-equivalent serine residue. This non-synonymous amino acid change, which occurred in the NADPH binding domain of NYC1, is the first example of such a mutation in an SDR protein inhibiting a physiological response in plants.

  5. Transformation of RDX and other energetic compounds by xenobiotic reductases XenA and XenB

    Science.gov (United States)

    McClay, Kevin; Hawari, Jalal; Paquet, Louise; Malone, Thomas E.; Fox, Brian G.; Steffan, Robert J.

    2017-01-01

    The transformation of explosives, including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), by xenobiotic reductases XenA and XenB (and the bacterial strains harboring these enzymes) under both aerobic and anaerobic conditions was assessed. Under anaerobic conditions, Pseudomonas fluorescens I-C (XenB) degraded RDX faster than Pseudomonas putida II-B (XenA), and transformation occurred when the cells were supplied with sources of both carbon (succinate) and nitrogen (NH4+), but not when only carbon was supplied. Transformation was always faster under anaerobic conditions compared to aerobic conditions, with both enzymes exhibiting a O2 concentration-dependent inhibition of RDX transformation. The primary degradation pathway for RDX was conversion to methylenedinitramine and then to formaldehyde, but a minor pathway that produced 4-nitro-2,4-diazabutanal (NDAB) also appeared to be active during transformation by whole cells of P. putida II-B and purified XenA. Both XenA and XenB also degraded the related nitramine explosives octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine and 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane. Purified XenB was found to have a broader substrate range than XenA, degrading more of the explosive compounds examined in this study. The results show that these two xenobiotic reductases (and their respective bacterial strains) have the capacity to transform RDX as well as a wide variety of explosive compounds, especially under low oxygen concentrations. PMID:19455327

  6. Effect of MK-906, a specific 5 alpha-reductase inhibitor, on serum androgens and androgen conjugates in normal men.

    Science.gov (United States)

    Rittmaster, R S; Stoner, E; Thompson, D L; Nance, D; Lasseter, K C

    1989-01-01

    To determine the hormonal effects of MK-906, an orally active 5 alpha-reductase inhibitor, on serum androgens and androgen conjugates, 12 healthy men were given 10, 20, 50, and 100 mg MK-906 2 weeks apart in randomized order in a 4-period crossover design. Serum testosterone (T), dihydrotestosterone (DHT), androstanediol glucuronide, and androsterone glucuronide were measured before and 24 hours after each dose. The effect of MK-906 on glucuronyl transferase activity, the enzyme responsible for androstanediol glucuronide and androsterone glucuronide formation, was assessed in vitro using rat prostate tissue. Serum T levels were unchanged after all doses. Serum DHT, androstanediol glucuronide, and androsterone glucuronide were suppressed by 70, 40, and 56%, respectively, after the 10-mg dose, and by 82, 52, and 66% after the 100-mg dose (P less than 0.02 for the comparison between the 10 and 100-mg doses for all three steroids), respectively. Baseline serum T and DHT levels were strongly correlated (R = 0.89, P = 0.0002), as were androstanediol glucuronide and androsterone glucuronide levels (R = 0.78, P = 0.003), but there was no correlation between DHT levels and the levels of either conjugated steroid. MK-906 had no effect on glucuronyl transferase activity in vitro. It was concluded that single doses of MK-906 suppress both conjugated and unconjugated 5 alpha-reduced androgens. While all three steroids appeared to be good markers of systemic 5 alpha-reductase inhibition, further research will be needed to determine which steroid best reflects tissue DHT levels in patients receiving these inhibitors.

  7. 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

  8. 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.

  9. Molecular bases of thioredoxin and thioredoxin reductase-mediated prooxidant actions of (-)-epigallocatechin-3-gallate.

    Science.gov (United States)

    Zhang, Huihui; Cao, Dong; Cui, Wei; Ji, Mingjuan; Qian, Xiaohong; Zhong, Liangwei

    2010-12-15

    Thioredoxin (Trx) and thioredoxin reductase (TrxR) function as antioxidant and anti-apoptotic proteins, which are often up-regulated in drug-resistant cancer cells. (-)-epigallocatechin-3-gallate (EGCG) is a naturally occurring antioxidant in green tea, but also exhibits prooxidant and apoptosis-inducing properties. We have previously showed a linkage between EGCG-induced inactivation of TrxR and decreased cell survival, revealing TrxR as a new target of EGCG. However, the molecular events underlying the importance of Trx/TrxR in EGCG-induced cytotoxicity remain unclear. Here, we show that the crosstalk between EGCG and Trx/TrxR occurred in a redox-dependent manner, and EGCG induced inactivation of Trx/TrxR in parallel with increased ROS levels in HeLa cells. Moreover, EGCG displayed great reactivity with Cys/Sec residues that have low pK(a) values. The structure of EGCG suggests that its quinone form would readily react with thiolate and selenolate nucleophiles. Using mass spectrometry, we have demonstrated the formation of EGCG-Trx1 (Cys(32)) and EGCG-TrxR (Cys/Sec) conjugates, confirming that EGCG quinone specifically conjugates with active-site Cys(32) in Trx or C-terminal Cys/Selenocysteine (Sec) couple in TrxR under conditions where Trx/TrxR are reduced. Non-reduced form of Trx/TrxR could escape from EGCG inhibition. These data reveal a potential mechanism for enhancing EGCG-induced cancer cell death by the NADPH-dependent reduction of Trx/TrxR. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. 9,10-Phenanthrenequinone promotes secretion of pulmonary aldo-keto reductases with surfactant.

    Science.gov (United States)

    Matsunaga, Toshiyuki; Haga, Mariko; Watanabe, Gou; Shinoda, Yuhki; Endo, Satoshi; Kajiwara, Yu; Tanaka, Hiroyuki; Inagaki, Naoki; El-Kabbani, Ossama; Hara, Akira

    2012-02-01

    9,10-Phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust particles, induces apoptosis via the generation of reactive oxygen species (ROS) because of 9,10-PQ redox cycling. We have found that intratracheal infusion of 9,10-PQ facilitates the secretion of surfactant into rat alveolus. In the cultured rat lung, treatment with 9,10-PQ results in an increase in a lower-density surfactant by ROS generation through redox cycling of the quinone. The surfactant contains aldo-keto reductase (AKR) 1C15, which reduces 9,10-PQ and the enzyme level in the surfactant increases on treatment with 9,10-PQ suggesting an involvement of AKR1C15 in the redox cycling of the quinone. In six human cell types (A549, MKN45, Caco2, Hela, Molt4 and U937) only type II epithelial A549 cells secrete three human AKR1C subfamily members (AKR1C1, AKR1C2 and AKR1C3) with the surfactant into the medium; this secretion is highly increased by 9,10-PQ treatment. Using in vitro enzyme inhibition analysis, we have identified AKR1C3 as the most abundantly secreted AKR1C member. The AKR1C enzymes in the medium efficiently reduce 9,10-PQ and initiate its redox cycling accompanied by ROS production. The exposure of A549 cells to 9,10-PQ provokes viability loss, which is significantly protected by the addition of the AKR1C3 inhibitor and antioxidant enzyme and by the removal of the surfactants from the culture medium. Thus, the AKR1C enzymes secreted in pulmonary surfactants probably participate in the toxic mechanism triggered by 9,10-PQ.

  11. A biophysically based mathematical model for the catalytic mechanism of glutathione reductase.

    Science.gov (United States)

    Pannala, Venkat R; Bazil, Jason N; Camara, Amadou K S; Dash, Ranjan K

    2013-12-01

    Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH as the reducing cofactor, and thereby maintains a constant GSH level in the system. GSH scavenges superoxide (O2(*-)) and hydroxyl radicals (OH) nonenzymatically or by serving as an electron donor to several enzymes involved in reactive oxygen species (ROS) detoxification. In either case, GSH oxidizes to GSSG and is subsequently regenerated by the catalytic action of GR. Although the GR kinetic mechanism has been extensively studied under various experimental conditions with variable substrates and products, the catalytic mechanism has not been studied in terms of a mechanistic model that accounts for the effects of the substrates and products on the reaction kinetics. The aim of this study is therefore to develop a comprehensive mathematical model for the catalytic mechanism of GR. We use available experimental data on GR kinetics from various species/sources to develop the mathematical model and estimate the associated model parameters. The model simulations are consistent with the experimental observation that GR operates via both ping-pong and sequential branching mechanisms based on relevant concentrations of its reaction substrate GSSG. Furthermore, we show the observed pH-dependent substrate inhibition of GR activity by GSSG and bimodal behavior of GR activity with pH. The model presents a unique opportunity to understand the effects of products on the kinetics of GR. The model simulations show that under physiological conditions, where both substrates and products are present, the flux distribution depends on the concentrations of both GSSG and NADP(+), with ping-pong flux operating at low levels and sequential flux dominating at higher levels. The kinetic model of GR may serve as a key module for the development of integrated models for ROS-scavenging systems to understand protection of cells under normal and oxidative stress

  12. Elucidating features that drive the design of selective antifolates using crystal structures of human dihydrofolate reductase.

    Science.gov (United States)

    Lamb, Kristen M; G-Dayanandan, Narendran; Wright, Dennis L; Anderson, Amy C

    2013-10-15

    The pursuit of antimicrobial drugs that target dihydrofolate reductase (DHFR) exploits differences in sequence and dynamics between the pathogenic and human enzymes. Here, we present five crystal structures of human DHFR bound to a new class of antimicrobial agents, the propargyl-linked antifolates (PLAs), with a range of potency (IC50 values of 0.045-1.07 μM) for human DHFR. These structures reveal that interactions between the ligands and Asn 64, Phe 31, and Phe 34 are important for increased affinity for human DHFR and that loop residues 58-64 undergo ligand-induced conformational changes. The utility of these structural studies was demonstrated through the design of three new ligands that reduce the number of contacts with Asn 64, Phe 31, and Phe 34. Synthesis and evaluation show that one of the designed inhibitors exhibits the lowest affinity for human DHFR of any of the PLAs (2.64 μM). Comparisons of structures of human and Staphylococcus aureus DHFR bound to the same PLA reveal a conformational change in the ligand that enhances interactions with residues Phe 92 (Val 115 in huDHFR) and Ile 50 (Ile 60 in huDHFR) in S. aureus DHFR, yielding selectivity. Likewise, comparisons of human and Candida glabrata DHFR bound to the same ligand show that hydrophobic interactions with residues Ile 121 and Phe 66 (Val 115 and Asn 64 in human DHFR) yield selective inhibitors. The identification of residue substitutions that are important for selectivity and the observation of active site flexibility will help guide antimicrobial antifolate development for the inhibition of pathogenic species.

  13. Human Biliverdin Reductase Suppresses Goodpasture Antigen-binding Protein (GPBP) Kinase Activity

    Science.gov (United States)

    Miralem, Tihomir; Gibbs, Peter E. M.; Revert, Fernando; Saus, Juan; Maines, Mahin D.

    2010-01-01

    The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-α). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-κB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-α-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-κB show the hBVR role in the initial stimulation of GPBP expression by TNF-α-activated NF-κB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the 281CX10C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC281, corresponding to the core of the consensus D(δ)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-α dependent NF-κB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-α-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis. PMID:20177069

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

    Directory of Open Access Journals (Sweden)

    Fu-Ming Tsai

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

  15. Human biliverdin reductase promotes EMT through the ERK1/2 signal pathway in breast cancer.

    Science.gov (United States)

    Zhang, Min; Song, Shasha; Yi, Zhi; Zhao, Xijuan; Fu, Li; Wang, Lin; Ma, Cui; Mao, Min; Xing, Yan; Zhu, Daling

    2016-10-05

    Epithelial-to-mesenchymal transition (EMT) plays an important role in the development of the invasive and metastatic potentials of breast cancer cells during progression. Human biliverdin reductase (hBVR), an enzyme in the heme metabolism pathway, is involved in hypoxia-induced renal tubular EMT. However, whether hBVR contributes to the EMT of breast cancer remains unclear. Here, we used breast cancer cell lines (MCF-7, T-47D) and normal breast epithelial cells (MCF-10A) to explore the potential role of hBVR in the EMT of breast cancer. Western blot, RT-PCR and immunofluorescence were employed to test the expression and location of hBVR in the cell lines. Small interfering RNA of hBVR (si-hBVR) was used to knockdown the expression of hBVR, and U0126 was applied to inhibit the ERK1/2 signaling in MCF-7, T-47D cells. We found that hBVR highly expressed in MCF-7 and T-47D cells compared with MCF-10A cells, and had different cellular locations between them. Our results revealed that EMT occurred in tissues from breast cancer patients and breast cancer cell lines. However, the EMT in MCF-7 and T-47D cells was suppressed by si-hBVR and U0126. Furthermore, the expression of phosphorylated ERK1/2 was down-regulated by si-hBVR. In addition, hBVR regulated EMT through the ERK1/2 signaling, but bilirubin, which is a product of hBVR in the heme metabolism pathway in breast cancer, did not. Taken together, these findings provide new evidence that hBVR plays an important role in promoting EMT in human breast cancer through the ERK1/2 signaling pathway, and hBVR may be a therapeutic target for this disease. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Al-Haidari, Amr A.; Syk, Ingvar; Thorlacius, Henrik, E-mail: henrik.thorlacius@med.lu.se

    2014-03-28

    Highlights: • Simvastatin blocked CCL17-induced and CCR4-dependent RhoA activation in HT29 cells. • CCL17/CCR4-mediated migration of colon cancer cells was antagonised by simvastatin. • Cell migration recovered by adding Mevalonate and geranylgeranyl pyrophosphate. • Targeting HMG-CoA reductase might be useful to inhibit colon cancer metastasis. - Abstract: Background: Simvastatin is widely used to lower cholesterol levels in patients with cardiovascular diseases, although accumulating evidence suggests that statins, such as simvastatin, also exert numerous anti-tumoral effects. Aim: The aim of this study was to examine the effect of simvastatin on colon cancer cell migration. Methods: Migration assays were performed to evaluate CCL17-induced colon cancer cell (HT-29) chemotaxis. In vitro tumor growth and apoptosis were assessed using a proliferation assay and annexin V assay, respectively. Active RhoA protein levels in CCL17-stimulated colon cancer cells were quantified using a G-LISA assay. Results: We found that simvastatin dose-dependently decreased CCL17-induced colon cancer cell migration. Simvastatin had no effect on colon cancer cell proliferation or apoptosis. Inhibition of beta chemokine receptor 4, CCR4, reduced CCL17-evoked activation of RhoA in colon cancer cells. Moreover, administration of mevalonate reversed the inhibitory effect of simvastatin on CCL17-induced colon cancer cell migration. Interestingly, co-incubation with geranylgeranyl pyrophosphate (GGPP) antagonized the inhibitory impact of simvastatin on colon cancer cell migration triggered by CCL17. Moreover, we observed that simvastatin decreased CCL17-induced activation of RhoA in colon cancer cells. Administration of mevalonate and GGPP reversed the inhibitory effect of simvastatin on CCL17-provoked RhoA activation in colon cancer cells. Conclusions: Taken together, our findings show for the first time that HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via

  17. Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

    Science.gov (United States)

    Bodra, Nandita; Young, David; Astolfi Rosado, Leonardo; Pallo, Anna; Wahni, Khadija; de Proft, Frank; Huang, Jingjing; van Breusegem, Frank; Messens, Joris

    2017-02-01

    Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.

  18. Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

    Science.gov (United States)

    Bodra, Nandita; Young, David; Astolfi Rosado, Leonardo; Pallo, Anna; Wahni, Khadija; De Proft, Frank; Huang, Jingjing; Van Breusegem, Frank; Messens, Joris

    2017-01-01

    Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release. PMID:28195196

  19. Solvent effects on catalysis by Escherichia coli dihydrofolate reductase.

    Science.gov (United States)

    Loveridge, E Joel; Tey, Lai-Hock; Allemann, Rudolf K

    2010-01-27

    Hydride transfer catalyzed by dihydrofolate reductase (DHFR) has been described previously within an environmentally coupled model of hydrogen tunneling, where protein motions control binding of substrate and cofactor to generate a tunneling ready conformation and modulate the width of the activation barrier and hence the reaction rate. Changes to the composition of the reaction medium are known to perturb protein motions. We have measured kinetic parameters of the reaction catalyzed by DHFR from Escherichia coli in the presence of various cosolvents and cosolutes and show that the dielectric constant, but not the viscosity, of the reaction medium affects the rate of reaction. Neither the primary kinetic isotope effect on the reaction nor its temperature dependence were affected by changes to the bulk solvent properties. These results are in agreement with our previous report on the effect of solvent composition on catalysis by DHFR from the hyperthermophile Thermotoga maritima. However, the effect of solvent on the temperature dependence of the kinetic isotope effect on hydride transfer catalyzed by E. coli DHFR is difficult to explain within a model, in which long-range motions couple to the chemical step of the reaction, but may indicate the existence of a short-range promoting vibration or the presence of multiple nearly isoenergetic conformational substates of enzymes with similar but distinct catalytic properties.

  20. Molecular Characterization of a Dehydroascorbate Reductase from Pinus bungeana

    Institute of Scientific and Technical Information of China (English)

    Hai-Ling Yang; Ying-Ru Zhao; Cai-Ling Wang; Zhi-Ling Yang; Qing-Yin Zeng; Hai Lu

    2009-01-01

    Dehydroascorbate reductase (DHAR) plays a critical role in the ascorbate-glutathione recycling reaction for most higher plants. To date, studies on DHAR in higher plants have focused largely on Arabidopsis and agricultural plants, and there is virtually no information on the molecular characteristics of DHAR in gymnosperms. The present study reports the cloning and characteristics of a DHAR (PbDHAR) from a pine, Pinus bungeana Zucc. ex Endl. The PbDHAR gene encodes a protein of 215 amino acid residues with a calculated molecular mass of 24.26 kDa. The predicted 3-D structure of PbDHAR showed a typical glutathione S-transferase fold. Reverse transcription-polymerase chain reaction revealed that the PbDHAR was a constitutive expression gene in P. bungeana. The expression level of PbDHAR mRNA in P. bungeana seedlings did not show significant change under high temperature stress. The recombinant PbDHAR was overexpressed in Escherichia coll following purification with affinity chromatography. The recombinant PbDHAR exhibited enzymatic activity (19.84μmol/min per mg) and high affinity (a K_m of 0.08 mM) towards the substrates dehydroascorbate (DHA). Moreover, the recombinant PbDHAR was a thermostable enzyme, and retained 77% of its initial activity at 55 ℃. The present study is the first to provide a detailed molecular characterization of the DHAR in P. bungeana.

  1. Structural Basis for the Thermostability of Sulfur Oxygenase Reductases

    Institute of Scientific and Technical Information of China (English)

    尤晓颜; 孟珍; 陈栋炜; 郭旭; Josef Zeyer; 刘双江; 姜成英

    2012-01-01

    The thermostability of three sulfur oxygenase reductases (SORs) was investigated from thermoacidophilic achaea Acidianus tengchongensis (SORAT) and Sulfolobus tokodaii (SORsT) as well as the moderately thermophilic bacterium Acidithiobacillus sp. SM-1 (SORsB). The optimal temperatures for catalyzing sulfur oxidation were 80 ℃ (SORAT), 85 ℃ (SORsT), and 70 ℃ (SORsB), respectively. The half-lives of the three SORs at their optimal catalytic conditions were 100 min (SORAT), 58 min (SORsT), and 37 min (SORsB). In order to reveal the structural basis of the thermostability of these SORs, three-dimensional structural models of them were generated by homology modeling using the previously reported high-resolution X-ray structure of SORAA (from Acidianus ambivalens) as a template. The results suggest that thermostability was dependent on: (a) high number of the charged amino acid glutamic acid and the flexible amino acid proline, (b) low number of the therraolabile amino acid glutamine, (c) increased number of ion pairs, (d) decreased ratio of hydrophobie accessible solvent surface area (ASA) to charged ASA, and (e) increased volumes of the cavity. The number of cavities and the number of hydrogen bonds did not signifieantly affect the thermostability of SORs, whereas the cavity volumes increased as the thermal stability increased.

  2. 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.

  3. 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.

  4. Functional characterization of methionine sulfoxide reductase A from Trypanosoma spp.

    Science.gov (United States)

    Arias, Diego G; Cabeza, Matías S; Erben, Esteban D; Carranza, Pedro G; Lujan, Hugo D; Téllez Iñón, María T; Iglesias, Alberto A; Guerrero, Sergio A

    2011-01-01

    Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems has been mainly focused on the involvement of trypanothione, a specific redox component in these organisms. However, no information is available concerning their mechanisms for repairing oxidized proteins, which would be relevant for the survival of these pathogens in the various stages of their life cycle. We report the molecular cloning of three genes encoding a putative A-type MSR in trypanosomatids. The genes were expressed in Escherichia coli, and the corresponding recombinant proteins were purified and functionally characterized. The enzymes were specific for L-Met(S)SO reduction, using Trypanosoma cruzi tryparedoxin I as the reducing substrate. Each enzyme migrated in electrophoresis with a particular profile reflecting the differences they exhibit in superficial charge. The in vivo presence of the enzymes was evidenced by immunological detection in replicative stages of T. cruzi and Trypanosoma brucei. The results support the occurrence of a metabolic pathway in Trypanosoma spp. involved in the critical function of repairing oxidized macromolecules.

  5. Kinetic, thermodynamic and X-ray structural insights into the interaction of melatonin and analogues with quinone reductase 2

    Energy Technology Data Exchange (ETDEWEB)

    Calamini, Barbara; Santarsiero, Bernard D.; Boutin, Jean A.; Mesecar, Andrew D. (IdRS); (UIC)

    2008-09-12

    Melatonin exerts its biological effects through at least two transmembrane G-protein-coupled receptors, MT1 and MT2, and a lower-affinity cytosolic binding site, designated MT3. MT3 has recently been identified as QR2 (quinone reductase 2) (EC 1.10.99.2) which is of significance since it links the antioxidant effects of melatonin to a mechanism of action. Initially, QR2 was believed to function analogously to QR1 in protecting cells from highly reactive quinones. However, recent studies indicate that QR2 may actually transform certain quinone substrates into more highly reactive compounds capable of causing cellular damage. Therefore it is hypothesized that inhibition of QR2 in certain cases may lead to protection of cells against these highly reactive species. Since melatonin is known to inhibit QR2 activity, but its binding site and mode of inhibition are not known, we determined the mechanism of inhibition of QR2 by melatonin and a series of melatonin and 5-hydroxytryptamine (serotonin) analogues, and we determined the X-ray structures of melatonin and 2-iodomelatonin in complex with QR2 to between 1.5 and 1.8 {angstrom} (1 {angstrom} = 0.1 nm) resolution. Finally, the thermodynamic binding constants for melatonin and 2-iodomelatonin were determined by ITC (isothermal titration calorimetry). The kinetic results indicate that melatonin is a competitive inhibitor against N-methyldihydronicotinamide (K{sub i} = 7.2 {mu}M) and uncompetitive against menadione (K{sub i} = 92 {mu}M), and the X-ray structures shows that melatonin binds in multiple orientations within the active sites of the QR2 dimer as opposed to an allosteric site. These results provide new insights into the binding mechanisms of melatonin and analogues to QR2.

  6. Rational design of broad spectrum antibacterial activity based on a clinically relevant enoyl-acyl carrier protein (ACP) reductase inhibitor.

    Science.gov (United States)

    Schiebel, Johannes; Chang, Andrew; Shah, Sonam; Lu, Yang; Liu, Li; Pan, Pan; Hirschbeck, Maria W; Tareilus, Mona; Eltschkner, Sandra; Yu, Weixuan; Cummings, Jason E; Knudson, Susan E; Bommineni, Gopal R; Walker, Stephen G; Slayden, Richard A; Sotriffer, Christoph A; Tonge, Peter J; Kisker, Caroline

    2014-06-06

    Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Rational Design of Broad Spectrum Antibacterial Activity Based on a Clinically Relevant Enoyl-Acyl Carrier Protein (ACP) Reductase Inhibitor*

    Science.gov (United States)

    Schiebel, Johannes; Chang, Andrew; Shah, Sonam; Lu, Yang; Liu, Li; Pan, Pan; Hirschbeck, Maria W.; Tareilus, Mona; Eltschkner, Sandra; Yu, Weixuan; Cummings, Jason E.; Knudson, Susan E.; Bommineni, Gopal R.; Walker, Stephen G.; Slayden, Richard A.; Sotriffer, Christoph A.; Tonge, Peter J.; Kisker, Caroline

    2014-01-01

    Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms. PMID:24739388

  8. Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities.

    Science.gov (United States)

    Kuzu, Muslum; Aslan, Abdulselam; Ahmed, Ishtiaq; Comakli, Veysel; Demirdag, Ramazan; Uzun, Naim

    2016-04-01

    Glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) are metabolically quite important enzymes. Within this study, these two enzymes were purified for the first time from the gills of Lake Van fish. In the purifying process, ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity column chromatography techniques for glucose-6-phosphate dehydrogenase, temperature degradation and 2',5'-ADP Sepharose 4B affinity column chromatography for glutathione reductase enzyme were used. The control of the enzyme purity and determination of molecular weight were done with sodium dodecyl sulfate polyacrylamide gel electrophoresis. K(M) and V(max) values were determined with Lineweaver-Burk plot. Besides, the effects of some chalcone derivatives on the purified enzymes were analyzed. For the ones showing inhibition effect, % activity-[I] figures were drawn and IC50 values were determined. K(i) value was calculated by using Cheng-Prusoff equation.

  9. Reduction of nitrates in Cucumis sativus L. seedlings II. Influence of tungsten and vanadium on nitrate reductase and adenosine triphosphatase activities

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-02-01

    Full Text Available ATPases isolated from the roots of cucumber seedlings activated by Mg+2 ions in experiments in vitro, were fairly distinctly inhibited by Ca-2 ions, very slightly inhibited by fluorides and molybdenum ions while NO3- anions had no effect on the level of ATPase activity studied. Introduction into the nutrient of 10-4 M Na2WO4 or 10-3 M Na VO3 (inhibitors of nitrate reductase NR distinctly inhibited activity of the ATPase under study especially of fractions IIa and III, and inhibited NR activity and lowered uptake of NO3-. WO4-2 and VO3 inhibited to the same extent absorption and reduction of NO3- in the initial phase of NR induction, whereas at a later stage both inhibitors checked reduction to a greater degree than uptake of NO3-. The results indicate the possibility of certain ATPase participation in assimilating nitrates, and suggest that in the initial stage of biosynthesis of the NR enzyme system, activity of the enzyme is distinctly dependent upon NO3- transport and the level of NR activity limited by the amount of nitrate taken up. At a later an additional mechanism of NO3- transport probably functions, not connected with simultaneous reduction of nitrates. On the basis of results the Butz and Jackson (1977 hypothesis concerning a model for the absorption and reduction of NO3- by plant tissues is discussed.

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

    Science.gov (United States)

    Giovanella, Patricia; Cabral, Lucélia; Bento, Fátima Menezes; Gianello, Clesio; Camargo, Flávio Anastácio Oliveira

    2016-01-25

    This study aimed to isolate mercury resistant bacteria, determine the minimum inhibitory concentration for Hg, estimate mercury removal by selected isolates, explore the mer genes, and detect and characterize the activity of the enzyme mercuric (II) reductase produced by a new strain of Pseudomonas sp. B50A. The Hg removal capacity of the isolates was determined by incubating the isolates in Luria Bertani broth and the remaining mercury quantified by atomic absorption spectrophotometry. A PCR reaction was carried out to detect the merA gene and the mercury (II) reductase activity was determined in a spectrophotometer at 340 nm. Eight Gram-negative bacterial isolates were resistant to high mercury concentrations and capable of removing mercury, and of these, five were positive for the gene merA. The isolate Pseudomonas sp. B50A removed 86% of the mercury present in the culture medium and was chosen for further analysis of its enzyme activity. Mercuric (II) reductase activity was detected in the crude extract of this strain. This enzyme showed optimal activity at pH 8 and at temperatures between 37 °C and 45 °C. The ions NH4(+), Ba(2+), Sn(2+), Ni(2+) and Cd(2+) neither inhibited nor stimulated the enzyme activity but it decreased in the presence of the ions Ca(2+), Cu(+) and K(+). The isolate and the enzyme detected were effective in reducing Hg(II) to Hg(0), showing the potential to develop bioremediation technologies and processes to clean-up the environment and waste contaminated with mercury.

  11. Effect of dihydrofolate reductase gene knock-down on the expression of heart and neural crest derivatives expressed transcript 2 in zebrafish cardiac development

    Institute of Scientific and Technical Information of China (English)

    SUN Shu-na; GUI Yong-hao; WANG Yue-xiang; QIAN Lin-xi; JIANG Qiu; LIU Dong; SONG Hou-yan

    2007-01-01

    Background Folic acid is very important for embryonic development and dihydrofolate reductase is one of the key enzymes in the process of folic acid performing its biological function. Therefore, the dysfunction of dihydrofolate reductase can inhibit the function of folic acid and finally cause the developmental malformations. In this study, we observed the abnormal cardiac phenotypes in dihydrofolate reductase (DHFR) gene knock-down zebrafish embryos,investigated the effect of DHFR on the expression of heart and neural crest derivatives expressed transcript 2 (HAND2)and explored the possible mechanism of DHFR knock-down inducing zebrafish cardiac malformations.Methods Morpholino oligonucleotides were microinjected into fertilized eggs to knock down the functions of DHFR or HAND2. Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpress HAND2. The cardiac morphologies, the heart rates and the ventricular shortening fraction were observed and recorded under the microscope at 48 hours post fertilization. Whole-mount in situ hybridization and real-time PCR were performed to detect HAND2 expression.Results DHFR or HAND2 knock-down caused the cardiac malformation in zebrafish. The expression of HAND2 was obviously reduced in DHFR knock-down embryos (P<0.05). Microinjecting HAND2 mRNA into fertilized eggs can induce HAND2 overexpression. HAND2 overexpression rescued the cardiac malformation phenotypes of DHFR knock-down embryos.Conclusions DHFR plays a crucial role in cardiac development. The down-regulation of HAND2 caused by DHFR knock-down is the possible mechanism of DHFR knock-down inducing the cardiac malformation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Serafini, P.; Lobo, R.A.

    1985-01-01

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

  13. Differing views of the role of selenium in thioredoxin reductase

    Science.gov (United States)

    Ruggles, Erik L.

    2010-01-01

    This review covers three different chemical explanations that could account for the requirement of selenium in the form of selenocysteine in the active site of mammalian thioredoxin reductase. These views are the following: (1) the traditional view of selenocysteine as a superior nucleophile relative to cysteine, (2) the superior leaving group ability of a selenol relative to a thiol due to its significantly lower pKa and, (3) the superior ability of selenium to accept electrons (electrophilicity) relative to sulfur. We term these chemical explanations as the “chemico-enzymatic” function of selenium in an enzyme. We formally define the chemico-enzymatic function of selenium as its specific chemical property that allows a selenoenzyme to catalyze its individual reaction. However we, and others, question whether selenocysteine is chemically necessary to catalyze an enzymatic reaction since cysteine-homologs of selenocysteine-containing enzymes catalyze their specific enzymatic reactions with high catalytic efficiency. There must be a unique chemical reason for the presence of selenocysteine in enzymes that explains the biological pressure on the genome to maintain the complex selenocysteine-insertion machinery. We term this biological pressure the “chemico-biological” function of selenocysteine. We discuss evidence that this chemico-biological function is the ability of selenoenzymes to resist inactivation by irreversible oxidation. The way in which selenocysteine confers resistance to oxidation could be due to the superior ability of the oxidized form of selenocysteine (Sec-SeO2−, seleninic acid) to be recycled back to its parent form (Sec-SeH, selenocysteine) in comparison to the same cycling of cysteine-sulfinic acid to cysteine (Cys-SO2− to Cys-SH). PMID:20397034

  14. Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase*

    Science.gov (United States)

    Wu, Sheng-Yi; Rothery, Richard A.; Weiner, Joel H.

    2015-01-01

    We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser719, NarG-His1163, and NarG-His1184); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His1092 and NarG-His1098). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔEm values of −88 and −36 mV, respectively). Ala variants of His1092 and His1098 also elicit large ΔEm values of −143 and −101 mV, respectively. An Arg variant of His1092 elicits a small ΔEm of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum Em value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis. PMID:26297003

  15. 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.

  16. Prokaryotic arsenate reductase enhances arsenate resistance in Mammalian cells.

    Science.gov (United States)

    Wu, Dan; Tao, Xuanyu; Wu, Gaofeng; Li, Xiangkai; Liu, Pu

    2014-01-01

    Arsenic is a well-known heavy metal toxicant in the environment. Bioremediation of heavy metals has been proposed as a low-cost and eco-friendly method. This article described some of recent patents on transgenic plants with enhanced heavy metal resistance. Further, to test whether genetic modification of mammalian cells could render higher arsenic resistance, a prokaryotic arsenic reductase gene arsC was transfected into human liver cancer cell HepG2. In the stably transfected cells, the expression level of arsC gene was determined by quantitative real-time PCR. Results showed that arsC was expressed in HepG2 cells and the expression was upregulated by 3 folds upon arsenate induction. To further test whether arsC has function in HepG2 cells, the viability of HepG2-pCI-ArsC cells exposed to arsenite or arsenate was compared to that of HepG2-pCI cells without arsC gene. The results indicated that arsC increased the viability of HepG2 cells by 25% in arsenate, but not in arsenite. And the test of reducing ability of stably transfected cells revealed that the concentration of accumulated trivalent arsenic increased by 25% in HepG2-pCI-ArsC cells. To determine the intracellular localization of ArsC, a fusion vector with fluorescent marker pEGFP-N1-ArsC was constructed and transfected into.HepG2. Laser confocal microscopy showed that EGFP-ArsC fusion protein was distributed throughout the cells. Taken together, these results demonstrated that prokaryotic arsenic resistant gene arsC integrated successfully into HepG2 genome and enhanced arsenate resistance of HepG2, which brought new insights of arsenic detoxification in mammalian cells.

  17. Rational Design of a Structural and Functional Nitric Oxide Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Yeung, N.; Lin, Y; Gao, Y; Zhao, X; Russell, B; Lei, L; Miner, L; Robinson, H; Lu, Y

    2009-01-01

    Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally, it is more difficult to design functional proteins. In comparison to recent successes in designing non-metalloproteins, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes. This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem FeB centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.

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

    Science.gov (United States)

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

    2012-01-01

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

  19. 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.

  20. Recombinant pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) catalyze opposite enantiospecific conversions.

    Science.gov (United States)

    Fujita, M; Gang, D R; Davin, L B; Lewis, N G

    1999-01-01

    Although the heartwood of woody plants represents the main source of fiber and solid wood products, essentially nothing is known about how the biological processes leading to its formation are initiated and regulated. Accordingly, a reverse transcription-polymerase chain reaction-guided cloning strategy was employed to obtain genes encoding pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) as a means to initiate the study of its heartwood formation. (+)-Pinoresinol-(+)-lariciresinol reductase from Forsythia intermedia was used as a template for primer construction for reverse transcription-polymerase chain reaction amplifications, which, when followed by homologous hybridization cloning, resulted in the isolation of two distinct classes of putative pinoresinol-lariciresinol reductase cDNA clones from western red cedar. A representative of each class was expressed as a fusion protein with beta-galactosidase and assayed for enzymatic activity. Using both deuterated and radiolabeled (+/-)-pinoresinols as substrates, it was established that each class of cDNA encoded a pinoresinol-lariciresinol reductase of different (opposite) enantiospecificity. Significantly, the protein from one class converted (+)-pinoresinol into (-)-secoisolariciresinol, whereas the other utilized the opposite (-)-enantiomer to give the corresponding (+)-form. This differential substrate specificity raises important questions about the role of each of these individual reductases in heartwood formation, such as whether they are expressed in different cells/tissues or at different stages during heartwood development.

  1. 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.

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

    Directory of Open Access Journals (Sweden)

    R. Seenivasagan

    2016-07-01

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

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

    Science.gov (United States)

    Montalvetti, A; Peña-Díaz, J; Hurtado, R; Ruiz-Pérez, L M; González-Pacanowska, D

    2000-07-01

    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 reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.

  4. E2F4 and ribonucleotide reductase mediate S-phase arrest in colon cancer cells treated with chlorophyllin.

    Science.gov (United States)

    Chimploy, Korakod; Díaz, G Dario; Li, Qingjie; Carter, Orianna; Dashwood, Wan-Mohaiza; Mathews, Christopher K; Williams, David E; Bailey, George S; Dashwood, Roderick H

    2009-11-01

    Chlorophyllin (CHL) is a water-soluble derivative of chlorophyll that exhibits cancer chemopreventive properties, but which also has been studied for its possible cancer therapeutic effects. We report here that human colon cancer cells treated with CHL accumulate in S-phase of the cell cycle, and this is associated with reduced expression levels of p53, p21, and other G(1)/S checkpoint controls. At the same time, E2F1 and E2F4 transcription factors become elevated and exhibit increased DNA binding activity. In CHL-treated colon cancer cells, bromodeoxyuridine pulse-chase experiments provided evidence for the inhibition of DNA synthesis. Ribonucleotide reductase (RR), a pivotal enzyme for DNA synthesis and repair, was reduced at the mRNA and protein level after CHL treatment, and the enzymatic activity was inhibited in a concentration-dependent manner both in vitro and in vivo. Immunoblotting revealed that expression levels of RR subunits R1, R2, and p53R2 were reduced by CHL treatment in HCT116 (p53(+/+)) and HCT116 (p53(-/-)) cells, supporting a p53-independent mechanism. Prior studies have shown that reduced levels of RR small subunits can increase the sensitivity of colon cancer cells to clinically used DNA-damaging agents and RR inhibitors. We conclude that by inhibiting R1, R2, and p53R2, CHL has the potential to be effective in the clinical setting, when used alone or in combination with currently available cancer therapeutic agents.

  5. Uncoupling of allosteric and oligomeric regulation in a functional hybrid enzyme constructed from Escherichia coli and human ribonucleotide reductase.

    Science.gov (United States)

    Fu, Yuan; Long, Marcus J C; Rigney, Mike; Parvez, Saba; Blessing, William A; Aye, Yimon

    2013-10-08

    An N-terminal-domain (NTD) and adjacent catalytic body (CB) make up subunit-α of ribonucleotide reductase (RNR), the rate-limiting enzyme for de novo dNTP biosynthesis. A strong linkage exists between ligand binding at the NTD and oligomerization-coupled RNR inhibition, inducible by both dATP and nucleotide chemotherapeutics. These observations have distinguished the NTD as an oligomeric regulation domain dictating the assembly of inactive RNR oligomers. Inactive states of RNR differ between eukaryotes and prokaryotes (α6 in human versus α4β4 in Escherichia coli , wherein β is RNR's other subunit); however, the NTD structurally interconnects individual α2 or α2 and β2 dimeric motifs within the respective α6 or α4β4 complexes. To elucidate the influence of NTD ligand binding on RNR allosteric and oligomeric regulation, we engineered a human- E. coli hybrid enzyme (HE) where human-NTD is fused to E. coli -CB. Both the NTD and the CB of the HE bind dATP. The HE specifically partners with E. coli -β to form an active holocomplex. However, although the NTD is the sole physical tether to support α2 and/or β2 associations in the dATP-bound α6 or α4β4 fully inhibited RNR complexes, the binding of dATP to the HE NTD only partially suppresses HE activity and fully precludes formation of higher-order HE oligomers. We postulate that oligomeric regulation is the ultimate mechanism for potent RNR inhibition, requiring species-specific NTD-CB interactions. Such interdomain cooperativity in RNR oligomerization is unexpected from structural studies alone or biochemical studies of point mutants.

  6. Impacts of some divalent cations on periplasmic nitrate reductase and dehydrogenase enzymes of Escherichia, Pseudomonas and Acinetobacter species

    Directory of Open Access Journals (Sweden)

    Christian E. Nwanyanwu

    2008-08-01

    Full Text Available The impacts of Hg2+, Cd2+ and Zn2+ on the activities of periplasmic nitrate reductase (NAP and dehydrogenase (DHA enzymes of three organisms isolated from soil and sediment-water interface were analysed in liquid culture studies. NAP and DHA activities were estimated from nitrite and triphenyl formazan were produced respectively after 4h incubation at 28 ± 2oC. Hg2+ completely inhibited NAP activity in Escherichia and Pseudomonas spp at all the concentrations (0.2 – 1mM while progressive inhibitions of NAP activity were observed in Escherichia and Pseudomonas spp with increasing concentrations of Zn2+ and Cd2+. Both metals were stimulatory to NAP of Acinetobacter sp at 0.2 – 1mM. Apart from stimulation of DHA activity by Zn2+ (0.2 – 1mM in Escherichia sp, Cd2+ (0.4 -1.0mM in Acinetobacter sp and (1.0mM in Pseudomonas sp, all the metals progressively inhibited DHA activities in the three organisms. In Escherichia sp, the activities of the two enzymes were negatively correlated on exposure to Zn2+ (r = -0.91 and positively correlated (r = >0.90 on exposure to Cd2+ and Hg2+. Based on IC50 values of the metals for the DHA and NAP enzymes, the most resistant of the three organisms were Escherichia sp and Acinetobacter sp respectively. Quantitatively, NAP with its lower IC50 values than DHA was a more sensitive toxicity measure for Hg2+ in all the organisms. The sensitivity of microbial metabolic enzymes to the toxic effects of metals varies with the type of enzyme, metal and the microorganism involved.

  7. Cloning of thioredoxin h reductase and characterization of the thioredoxin reductase-thioredoxin h system from wheat.

    Science.gov (United States)

    Serrato, Antonio J; Pérez-Ruiz, Juan M; Cejudo, Francisco J

    2002-10-15

    Thioredoxins h are ubiquitous proteins reduced by NADPH- thioredoxin reductase (NTR). They are able to reduce disulphides in target proteins. In monocots, thioredoxins h accumulate at high level in seeds and show a predominant localization in the nucleus of seed cells. These results suggest that the NTR-thioredoxin h system probably plays an important role in seed physiology. To date, the study of this system in monocots is limited by the lack of information about NTR. In the present study, we describe the cloning of a full-length cDNA encoding NTR from wheat ( Triticum aestivum ). The polypeptide deduced from this cDNA shows close similarity to NTRs from Arabidopsis, contains FAD- and NADPH-binding domains and a disulphide probably interacting with the disulphide at the active site of thioredoxin h. Wheat NTR was expressed in Escherichia coli as a His-tagged protein. The absorption spectrum of the purified recombinant protein is typical of flavoenzymes. Furthermore, it showed NADPH-dependent thioredoxin h reduction activity, thus confirming that the cDNA clone reported in the present study encodes wheat NTR. Using the His-tagged NTR and TRXhA (wheat thioredoxin h ), we successfully reconstituted the wheat NTR-thioredoxin h system in vitro, as shown by the insulin reduction assay. A polyclonal antibody was raised against wheat NTR after immunization of rabbits with the purified His-tagged protein. This antibody efficiently detected a single polypeptide of the corresponding molecular mass in seed extracts and it allowed the analysis of the pattern of accumulation of NTR in different wheat organs and developmental stages. NTR shows a wide distribution in wheat, but, surprisingly, its accumulation in seeds is low, in contrast with the level of thioredoxins h.

  8. Characterization and gene cloning of l-xylulose reductase involved in l-arabinose catabolism from the pentose-fermenting fungus Rhizomucor pusillus.

    Science.gov (United States)

    Yamasaki-Yashiki, Shino; Komeda, Hidenobu; Hoshino, Kazuhiro; Asano, Yasuhisa

    2017-08-01

    l-Xylulose reductase (LXR) catalyzes the reduction of l-xylulose to xylitol in the fungal l-arabinose catabolic pathway. LXR (RpLXR) was purified from the pentose-fermenting zygomycetous fungus Rhizomucor pusillus NBRC 4578. The native RpLXR is a homotetramer composed of 29 kDa subunits and preferred NADPH as a coenzyme. The Km values were 8.71 mM for l-xylulose and 3.89 mM for dihydroxyacetone. The lxr3 (Rplxr3) gene encoding RpLXR consists of 792 bp and encodes a putative 263 amino acid protein (Mr = 28,341). The amino acid sequence of RpLXR showed high similarity to 3-oxoacyl-(acyl-carrier-protein) reductase. The Rplxr3 gene was expressed in Escherichia coli and the recombinant RpLXR exhibited properties similar to those of native RpLXR. Transcription of the Rplxr3 gene in R. pusillus NBRC 4578 was induced in the presence of l-arabinose and inhibited in the presence of d-glucose, d-xylose, and d-mannitol, indicating that RpLXR is involved in the l-arabinose catabolic pathway.

  9. Renal reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase-mediated metabolism of the carcinogen N-(4-(5-nitro-2-furyl)-2-thiazolyl)acetamide

    Energy Technology Data Exchange (ETDEWEB)

    Mattammal, M.B.; Zenser, T.V.; Palmier, M.O.; Davis, B.B.

    1985-01-01

    N-(4-(5-Nitro-2-furyl)-2-thiazolyl)acetamide (NFTA) metabolism was examined in vitro using microsomes prepared from rat liver and renal cortex and from rabbit liver and renal cortex and outer and inner medulla. NFTA nitroreduction was observed with each tissue. Three mol of NADPH were used per mol of NFTA reduced. Substrate and inhibitor specificity suggested that the microsomal nitroreduction was due to NADPH:cytochrome c reductase. Metabolite(s) formed bound to protein, RNA, DNA, and synthetic polyribonucleotides. Maximum covalent binding was seen with polyguanylic acid. A guanosine-NFTA adduct was isolated. Binding was inhibited by sulfhydryl compounds and vitamin E. The (/sup 14/C)NFTA:glutathione or (/sup 3/H)glutathione:NFTA conjugates obtained from microsomal incubations showed identical chromatographic properties as the product obtained by the reaction of synthetic N-hydroxy-NFTA with (/sup 3/H)glutathione. Structures of synthetic N-hydroxy-NFTA and the microsomal reduction product 1-(4-(2-acetylaminothiazolyl))-3-cyano-1-propanone were established by mass spectrometry. The latter reduction product did not bind macromolecules. These results suggest that renal NADPH:cytochrome c reductase reduces NFTA to an N-hydroxy-NFTA intermediate that binds nucleophilic sites on macromolecules.

  10. Crystal Structure of Human Liver [delta][superscript 4]-3-Ketosteroid 5[beta]-Reductase (AKR1D1) and Implications for Substrate Binding and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Di Costanzo, Luigi; Drury, Jason E.; Penning, Trevor M.; Christianson, David W. (UPENN); (UPENN-MED)

    2008-07-15

    AKR1D1 (steroid 5{beta}-reductase) reduces all {Delta}{sup 4}-3-ketosteroids to form 5{beta}-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an {alpha}{beta}-unsaturated ketone by 5{beta}-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the {beta}-face of a {Delta}{sup 4}-3-ketosteroid yields a cis-A/B-ring configuration with an {approx}90{sup o} bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human {Delta}{sup 4}-3-ketosteroid 5{beta}-reductase (AKR1D1), and its complexes with intact substrates. We have determined the structures of AKR1D1 complexes with NADP{sup +} at 1.79- and 1.35-{angstrom} resolution (HEPES bound in the active site), NADP{sup +} and cortisone at 1.90-{angstrom} resolution, NADP{sup +} and progesterone at 2.03-{angstrom} resolution, and NADP{sup +} and testosterone at 1.62-{angstrom} resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP{sup +}. This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr{sup 58} and Glu{sup 120}. The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.

  11. Side chain conformational averaging in human dihydrofolate reductase.

    Science.gov (United States)

    Tuttle, Lisa M; Dyson, H Jane; Wright, Peter E

    2014-02-25

    The three-dimensional structures of the dihydrofolate reductase enzymes from Escherichia coli (ecDHFR or ecE) and Homo sapiens (hDHFR or hE) are very similar, despite a rather low level of sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and E. coli enzymes, we conducted a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations cannot be detected for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that helix αF changes position by sliding between the two states. Analysis of χ1 rotamer populations derived from measurements of (3)JCγCO and (3)JCγN couplings indicates that many of the side chains that contact helix αF exhibit rotamer averaging that may facilitate the conformational change. The χ1 rotamer adopted by the Phe31 side chain depends upon whether the active site contains the substrate or product. In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 χ1 rotamer opens the active site to facilitate entry of the substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as

  12. RESISTANCE LEVEL OF Pseudomonas stutzeri AGAINST MERCURY AND ITS ABILITY IN PRODUCTION OF MERCURY REDUCTASE ENZYME

    Directory of Open Access Journals (Sweden)

    Purkan Purkan

    2016-11-01

    Full Text Available Mercury reductase is an enzyme that is able to reduce Hg2+ to Hg0 non toxic. This enzyme is usually produced by mercury resistant bacteria. The research wanted to determine the resistance of indigenous Pseudomonas stutzeri isolate toward mercury and to explore the mercury reductase activity which is produced by the bacteria. The results of resistance assay of the Pseudomonas stutzeri toward mercury ion showed that the isolate could survive in media containing HgCl2 up to a concentration of 80 µM. The bacteria could produce mercury reductase optimally at the 24th of fermentation time. The enzyme showed optimum activity at pH 7 and temperature of 45 oC

  13. A substrate-bound structure of cyanobacterial biliverdin reductase identifies stacked substrates as critical for activity

    Science.gov (United States)

    Takao, Haruna; Hirabayashi, Kei; Nishigaya, Yuki; Kouriki, Haruna; Nakaniwa, Tetsuko; Hagiwara, Yoshinori; Harada, Jiro; Sato, Hideaki; Yamazaki, Toshimasa; Sakakibara, Yoichi; Suiko, Masahito; Asada, Yujiro; Takahashi, Yasuhiro; Yamamoto, Ken; Fukuyama, Keiichi; Sugishima, Masakazu; Wada, Kei

    2017-01-01

    Biliverdin reductase catalyses the last step in haem degradation and produces the major lipophilic antioxidant bilirubin via reduction of biliverdin, using NAD(P)H as a cofactor. Despite the importance of biliverdin reductase in maintaining the redox balance, the molecular details of the reaction it catalyses remain unknown. Here we present the crystal structure of biliverdin reductase in complex with biliverdin and NADP+. Unexpectedly, two biliverdin molecules, which we designated the proximal and distal biliverdins, bind with stacked geometry in the active site. The nicotinamide ring of the NADP+ is located close to the reaction site on the proximal biliverdin, supporting that the hydride directly attacks this position of the proximal biliverdin. The results of mutagenesis studies suggest that a conserved Arg185 is essential for the catalysis. The distal biliverdin probably acts as a conduit to deliver the proton from Arg185 to the proximal biliverdin, thus yielding bilirubin. PMID:28169272

  14. Denitrification by plant roots? New aspects of plant plasma membrane-bound nitrate reductase.

    Science.gov (United States)

    Eick, Manuela; Stöhr, Christine

    2012-10-01

    A specific form of plasma membrane-bound nitrate reductase in plants is restricted to roots. Two peptides originated from plasma membrane integral proteins isolated from Hordeum vulgare have been assigned as homologues to the subunit NarH of respiratory nitrate reductase of Escherichia coli. Corresponding sequences have been detected for predicted proteins of Populus trichocarpa with high degree of identities for the subunits NarH (75%) and NarG (65%), however, with less accordance for the subunit NarI. These findings coincide with biochemical properties, particularly in regard to the electron donors menadione and succinate. Together with the root-specific and plasma membrane-bound nitrite/NO reductase, nitric oxide is produced under hypoxic conditions in the presence of nitrate. In this context, a possible function in nitrate respiration of plant roots and an involvement of plants in denitrification processes are discussed.

  15. Directed Molecular Evolution of Nitrite Oxido-reductase by DNA-shuffling

    Institute of Scientific and Technical Information of China (English)

    JUN-WEN LI; JIN-LAI ZHENG; XIN-WEI WANG; MIN JIN; FU-HUAN CHAO

    2007-01-01

    Objective To develtop directly molecular evolution of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremely slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatment. Methods The norB gene coding the nitrite oxido-reductase in nitrobacteria was cloned and sequenced. Then, directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria. Results After DNA-shuffling with sexual PCR and staggered extension process PCR, the sequence was different from its parental DNA fragments and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures.Conclusion DNA-shuffling was successfully used to engineer E. coli, which had norB gene and could degrade inorganic nitrogen effectively.

  16. Structure of Physarum polycephalum cytochrome b5 reductase at 1.56 A resolution.

    Science.gov (United States)

    Kim, Sangwoo; Suga, Michihiro; Ogasahara, Kyoko; Ikegami, Terumi; Minami, Yoshiko; Yubisui, Toshitsugu; Tsukihara, Tomitake

    2007-04-01

    Physarum polycephalum cytochrome b(5) reductase catalyzes the reduction of cytochrome b(5) by NADH. The structure of P. polycephalum cytochrome b(5) reductase was determined at a resolution of 1.56 A. The molecular structure was compared with that of human cytochrome b(5) reductase, which had previously been determined at 1.75 A resolution [Bando et al. (2004), Acta Cryst. D60, 1929-1934]. The high-resolution structure revealed conformational differences between the two enzymes in the adenosine moiety of the FAD, the lid region and the linker region. The structural properties of both proteins were inspected in terms of hydrogen bonding, ion pairs, accessible surface area and cavity volume. The differences in these structural properties between the two proteins were consistent with estimates of their thermostabilities obtained from differential scanning calorimetry data.

  17. Cuminaldehyde: Aldose Reductase and alpha-Glucosidase Inhibitor Derived from Cuminum cyminum L. Seeds.

    Science.gov (United States)

    Lee, Hoi-Seon

    2005-04-06

    The inhibitory activity of Cuminum cyminum seed-isolated component was evaluated against lens aldose reductase and alpha-glucosidase isolated from Sprague-Dawley male rats and compared to that of 11 commercially available components derived from C. cyminum seed oil, as well as quercitrin as an aldose reductase inhibitor and acarbose as an alpha-glucosidase inhibitor. The biologically active constituent of C. cyminum seed oil was characterized as cuminaldehyde by various spectral analyses. The IC(50) value of cuminaldehyde is 0.00085 mg/mL against aldose reductase and 0.5 mg/mL against alpha-glucosidase, respectively. Cuminaldehyde was about 1.8 and 1.6 times less in inhibitory activity than acarbose and quercitin, respectively. Nonetheless, cuminaldehyde may be useful as a lead compound and a new agent for antidiabetic therapeutics.

  18. Circadian variation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in swine liver and ileum.

    Science.gov (United States)

    Rogers, D H; Kim, D N; Lee, K T; Reiner, J M; Thomas, W A

    1981-07-01

    The temporal variation of HMG-CoA reductase activity in the liver and intestine of swine was investigated. The thin-layer chromatographic method widely used in the assay of the reductase was successfully applied to the porcine enzymes. Parallel circadian rhythms were demonstrated in both hepatic and ileal reductases from mash-fed animals. Peak activity occurred approximately 6 hr after feeding, 2.7-fold over the basal level in the liver, and 1.6-fold in the ileum. A milk-cholesterol diet caused a marked depression of both rhythms (90% in liver, 50% in ileum); however, the hourly variation in activity persisted in both organs. Cholestyramine was found to elevate hepatic activity (2.7-fold throughout the rhythm) without affecting that of the intestine. Clofibrate had no effect on either enzyme at any time during the cycle despite a 34% reduction in serum cholesterol concentrations.

  19. 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.

  20. 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.

  1. 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.

  2. 5{alpha}-reductase expression by prostate cancer cell lines and benign prostatic hyperplasia in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Smith, C.M.; Masters, J.R.W. [Univ. College of London (United Kingdom)]|[Pfizer Central Research, Kent (United Kingdom); Ballard, S.A.; Worman, N. [Pfizer Central Research, Sandwich (United Kingdom)

    1996-04-01

    5{alpha}-Reductase (5{alpha}R) activity in two human prostate cancer cell lines was compared to that in benign prostatic hyperplasia (BPH) tissue and COS cells transfected with and expressing the human genes for 5{alpha}-reductase type 1 (5{alpha}R1) and type 2 (5{alpha}R2). Comparisons were based on pH profiles and sensitivities to selective inhibitors of 5{alpha}-reductase. In the cancer lines, activity was greatest over the pH range 7-8, compared to a sharp peak of activity between pH 5-5.5 in BPH tissue and COS cells expressing 5{alpha}R2. Finasteride and SKF105,657 were potent inhibitors of 5{alpha}-reductase activity in BPH tissue and COS cells expressing 5{alpha}R2, but weak inhibitors in the cancer lines and in COS cells expressing 5{alpha}R1. In contrast, LTK1 17,026 was a more potent inhibitor of 5{alpha}-reductase activity in the prostate cancer cell lines and in COS cells expressing 5{alpha}R1. These data indicate that human prostate cancer cell lines express 5{alpha}-reductase activity similar to that in COS cells transfected with 5{alpha}R1, but different from that in BPH tissue. This may be a consequence of in vitro culture. Alternatively, it may reflect a change occurring as a result of neoplastic transformation, in which case it will be important to select appropriate inhibitors in the clinic. 29 refs., 3 figs., 2 tabs.

  3. Adverse Effects and Safety of 5-alpha Reductase Inhibitors (Finasteride, Dutasteride): A Systematic Review

    Science.gov (United States)

    Hirshburg, Jason M.; Kelsey, Petra A.; Therrien, Chelsea A.; Gavino, A. Carlo; Reichenberg, Jason S.

    2016-01-01

    Finasteride and dutasteride, both 5-alpha reductase inhibitors, are considered first-line treatment for androgenetic hair loss in men and used increasingly in women. In each case, patients are expected to take the medications indefinitely despite the lack of research regarding long-term adverse effects. Concerns regarding the adverse effects of these medications has led the United States National Institutes of Health to add a link for post-finasteride syndrome to its Genetic and Rare Disease Information Center. Herein, the authors report the results of a literature search reviewing adverse events of 5-alpha reductase inhibitors as they relate to prostate cancer, psychological effects, sexual health, and use in women. Several large studies found no increase in incidence of prostate cancer, a possible increase of high-grade cancer when detected, and no change in survival rate with 5-alpha reductase inhibitor use. Currently, there is no direct link between 5-alpha reductase inhibitor use and depression; however, several small studies have led to depression being listed as a side effect on the medication packaging. Sexual effects including erectile dysfunction and decreased libido and ejaculate were reported in as many as 3.4 to 15.8 percent of men. To date, there are very few studies evaluating 5-alpha reductase inhibitor use in women. Risks include birth defects in male fetuses if used in pregnancy, decreased libido, headache, gastrointestinal discomfort, and isolated reports of changes in menstruation, acne, and dizziness. Overall, 5-alpha reductase inhibitors were well-tolerated in both men and women, but not without risk, highlighting the importance of patient education prior to treatment. PMID:27672412

  4. Inhibition of a plant sesquiterpene cyclase by mevinolin.

    Science.gov (United States)

    Vögeli, U; Chappell, J

    1991-07-01

    The specificity of mevinolin as an inhibitor of sterol and sesquiterpene metabolism in tobacco cell suspension cultures was examined. Exogenous mevinolin inhibited [14C]acetate, but not [3H]mevalonate incorporation into free sterols. In contrast, mevinolin inhibited the incorporation of both [14C]acetate and [3H]mevalonate into capsidiol, an extracellular sesquiterpene. Microsomal 3-hydroxy-3-methylglutaryl Coenzyme A reductase was inhibited greater than 90% by microM mevinolin, while squalene synthetase was insensitive to even 600 microM mevinolin. Sesquiterpene cyclase, the first branch point enzyme specific for sesquiterpene biosynthesis, was inhibited in a dose-dependent manner by mevinolin with a 50% reduction in activity at 100 microM. Kinetic analysis indicated that the mechanism for inhibition was complex with mevinolin acting as both a competitive and noncompetitive inhibitor. The results suggest that the mevinolin inhibition of [3H]mevalonate incorporation into extracellular sesquiterpenes can, in part, be attributed to a secondary, but specific, site of inhibition, the sesquiterpene cyclase.

  5. The omega-hydroxlyation of lauric acid: oxidation of 12-hydroxlauric acid to dodecanedioic acid by a purified recombinant fusion protein containing P450 4A1 and NADPH-P450 reductase.

    Science.gov (United States)

    Shet, M s; Fisher, C W; Holmans, P L; Estabrook, R W

    1996-06-01

    The recombinant fusion protein rF450[mRat4Al/mRatOR]L1, containing the heme domain of P450 4A1 and the flavin domains of NADPH-P450 reductase, when incubated with dilaurylphosphatidylcholine (DLPC), Chaps, cytochrome b5, and a 20-fold excess of purified NADPH-P450 reductase, catalyzes the omega- oxidation of lauric acid at a rate of about 300 nmol/min/nmol P450. This is the first report of a mammalian P450 enzyme with such a high turnover number. The resultant 12-hydroxydodecanoic acid [12-hydroxylauric acid (12-OH LA)] is further oxidized by the P450 oxygenase reaction to dodecanedioic acid (decane-1,10-dicarboxylic acid) via 12,12-dihydroxydodecanoic acid. Spectral binding studies show that 12-OH LA inhibits the binding of lauric acid to the active site of P450 with a Ki of about 1.9 microM. The construction and expression of recombinant P450 4A1 containing a six-member polyhistidine domain at the carboxy-terminus of the protein is described. Reconstitution experiments with this purified recombinant P450 4A1, DLPC, Chaps, b5, and purified NADPH-P450 reductase show results similar to those obtained with the purified fusion protein, albeit at lower turnover rates. The requirement for normal-phase HPLC in resolving the metabolites formed during lauric acid metabolism is demonstrated.

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

    DEFF Research Database (Denmark)

    Kirkensgaard, Kristine Groth; Hägglund, Per; Finnie, Christine

    2009-01-01

    Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs...... relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead...

  8. Subcellular localization of the five members of the human steroid 5α-reductase family

    Directory of Open Access Journals (Sweden)

    Antonella Scaglione

    2017-06-01

    We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.

  9. Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone

    OpenAIRE

    Dow, J. M.; Grahl, S.; Ward, R; Evans, R.; Byron, O; Norman, D. G.; Palmer, T; Sargent, F

    2013-01-01

    Escherichia coli is a Gram‐negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase NapA contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin‐arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twin‐arginine signal peptide of NapA. Here we show, using spin labelli...

  10. 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

    of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5' flanking region by cDNA analysis and establishment...... cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly...

  11. 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...... of 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...... or unwilling to undergo surgical resection of the prostate will benefit from such therapy....

  12. 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).

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

    NARCIS (Netherlands)

    Sanchez-Riego, Ana M.; Mata-Cabana, Alejandro; Galmozzi, CarlaV.; Florencio, Francisco J.

    2016-01-01

    NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of t

  14. Rubredoxin Reductase of Pseudomonas oleovorans. Structural Relationship to Other Flavoprotein Oxidoreductases Based on One NAD and Two FAD Fingerprints

    NARCIS (Netherlands)

    Eggink, Gerrit; Engel, Henk; Vriend, Gert; Terpstra, Peter; Witholt, Bernard

    1990-01-01

    The oxidation of alkanes to alkanols by Pseudomonas oleovorans involves a three-component enzyme system: alkane hydroxylase, rubredoxin and rubredoxin reductase. Alkane hydroxylase and rubredoxin are encoded by the alkBPGHJKL operon, while previous studies indicated that rubredoxin reductase is most

  15. Self-suppression of corticosteroidogenesis: evidence for a role of adrenal 5 alpha-reductase.

    Science.gov (United States)

    Carsia, R V; Scanes, C G; Malamed, S

    1984-12-01

    Exogenous corticosterone (B), the natural glucocorticoid product of rats, suppressed endogenous B production of isolated rat adrenocortical cells induced by alpha ACTH-(1-24), [9-tryptophan (O-nitrophenylsulfenyl)]ACTH-(1-24) [( Trp (Nps)9]ACTH-(1-24], and cAMP as well as pregnenolone supported-steroidogenesis. This self-suppression occurred within 2 h. It was dependent on the concentration of exogenous B. However, self-suppression did not alter the half-maximal steroidogenic concentration (ED50) of each steroidogenic agent. In addition, exogenous B did not suppress ACTH-induced cAMP production or gross protein synthesis, as measured by leucine incorporation into bulk cellular proteins. These results with isolated cells suggested at least two mechanisms for self-suppression: 1) exogenous B inhibited steroidogenic steps in a noncompetitive manner, and/or 2) exogenous B induced B degradation. In this study we examined the effect of exogenous B on the degradation of B. Accordingly, we measured the adrenal 5 alpha-reductase activity (5 alpha RA) of cell homogenates prepared from treated cells. Isolated adrenocortical cells were incubated for 2 h with alpha ACTH-(1-24), ovine PRL (oPRL), and B. They were then homogenized and assayed for 5 alpha RA, as indicated by the disappearance of exogenous B, as shown by RIA. In addition, the percentage of exogenous tritium-labeled B [( 3H]B) converted to 5 alpha-dihydrocorticosterone (DHB), the principal reduced metabolite of B, was determined by TLC. Isolated adrenocortical cells from intact rats showed insignificant 5 alpha RA and DHB formation when incubated with or without alpha ACTH-(1-24) and with or without oPRL. However, with exogenous B, there was significant 5 alpha RA and DHB formation. oPRL plus B decreased DHB formation. The effects of B and oPRL were more demonstrable with cells from hypophysectomized rats. These cells exhibited high 5 alpha RA and DHB formation; exogenous B increased these values, whereas o

  16. Physarum polycephalum expresses a dihydropteridine reductase with selectivity for pterin substrates with a 6-(1', 2'-dihydroxypropyl) substitution.

    Science.gov (United States)

    Wild, Claudia; Golderer, Georg; Gröbner, Peter; Werner-Felmayer, Gabriele; Werner, Ernst R

    2003-07-01

    Physarum polycephalum is one of few non-animal organisms capable of synthesizing tetrahydrobiopterin from GTP. Here we demonstrate developmentally regulated expression of quinoid dihydropteridine reductase (EC 1.6.99.7), an enzyme required for recycling 6,7-[8H]-dihydrobiopterin. Physarum also expresses phenylalanine-4-hydroxylase activity, an enzyme that depends on dihydropteridine reductase. The 24.4 kDa Physarum dihydropteridine reductase shares 43% amino acid identity with the human protein. A number of residues important for function of the mammalian enzyme are also conserved in the Physarum sequence. In comparison to sheep liver dihydropteridine reductase, purified recombinant Physarum dihydropteridine reductase prefers pterin substrates with a 6-(1', 2'-dihydroxypropyl) group. Our results demonstrate that Physarum synthesizes, utilizes and metabolizes tetrahydrobiopterin in a way hitherto thought to be restricted to the animal kingdom.

  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.

    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

  18. meta-Tyrosine induces modification of reactive nitrogen species level, protein nitration and nitrosoglutathione reductase in tomato roots.

    Science.gov (United States)

    Krasuska, Urszula; Andrzejczak, Olga; Staszek, Paweł; Borucki, Wojciech; Gniazdowska, Agnieszka

    2017-08-01

    A non-protein amino acid (NPAA) - meta-Tyrosine (m-Tyr), is a harmful compound produced by fescue roots. Young (3-4 days old) tomato (Solanum lycopersicum L.) seedlings were supplemented for 24-72 h with m-Tyr (50 or 250 μM) inhibiting root growth by 50 or 100%, without lethal effect. Fluorescence of DAF-FM and APF derivatives was determined to show reactive nitrogen species (RNS) localization and level in roots of tomato plants. m-Tyr-induced restriction of root elongation growth was related to formation of nitrated proteins described as content of 3-nitrotyrosine. Supplementation with m-Tyr enhanced superoxide radicals generation in extracts of tomato roots and stimulated protein nitration. It correlated well to increase of fluorescence of DAF-FM derivatives, and transiently stimulated fluorescence of APF derivatives corresponding respectively to NO and ONOO(-) formation. Alterations in RNS formation induced by m-Tyr were linked to metabolism of nitrosoglutathione (GSNO). Activity of nitrosoglutatione reductase (GSNOR), catalyzing degradation of GSNO was enhanced by long term plant supplementation with m-Tyr, similarly as protein abundance, while transcripts level were only slightly altered by tested NPAA. We conclude, that although in animal cells m-Tyr is considered as a marker of oxidative stress, its secondary mode of action in tomato plants involves perturbation in RNS formation, alteration in GSNO metabolism and modification of protein nitration level. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer siRNAs to tristetraprolin blocks transcription of HMGR in vivo in rat liver. Black-Right-Pointing-Pointer siRNAs to tristetraprolin inhibits insulin activation of HMGR transcription. Black-Right-Pointing-Pointer Insulin acts to rapidly increase tristetraprolin in liver nuclear extracts. -- Abstract: Several AU-rich RNA binding element (ARE) proteins were investigated for their possible effects on transcription of hepatic 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR) in normal rats. Using in vivo electroporation, four different siRNAs to each ARE protein were introduced together with HMGR promoter (-325 to +20) luciferase construct and compared to saline controls. All four siRNAs to tristetraprolin (TTP) completely eliminated transcription from the HMGR promoter construct. Since insulin acts to rapidly increase hepatic HMGR transcription, the effect of TTP siRNA on induction by insulin was tested. The 3-fold stimulation by insulin was eliminated by this treatment. In comparison, siRNA to AU RNA binding protein/enoyl coenzyme A hydratase (AUH) had no effect. These findings indicate a role for TTP in the insulin-mediated activation of hepatic HMGR transcription.

  20. Towards the Understanding of Resistance Mechanisms in Clinically Isolated Trimethoprim-resistant, Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Frey, K.; Lombardo, M; Wright, D; Anderson, A

    2010-01-01

    Resistance to therapeutics such as trimethoprim-sulfamethoxazole has become an increasing problem in strains of methicillin-resistant Staphylococcus aureus (MRSA). Clinically isolated trimethoprim-resistant strains reveal a double mutation, H30N/F98Y, in dihydrofolate reductase (DHFR). In order to develop novel and effective therapeutics against these resistant strains, we evaluated a series of propargyl-linked antifolate lead compounds for inhibition of the mutant enzyme. For the propargyl-linked antifolates, the F98Y mutation generates minimal (between 1.2- and 6-fold) losses of affinity and the H30N mutation generates greater losses (between 2.4- and 48-fold). Conversely, trimethoprim affinity is largely diminished by the F98Y mutation (36-fold) and is not affected by the H30N mutation. In order to elucidate a mechanism of resistance, we determined a crystal structure of a complex of this double mutant with a lead propargyl-linked antifolate. This structure suggests a resistance mechanism consistent both for the propargyl-linked class of antifolates and for trimethoprim that is based on the loss of a conserved water-mediated hydrogen bond.

  1. Structure-based de novo design, molecular docking and molecular dynamics of primaquine analogues acting as quinone reductase II inhibitors.

    Science.gov (United States)

    Murce, Erika; Cuya-Guizado, Teobaldo Ricardo; Padilla-Chavarria, Helmut Isaac; França, Tanos Celmar Costa; Pimentel, Andre Silva

    2015-11-01

    Primaquine is a traditional antimalarial drug with low parasitic resistance and generally good acceptance at higher doses, which has been used for over 60 years in malaria treatment. However, several limitations related to its hematotoxicity have been reported. It is believed that this toxicity comes from the hydroxylation of the C-5 and C-6 positions of its 8-aminoquinoline ring before binding to the molecular target: the quinone reductase II (NQO2) human protein. In this study we propose primaquine derivatives, with substitution at position C-6 of the 8-aminoquinoline ring, planned to have better binding to NQO2, compared to primaquine, but with a reduced toxicity related to the C-5 position being possible to be oxidized. On this sense the proposed analogues were suggested in order to reduce or inhibit hydroxylation and further oxidation to hemotoxic metabolites. Five C-6 substituted primaquine analogues were selected by de novo design and further submitted to docking and molecular dynamics simulations. Our results suggest that all analogues bind better to NQO2 than primaquine and may become better antimalarials. However, the analogues 3 and 4 are predicted to have a better activity/toxicity balance.

  2. Design, synthesis, and biological evaluation of resveratrol analogues as aromatase and quinone reductase 2 inhibitors for chemoprevention of cancer

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Bin; Hoshino, Juma; Jermihov, Katie; Marler, Laura; Pezzuto, John M.; Mesecar, Andrew D.; Cushman, Mark (Hawaii); (Purdue); (UIC)

    2012-07-11

    A series of new resveratrol analogues were designed and synthesized and their inhibitory activities against aromatase were evaluated. The crystal structure of human aromatase (PDB 3eqm) was used to rationalize the mechanism of action of the aromatase inhibitor 32 (IC{sub 50} 0.59 {mu}M) through docking, molecular mechanics energy minimization, and computer graphics molecular modeling, and the information was utilized to design several very potent inhibitors, including compounds 82 (IC{sub 50} 70 nM) and 84 (IC{sub 50} 36 nM). The aromatase inhibitory activities of these compounds are much more potent than that for the lead compound resveratrol, which has an IC{sub 50} of 80 {mu}M. In addition to aromatase inhibitory activity, compounds 32 and 44 also displayed potent QR2 inhibitory activity (IC{sub 50} 1.7 {mu}M and 0.27 {mu}M, respectively) and the high-resolution X-ray structures of QR2 in complex with these two compounds provide insight into their mechanism of QR2 inhibition. The aromatase and quinone reductase inhibitors resulting from these studies have potential value in the treatment and prevention of cancer.

  3. Characterization of a novel methionine sulfoxide reductase A from tomato (Solanum lycopersicum ), and its protecting role in Escherichia coli.

    Science.gov (United States)

    Dai, Changbo; Singh, Naresh Kumar; Park, Myungho

    2011-12-01

    Methionine sulfoxide reductase A (MSRA) is a ubiquitous enzyme that has been demonstrated to reduce the S enantiomer of methionine sulfoxide (MetSO) to methionine (Met) and can protect cells against oxidative damage. In this study, we isolated a novel MSRA (SlMSRA2) from Micro-Tom (Solanum lycopersicum L. cv. Micro-Tom) and characterized it by subcloning the coding sequence into a pET expression system. Purified recombinant protein was assayed by HPLC after expression and refolding. This analysis revealed the absolute specificity for methionine-S-sulfoxide and the enzyme was able to convert both free and protein-bound MetSO to Met in the presence of DTT. In addition, the optimal pH, appropriate temperature, and Km and Kcat values for MSRA2 were observed as 8.5, 25oC, 352 ± 25 μM, and 0.066 ± 0.009 S(-1), respectively. Disk inhibition and growth rate assays indicated that SlMSRA2 may play an essential function in protecting E. coli against oxidative damage.

  4. Glucocorticoid-induced androgen inactivation by aldo-keto reductase 1C2 promotes adipogenesis in human preadipocytes.

    Science.gov (United States)

    Veilleux, Alain; Côté, Julie-Anne; Blouin, Karine; Nadeau, Mélanie; Pelletier, Mélissa; Marceau, Picard; Laberge, Philippe Y; Luu-The, Van; Tchernof, André

    2012-04-15

    Adipogenesis and lipid storage in human adipose tissue are inhibited by androgens such as DHT. Inactivation of DHT to 3α-diol is stimulated by glucocorticoids in human preadipocytes. We sought to characterize glucocorticoid-induced androgen inactivation in human preadipocytes and to establish its role in the antiadipogenic action of DHT. Subcutaneous and omental primary preadipocyte cultures were established from fat samples obtained in subjects undergoing abdominal surgeries. Inactivation of DHT to 3α/β-diol for 24 h was measured in dexamethasone- or vehicle-treated cells. Specific downregulation of aldo-keto reductase 1C (AKR1C) enzymes in human preadipocytes was achieved using RNA interference. In whole adipose tissue sample, cortisol production was positively correlated with androgen inactivation in both subcutaneous and omental adipose tissue (P adipose tissue of men and women (r = 0.24, n = 26, P effective in omental preadipocytes of obese men. The interplay between glucocorticoids and AKR1C2-dependent androgen inactivation may locally modulate adipogenesis and lipid accumulation in a depot-specific manner.

  5. Geometric restraint drives on- and off-pathway catalysis by the Escherichia coli menaquinol:fumarate reductase.

    Science.gov (United States)

    Tomasiak, Thomas M; Archuleta, Tara L; Andréll, Juni; Luna-Chávez, César; Davis, Tyler A; Sarwar, Maruf; Ham, Amy J; McDonald, W Hayes; Yankovskaya, Victoria; Stern, Harry A; Johnston, Jeffrey N; Maklashina, Elena; Cecchini, Gary; Iverson, Tina M

    2011-01-28

    Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.

  6. Biochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

    Science.gov (United States)

    Sonawane, Prashant; Vishwakarma, Rishi Kishore; Khan, Bashir M

    2013-07-01

    Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25°C for 90 min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×10(6) M(-1) s(-1)), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20-50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm(3) with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme.

  7. Predicting the Cytotoxic Potency of Cigarette Smoke by Assessing the Thioredoxin Reductase Inhibitory Capacity of Cigarette Smoke Extract.

    Science.gov (United States)

    Zhang, Longjie; Ning, Min; Xu, Yingbo; Wang, Chenghui; Zhao, Guangshan; Cao, Qingqing; Zhang, Jinsong

    2016-03-21

    The present study investigated the influence of the cigarette smoke extract (CSE) on mammalian thioredoxin reductase (TrxR) activity. TrxR is a selenoenzyme with a selenocysteine (Sec) residue exposed on the enzyme's surface. This unique Sec residue is particularly susceptible to modification by numerous types of electrophiles, leading to inactivation of TrxR and consequent cytotoxicity. Cigarette smoke contains various electrophiles, and the present study showed that CSE could inhibit intracellular TrxR through causing crosslinking and alkylation of TrxR1. TrxR inhibitory capacities of various CSEs were evaluated by using mouse-liver homogenate. Among the CSEs prepared from 18 commercial cigarette brands, TrxR inhibitory capacities of the maximum and the minimum had a 2.5-fold difference. Importantly, CSE's inhibitory capacity greatly paralleled its cytotoxic potency in all cell lines used. Compared to cytotoxic assays, which have been widely used for evaluating cigarette toxicity but are not suitable for simultaneously examining a large number of cigarette samples, the present method was simple and rapid with a high-throughput feature and thus could be used as an auxiliary means to predict the cytotoxicity of a large number of cigarette samples, making it possible to extensively screen numerous agricultural and industrial measures that potentially affect cigarette safety.

  8. Arabidopsis dehydroascorbate reductase 1 and 2 modulate redox states of ascorbate-glutathione cycle in the cytosol in response to photooxidative stress.

    Science.gov (United States)

    Noshi, Masahiro; Yamada, Hiroki; Hatanaka, Risa; Tanabe, Noriaki; Tamoi, Masahiro; Shigeoka, Shigeru

    2017-03-01

    Ascorbate and glutathione are indispensable cellular redox buffers and allow plants to acclimate stressful conditions. Arabidopsis contains three functional dehydroascorbate reductases (DHAR1-3), which catalyzes the conversion of dehydroascorbate into its reduced form using glutathione as a reductant. We herein attempted to elucidate the physiological role in DHAR1 and DHAR2 in stress responses. The total DHAR activities in DHAR knockout Arabidopsis plants, dhar1 and dhar2, were 22 and 92%, respectively, that in wild-type leaves. Under high light (HL), the levels of total ascorbate and dehydroascorbate were only reduced and increased, respectively, in dhar1. The oxidation of glutathione under HL was significantly inhibited in both dhar1 and dhar2, while glutathione contents were only enhanced in dhar1. The dhar1 showed stronger visible symptoms than the dhar2 under photooxidative stress conditions. Our results demonstrated a pivotal role of DHAR1 in the modulation of cellular redox states under photooxidative stress.

  9. Synthesis and highly potent hypolipidemic activity of alpha-asarone- and fibrate-based 2-acyl and 2-alkyl phenols as HMG-CoA reductase inhibitors.

    Science.gov (United States)

    Mendieta, Aarón; Jiménez, Fabiola; Garduño-Siciliano, Leticia; Mojica-Villegas, Angélica; Rosales-Acosta, Blanca; Villa-Tanaca, Lourdes; Chamorro-Cevallos, Germán; Medina-Franco, José L; Meurice, Nathalie; Gutiérrez, Rsuini U; Montiel, Luisa E; Cruz, María Del Carmen; Tamariz, Joaquín

    2014-11-01

    In the search for new potential hypolipidemic agents, the present study focused on the synthesis of 2-acyl phenols (6a-c and 7a-c) and their saturated side-chain alkyl phenols (4a-c and 5a-c), and on the evaluation of their hypolipidemic activity using a murine Tyloxapol-induced hyperlipidemic protocol. The whole series of compounds 4-7 greatly and significantly reduced elevated serum levels of total cholesterol, LDL-cholesterol, and triglycerides, with series 6 and 7 showing the greatest potency ever found in our laboratory. At the minimum dose (25mg/kg/day), the latter compounds lowered cholesterol by 68-81%, LDL by 72-86%, and triglycerides by 59-80%. This represents a comparable performance than that shown by simvastatin. Experimental evidence and docking studies suggest that the activity of these derivatives is associated with the inhibition of HMG-CoA reductase.

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

    Science.gov (United States)

    Zhang, Ranran; Du, Shaoting

    2016-01-01

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

  11. Sites of inhibition of mitochondrial electron transport in macrophage-injured neoplastic cells.

    Science.gov (United States)

    Granger, D L; Lehninger, A L

    1982-11-01

    Previous work has shown that injury of neoplastic cells by cytotoxic macrophages (CM) in cell culture is accompanied by inhibition of mitochondrial respiration. We have investigated the nature of this inhibition by studying mitochondrial respiration in CM-injured leukemia L1210 cells permeabilized with digitonin. CM-induced injury affects the mitochondrial respiratory chain proper. Complex I (NADH-coenzyme Q reductase) and complex II (succinate-coenzyme Q reductase) are markedly inhibited. In addition a minor inhibition of cytochrome oxidase was found. Electron transport from alpha-glycerophosphate through the respiratory chain to oxygen is unaffected and permeabilized CM-injured L1210 cells oxidizing this substrate exhibit acceptor control. However, glycerophosphate shuttle activity was found not to occur within CM-injured or uninjured L1210 cells in culture hence, alpha-glycerophosphate is apparently unavailable for mitochondrial oxidation in the intact cell. It is concluded that the failure of respiration of intact neoplastic cells injured by CM is caused by the nearly complete inhibition of complexes I and II of the mitochondrial electron transport chain. The time courses of CM-induced electron transport inhibition and arrest of L1210 cell division are examined and the possible relationship between these phenomena is discussed.

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

    Directory of Open Access Journals (Sweden)

    Célio Damacena-Angelis

    2017-08-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-05-13

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

  14. Diabetes-induced impairment in visual function in mice: contributions of p38 MAPK, rage, leukocytes, and aldose reductase.

    Science.gov (United States)

    Lee, Chieh Allen; Li, Guangyuan; Patel, Mansi D; Petrash, J Mark; Benetz, Beth Ann; Veenstra, Alex; Amengual, Jaume; von Lintig, Johannes; Burant, Christopher J; Tang, Johnny; Kern, Timothy S

    2014-05-02

    Visual function is impaired in diabetes, but molecular causes of this dysfunction are not clear. We assessed effects of diabetes on visual psychophysics in mice, and tested the effect of therapeutic approaches reported previously to inhibit vascular lesions of the retinopathy. We used the optokinetic test to assess contrast sensitivity and spatial frequency threshold in diabetic C57Bl/6J mice and age-matched nondiabetic controls between 2 and 10 months of diabetes. Contributions of p38 MAP kinase (MAPK), receptor for advanced glycation end products (RAGE), leukocytes, and aldose reductase (AR) to the defect in contrast sensitivity were investigated. Cataract, a potential contributor to reductions in vision, was scored. Diabetes of 2 months' duration impaired contrast sensitivity and spatial frequency threshold in mice. The defect in contrast sensitivity persisted for at least 10 months, and cataract did not account for this impairment. Diabetic mice deficient in AR were protected significantly from development of the diabetes-induced defects in contrast sensitivity and spatial frequency threshold. In contrast, pharmacologic inhibition of p38 MAPK or RAGE, or deletion of inducible nitrous oxide synthase (iNOS) from bone marrow-derived cells did not protect the visual function in diabetes. Diabetes reduces spatial frequency threshold and contrast sensitivity in mice, and the mechanism leading to development of these defects involves AR. The mechanism by which AR contributes to the diabetes-induced defect in visual function can be probed by identifying which molecular abnormalities are corrected by AR deletion, but not other therapies that do not correct the defect in visual function.

  15. The isolation and characterization of β-glucogallin as a novel aldose reductase inhibitor from Emblica officinalis.

    Directory of Open Access Journals (Sweden)

    Muthenna Puppala

    Full Text Available Diabetes mellitus is recognized as a leading cause of new cases of blindness. The prevalence of diabetic eye disease is expected to continue to increase worldwide as a result of the dramatic increase in the number of people with diabetes. At present, there is no medical treatment to delay or prevent the onset and progression of cataract or retinopathy, the most common causes of vision loss in diabetics. The plant Emblica officinalis (gooseberry has been used for thousands of years as a traditional Indian Ayurvedic preparation for the treatment of diabetes in humans. Extracts from this plant have been shown to be efficacious against the progression of cataract in a diabetic rat model. Aldose reductase (ALR2 is implicated in the development of secondary complications of diabetes including cataract and, therefore, has been a major drug target for the development of therapies to treat diabetic disease. Herein, we present the bioassay-guided isolation and structure elucidation of 1-O-galloyl-β-D-glucose (β-glucogallin, a major component from the fruit of the gooseberry that displays selective as well as relatively potent inhibition (IC(50 = 17 µM of AKR1B1 in vitro. Molecular modeling demonstrates that this inhibitor is able to favorably bind in the active site. Further, we show that β-glucogallin effectively inhibits sorbitol accumulation by 73% at 30 µM under hyperglycemic conditions in an ex-vivo organ culture model of lenses excised from transgenic mice overexpressing human ALR2 in the lens. This study supports the continued development of natural products such as β-glucogallin as therapeutic leads in the development of novel therapies to treat diabetic complications such as cataract.

  16. Catalase as a sulfide-sulfur oxido-reductase: An ancient (and modern? regulator of reactive sulfur species (RSS

    Directory of Open Access Journals (Sweden)

    Kenneth R. Olson

    2017-08-01

    Full Text Available Catalase is well-known as an antioxidant dismutating H2O2 to O2 and H2O. However, catalases evolved when metabolism was largely sulfur-based, long before O2 and reactive oxygen species (ROS became abundant, suggesting catalase metabolizes reactive sulfide species (RSS. Here we examine catalase metabolism of H2Sn, the sulfur analog of H2O2, hydrogen sulfide (H2S and other sulfur-bearing molecules using H2S-specific amperometric electrodes and fluorophores to measure polysulfides (H2Sn; SSP4 and ROS (dichlorofluorescein, DCF. Catalase eliminated H2Sn, but did not anaerobically generate H2S, the expected product of dismutation. Instead, catalase concentration- and oxygen-dependently metabolized H2S and in so doing acted as a sulfide oxidase with a P50 of 20 mmHg. H2O2 had little effect on catalase-mediated H2S metabolism but in the presence of the catalase inhibitor, sodium azide (Az, H2O2 rapidly and efficiently expedited H2S metabolism in both normoxia and hypoxia suggesting H2O2 is an effective electron acceptor in this reaction. Unexpectedly, catalase concentration-dependently generated H2S from dithiothreitol (DTT in both normoxia and hypoxia, concomitantly oxidizing H2S in the presence of O2. H2S production from DTT was inhibited by carbon monoxide and augmented by NADPH suggesting that catalase heme-iron is the catalytic site and that NADPH provides reducing equivalents. Catalase also generated H2S from garlic oil, diallyltrisulfide, thioredoxin and sulfur dioxide, but not from sulfite, metabisulfite, carbonyl sulfide, cysteine, cystine, glutathione or oxidized glutathione. Oxidase activity was also present in catalase from Aspergillus niger. These results show that catalase can act as either a sulfide oxidase or sulfur reductase and they suggest that these activities likely played a prominent role in sulfur metabolism during evolution and may continue do so in modern cells as well. This also appears to be the first observation of catalase

  17. 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...

  18. 5 Alpha-reductase inhibitory and antiandrogenic activities of novel steroids in hamster seminal vesicles.

    Science.gov (United States)

    Cabeza, Marisa; Bratoeff, Eugene; Flores, Eugenio; Ramírez, Elena; Calleros, Jorge; Montes, Diana; Quiroz, Alexandra; Heuze, Ivonne

    2002-11-01

    The pharmacological activity of several 16-bromosubstituted trienediones 4 and 5, 16-methyl substituted dienediones 6 and 7 and the 16-methyl substituted trienedione 8 was determined on gonadectomized hamster seminal vesicles by measuring the in vitro conversion of testosterone (T) to dihydrotestosterone (DHT) as 5alpha-reductase inhibitors and also the ability of these steroids to bind to the androgen receptor. Steroids 6 and 7 when injected together with T decreased the weight of the seminal vesicles thus showing an antiandrogenic effect. Compounds 5 and 6 reduced substantially the conversion of T to DHT and therefore can be considered good inhibitors for the enzyme 5alpha-reductase; however both steroids failed to form a complex with the androgen receptor. On the other hand compound 7 which showed a very small inhibitory activity for the enzyme 5alpha-reductase, exhibited a very high affinity for the androgen receptor and thus can be considered an effective antiandrogen. This compound also reduced substantially the weight of the seminal vesicles. Steroids 4 and 8 did not reduce the weight of the seminal vesicles and exhibited a low affinity for the androgen receptor; 8 showed a weak 5alpha-reductase inhibitory activity, whereas 4 exhibited a weak androgenic effect.

  19. 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...

  20. pH dependence of copper geometry, reduction potential, and nitrite affinity in nitrite reductase.

    NARCIS (Netherlands)

    Jacobson, F.; Pistorius, A.M.A.; Farkas, D.; Grip, W.J. de; Hansson, O.; Sjolin, L.; Neutze, R.

    2007-01-01

    Many properties of copper-containing nitrite reductase are pH-dependent, such as gene expression, enzyme activity, and substrate affinity. Here we use x-ray diffraction to investigate the structural basis for the pH dependence of activity and nitrite affinity by examining the type 2 copper site and

  1. 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

  2. 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...

  3. Proximal hypospadias in a male patient with 5α-reductase deficiency: A case reports

    Directory of Open Access Journals (Sweden)

    Erol Basuguy

    2014-01-01

    Full Text Available Hypospadias is a congenital disorder of male external genital. The newborn showed penoscrotal hypospadias with chordee and microphallus. Endocrine data and a normal male karyotype were suggestive of 5α-reductase deficiency. Penoscrotalhypospadias repair of the patient was made.

  4. Structure of Physarum polycephalum cytochrome b{sub 5} reductase at 1.56 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sangwoo; Suga, Michihiro; Ogasahara, Kyoko [Institute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita, Osaka (Japan); Ikegami, Terumi; Minami, Yoshiko; Yubisui, Toshitsugu [Department of Biochemistry, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005 (Japan); Tsukihara, Tomitake, E-mail: tsuki@protein.osaka-u.ac.jp [Institute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita, Osaka (Japan)

    2007-04-01

    The structure of P. polycephalum cytochrome b{sub 5} reductase, an enzyme which catalyzes the reduction of cytochrome b{sub 5} by NADH, was determined at a resolution of 1.56 Å. Physarum polycephalum cytochrome b{sub 5} reductase catalyzes the reduction of cytochrome b{sub 5} by NADH. The structure of P. polycephalum cytochrome b{sub 5} reductase was determined at a resolution of 1.56 Å. The molecular structure was compared with that of human cytochrome b{sub 5} reductase, which had previously been determined at 1.75 Å resolution [Bando et al. (2004 ▶), Acta Cryst. D60, 1929–1934]. The high-resolution structure revealed conformational differences between the two enzymes in the adenosine moiety of the FAD, the lid region and the linker region. The structural properties of both proteins were inspected in terms of hydrogen bonding, ion pairs, accessible surface area and cavity volume. The differences in these structural properties between the two proteins were consistent with estimates of their thermostabilities obtained from differential scanning calorimetry data.

  5. Structural characterization and functional validation of aldose reductase from the resurrection plant Xerophyta viscosa.

    Science.gov (United States)

    Singh, Preeti; Sarin, Neera Bhalla

    2014-11-01

    Aldose reductases are key enzymes in the detoxification of reactive aldehyde compounds like methylglyoxal (MG) and malondialdehyde. The present study describes for first time the preliminary biochemical and structural characterization of the aldose reductase (ALDRXV4) enzyme from the resurrection plant Xerophyta viscosa. The ALDRXV4 cDNA was expressed in E. coli using pET28a expression vector, and the protein was purified using affinity chromatography. The recombinant protein showed a molecular mass of ~36 kDa. The K M (1.2 mM) and k cat (14.5 s(-1)) of the protein determined using MG as substrate was found to be comparable with other reported homologs. Three-dimensional structure prediction based on homology modeling suggested several similarities with the other aldose reductases reported from plants. Circular dichroism spectroscopy results supported the bioinformatic prediction of alpha-beta helix nature of aldose reductase proteins. Subcellular localization studies revealed that the ALDRXV4-GFP fusion protein was localized both in the nucleus and the cytoplasm. The E. coli cells overexpressing ALDRXV4 exhibited improved growth and showed tolerance against diverse abiotic stresses induced by high salt (500 mM NaCl), osmoticum (10 % PEG 6000), heavy metal (20 mM CdCl2), and MG (5 mM). Based on these results, we propose that ALDRXV4 gene from X. viscosa could be a potential candidate for developing stress-tolerant crop plants.

  6. Spectrophotometric activity microassay for pure and recombinant cytochrome P450-type nitric oxide reductase

    CSIR Research Space (South Africa)

    Garny, S

    2014-02-01

    Full Text Available Nitric oxide reductase (NOR) of the P450 oxidoreductase family accepts electrons directly from its cofactor, NADH, to reduce two nitric oxide (NO) molecules to one nitrous oxide molecule and water. The enzyme plays a key role in removal of radical...

  7. Primary △4-3-oxosteroid 5β-reductase deficiency: Two cases in China

    Institute of Scientific and Technical Information of China (English)

    Jing Zhao; Ling-Juan Fang; Kenneth DR Setchell; Rui Chen; Li-Ting Li; Jian-She Wang

    2012-01-01

    Aldo-keto reductase 1D1 (AKR1D1) deficiency,a rare but life-threatening form of bile acid deficiency,has not been previously described in China.Here,we describe the first two primary △4-3-oxosteroid 5β-reductase deficiency patients in Mainland China diagnosed by fast atom bombardment-mass spectroscopy of urinary bile acids and confirmed by genetic analysis.A high proportion of atypical 3-oxo-A4-bile acids in the urine indicated a deficiency in A4-3-oxosteroid 5β-reductase.All of the coding exons and adjacent intronic sequence of the AKR1D1 gene were sequenced using peripheral lymphocyte genomic DNA of two patients and one of the patient's parents.One patient exhibited compound heterozygous mutations:c.396C>A and c.722A>T,while the other was heterozygous for the mutation c.797G>A.Based on these mutations,a diagnosis of primary △4-3-oxosteroid 5β-reductase deficiency could be confirmed.With ursodeoxycholic acid treatment and fat-soluble vitamin supplements,liver function tests normalized rapidly,and the degree of hepatomegaly was markedly reduced in both patients.

  8. 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...

  9. Cloning, expression and antigenicity of the L. donovani reductase

    DEFF Research Database (Denmark)

    Jensen, A T; Kemp, K; Theander, T G

    2001-01-01

    (K). Only 2 of 22 plasma samples from patients with visceral leishmaniasis were found to have detectable anti-reductase antibodies and peripheral blood mononuclear cells (PBMC) from one of three individuals previously infected with visceral leishmaniasis proliferated in the presence of recombinant...

  10. 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

  11. Part of respiratory nitrate reductase of Klebsiella aerogenes is intimately associated with the peptidoglycan.

    Science.gov (United States)

    Abraham, P R; Wientjes, F B; Nanninga, N; Van't Riet, J

    1987-02-01

    Lysozyme digestion and sonication of sodium dodecyl sulfate (SDS)-purified Klebsiella aerogenes murein sacculi resulted in the quantitative release of both subunits of nitrate reductase, as well as a number of other cytoplasmic membrane polypeptides (5.2%, by weight, of the total membrane proteins). Similar results were obtained after lysozyme digestion of SDS-prepared peptidoglycan fragments, which excluded the phenomenon of simple trapping of the polypeptides by the surrounding peptidoglycan matrix. About 28% of membrane-bound nitrate reductase appears to be tightly associated with the peptidoglycan. Additional evidence for this association was demonstrated by positive immunogold labeling of SDS-murein sacculi and thin sections of plasmolyzed bacteria. Qualitative amino acid analysis of trypsin-treated sacculi, a tryptic product of holo-nitrate reductase, and amino- and carboxypeptidase digests of both nitrate reductase subunits indicated the possible existence of a terminal anchoring peptide containing the following amino acids: (Gly)n, Trp, Ser, Pro, Ile, Leu, Phe, Cys, Tyr, Asp, and Lys.

  12. 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

  13. Functional characterization of a soluble NADPH-cytochrome P450 reductase from Fusarium graminearum.

    Science.gov (United States)

    Etzerodt, Thomas; Wetterhorn, Karl; Dionisio, Giuseppe; Rayment, Ivan

    2017-10-01

    Fusarium head blight is a devastating disease in wheat caused by some fungal pathogens of the Fusarium genus mainly F. graminearum, due to accumulation of toxic trichothecenes. Most of the trichothecene biosynthetic pathway has been mapped, although some proteins of the pathway remain uncharacterized, including an NADPH-cytochrome P450 reductase. We subcloned a F. graminearum cytochrome P450 reductase that might be involved in the trichothecene biosynthesis. It was expressed heterologously in E. coli as N-terminal truncated form with an octahistidine tag for purification. The construct yielded a soluble apoprotein and its incubation with flavins yielded the corresponding monomeric holoprotein. It was characterized for activity in the pH range 5.5-9.5, using thiazolyl blue tetrazolium bromide (MTT) or cytochrome c as substrates. Binding of the small molecule MTT was weaker than for cytochrome c, however, the rate of MTT reduction was faster. Contrary to other studies of cytochrome reductase proteins, MTT reduction proceeded in a cooperative manner in our studies. Optimum kinetic activity was found at pH 7.5-8.5 for bothMTT and cytochrome c. This is the first paper presenting characterization of a cytochrome P450 reductase from F. graminearum which most likely is involved in mycotoxin biosynthesis or some primary metabolic pathway such as sterol biosynthesis in F. graminearum. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. 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

  15. Reduced bone mass and muscle strength in male 5α-reductase type 1 inactivated mice.

    Directory of Open Access Journals (Sweden)

    Sara H Windahl

    Full Text Available Androgens are important regulators of bone mass but the relative importance of testosterone (T versus dihydrotestosterone (DHT for the activation of the androgen receptor (AR in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2, encoded by separate genes (Srd5a1 and Srd5a2. 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1⁻/⁻ mice. Four-month-old male Srd5a1⁻/⁻ mice had reduced trabecular bone mineral density (-36%, p<0.05 and cortical bone mineral content (-15%, p<0.05 but unchanged serum androgen levels compared with wild type (WT mice. The cortical bone dimensions were reduced in the male Srd5a1⁻/⁻ mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05 in orchidectomized WT mice but not in orchidectomized Srd5a1⁻/⁻ mice. Male Srd5a1⁻/⁻ mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05. Female Srd5a1⁻/⁻ mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1⁻/⁻ mice, is an indirect effect mediated by elevated circulating androgen levels.

  16. Monoterpene metabolism. Cloning, expression, and characterization of menthone reductases from peppermint.

    Science.gov (United States)

    Davis, Edward M; Ringer, Kerry L; McConkey, Marie E; Croteau, Rodney

    2005-03-01

    (-)-Menthone is the predominant monoterpene produced in the essential oil of maturing peppermint (Mentha x piperita) leaves during the filling of epidermal oil glands. This early biosynthetic process is followed by a second, later oil maturation program (approximately coincident with flower initiation) in which the C3-carbonyl of menthone is reduced to yield (-)-(3R)-menthol and (+)-(3S)-neomenthol by two distinct NADPH-dependent ketoreductases. An activity-based in situ screen, by expression in Escherichia coli of 23 putative redox enzymes from an immature peppermint oil gland expressed sequence tag library, was used to isolate a cDNA encoding the latter menthone:(+)-(3S)-neomenthol reductase. Reverse transcription-PCR amplification and RACE were used to acquire the former menthone:(-)-(3R)-menthol reductase directly from mRNA isolated from the oil gland secretory cells of mature leaves. The deduced amino acid sequences of these two reductases share 73% identity, provide no apparent subcellular targeting information, and predict inclusion in the short-chain dehydrogenase/reductase family of enzymes. The menthone:(+)-(3S)-neomenthol reductase cDNA encodes a 35,722-D protein, and the recombinant enzyme yields 94% (+)-(3S)-neomenthol and 6% (-)-(3R)-menthol from (-)-menthone as substrate, and 86% (+)-(3S)-isomenthol and 14% (+)-(3R)-neoisomenthol from (+)-isomenthone as substrate, has a pH optimum of 9.3, and K(m) values of 674 mum, > 1 mm, and 10 mum for menthone, isomenthone, and NADPH, respectively, with a k(cat) of 0.06 s(-1). The recombinant menthone:(-)-(3R)-menthol reductase has a deduced size of 34,070 D and converts (-)-menthone to 95% (-)-(3R)-menthol and 5% (+)-(3S)-neomenthol, and (+)-isomenthone to 87% (+)-(3R)-neoisomenthol and 13% (+)-(3S)-isomenthol, displays optimum activity at neutral pH, and has K(m) values of 3.0 mum, 41 mum, and 0.12 mum for menthone, isomenthone, and NADPH, respectively, with a k(cat) of 0.6 s(-1). The respective activities of

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

    Science.gov (United States)

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

    2013-11-20

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

  18. Protein method for investigating mercuric reductase gene expression in aquatic environments.

    Science.gov (United States)

    Ogunseitan, O A

    1998-02-01

    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 NADPH oxidation. Residual NADPH was determined by titration with phenazine methosulfate-catalyzed reduction of methyl thiazolyl tetrazolium to produce visible formazan. Spectrophotometric determination of formazan concentration showed a positive correlation with the amount of NADPH remaining in the reaction mixture (r2 = 0.99). Mercuric reductase activity in the protein extracts was inversely related to the amount of NADPH remaining and to the amount of formazan produced. A qualitative nitrocellulose membrane-based version of the method was also developed, where regions of mercuric reductase activity remained colorless against a stained-membrane background. The assay detected induced mercuric reductase activity from 10(2) CFU, and up to threefold signal intensity was detected in seeded freshwater samples amended with mercury compared to that in mercury-free samples. The efficiency of extraction of bacterial proteins from the freshwater samples was (97 +/- 2)% over the range of population densities investigated (10(2) to 10(8) CFU/ml). The method was validated by detection of enzyme activity in protein extracts of water samples from a polluted site harboring naturally occurring mercury-resistant bacteria. The new method is proposed as a supplement to the repertoire of molecular techniques available for assessing specific gene expression in heterogeneous microbial communities impacted by mercury pollution.

  19. Isolation and characterization of cloned cDNAs as encoding human liver chlordecone reductase

    Energy Technology Data Exchange (ETDEWEB)

    Winters, C.J.; Molowa, D.T.; Guzelian, P.S. (Medical College of Virginia, Richmond (USA))

    1990-01-30

    Chlordecone (Kepone), a toxic organochlorine pesticide, undergoes bioreduction to chlorodecone alcohol in human liver. This reaction is controlled by a cytosolic enzyme, chlordecone reductase (CDR), which may be of the aldo-keto reductase family of xenobiotic metabolizing enzymes. To further investigate the primary structure and expression of CDR, the authors screened a library of human liver cDNAs cloned in the expression vector {lambda}gt11 and isolated an 800 bp cDNA that directed synthesis of a fusion protein recognized by polyclonal anti-CDR antibodies. Using this cDNA as a probe, they screened two human liver cDNA libraries and found several 1.2-kb cDNAs which would code for polypeptide with 308 residues (35.8 kDa). However, a similar full-length cDNA, possibly the transcript of a pseudogene, contained an in-frame nonsense codon. The deduced protein sequence of CDR showed 65% similarity to the primary structure of human liver aldehyde reductase and 66% similarity to the inferred protein sequence of rat lens aldose reductase. A search of GenBank revealed significant nucleotide similarity to a cDNA coding for bovine lung prostaglandin f synthase and to a partial cDNA coding for frog lens {rho}-crystallin. RNA from adult but not fetal human liver, and from the human hepatoma cell-line Hep G2, contained major (1.6 kb) and minor (2.8 kb) species hybridizable to a CDR cDNA. The relative amounts of these RNAs varied markedly among nine subjects. From this initial description of the nucleotide sequence for a human carbonyl reductase, they conclude that CDR and several related enzymes are part of a novel multigene family involved in the metabolism of such xenobiotics as chlordecone and possibly endogenous substrates.

  20. Selective peptide inhibitors of bifunctional thymidylate synthase-dihydrofolate reductase from Toxoplasma gondii provide insights into domain-domain communication and allosteric regulation.

    Science.gov (United States)

    Landau, Mark J; Sharma, Hitesh; Anderson, Karen S

    2013-09-01

    The bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) plays an essential role in DNA synthesis and is unique to several species of pathogenic protozoans, including the parasite Toxoplasma gondii. Infection by T. gondii causes the prevalent disease toxoplasmosis, for which TS-DHFR is a major therapeutic target. Here, we design peptides that target the dimer interface between the TS domains of bifunctional T. gondii TS-DHFR by mimicking β-strands at the interface, revealing a previously unknown allosteric target. The current study shows that these β-strand mimetic peptides bind to the apo-enzyme in a species-selective manner to inhibit both the TS and distal DHFR. Fluorescence spectroscopy was used to monitor conformational switching of the TS domain and demonstrate that these peptides induce a conformational change in the enzyme. Using structure-guided mutagenesis, nonconserved residues in the linker between TS and DHFR were identified that play a key role in domain-domain communication and in peptide inhibition of the DHFR domain. These studies validate allosteric inhibition of apo-TS, specifically at the TS-TS interface, as a potential target for novel, species-specific therapeutics for treating T. gondii parasitic infections and overcoming drug resistance. © 2013 The Protein Society.

  1. Study on the 3-hydroxy-3-methyl-glutaryl CoA reductase inhibitory properties of Agaricus bisporus and extraction of bioactive fractions using pressurised solvent technologies.

    Science.gov (United States)

    Gil-Ramírez, Alicia; Clavijo, Cristina; Palanisamy, Marimuthu; Ruiz-Rodríguez, Alejandro; Navarro-Rubio, María; Pérez, Margarita; Marín, Francisco R; Reglero, Guillermo; Soler-Rivas, Cristina

    2013-08-30

    Agaricus bisporus mushrooms were able to lower cholesterol levels in hypercholesterolaemic rats and it was suggested that dietary fibre might inhibit cholesterol absorption. However, A. bisporus extracts were also able to inhibit the 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGCR, the key enzyme in the cholesterol biosynthetic pathway) and this might also contribute to the observed lowering of cholesterol levels in serum. The methanol-water extracts obtained from A. bisporus were able to inhibit up to 60% the HMGCR activity using an in vitro assay. The HMGCR inhibitory capacities depended on cultivation conditions, strains, etc. The potential inhibitors were not statins, they might be β-glucans able to scavenge the substrate and impair the enzymatic reaction. They were present during all mushroom developmental stages and similarly distributed through all the tissues including the parts discarded as a by-product. Accelerated solvent extractions using 1:1 ethanol-water as pressurised solvent (10.7 MPa, 25°C, five cycles of 5 min) were more effective in the extraction of the HMGCiR inhibitor(s) than supercritical fluid extractions (9 MPa, 40°C) using CO2 with 10% ethanol. A mushroom cultivation and two extraction procedures were optimised to obtain fractions from A. bisporus with high HMGCR inhibitory activities to design novel ingredients for hypocholesterolaemic functional foodstuffs. © 2013 Society of Chemical Industry.

  2. Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Song, Shasha [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Wang, Shuang [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China); Ma, Jun; Yao, Lan; Xing, Hao; Zhang, Lei; Liao, Lin [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Zhu, Daling, E-mail: dalingz@yahoo.com [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China)

    2013-08-01

    Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway. Highlights: • BVR expresses in PASMC and is up-regulated by hypoxia in protein and mRNA levels. • BVR/bilirubin contribute to the inhibitive process of hypoxia on PASMC apoptosis. • Bilirubin protects PASMC from apoptosis under hypoxia via ERK1/2 pathway.

  3. Statins inhibit protein lipidation and induce the unfolded protein response in the non-sterol producing nematode Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Mörck, Catarina; Olsen, Louise Cathrine Braun; Kurth, Caroline;

    2009-01-01

    of lipid moieties for protein prenylation. The nematode Caenorhabditis elegans possesses a mevalonate pathway that lacks the branch leading to cholesterol synthesis, and thus represents an ideal organism to specifically study the noncholesterol roles of the pathway. Inhibiting HMG-CoA reductase in C....... elegans using statins or RNAi leads to developmental arrest and loss of membrane association of a GFP-based prenylation reporter. The unfolded protein response (UPR) is also strongly activated, suggesting that impaired prenylation of small GTPases leads to the accumulation of unfolded proteins and ER...... and fatty acid composition were unaffected in statin-treated worms, even though they showed reduced staining with Nile red. We conclude that inhibitors of HMG-CoA reductase or of farnesyl transferases induce the UPR by inhibiting the prenylation of M57.2 substrates, resulting in developmental arrest in C...

  4. Simvastatin inhibits interferon-γ-induced MHC class II up-regulation in cultured astrocytes

    Directory of Open Access Journals (Sweden)

    Glazenburg Lisa

    2006-07-01

    Full Text Available Abstract Based on their potent anti-inflammatory properties and a preliminary clinical trial, statins (HMG-CoA reductase inhibitors are being studied as possible candidates for multiple sclerosis (MS therapy. The pathogenesis of MS is unclear. One theory suggests that the development of autoimmune lesions in the central nervous system may be due to a failure of endogenous inhibitory control of MHC class II expression on astrocytes, allowing these cells to adapt an interferon (IFN-γ-induced antigen presenting phenotype. By using immunocytochemistry in cultured astrocytes derived from newborn Wistar rats we found that simvastatin at nanomolar concentrations inhibited, in a dose-response fashion, up to 70% of IFN-γ-induced MHC class II expression. This effect was reversed by the HMG-CoA reductase product mevalonate. Suppression of the antigen presenting function of astrocytes might contribute to the beneficial effects of statins in MS.

  5. Molecular cloning and characterization of a birch pollen minor allergen, Bet v 5, belonging to a family of isoflavone reductase-related proteins.

    Science.gov (United States)

    Karamloo, F; Schmitz, N; Scheurer, S; Foetisch, K; Hoffmann, A; Haustein, D; Vieths, S

    1999-11-01

    Birch pollen is a major cause of pollinosis and is responsible for cross-reactive oral allergies to fruits, nuts, and vegetables. The major allergen, Bet v 1, has been extensively characterized, and 3 minor allergens, Bet v 2, Bet v 3, and Bet v 4, have been cloned and sequenced. Recently, another birch pollen protein with an apparent mass of 35 kd was described as a new IgE-binding protein in birch pollen with cross-reacting homologues in plant foods. The aim of this study was to determine the primary structure of the 35-kd birch pollen allergen and to investigate its immunologic properties. On the basis of a known complementary DNA fragment, a PCR strategy was applied to obtain the full-length nucleotide sequence of the coding region. The protein was expressed as His-Tag fusion protein in Escherichia coli and purified by Ni-chelate affinity chromatography. Nonfusion protein was obtained by cyanogen bromide treatment of the fusion protein. IgE-binding characteristics and potential allergenicity were investigated by immunoblot, immunoblot inhibition analysis, rat basophil leukemia-cell mediator release assay, and basophil histamine release and compared with those of natural (n) Bet v 5, recombinant (r)Bet v 1, and rBet v 2. Recombinant Bet v 5 has a mass of 33 kd, an isoelectric point of 9.0, and sequence identity of 60% to 80% to isoflavone reductase homologue proteins from various plants. On immunoblots the recombinant Bet v 5 bound IgE from 9 (32%) of 28 sera from patients allergic to birch pollen with a CAP class of at least 3; Bet v 1 was detected by 89% of these patients. IgE immunoblot and inhibition experiments showed that nBet v 5 and rBet v 5 shared identical epitopes. A rabbit antiserum raised against pea isoflavone reductase and patients' IgE reacted with Bet v 5 and proteins of similar size in several vegetable foods, including exotic fruits. A similar reaction pattern was obtained with 2 Bet v 5-specific mAbs. Furthermore, Bet v 5 triggered a dose

  6. Sexual dimorphism of growth plate prehypertrophic and hypertrophic chondrocytes in response to testosterone requires metabolism to dihydrotestosterone (DHT) by steroid 5-alpha reductase type 1.

    Science.gov (United States)

    Raz, P; Nasatzky, E; Boyan, B D; Ornoy, A; Schwartz, Z

    2005-05-01

    Rat costochondral growth plate chondrocytes exhibit sex-specific and cell maturation dependent responses to testosterone. Only male cells respond to testosterone, although testosterone receptors are present in both male and female cells, suggesting other mechanisms are involved. We examined the hypothesis that the sex-specific response of rat costochondral cartilage cells to testosterone requires further metabolism of the hormone to dihydrotestosterone (DHT). Resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) chondrocytes from male and female Sabra strain rats exhibited sex-specific responses to testosterone and DHT: only male cells were responsive. Testosterone and DHT treatment for 24 h caused a comparable dose-dependent increase in [3H]-thymidine incorporation in quiescent preconfluent cultures of male GC cells, and a comparable increase in alkaline phosphatase specific activity in confluent cultures. RC cells responded in a differential manner to testosterone and DHT. Testosterone decreased DNA synthesis in male RC cells but DHT had no effect and alkaline phosphatase specific activity of male RC cells was unaffected by either hormone. Inhibition of steroid 5alpha-reductase activity with finasteride (1, 5, or 10 microg/ml), reduced the response of male GC cells to testosterone in a dose-dependent manner, indicating that metabolism to DHT was required. RT-PCR showed that both male and female cells expressed mRNAs for steroid 5alpha-reductase type 1 but lacked mRNAs for the type 2 form of the enzyme. Male cells also exhibited 5alpha-reductase activity but activity of this enzyme was undetectable in female cells. These observations show that sex-specific responses of rat growth zone chondrocytes to testosterone requires the further metabolism of the hormone to DHT and that the effect of DHT in the male growth plate is maturation-state dependent. Failure of female chondrocytes to respond to testosterone may reflect differences in

  7. Induction of quinone reductase (QR) by withanolides isolated from Physalis pubescens L. (Solanaceae).

    Science.gov (United States)

    Ji, Long; Yuan, Yonglei; Ma, Zhongjun; Chen, Zhe; Gan, Lishe; Ma, Xiaoqiong; Huang, Dongsheng

    2013-09-01

    In the present study, it was demonstrated that the dichloromethane extract of Physalis pubescens L. (DEPP) had weak potential quinone reductase (QR) inducing activity, but an UPLC-ESI-MS method with glutathione (GSH) as the substrate revealed that the DEPP had electrophiles (with an α,β-unsaturated ketone moiety). These electrophiles could induce quinone reductase (QR) activity, which might be attributed to the modification of the highly reactive cysteine residues in Keap1. Herein, four withanolides, including three new compounds physapubescin B (2), physapubescin C (3), physapubescin D (4), together with one known steroidal compound physapubescin (1) were isolated. Structures of these compounds were determined by spectroscopic analysis and that of physapubescin C (3) was confirmed by a combination of molecular modeling and quantum chemical DFT-GIAO calculations. Evaluation of the QR inducing activities of all withanolides indicated potent activities of compounds 1 and 2, which had a common α,β-unsaturated ketone moiety.

  8. 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.

  9. 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.

  10. Synthesis of 3-[(N-Carboalkoxy)ethylamino]-indazole-dione Derivatives and Their Biological Activities on Human Liver Carbonyl Reductase

    OpenAIRE

    Berhe, Solomon; Slupe, Andrew; Luster, Choice; Charlier, Henry A.; Warner, Don L.; Zalkow, Leon H.; Burgess, Edward M.; Enwerem, Nkechi M.; Bakare, Oladapo

    2009-01-01

    A series of indazole-dione derivatives were synthesized by the 1,3-dipolar cycloaddition reaction of appropriate substituted benzoquinones or naphthoquinones and N-carboalkoxyamino diazopropane derivatives. These compounds were evaluated for their effects on human carbonyl reductase. Several of the analogs were found to serve as substrates for carbonyl reductase with a wide range of catalytic efficiencies, while four analogs display inhibitory activities with IC50 values ranging from 3 – 5 μM...

  11. High dose androgen therapy in male pseudohermaphroditism due to 5 alpha-reductase deficiency and disorders of the androgen receptor.

    OpenAIRE

    Price, P; Wass, J. A.; Griffin, J E; Leshin, M; Savage, M O; Large, D. M.; Bu'Lock, D E; Anderson, D. C.; Wilson, J. D.; Besser, G M

    1984-01-01

    We describe the clinical and biochemical features of six men with male pseudohermaphroditism due to androgen resistance. Each of the subjects had male-gender behavior but incomplete virilization. The underlying defects in androgen metabolism were defined by studies of the 5 alpha-reductase enzyme and the androgen receptor in fibroblasts cultured from biopsies of genital skin. Four of the six have 5 alpha-reductase deficiency, and two have defects of the androgen receptor (the Reifenstein synd...

  12. A conservative region of the mercuric reductase gene (mera) as a molecular marker of bacterial mercury resistance

    Science.gov (United States)

    Sotero-Martins, Adriana; de Jesus, Michele Silva; Lacerda, Michele; Moreira, Josino Costa; Filgueiras, Ana Luzia Lauria; Barrocas, Paulo Rubens Guimarães

    2008-01-01

    The most common bacterial mercury resistance mechanism is based on the reduction of Hg(II) to Hg0, which is dependent of the mercuric reductase enzyme (MerA) activity. The use of a 431 bp fragment of a conservative region of the mercuric reductase (merA) gene was applied as a molecular marker of this mechanism, allowing the identification of mercury resistant bacterial strains. PMID:24031221

  13. Substrate Specificity, Inhibitor Selectivity and Structure-Function Relationships of Aldo-Keto Reductase 1B15: A Novel Human Retinaldehyde Reductase.

    Directory of Open Access Journals (Sweden)

    Joan Giménez-Dejoz

    Full Text Available Human aldo-keto reductase 1B15 (AKR1B15 is a newly discovered enzyme which shares 92% amino acid sequence identity with AKR1B10. While AKR1B10 is a well characterized enzyme with high retinaldehyde reductase activity, involved in the development of several cancer types, the enzymatic activity and physiological role of AKR1B15 are still poorly known. Here, the purified recombinant enzyme has been subjected to substrate specificity characterization, kinetic analysis and inhibitor screening, combined with structural modeling. AKR1B15 is active towards a variety of carbonyl substrates, including retinoids, with lower kcat and Km values than AKR1B10. In contrast to AKR1B10, which strongly prefers all-trans-retinaldehyde, AKR1B15 exhibits superior catalytic efficiency with 9-cis-retinaldehyde, the best substrate found for this enzyme. With ketone and dicarbonyl substrates, AKR1B15 also shows higher catalytic activity than AKR1B10. Several typical AKR inhibitors do not significantly affect AKR1B15 activity. Amino acid substitutions clustered in loops A and C result in a smaller, more hydrophobic and more rigid active site in AKR1B15 compared with the AKR1B10 pocket, consistent with distinct substrate specificity and narrower inhibitor selectivity for AKR1B15.

  14. 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

    2016-01-01

    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.

  15. Gene expression of monodehydroascorbate reductase and dehydroascorbate reductase during fruit ripening and in response to environmental stresses in acerola (Malpighia glabra).

    Science.gov (United States)

    Eltelib, Hani A; Badejo, Adebanjo A; Fujikawa, Yukichi; Esaka, Muneharu

    2011-04-15

    Acerola (Malpighia glabra) is an exotic fruit cultivated primarily for its abundant ascorbic acid (AsA) content. The molecular mechanisms that regulate the metabolism of AsA in acerola have yet to be defined. Monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) are key enzymes of the ascorbate-glutathione cycle that maintain reduced pools of ascorbic acid and serve as important antioxidants. cDNAs encoding MDHAR and DHAR were isolated from acerola using RT-PCR and RACE. Phylogenetic trees associated acerola MDHAR and DHAR with other plant cytosolic MDHARs and DHARs. Expressions of the two genes correlated with their enzymatic activities and were differentially regulated during fruit ripening. Interestingly, MDHAR expression was only detected in overripe fruits, whereas the transcript level of DHAR was highest at the intermediate stage of fruit ripening. Under dark conditions, there was a sharp and significant decline in the total and reduced ascorbate contents, accompanied by a decrease in the level of transcripts and enzyme activities of the two genes in acerola leaves. MDHAR and DHAR transcripts and enzyme activities were significantly up-regulated in the leaves of acerola under cold and salt stress conditions, indicating that expression of both genes are transcriptionally regulated under these stresses.

  16. Separation and distribution of thiosulfate-oxidizing enzyme, tetrathionate reductase, and thiosulfate reductase in extracts of marine heterotroph strain 16B.

    Science.gov (United States)

    Whited, G M; Tuttle, J H

    1983-11-01

    Thiosulfate-oxidizing enzyme (TSO), tetrathionate reductase (TTR), and thiosulfate reductase (TSR) were demonstrated in cell-free extracts of the marine heterotrophic thiosulfate-oxidizing bacterium strain 16B. Extracts prepared from cells cultured aerobically in the absence of thiosulfate or tetrathionate exhibited constitutive TSO and TTR activity which resided in the soluble fraction of ultracentrifuged crude extracts. Constitutive TSO and TTR cochromatographed on DEAE-Sephadex A-50, Cellex D, Sephadex G-150, and orange A dye-ligand affinity gels. Extracts prepared from cells cultured anaerobically with tetrathionate or aerobically with thiosulfate followed by oxygen deprivation showed an 11- to 30-fold increase in TTR activity, with no increase in TSO activity. The inducible TTR resided in both the ultracentrifuge pellet and supernatant fractions and was readily separated from constitutive TSO and TTR in the latter by DEAE-Sephadex chromatography. Inducible TTR exhibited TSR activity, which was also located in both membrane and soluble extract fractions and which cochromatographed with inducible TTR. The results indicate that constitutive TSO and TTR in marine heterotroph 16B represent reverse activities of the same enzyme whose major physiological function is thiosulfate oxidation. Evidence is also presented which suggests a possible association of inducible TTR and TSR in strain 16B.

  17. Transgenic Tobacco Overexpressing Tea cDNA Encoding Dihydroflavonol 4-Reductase and Anthocyanidin Reductase Induces Early Flowering and Provides Biotic Stress Tolerance.

    Directory of Open Access Journals (Sweden)

    Vinay Kumar

    Full Text Available Flavan-3-ols contribute significantly to flavonoid content of tea (Camellia sinensis L.. Dihydroflavonol 4-reductase (DFR and anthocyanidin reductase (ANR are known to be key regulatory enzymes of flavan-3-ols biosynthesis. In this study, we have generated the transgenic tobacco overexpressing individually tea cDNA CsDFR and CsANR encoding for DFR and ANR to evaluate their influence on developmental and protective abilities of plant against biotic stress. The transgenic lines of CsDFR and CsANR produced early flowering and better seed yield. Both types of transgenic tobacco showed higher content of flavonoids than control. Flavan-3-ols such as catechin, epicatechin and epicatechingallate were found to be increased in transgenic lines. The free radical scavenging activity of CsDFR and CsANR transgenic lines was improved. Oxidative stress was observed to induce lesser cell death in transgenic lines compared to control tobacco plants. Transgenic tobacco overexpressing CsDFR and CsANR also showed resistance against infestation by a tobacco leaf cutworm Spodoptera litura. Results suggested that the overexpression of CsDFR and CsANR cDNA in tobacco has improved flavonoids content and antioxidant potential. These attributes in transgenic tobacco have ultimately improved their growth and development, and biotic stress tolerance.

  18. 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.