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Sample records for aldo-keto reductase akr1b10

  1. 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.

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

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    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.

  3. Aldo-keto reductase 1B10 and its role in proliferation capacity of drug-resistant cancers

    Directory of Open Access Journals (Sweden)

    Toshiyuki eMatsunaga

    2012-01-01

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

  4. The aldo-keto reductase superfamily homepage.

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    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.

  5. [Preparation and identification of rabbit anti-AKR1B10 polyclonal antibody].

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    Hu, Jian; Wang, Qingmei; Huang, Li; Zeng, Yuanqing; Hu, Zheng; Luo, Dixian

    2016-11-01

    Objective To prepare rabbit anti-aldo-keto reductase family 1 member B10 (AKR1B10) polyclonal antibody and identify its specificity. Methods AKR1B10 cDNA was amplified by reverse transcription PCR (RT-PCR) and inserted into prokaryotic expression vector pET-15b to form recombinant plasmid pET-15b-AKR1B10. The recombinant plasmid pET-15b-AKR1B10 was transformed into E.coli DH5α. Isopropylthio-β-D-galactoside (IPTG) was used to induce the expression of the recombinant protein His-tagged AKR1B10 in E.coli DH5α. The expression products from different clones of E.coli DH5α were identified by SDS-PAGE. The positive bacteria were picked out and amplified. His-Tag-AKR1B10 protein was purified from the expression product of the positive clones by His-tagged purification column. The purified recombinant protein His-Tag-AKR1B10 was used to immunize New Zealand white rabbits. Antisera were acquired after two months. Anti-AKR1B10 polyclonal antibodies were purified by antigen purification column with AKR1B10 recombinant protein. Lastly, the purified polyclonal antibodies were identified by SDS-PAGE, ELISA, Western blotting. Results The recombinant plasmid pET-15b-AKR1B10 was constructed successfully, and the recombinant protein His-Tag-AKR1B10 with high purity was acquired. The purified polyclonal antibodies were able to specifically recognize AKR1B10 protein. Conclusion The rabbit anti-AKR1B10 polyclonal antibodies is prepared successfully with high specificity.

  6. Overexpression and enhanced specific activity of aldoketo reductases (AKR1B1 & AKR1B10) in human breast cancers.

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    Reddy, K Ashok; Kumar, P Uday; Srinivasulu, M; Triveni, B; Sharada, K; Ismail, Ayesha; Reddy, G Bhanuprakash

    2017-02-01

    The incidence of breast cancer in India is on the rise and is rapidly becoming the primary cancer in Indian women. The aldoketo reductase (AKR) family has more than 190 proteins including aldose reductase (AKR1B1) and aldose reductase like protein (AKR1B10). Apart from liver cancer, the status of AKR1B1 and AKR1B10 with respect to their expression and activity has not been reported in other human cancers. We studied the specific activity and expression of AKR1B1 and AKR1B10 in breast non tumor and tumor tissues and in the blood. Fresh post-surgical breast cancer and non-cancer tissues and blood were collected from the subjects who were admitted for surgical therapy. Malignant, benign and pre-surgical chemotherapy samples were evaluated by histopathology scoring. Expression of AKR1B1 and AKR1B10 was carried out by immunoblotting and immunohistochemistry (IHC) while specific activity was determined spectrophotometrically. The specific activity of AKR1B1 was significantly higher in red blood cells (RBC) in all three grades of primary surgical and post-chemotherapy samples. Specific activity of both AKR1B1 and AKR1B10 increased in tumor samples compared to their corresponding non tumor samples (primary surgical and post-chemotherapy). Immunoblotting and IHC data also indicated overexpression of AKR1B1 in all grades of tumors compared to their corresponding non tumor samples. There was no change in the specific activity of AKR1B1 in benign samples compared to all grades of tumor and non-tumors.

  7. Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels

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    Zhong, Linlin [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States); Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001 (China); Liu, Ziwen [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States); Department of Surgery, Peking Union Medical College Hospital, Beijing 100730 (China); Yan, Ruilan [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States); Johnson, Stephen [Carbon Dynamics Institute, LLC, 2835 via Verde Drive, Springfield, IL 62703-4325 (United States); Zhao, Yupei [Department of Surgery, Peking Union Medical College Hospital, Beijing 100730 (China); Fang, Xiubin [Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001 (China); Cao, Deliang, E-mail: dcao@siumed.edu [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States)

    2009-09-18

    Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 {mu}M, 4-hydroxynonenal (HNE) at 0.10 {mu}M, trans-2-hexanal at 0.10 {mu}M, and trans-2,4-hexadienal at 0.05 {mu}M, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 {mu}M (toxic) by converting to 1,4-dihydroxynonene, protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.

  8. 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.

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    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.

  9. Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10

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    Matsunaga, Toshiyuki, E-mail: matsunagat@gifu-pu.ac.jp [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Morikawa, Yoshifumi; Haga, Mariko; Endo, Satoshi [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Soda, Midori; Yamamura, Keiko [Laboratory of Clinical Pharmacy, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650 (Japan); El-Kabbani, Ossama [Monash Institute of Pharmaceutical Sciences, Monash University, Victoria 3052 (Australia); Tajima, Kazuo [Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181 (Japan); Ikari, Akira [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Hara, Akira [Faculty of Engineering, Gifu University, Gifu 501-1193 (Japan)

    2014-07-15

    Inhalation of 9,10-phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust, exerts fatal damage against a variety of cells involved in respiratory function. Here, we show that treatment with high concentrations of 9,10-PQ evokes apoptosis of lung cancer A549 cells through production of reactive oxygen species (ROS). In contrast, 9,10-PQ at its concentrations of 2 and 5 μM elevated the potentials for proliferation, invasion, metastasis and tumorigenesis, all of which were almost completely inhibited by addition of an antioxidant N-acetyl-L-cysteine, inferring a crucial role of ROS in the overgrowth and malignant progression of lung cancer cells. Comparison of mRNA expression levels of six aldo-keto reductases (AKRs) in the 9,10-PQ-treated cells advocated up-regulation of AKR1B10 as a major cause contributing to the lung cancer malignancy. In support of this, the elevation of invasive, metastatic and tumorigenic activities in the 9,10-PQ-treated cells was significantly abolished by the addition of a selective AKR1B10 inhibitor oleanolic acid. Intriguingly, zymographic and real-time PCR analyses revealed remarkable increases in secretion and expression, respectively, of matrix metalloproteinase 2 during the 9,10-PQ treatment, and suggested that the AKR1B10 up-regulation and resultant activation of mitogen-activated protein kinase cascade are predominant mechanisms underlying the metalloproteinase induction. In addition, HPLC analysis and cytochrome c reduction assay in in vitro 9,10-PQ reduction by AKR1B10 demonstrated that the enzyme catalyzes redox-cycling of this quinone, by which ROS are produced. Collectively, these results suggest that AKR1B10 is a key regulator involved in overgrowth and malignant progression of the lung cancer cells through ROS production due to 9,10-PQ redox-cycling. - Highlights: • 9,10-PQ promotes invasion, metastasis and tumorigenicity in lung cancer cells. • The 9,10-PQ-elicited promotion is possibly due to AKR1B10 up

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

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    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.

  11. High expression of AKR1B10 predicts low risk of early tumor recurrence in patients with hepatitis B virus-related hepatocellular carcinoma

    Science.gov (United States)

    Wang, Yan-Yan; Qi, Lu-Nan; Zhong, Jian-Hong; Qin, Hong-Gui; Ye, Jia-Zhou; Lu, Shi-Dong; Ma, Liang; Xiang, Bang-De; Li, Le-Qun; You, Xue-Mei

    2017-01-01

    To clarify the relationship between aldo-keto reductase family 1 member B10 (AKR1B10) expression and early hepatocellular carcinoma (HCC) recurrence, this study detected AKR1B10 expression in tumor and adjacent non-tumor tissues from 110 patients with hepatitis B virus (HBV)-related HCC underwent liver resection and analyzed its correlations with clinicopathological characteristics and prognosis of these patients. Detected by quantitative reverse transcription polymerase chain reaction, AKR1B10 mRNA expression showed significantly higher in HCC tissues than in adjacent non-tumor tissues, with a low level in normal liver tissues. Similar results was confirmed at the protein level using immunohistochemistry and Western blotting. High AKR1B10 expression was negatively correlated with serum alpha-fetoprotein level and positively correlated with HBV-DNA level. Patients with high AKR1B10 expression had significantly higher disease-free survival than those with low expression within 2 years after liver resection. Multivariate analysis also confirmed high AKR1B10 expression to be a predictor of low risk of early HCC recurrence. In addition, high AKR1B10 expression was found to be a favorable factor of overall survival. These results suggest that AKR1B10 is involved in HBV-related hepatocarcinogenesis, but its high expression could predict low risk of early tumor recurrence in patients with HBV-related HCC after liver resection. PMID:28181486

  12. 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.

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

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    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.

  14. Pretreatment AKR1B10 expression predicts the risk of hepatocellular carcinoma development after hepatitis C virus eradication

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    Murata, Ayato; Genda, Takuya; Ichida, Takafumi; Amano, Nozomi; Sato, Sho; Tsuzura, Hironori; Sato, Shunsuke; Narita, Yutaka; Kanemitsu, Yoshio; Shimada, Yuji; Hirano, Katsuharu; Iijima, Katsuyori; Wada, Ryo; Nagahara, Akihito; Watanabe, Sumio

    2016-01-01

    AIM To clarify the association between aldo-keto reductase family 1 member B10 (AKR1B10) expression and hepatocarcinogenesis after hepatitis C virus eradication. METHODS In this study, we enrolled 303 chronic hepatitis C patients who had achieved sustained virological response (SVR) through interferon-based antiviral therapy. Pretreatment AKR1B10 expression in the liver was immunohistochemically assessed and quantified as a percentage of positive staining area by using image-analysis software. A multivariate Cox analysis was used to estimate the hazard ratios (HRs) of AKR1B10 expression for hepatocellular carcinoma (HCC) development after achieving SVR. The cumulative incidences of HCC development were evaluated using Kaplan-Meier analysis and the log-rank test. RESULTS Of the 303 chronic hepatitis C patients, 153 (50.5%) showed scarce hepatic AKR1B10 expression, quantified as 0%, which was similar to the expression in control normal liver tissues. However, the remaining 150 patients (49.5%) exhibited various degrees of AKR1B10 expression in the liver, with a maximal AKR1B10 expression of 73%. During the median follow-up time of 3.6 years (range 1.0-10.0 years), 8/303 patients developed HCC. Multivariate analysis revealed that only high AKR1B10 expression (≥ 8%) was an independent risk factor for HCC development (HR = 15.4, 95%CI: 1.8-132.5, P = 0.012). The 5-year cumulative incidences of HCC development were 13.7% and 0.5% in patients with high and low AKR1B10 expression, respectively (P < 0.001). During the follow-up period after viral eradication, patients expressing high levels of AKR1B10 expressed markedly higher levels of alanine aminotransferase and α-fetoprotein than did patients exhibiting low AKR1B10 expression. CONCLUSION Chronic hepatitis C patients expressing high levels of hepatic AKR1B10 had an increased risk of HCC development even after SVR. PMID:27672277

  15. Enzymatic detection of γ-hydroxybutyrate using aldo-keto reductase 7A2.

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    Bendinskas, Kestutis; Sattelberg, Patricia; Crossett, Daniel; Banyikwa, Andrew; Dempsey, Daniel; MacKenzie, James A

    2011-05-01

    Gamma-hydroxybutyrate (GHB) is a prescribed medication as well as a drug of abuse. Its detection in various matrices for in-field forensic scientists remains a challenge. We have developed an assay that uses aldo-keto reductase 7A2 (AKR7A2) for the specific determination of GHB in various drinks. AKR7A2 was purified using Ni-affinity chromatography. The Michaelis-Menten constant for the GHB oxidation reaction was 10 mM, and the minimum detection limit was 4 mM. Ethanol was not a substrate for AKR7A2. In a coupled reaction with NADP(+), phenazine methosulfate (PMS), and 2,6-dichlorophenolindophenol, various beverages (orange juice, milk, soda, and numerous alcoholic drinks) containing GHB turned from blue to light yellow. In a second coupled reaction where diaphorase replaced PMS, the presence of GHB also caused the expected change of color in various beers.

  16. Lignases and aldo-keto reductases for conversion of lignin-containing materials to fermentable products

    Energy Technology Data Exchange (ETDEWEB)

    Scharf, Michael; Sethi, Amit

    2016-09-13

    Termites have specialized digestive systems that overcome the lignin barrier in wood to release fermentable simple sugars. Using the termite Reticulitermes flavipes and its gut symbionts, high-throughput titanium pyrosequencing and proteomics approaches experimentally compared the effects of lignin-containing diets on host-symbiont digestome composition. Proteomic investigations and functional digestive studies with recombinant lignocellulases conducted in parallel provided strong evidence of congruence at the transcription and translational levels and provide enzymatic strategies for overcoming recalcitrant lignin barriers in biofuel feedstocks. Briefly described, therefore, the disclosure provides a system for generating a fermentable product from a lignified plant material, the system comprising a cooperating series of at least two catalytically active polypeptides, where said catalytically active polypeptides are selected from the group consisting of: cellulase Cell-1, .beta.-glu cellulase, an aldo-keto-reductase, a catalase, a laccase, and an endo-xylanase.

  17. Aldo-keto Reductase Family 1 B10 as a Novel Target for Breast Cancer Treatment

    Science.gov (United States)

    2010-08-01

    cells via identifying the functional domain(s). Body 1) AKR1B10 silencing inhibits breast cancer cells BT-20 growth in culture and...Laboratory of Chemical Biology, Guangdong Province, Tsinghua University Graduate School at Shenzhen , Guangdong 518055 and 6School of Medicine, Tsinghua...breast cancer. Silencing of AKR1B10 in BT-20 human breast cancer cells inhibited cell growth in culture and tumorigenesis in female nude mice. Taken

  18. 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.

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

    Science.gov (United States)

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

    2016-04-20

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

  20. 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; Udayakumar, M

    2016-09-09

    In recent years, concerns about the use of glyphosate-resistant (GR) 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 homologs 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 seedlings 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. This article is protected by copyright. All rights reserved.

  1. 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.

  2. 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

  3. Cisplatin resistance by induction of aldo-keto reductase family 1 member C2 in human bladder cancer cells

    OpenAIRE

    Shirato, Akitomi; KIKUGAWA, TADAHIKO; Miura, Noriyoshi; Tanji, Nozomu; Takemori, Nobuaki; Higashiyama, Shigeki; Yokoyama, Masayoshi

    2013-01-01

    Cisplatin is currently the most effective anti-tumor agent available against bladder cancer. To clarify the mechanism underlying cisplatin resistance in bladder cancer, the present study examined the role of the aldo-keto reductase family 1 member C2 (AKR1C2) protein on chemoresistance using a human bladder cancer cell line. The function of AKR1C2 in chemoresistance was studied using the human HT1376 bladder cancer cell line and the cisplatin-resistant HT1376-CisR subline. AKR1C2 was expresse...

  4. The aldo-keto reductase AKR1B7 coexpresses with renin without influencing renin production and secretion.

    Science.gov (United States)

    Machura, Katharina; Iankilevitch, Elina; Neubauer, Björn; Theuring, Franz; Kurtz, Armin

    2013-03-01

    On the basis of evidence that within the adult kidney, the aldo-keto reductase AKR1B7 (aldo-keto reductase family 1, member 7, also known as mouse vas deferens protein, MVDP) is selectively expressed in renin-producing cells, we aimed to define a possible role of AKR1B7 for the regulation and function of renin cells in the kidney. We could confirm colocalization and corecruitment of renin and of AKR1B7 in wild-type kidneys. Renin cells in AKR1B7-deficient kidneys showed normal morphology, numbers, and intrarenal distribution. Plasma renin concentration (PRC) and renin mRNA levels of AKR1B7-deficient mice were normal at standard chow and were lowered by a high-salt diet directly comparable to wild-type mice. Treatment with a low-salt diet in combination with an angiotensin-converting enzyme inhibitor strongly increased PRC and renin mRNA in a similar fashion both in AKR1B7-deficient and wild-type mice. Under this condition, we also observed a strong retrograde recruitment of renin-expressing cell along the preglomerular vessels, however, without a difference between AKR1B7-deficient and wild-type mice. The isolated perfused mouse kidney model was used to study the acute regulation of renin secretion by ANG II and by perfusion pressure. Regarding these parameters, no differences were observed between AKR1B7-deficient and wild-type kidneys. In summary, our data suggest that AKR1B7 is not of major relevance for the regulation of renin production and secretion in spite of its striking coregulation with renin expression.

  5. The Role of Human Aldo-Keto Reductases in the Metabolic Activation and Detoxication of Polycyclic Aromatic Hydrocarbons: Interconversion of PAH Catechols and PAH o-Quinones

    OpenAIRE

    Li eZhang; Yi eJin; Meng eHuang; Penning, Trevor M.

    2012-01-01

    Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants. They are procarcinogens requiring metabolic activation to elicit their deleterious effects. Aldo-keto reductases (AKR) catalyze the oxidation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active PAH o-quinones. AKRs are also found to be capable of reducing PAH o-quinones to form PAH catechols. The interconversion of o-quinones and catechols results in the redox-cycling of PAH o-...

  6. Anthracycline resistance mediated by reductive metabolism in cancer cells: The role of aldo-keto reductase 1C3

    Energy Technology Data Exchange (ETDEWEB)

    Hofman, Jakub; Malcekova, Beata; Skarka, Adam; Novotna, Eva; Wsol, Vladimir, E-mail: wsol@faf.cuni.cz

    2014-08-01

    Pharmacokinetic drug resistance is a serious obstacle that emerges during cancer chemotherapy. In this study, we investigated the possible role of aldo-keto reductase 1C3 (AKR1C3) in the resistance of cancer cells to anthracyclines. First, the reducing activity of AKR1C3 toward anthracyclines was tested using incubations with a purified recombinant enzyme. Furthermore, the intracellular reduction of daunorubicin and idarubicin was examined by employing the transfection of A549, HeLa, MCF7 and HCT 116 cancer cells with an AKR1C3 encoding vector. To investigate the participation of AKR1C3 in anthracycline resistance, we conducted MTT cytotoxicity assays with these cells, and observed that AKR1C3 significantly contributes to the resistance of cancer cells to daunorubicin and idarubicin, whereas this resistance was reversible by the simultaneous administration of 2′-hydroxyflavanone, a specific AKR1C3 inhibitor. In the final part of our work, we tracked the changes in AKR1C3 expression after anthracycline exposure. Interestingly, a reciprocal correlation between the extent of induction and endogenous levels of AKR1C3 was recorded in particular cell lines. Therefore, we suggest that the induction of AKR1C3 following exposure to daunorubicin and idarubicin, which seems to be dependent on endogenous AKR1C3 expression, eventually might potentiate an intrinsic resistance given by the normal expression of AKR1C3. In conclusion, our data suggest a substantial impact of AKR1C3 on the metabolism of daunorubicin and idarubicin, which affects their pharmacokinetic and pharmacodynamic behavior. In addition, we demonstrate that the reduction of daunorubicin and idarubicin, which is catalyzed by AKR1C3, contributes to the resistance of cancer cells to anthracycline treatment. - Highlights: • Metabolism of anthracyclines by AKR1C3 was studied at enzyme and cellular levels. • Anthracycline resistance mediated by AKR1C3 was demonstrated in cancer cells. • Induction of AKR1C3

  7. Characterization of the aldo-keto reductase 1C gene cluster on pig chromosome 10: possible associations with reproductive traits

    Directory of Open Access Journals (Sweden)

    Nonneman Dan J

    2006-09-01

    Full Text Available Abstract Background The rate of pubertal development and weaning to estrus interval are correlated and affect reproductive efficiency of swine. Quantitative trait loci (QTL for age of puberty, nipple number and ovulation rate have been identified in Meishan crosses on pig chromosome 10q (SSC10 near the telomere, which is homologous to human chromosome 10p15 and contains an aldo-keto reductase (AKR gene cluster with at least six family members. AKRs are tissue-specific hydroxysteroid dehydrogenases that interconvert weak steroid hormones to their more potent counterparts and regulate processes involved in development, homeostasis and reproduction. Because of their location in the swine genome and their implication in reproductive physiology, this gene cluster was characterized and evaluated for effects on reproductive traits in swine. Results Screening the porcine CHORI-242 BAC library with a full-length AKR1C4 cDNA identified 7 positive clones and sample sequencing of 5 BAC clones revealed 5 distinct AKR1C genes (AKR1CL2 and AKR1C1 through 4, which mapped to 126–128 cM on SSC10. Using the IMpRH7000rad and IMNpRH212000rad radiation hybrid panels, these 5 genes mapped between microsatellite markers SWR67 and SW2067. Comparison of sequence data with the porcine BAC fingerprint map show that the cluster of genes resides in a 300 kb region. Twelve SNPs were genotyped in gilts observed for age at first estrus and ovulation rate from the F8 and F10 generations of one-quarter Meishan descendants of the USMARC resource population. Age at puberty, nipple number and ovulation rate data were analyzed for association with genotypes by MTDFREML using an animal model. One SNP, a phenylalanine to isoleucine substitution in AKR1C2, was associated with age of puberty (p = 0.07 and possibly ovulation rate (p = 0.102. Two SNP in AKR1C4 were significantly associated with nipple number (p ≤ 0.03 and another possibly associated with age at puberty (p = 0

  8. The Role of Human Aldo-Keto Reductases in the Metabolic Activation and Detoxication of Polycyclic Aromatic Hydrocarbons: Interconversion of PAH Catechols and PAH o-Quinones.

    Science.gov (United States)

    Zhang, Li; Jin, Yi; Huang, Meng; Penning, Trevor M

    2012-01-01

    Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants. They are procarcinogens requiring metabolic activation to elicit their deleterious effects. Aldo-keto reductases (AKR) catalyze the oxidation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active PAH o-quinones. AKRs are also found to be capable of reducing PAH o-quinones to form PAH catechols. The interconversion of o-quinones and catechols results in the redox-cycling of PAH o-quinones to give rise to the generation of reactive oxygen species and subsequent oxidative DNA damage. On the other hand, PAH catechols can be intercepted through phase II metabolism by which PAH o-quinones could be detoxified and eliminated. The aim of the present review is to summarize the role of human AKRs in the metabolic activation/detoxication of PAH and the relevance of phase II conjugation reactions to human lung carcinogenesis.

  9. Aldo-keto reductase 1C1 induced by interleukin-1β mediates the invasive potential and drug resistance of metastatic bladder cancer cells

    Science.gov (United States)

    Matsumoto, Ryuji; Tsuda, Masumi; Yoshida, Kazuhiko; Tanino, Mishie; Kimura, Taichi; Nishihara, Hiroshi; Abe, Takashige; Shinohara, Nobuo; Nonomura, Katsuya; Tanaka, Shinya

    2016-01-01

    In treating bladder cancer, determining the molecular mechanisms of tumor invasion, metastasis, and drug resistance are urgent to improving long-term patient survival. One of the metabolic enzymes, aldo-keto reductase 1C1 (AKR1C1), plays an essential role in cancer invasion/metastasis and chemoresistance. In orthotopic xenograft models of a human bladder cancer cell line, UM-UC-3, metastatic sublines were established from tumors in the liver, lung, and bone. These cells possessed elevated levels of EMT-associated markers, such as Snail, Slug, or CD44, and exhibited enhanced invasion. By microarray analysis, AKR1C1 was found to be up-regulated in metastatic lesions, which was verified in metastatic human bladder cancer specimens. Decreased invasion caused by AKR1C1 knockdown suggests a novel role of AKR1C1 in cancer invasion, which is probably due to the regulation of Rac1, Src, or Akt. An inflammatory cytokine, interleukin-1β, was found to increase AKR1C1 in bladder cancer cell lines. One particular non-steroidal anti-inflammatory drug, flufenamic acid, antagonized AKR1C1 and decreased the cisplatin-resistance and invasion potential of metastatic sublines. These data uncover the crucial role of AKR1C1 in regulating both metastasis and drug resistance; as a result, AKR1C1 should be a potent molecular target in invasive bladder cancer treatment. PMID:27698389

  10. Discovery of (R)-2-(6-Methoxynaphthalen-2-yl)butanoic Acid as a Potent and Selective Aldo-keto Reductase 1C3 Inhibitor.

    Science.gov (United States)

    Adeniji, Adegoke; Uddin, Md Jashim; Zang, Tianzhu; Tamae, Daniel; Wangtrakuldee, Phumvadee; Marnett, Lawrence J; Penning, Trevor M

    2016-08-25

    Type 5 17β-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) converts Δ(4)-androstene-3,17-dione and 5α-androstane-3,17-dione to testosterone (T) and 5α-dihydrotestosterone, respectively, in castration resistant prostate cancer (CRPC). In CRPC, AKR1C3 is implicated in drug resistance, and enzalutamide drug resistance can be surmounted by indomethacin a potent inhibitor of AKR1C3. We examined a series of naproxen analogues and find that (R)-2-(6-methoxynaphthalen-2-yl)butanoic acid (in which the methyl group of R-naproxen was replaced by an ethyl group) acts as a potent AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound was devoid of inhibitory activity on COX isozymes and blocked AKR1C3 mediated production of T and induction of PSA in LNCaP-AKR1C3 cells as a model of a CRPC cell line. R-Profens are substrate selective COX-2 inhibitors and block the oxygenation of endocannabinoids and in the context of advanced prostate cancer R-profens could inhibit intratumoral androgen synthesis and act as analgesics for metastatic disease.

  11. A novel aldo-keto reductase from Jatropha curcas L. (JcAKR) plays a crucial role in the detoxification of methylglyoxal, a potent electrophile.

    Science.gov (United States)

    Mudalkar, Shalini; Sreeharsha, Rachapudi Venkata; Reddy, Attipalli Ramachandra

    2016-05-20

    Abiotic stress leads to the generation of reactive oxygen species (ROS) which further results in the production of reactive carbonyls (RCs) including methylglyoxal (MG). MG, an α, β-dicarbonyl aldehyde, is highly toxic to plants and the mechanism behind its detoxification is not well understood. Aldo-keto reductases (AKRs) play a role in detoxification of reactive aldehydes and ketones. In the present study, we cloned and characterised a putative AKR from Jatropha curcas (JcAKR). Phylogenetically, it forms a small clade with AKRs of Glycine max and Rauwolfia serpentina. JcAKR was heterologously expressed in Escherichia coli BL-21(DE3) cells and the identity of the purified protein was confirmed through MALDI-TOF analysis. The recombinant protein had high enzyme activity and catalytic efficiency in assays containing MG as the substrate. Protein modelling and docking studies revealed MG was efficiently bound to JcAKR. Under progressive drought and salinity stress, the enzyme and transcript levels of JcAKR were higher in leaves compared to roots. Further, the bacterial and yeast cells expressing JcAKR showed more tolerance towards PEG (5%), NaCl (200mM) and MG (5mM) treatments compared to controls. In conclusion, our results project JcAKR as a possible and potential target in crop improvement for abiotic stress tolerance.

  12. Overexpression of Aldo-Keto-Reductase in Azole-resistant Clinical Isolates of Candida Glabrata Determined by cDNA-AFLP

    Directory of Open Access Journals (Sweden)

    Mansour Heidari

    2013-01-01

    Full Text Available Background: Candida glabrata causes significant medical problems in immunocompromised patients. Many strains of this yeast are intrinsically resistant to azole antifungal agents, and treatment is problematic, leading to high morbidity and mortality rates in immunosuppressed individuals. The primary goal of this study was to investigate the genes involved in the drug resistance of clinical isolates of C. glabrata.Methods: The clinical isolates of C. glabrata were collected in an epidemiological survey of candidal infection inimmunocompromised patients and consisted of four fluconazole and itraconazole resistant isolates, two fluconazoleand itraconazole sensitive isolates, and C. glabrata CBS 138 as reference strain. Antifungal susceptibility patterns ofthe organisms were determined beforehand by the Clinical and Laboratory Standards Institute (CLSI. The potentialgene(s implicated in antifungal resistance were investigated using complementary DNA- Amplified Fragment Length Polymorphism (cDNA-AFLP. Semi-quantitative RT-PCR was carried out to evaluate the expression of gene(s in resistant isolates as compared to sensitive and reference strains.Results and conclusions: The aldo-keto-reductase superfamily (AKR gene was upregulated in the resistant clinicalisolates as assessed by cDNA-AFLP. Semi-quantitative RT-PCR revealed AKR mRNA expression approximately twice that seen in the sensitive isolates. Overexpression of the AKR gene was associated with increased fluconazole and itraconazole resistance in C. glabrata. The data suggest that upregulation of the AKR gene might give a new insight into the mechanism of azole resistance.

  13. Expression of aldo-keto reductase family 1 member C1 (AKR1C1 gene in porcine ovary and uterine endometrium during the estrous cycle and pregnancy

    Directory of Open Access Journals (Sweden)

    Hwang Sue-Yun

    2011-10-01

    Full Text Available Abstract Background The aldo-keto reductase family 1 member C1 (AKR1C1 belongs to a superfamily of NADPH-dependent reductases that convert a wide range of substrates, including carbohydrates, steroid hormones, and endogenous prostaglandins. The 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD is a member of AKR family. The aims of this study were to determine its expression in the ovary and uterus endometrium during the estrous cycle and pregnancy. Methods Rapid amplification of cDNA ends (RACE experiments were performed to obtain the 5' and 3' ends of the porcine 20alpha-HSD cDNA. Reverse-transcriptase-PCR (RT-PCR, real-time PCR, northern blot analysis, and western blot analysis were performed to examine the expression of porcine 20alpha-HSD. Immunohistochemical analysis was also performed to determine the localization in the ovary. Results The porcine 20alpha-HSD cDNA is 957 bp in length and encodes a protein of 319 amino acids. The cloned cDNA was virtually the same as the porcine AKR1C1 gene (337 amino acids reported recently, and only differed in the C-terminal region (the AKR1C1 gene has a longer C-terminal region than our sequence. The 20alpha-HSD gene (from now on referred to as AKR1C1 cloned in this paper encodes a deletion of 4 amino acids, compared with the C-terminal region of AKR1C1 genes from other animals. Porcine AKR1C1 mRNA was expressed on day 5, 10, 12, 15 of the cycle and 0-60 of pregnancy in the ovary. The mRNA was also specifically detected in the uterine endometrium on day 30 of pregnancy. Western blot analysis indicated that the pattern of AKR1C1 protein in the ovary during the estrous cycle and uterus during early pregnancy was similar to that of AKR1C1 mRNA expression. The recombinant protein produced in CHO cells was detected at approximately 37 kDa. Immunohistochemical analysis also revealed that pig AKR1C1 protein was localized in the large luteal cells in the early stages of the estrous cycle and before parturition

  14. Crystal structures of three classes of non-steroidal anti-inflammatory drugs in complex with aldo-keto reductase 1C3.

    Directory of Open Access Journals (Sweden)

    Jack U Flanagan

    Full Text Available Aldo-keto reductase 1C3 (AKR1C3 catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid, arylpropionic acids (flurbiprofen, ibuprofen, naproxen, and indomethacin analogues (indomethacin, sulindac, zomepirac. The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens

  15. Bioequivalence studies of tibolone in premenopausal women and effects on expression of the tibolone-metabolizing enzyme AKR1C (aldo-keto reductase) family caused by estradiol.

    Science.gov (United States)

    Kang, Keon W; Kim, Yoon G

    2008-12-01

    This study aimed to investigate the bioequivalence of a test formulation of tibolone with the marketed reference formulation in 24 young healthy female volunteers. Tibolone is a synthetic steroid hormone for menopausal women. Volunteers were treated with the 2 formulations of tibolone (total dose of active ingredient 2.5 mg) according to a 2 x 2 crossover design with a 1-week washout period. Plasma concentrations of 3alpha- and 3beta-hydroxytibolone, which are major metabolites of tibolone, were assayed in timed samples over a 24-hour period with a validated gas chromatography/mass spectrometry (GC/MS) method that had a lower limit of quantification of 0.5 ng/mL. The reference and test formulations gave a mean 3alpha-hydroxytibolone C(max) of 5.0 and 5.2 ng/mL, respectively, and a mean 3beta-hydroxytibolone C(max) of 16.4 and 16.5 ng/mL, respectively. The mean AUC(t) of 3alpha-hydroxytibolone was 24.7 and 24.3 ng h/mL, whereas the mean AUC(t) of 3beta-hydroxytibolone was 57.6 and 54.8 ng h/mL for the test and reference formulations, respectively. The authors did not find significant differences in pharmacokinetic parameters between the 2 formulations, but metabolite formation was different from reports in postmenopausal women. The authors therefore measured the effects of estradiol on the expression of the tibolone-metabolizing enzymes, from the aldo-keto reductase (AKR1C) family, using HepG2 cell (human hepatoma cells) and MCF-7 cell (human breast cancer cells). Estradiol increased mRNA levels of AKR1C1, AKR1C2, and AKR1C3 and protein levels of total AKR1C in HepG2 cells. Estradiol selectively enhanced levels of AKR1C2 mRNA in MCF-7 cells. Thus, changes in the major metabolites of tibolone might result from changes in AKR1C family expression by patient estrogen status.

  16. The Role of Human Aldo-Keto Reductases (AKRs in the Metabolic Activation and Detoxication of Polycyclic Aromatic Hydrocarbons: Interconversion of PAH-catechols and PAH o-Quinones

    Directory of Open Access Journals (Sweden)

    Li eZhang

    2012-11-01

    Full Text Available Polycyclic aromatic hydrocarbons (PAH are ubiquitous environmental pollutants. They are procarcinogens requiring metabolic activation to elicit their deleterious effects. Aldo-keto reductases (AKR catalyze the oxidation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active PAH o-quiniones. AKRs are also found to be capable of reducing PAH o-quinones to form PAH catechols. The interconversion of o-quinones and catechols results in the redox cycling of PAH o-quinones to give rise to the generation of reactive oxygen species and subsequent oxidative DNA damage. On the other hand, PAH catechols can be intercepted through phase II metabolism by which PAH o-quinones could be detoxified and eliminated. The aim of the present review is to summarize the role of human AKRs in the metabolic activation/detoxication of PAH and the relevance of phase II conjugation reactions to human lung carcinogenesis.

  17. A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli.

    Science.gov (United States)

    Agrawal, Chhavi; Sen, Sonia; Yadav, Shivam; Rai, Shweta; Rai, Lal Chand

    2015-01-01

    Present study deals with the identification of a novel aldo/keto reductase, AKR17A1 from Anabaena sp. PCC7120 and adds on as 17th family of AKR superfamily drawn from a wide variety of organisms. AKR17A1 shares many characteristics of a typical AKR such as- (i) conferring tolerance to multiple stresses like heat, UV-B, and cadmium, (ii) excellent activity towards known AKR substrates (isatin and 2-nitrobenzaldehyde), and (iii) obligate dependence on NADPH as a cofactor for enzyme activity. The most novel attribute of AKR17A1, first reported in this study, is its capability to metabolize butachlor, a persistent rice field herbicide that adversely affects agro-ecosystem and non-target organisms. The AKR17A1 catalyzed- degradation of butachlor resulted into formation of 1,2-benzene dicarboxylic acid and 2,6 bis (1,1, dimethylethyl) 4,-methyl phenol as the major products confirmed by GC-MS analysis.

  18. A Novel Aldo-Keto Reductase (AKR17A1 of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli.

    Directory of Open Access Journals (Sweden)

    Chhavi Agrawal

    Full Text Available Present study deals with the identification of a novel aldo/keto reductase, AKR17A1 from Anabaena sp. PCC7120 and adds on as 17th family of AKR superfamily drawn from a wide variety of organisms. AKR17A1 shares many characteristics of a typical AKR such as- (i conferring tolerance to multiple stresses like heat, UV-B, and cadmium, (ii excellent activity towards known AKR substrates (isatin and 2-nitrobenzaldehyde, and (iii obligate dependence on NADPH as a cofactor for enzyme activity. The most novel attribute of AKR17A1, first reported in this study, is its capability to metabolize butachlor, a persistent rice field herbicide that adversely affects agro-ecosystem and non-target organisms. The AKR17A1 catalyzed- degradation of butachlor resulted into formation of 1,2-benzene dicarboxylic acid and 2,6 bis (1,1, dimethylethyl 4,-methyl phenol as the major products confirmed by GC-MS analysis.

  19. STAR and AKR1B10 are down-regulated in high-grade endometrial cancer.

    Science.gov (United States)

    Sinreih, Maša; Štupar, Saša; Čemažar, Luka; Verdenik, Ivan; Grazio-Frković, Snježana; Smrkolj, Špela; Rižner, Tea Lanišnik

    2017-02-20

    Endometrial cancer is the most frequent gynecological malignancy in the developed world. The majority of cases are estrogen dependent, and are associated with diminished protective effects of progesterone. Endometrial cancer is also related to enhanced inflammation and decreased differentiation. In our previous studies, we examined the expression of genes involved in estrogen and progesterone actions, in inflammation and tumor differentiation, in tissue samples from endometrial cancer and adjacent control endometrium. The aims of the current study were to examine correlations between gene expression and several demographic characteristics, and to evaluate changes in gene expression with regard to histopathological and clinical characteristics of 51 patients. We studied correlations and differences in expression of 38 genes involved in five pathophysiological processes: (i) estrogen-stimulated proliferation; (ii) estrogen-dependent carcinogenesis; (iii) diminished biosynthesis of progesterone: (iv) enhanced formation of progesterone metabolites; and (v) increased inflammation and decreased differentiation. Spearman correlation coefficient analysis shows that expression of PAQR7 correlates with age, expression of SRD5A1, AKR1B1 and AKR1B10 correlate with body mass, while expression of SRD5A1 and AKR1B10 correlate with body mass index. When patients with endometrial cancer were stratified based on menopausal status, histological grade, myometrial invasion, lymphovascular invasion, and FIGO stage, Mann-Whitney U tests revealed significantly decreased expression of STAR (4.4-fold; adjusted p=0.009) and AKR1B10 (9-fold; adjusted p=0.003) in high grade versus low grade tumors. Lower levels of STAR might lead to decreased de-novo steroid hormone synthesis and tumor differentiation, and lower levels of AKR1B10 to diminished elimination of toxic electrophilic carbonyl compounds in high-grade endometrial cancer. These data thus reveal the potential STAR and AKR1B10 as

  20. Aldo-keto synthesis effect on Eu3+fluorescence in YBO3 compared with solid state diffusion

    Institute of Scientific and Technical Information of China (English)

    K.A. Koparkar; N.S. Bajaj; S.K. Omanwar

    2015-01-01

    The red-orange emitting phosphor YBO3:Eu3+was prepared by aldo-keto method and solid state diffusion. Aldo-keto method implied to decrease the processing time and heating temperature. The red-orange emitting phosphor was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), as well as emission and excitation photoluminescence spectra re-corded at room temperature. The result of aldo-keto method showed that the phosphor YBO3:Eu3+could be obtained at 900 °C in less time~60%as compared to solid state diffusion (SSD). The material showed that the strongest emission peak at 595 nm under excitation at 233 nm was only due to forced magnetic dipole 5D0→7F1 transition of Eu3+ions. Significantly, the emission inten-sity of YBO3:Eu3+phosphor prepared by aldo-keto method was relatively higher as compared to that obtained by the solid state diffusion.

  1. Pharmacological Characterization of a Novel Bifunctional Aldo-Keto Reductase 1C3 Inhibitor and Androgen Receptor Antagonist

    Science.gov (United States)

    2013-10-01

    efficacious therapy for APC. References 1. Altekruse SF , K. C., Krapcho M, Neyman N, Aminou R, Waldron W, Ruhl J, Howlader N...cancer. Br J Cancer 2004, 90, 2317- 2325. 9. Reid, A. H.; Attard, G.; Danila, D. C.; Oommen, N. B.; Olmos, D.; Fong , P. C.; Molife, L. R.; Hunt, J

  2. Long-term In Vitro Treatment of Human Glioblastoma Cells with Temozolomide Increases Resistance In Vivo through Up-regulation of GLUT Transporter and Aldo-Keto Reductase Enzyme AKR1C Expression

    Directory of Open Access Journals (Sweden)

    Benjamin Le Calvé

    2010-09-01

    Full Text Available Glioblastoma (GBM is the most frequent malignant glioma. Treatment of GBM patients is multimodal with maximum surgical resection, followed by concurrent radiation and chemotherapy with the alkylating drug temozolomide (TMZ. The present study aims to identify genes implicated in the acquired resistance of two human GBM cells of astrocytic origin, T98G and U373, to TMZ. Resistance to TMZ was induced by culturing these cells in vitro for months with incremental TMZ concentrations up to 1 mM. Only partial resistance to TMZ has been achieved and was demonstrated in vivo in immunocompromised mice bearing orthotopic U373 and T98G xenografts. Our data show that long-term treatment of human astroglioma cells with TMZ induces increased expression of facilitative glucose transporter/solute carrier GLUT/SLC2A family members, mainly GLUT-3, and of the AKR1C family of proteins. The latter proteins are phase 1 drug-metabolizing enzymes involved in the maintenance of steroid homeostasis, prostaglandin metabolism, and metabolic activation of polycyclic aromatic hydrocarbons. GLUT-3 has been previously suggested to exert roles in GBM neovascularization processes, and TMZ was found to exert antiangiogenic effects in experimental gliomas. AKR1C1 was previously shown to be associated with oncogenic potential, with proproliferative effects similar to AKR1C3 in the latter case. Both AKR1C1 and AKR1C2 proteins are involved in cancer pro-proliferative cell chemoresistance. Selective targeting of GLUT-3 in GBM and/or AKR1C proteins (by means of jasmonates, for example could thus delay the acquisition of resistance to TMZ of astroglioma cells in the context of prolonged treatment with this drug.

  3. 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.

  4. Pregnane X receptor activation and silencing promote steatosis of human hepatic cells by distinct lipogenic mechanisms.

    Science.gov (United States)

    Bitter, Andreas; Rümmele, Petra; Klein, Kathrin; Kandel, Benjamin A; Rieger, Jessica K; Nüssler, Andreas K; Zanger, Ulrich M; Trauner, Michael; Schwab, Matthias; Burk, Oliver

    2015-11-01

    In addition to its well-characterized role in the regulation of drug metabolism and transport by xenobiotics, pregnane X receptor (PXR) critically impacts on lipid homeostasis. In mice, both ligand-dependent activation and knockout of PXR were previously shown to promote hepatic steatosis. To elucidate the respective pathways in human liver, we generated clones of human hepatoma HepG2 cells exhibiting different PXR protein levels, and analyzed effects of PXR activation and knockdown on steatosis and expression of lipogenic genes. Ligand-dependent activation as well as knockdown of PXR resulted in increased steatosis in HepG2 cells. Activation of PXR induced the sterol regulatory element-binding protein (SREBP) 1-dependent lipogenic pathway via PXR-dependent induction of SREBP1a, which was confirmed in primary human hepatocytes. Inhibiting SREBP1 activity by blocking the cleavage-dependent maturation of SREBP1 protein impaired the induction of lipogenic SREBP1 target genes and triglyceride accumulation by PXR activation. On the other hand, PXR knockdown resulted in up-regulation of aldo-keto reductase (AKR) 1B10, which enhanced the acetyl-CoA carboxylase (ACC)-catalyzed reaction step of de novo lipogenesis. In a cohort of human liver samples histologically classified for non-alcoholic fatty liver disease, AKR1B10, SREBP1a and SREBP1 lipogenic target genes proved to be up-regulated in steatohepatitis, while PXR protein was reduced. In summary, our data suggest that activation and knockdown of PXR in human hepatic cells promote de novo lipogenesis and steatosis by induction of the SREBP1 pathway and AKR1B10-mediated increase of ACC activity, respectively, thus providing mechanistic explanations for a putative dual role of PXR in the pathogenesis of steatohepatitis.

  5. 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.

  6. Reductive detoxification of acrolein as a potential role for aldehyde reductase (AKR1A) in mammals.

    Science.gov (United States)

    Kurahashi, Toshihiro; Kwon, Myoungsu; Homma, Takujiro; Saito, Yuka; Lee, Jaeyong; Takahashi, Motoko; Yamada, Ken-Ichi; Miyata, Satoshi; Fujii, Junichi

    2014-09-12

    Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20% that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects.

  7. 5β-Reduced steroids and human Δ(4)-3-ketosteroid 5β-reductase (AKR1D1).

    Science.gov (United States)

    Chen, Mo; Penning, Trevor M

    2014-05-01

    5β-Reduced steroids are non-planar steroids that have a 90° bend in their structure to create an A/B cis-ring junction. This novel property is required for bile-acids to act as emulsifiers, but in addition 5β-reduced steroids have remarkable physiology and may act as potent tocolytic agents, endogenous cardiac glycosides, neurosteroids, and can act as ligands for orphan and membrane bound receptors. In humans there is only a single 5β-reductase gene AKR1D1, which encodes Δ(4)-3-ketosteroid-5β-reductase (AKR1D1). This enzyme is a member of the aldo-keto reductase superfamily, but possesses an altered catalytic tetrad, in which Glu120 replaces the conserved His residue. This predominant liver enzyme generates all 5β-dihydrosteroids in the C19-C27 steroid series. Mutations exist in the AKR1D1 gene, which result in loss of protein stability and are causative in bile-acid deficiency.

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

    Science.gov (United States)

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

    2016-01-01

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

  9. Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae.

    Science.gov (United States)

    Zhang, Min; Jiang, Shao-tong; Zheng, Zhi; Li, Xing-jiang; Luo, Shui-zhong; Wu, Xue-feng

    2015-07-01

    Rhizopus oryzae is valuable as a producer of organic acids via lignocellulose catalysis. R. oryzae metabolizes xylose, which is one component of lignocellulose hydrolysate. In this study, a novel NADPH-dependent xylose reductase gene from R. oryzae AS 3.819 (Roxr) was cloned and expressed in Pichia pastoris GS115. Homology alignment suggested that the 320-residue protein contained domains and active sites belonging to the aldo/keto reductase family. SDS-PAGE demonstrated that the recombinant xylose reductase has a molecular weight of approximately 37 kDa. The optimal catalytic pH and temperature of the purified recombinant protein were 5.8 and 50 °C, respectively. The recombinant protein was stable from pH 4.4 to 6.5 and at temperatures below 42 °C. The recombinant enzyme has bias for D-xylose and L-arabinose as substrates and NADPH as its coenzyme. Real-time quantitative reverse transcription PCR tests suggested that native Roxr expression is regulated by a carbon catabolite repression mechanism. Site-directed mutagenesis at two possible key sites involved in coenzyme binding, Thr(226)  → Glu(226) and Val(274)  → Asn(274), were performed, respectively. The coenzyme specificity constants of the resulted RoXR(T226E) and RoXR(V274N) for NADH increased 18.2-fold and 2.4-fold, which suggested possibility to improve the NADH preference of this enzyme through genetic modification.

  10. Enzymes of the AKR1B and AKR1C subfamilies and uterine diseases

    Directory of Open Access Journals (Sweden)

    Tea eLanisnik Rizner

    2012-03-01

    Full Text Available Endometrial and cervical cancers, uterine myoma, and endometriosis are very common uterine diseases. Worldwide, more than 800,000 women are affected annually by gynecological cancers, as a result of which, more than 360,000 die. During their reproductive age, about 70% of women develop uterine myomas, 10% to 15% suffer from endometriosis, and 35% to 50% from infertility associated with endometriosis. Uterine diseases are associated with aberrant inflammatory responses and concomitant increased production of prostaglandins (PG. They are also related to decreased differentiation, due to low levels of protective progesterone and retinoic acid, and to enhanced proliferation, due to high local concentrations of estrogens. The pathogenesis of these diseases can thus be attributed to disturbed PG, estrogen and retinoid metabolism and actions. Five human members of the aldo-keto reductase 1B (AKR1B and 1C (AKR1C superfamilies, i.e., AKR1B1, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, have roles in these processes and can thus be implicated in uterine diseases. AKR1B1 and AKR1C3 catalyze the formation of PGF2alpha which stimulates cell proliferation. AKR1C3 converts PGD2 to 9alpha,11beta-PGF2, and thus counteracts the formation of 15deoxy-PGJ2, which can activate pro-apoptotic peroxisome-proliferator-activated receptor beta. AKR1B10 catalyzes the reduction of retinal to retinol, and in thus lessens the formation of retinoic acid, with potential pro-differentiating actions. The AKR1C1-AKR1C3 enzymes also act as 17-keto- and 20-ketosteroid reductases to varying extents, and are implicated in increased estradiol and decreased progesterone levels. This review comprises a short introduction to uterine diseases, followed by an overview of the current literature on the AKR1B and AKR1C expression in the uterus and in uterine diseases. The potential implications of the AKR1B and AKR1C enzymes and their pathophysiologies are then discussed, followed by conclusions and

  11. Insights from modeling the 3D structure of NAD(P)H-dependent D-xylose reductase of Pichia stipitis and its binding interactions with NAD and NADP.

    Science.gov (United States)

    Wang, Jing-Fang; Wei, Dong-Qing; Lin, Ying; Wang, Yong-Hua; Du, Hong-Li; Li, Yi-Xve; Chou, Kuo-Chen

    2007-07-27

    NAD(P)H-dependent d-xylose reductase is a homodimeric oxidoreductase that belongs to the aldo-keto reductase superfamily. The enzyme has the special function to catalyze the first step in the assimilation of xylose into yeast metabolic pathways. Performing this function via reducing the open chain xylose to xylitol, the xylose reductase of Pichia stipitis is one of the most important enzymes that can be used to construct recombinant Saccharomyces cerevisiae strain for utilizing xylose and producing alcohol. To investigate into the interaction mechanism of the enzyme with its ligand NAD and NADP, the 3D structure was developed for the NAD(P)H-dependent d-xylose reductase from P. stipitis. With the 3D structure, the molecular docking operations were conducted to find the most stable bindings of the enzyme with NAD and NADP, respectively. Based on these results, the binding pockets of the enzyme for NAD and NADP have been explicitly defined. It has been found that the residues in forming the binding pockets for both NAD and NADP are almost the same and mainly hydrophilic. These findings may be used to guide mutagenesis studies, providing useful clues to modify the enzyme to improve the utilization of xylose for producing alcohol. Also, because human aldose reductases have the function to reduce the open chain form of glucose to sorbitol, a process physiologically significant for diabetic patients at the time that their blood glucose levels are elevated, the information gained through this study may also stimulate the development of new strategies for therapeutic treatment of diabetes.

  12. Detoxifying enzymes at the cross-roads of inflammation, oxidative stress and drug hypersensitivity: role of glutathione transferase P1-1 and aldose reductase

    Directory of Open Access Journals (Sweden)

    Francisco J Sánchez-Gómez

    2016-08-01

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

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

    Directory of Open Access Journals (Sweden)

    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

  14. Conversion of Human Steroid 5[beta]-Reductase (AKR1D1) into 3[beta]-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H: Example of Perfect Enzyme Engineering

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Mo; Drury, Jason E.; Christianson, David W.; Penning, Trevor M. (UPENN)

    2012-10-10

    Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5{beta}-reduction of {Delta}{sup 4}-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His120 (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5{beta}-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5{alpha}-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3{beta}-HSD as opposed to a 3{alpha}-HSD. The catalytic efficiency achieved for 3{beta}-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5{beta}-dihydrotestosterone, and {Delta}{sup 4}-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the {Delta}{sup 4}-double bond and confers 3{beta}-HSD activity on the 5{beta}-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its {alpha}-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.

  15. CHARACTERIZATION OF STABLE BENZOLALPYRENE-7,8-QUINONE-DNA ADDUCTS IN CALF THYMUS DNA AND POLYDEOXYNUCLEOTIDES

    Science.gov (United States)

    Bcnzo[a]pyrene-7,8-dione (BPQ) is a reactive aldo-keto reductase-mediated product of B[a]P-7,8-diol, a major P450/epoxide hydrolase metabolite of the multi-species carcinogen, B[a]P. The role of BPQ in B[a]P's genotoxicity and carcinogenesis is evolving. Toxicity pathways involvi...

  16. CHARACTERIZATION OF STABLE BENZO(A)PYRENE-7,8-QUINONE-DNA ADDUCTS IN CALF THYMUS DNA

    Science.gov (United States)

    Benzo[alpyrene-7,8-dione (BPQ) is a reactive aldo-keto reductase-mediated product of B[a]P-7,8-diol, a major P450/epoxide hydrolase metabolite of the multi-species carcinogen, B[a]P. The role of BPQ in B[a]P's genotoxicity and carcinogenesis is evolving. Toxicity pathways involvi...

  17. Maternal and offspring genetic variants of AKR1C3 and the risk of childhood leukemia

    OpenAIRE

    Liu, Chen-Yu; Hsu, Yi-Hsiang; Pan, Pi-Chen; Wu, Ming-Tsang; Ho, Chi-Kung; Su, Li; Xu, Xin; Li, Yi; Christiani, David C.

    2008-01-01

    The aldo-keto reductase 1C3 (AKR1C3) gene located on chromosome 10p15-p14, a regulator of myeloid cell proliferation and differentiation, represents an important candidate gene for studying human carcinogenesis. In a prospectively enrolled population-based case–control study of Han Chinese conducted in Kaohsiung in southern Taiwan, a total of 114 leukemia cases and 221 controls

  18. AcEST: DK957526 [AcEST

    Lifescience Database Archive (English)

    Full Text Available WD-40 repeat-containing protein MSI5 OS=Ara... 32 1.9 sp|Q5T2L2|AKCL1_HUMAN Aldo-keto reductase family 1 mem...EDVAFCPSSA--- 285 Query: 235 QQTCTVSSDSCLPL-DARQKRAP 300 Q+ C+V DSCL L DAR +P Sbjct: 286 QEFCSVGDDSCLMLWDARTGTSP 308 >sp|Q5T2L2

  19. 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.

  20. 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.

  1. Drug interaction of boceprevir and amlodipine in a patient with hepatitis C: A cardiovascular follow-up

    Directory of Open Access Journals (Sweden)

    Çiğdem Kader

    2015-03-01

    Full Text Available Boceprevir is a NS3/4A hepatitis C virus (HCV protease inhibitor, used in combination with peginterferon and ribavirin to treat HCV. Boceprevir undergoes extensive metabolism via cytochrome P450-mediated oxidation and ketoreduction by cytosolic aldo-keto reductases. Amlodipine has been used for the treatment of patients with hypertension and also metabolised through cytochrome P450 pathway. Here, we presented a case of boceprevir and amlodipine interaction in a patient with chronic HCV and her echocardiography and electrocardiographic follow-up results. J Microbiol Infect Dis 2015;5(1: 32-35

  2. 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

  3. Metabolism of bupropion by baboon hepatic and placental microsomes.

    Science.gov (United States)

    Wang, Xiaoming; Abdelrahman, Doaa R; Fokina, Valentina M; Hankins, Gary D V; Ahmed, Mahmoud S; Nanovskaya, Tatiana N

    2011-08-01

    The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (Km 36±6 μM, Vmax 258±32 pmol mg protein(-1) min(-1)), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent Km values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11β-hydroxysteroid dehydrogenases (18β-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB.

  4. 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.

  5. 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.

  6. Depressed levels of prostaglandin F2α in mice lacking Akr1b7 increase basal adiposity and predispose to diet-induced obesity.

    Science.gov (United States)

    Volat, Fanny E; Pointud, Jean-Christophe; Pastel, Emilie; Morio, Béatrice; Sion, Benoit; Hamard, Ghislaine; Guichardant, Michel; Colas, Romain; Lefrançois-Martinez, Anne-Marie; Martinez, Antoine

    2012-11-01

    Negative regulators of white adipose tissue (WAT) expansion are poorly documented in vivo. Prostaglandin F(2α) (PGF(2α)) is a potent antiadipogenic factor in cultured preadipocytes, but evidence for its involvement in physiological context is lacking. We previously reported that Akr1b7, an aldo-keto reductase enriched in adipose stromal vascular fraction but absent from mature adipocytes, has antiadipogenic properties possibly supported by PGF(2α) synthase activity. To test whether lack of Akr1b7 could influence WAT homeostasis in vivo, we generated Akr1b7(-/-) mice in 129/Sv background. Akr1b7(-/-) mice displayed excessive basal adiposity resulting from adipocyte hyperplasia/hypertrophy and exhibited greater sensitivity to diet-induced obesity. Following adipose enlargement and irrespective of the diet, they developed liver steatosis and progressive insulin resistance. Akr1b7 loss was associated with decreased PGF(2α) WAT contents. Cloprostenol (PGF(2α) agonist) administration to Akr1b7(-/-) mice normalized WAT expansion by affecting both de novo adipocyte differentiation and size. Treatment of 3T3-L1 adipocytes and Akr1b7(-/-) mice with cloprostenol suggested that decreased adipocyte size resulted from inhibition of lipogenic gene expression. Hence, Akr1b7 is a major regulator of WAT development through at least two PGF(2α)-dependent mechanisms: inhibition of adipogenesis and lipogenesis. These findings provide molecular rationale to explore the status of aldo-keto reductases in dysregulations of adipose tissue homeostasis.

  7. Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae.

    Science.gov (United States)

    Jirschitzka, Jan; Schmidt, Gregor W; Reichelt, Michael; Schneider, Bernd; Gershenzon, Jonathan; D'Auria, John Charles

    2012-06-26

    The pharmacologically important tropane alkaloids have a scattered distribution among angiosperm families, like many other groups of secondary metabolites. To determine whether tropane alkaloids have evolved repeatedly in different lineages or arise from an ancestral pathway that has been lost in most lines, we investigated the tropinone-reduction step of their biosynthesis. In species of the Solanaceae, which produce compounds such as atropine and scopolamine, this reaction is known to be catalyzed by enzymes of the short-chain dehydrogenase/reductase family. However, in Erythroxylum coca (Erythroxylaceae), which accumulates cocaine and other tropane alkaloids, no proteins of the short-chain dehydrogenase/reductase family were found that could catalyze this reaction. Instead, purification of E. coca tropinone-reduction activity and cloning of the corresponding gene revealed that a protein of the aldo-keto reductase family carries out this reaction in E. coca. This protein, designated methylecgonone reductase, converts methylecgonone to methylecgonine, the penultimate step in cocaine biosynthesis. The protein has highest sequence similarity to other aldo-keto reductases, such as chalcone reductase, an enzyme of flavonoid biosynthesis, and codeinone reductase, an enzyme of morphine alkaloid biosynthesis. Methylecgonone reductase reduces methylecgonone (2-carbomethoxy-3-tropinone) stereospecifically to 2-carbomethoxy-3β-tropine (methylecgonine), and has its highest activity, protein level, and gene transcript level in young, expanding leaves of E. coca. This enzyme is not found at all in root tissues, which are the site of tropane alkaloid biosynthesis in the Solanaceae. This evidence supports the theory that the ability to produce tropane alkaloids has arisen more than once during the evolution of the angiosperms.

  8. 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.

  9. 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.

  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. Plant science. Morphinan biosynthesis in opium poppy requires a P450-oxidoreductase fusion protein.

    Science.gov (United States)

    Winzer, Thilo; Kern, Marcelo; King, Andrew J; Larson, Tony R; Teodor, Roxana I; Donninger, Samantha L; Li, Yi; Dowle, Adam A; Cartwright, Jared; Bates, Rachel; Ashford, David; Thomas, Jerry; Walker, Carol; Bowser, Tim A; Graham, Ian A

    2015-07-17

    Morphinan alkaloids from the opium poppy are used for pain relief. The direction of metabolites to morphinan biosynthesis requires isomerization of (S)- to (R)-reticuline. Characterization of high-reticuline poppy mutants revealed a genetic locus, designated STORR [(S)- to (R)-reticuline] that encodes both cytochrome P450 and oxidoreductase modules, the latter belonging to the aldo-keto reductase family. Metabolite analysis of mutant alleles and heterologous expression demonstrate that the P450 module is responsible for the conversion of (S)-reticuline to 1,2-dehydroreticuline, whereas the oxidoreductase module converts 1,2-dehydroreticuline to (R)-reticuline rather than functioning as a P450 redox partner. Proteomic analysis confirmed that these two modules are contained on a single polypeptide in vivo. This modular assembly implies a selection pressure favoring substrate channeling. The fusion protein STORR may enable microbial-based morphinan production.

  12. Cold acclimation induced genes of trifoliate orange (Poncirus trifoliata).

    Science.gov (United States)

    Zhang, Can-kui; Lang, Ping; Dane, Fenny; Ebel, Robert C; Singh, Narendra K; Locy, Robert D; Dozier, William A

    2005-03-01

    Commercial citrus varieties are sensitive to low temperature. Poncirus trifoliata is a close relative of Citrus species and has been widely used as a cold-hardy rootstock for citrus production in low-temperature environments. mRNA differential display-reverse transcription (DDRT)-PCR and quantitative relative-RT-PCR were used to study gene expression of P. trifoliata under a gradual cold-acclimation temperature regime. Eight up-regulated cDNA fragments were isolated and sequenced. These fragments showed high similarities at the amino acid level to the following genes with known functions: betaine/proline transporter, water channel protein, aldo-keto reductase, early light-induced protein, nitrate transporter, tetratricopeptide-repeat protein, F-box protein, and ribosomal protein L15. These cold-acclimation up-regulated genes in P. trifoliata are also regulated by osmotic and photo-oxidative signals in other plants.

  13. 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.

  14. Biliverdin Reductase: a Target for Cancer Therapy?

    Directory of Open Access Journals (Sweden)

    Peter eGibbs

    2015-06-01

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

  15. 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

  16. 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.

  17. 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...

  18. 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.

  19. 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.

  20. 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.

  1. Methylenetetrahydrofolate Reductase Activity and Folate Metabolism

    Directory of Open Access Journals (Sweden)

    Nursen Keser

    2014-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Yeon Bok Kim

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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...

  12. 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

  13. 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...

  14. 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

  15. 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...

  16. 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.

  17. 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.

  18. 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.

  19. (+)-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.

  20. 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.

  1. Development of a CART Model to Predict the Synthesis of Cardiotoxic Daunorubicinol in Heart Tissue Samples From Donors With and Without Down Syndrome.

    Science.gov (United States)

    Hoefer, Carrie C; Blair, Rachael Hageman; Blanco, Javier G

    2016-06-01

    Daunorubicin (DAUN) and doxorubicin (DOX) are used to treat a variety of cancers. The use of DAUN and DOX is hampered by the development of cardiotoxicity. Clinical evidence suggests that patients with leukemia and Down syndrome are at increased risk for anthracycline-related cardiotoxicity. Carbonyl reductases and aldo-keto reductases (AKRs) catalyze the reduction of DAUN and DOX into cardiotoxic C-13 alcohol metabolites. Anthracyclines also exert cardiotoxicity by triggering mitochondrial dysfunction. In recent studies, a collection of heart samples from donors with and without Down syndrome was used to investigate determinants for anthracycline-related cardiotoxicity including cardiac daunorubicin reductase activity (DA), carbonyl reductase/AKRs protein expression, mitochondrial DNA content (mtDNA), and AKR7A2 DNA methylation status. In this study, the available demographic, biochemical, genetic, and epigenetic data were integrated through classification and regression trees analysis with the aim of pinpointing the most relevant variables for the synthesis of cardiotoxic daunorubicinol (i.e., DA). Seventeen variables were considered as potential predictors. Leave-one-out-cross-validation was performed for model selection and to estimate the generalization error. The classification and regression trees analysis model and variable importance measures suggest that cardiac mtDNA content, mtDNA(4977) deletion frequency, and AKR7A2 protein content are the most important variables in determining DA.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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 ...

  14. 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...

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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...

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Multiple abiotic stress tolerance in Vigna mungo is altered by overexpression of ALDRXV4 gene via reactive carbonyl detoxification.

    Science.gov (United States)

    Singh, Preeti; Kumar, Deepak; Sarin, Neera Bhalla

    2016-06-01

    Vigna mungo (blackgram) is an important leguminous pulse crop, which is grown for its protein rich edible seeds. Drought and salinity are the major abiotic stresses which adversely affect the growth and productivity of crop plants including blackgram. The ALDRXV4 belongs to the aldo-keto reductase superfamily of enzymes that catalyze the reduction of carbonyl metabolites in the cells and plays an important role in the osmoprotection and detoxification of the reactive carbonyl species. In the present study, we developed transgenic plants of V. mungo using Agrobacterium mediated transformation. The transgene integration was confirmed by Southern blot analysis whereas the expression was confirmed by RT-PCR, Western blot and enzyme activity. The T1 generation transgenic plants displayed improved tolerance to various environmental stresses, including drought, salt, methyl viologen and H2O2 induced oxidative stress. The increased aldose reductase activity, higher sorbitol content and less accumulation of the toxic metabolite, methylglyoxal in the transgenic lines under non-stress and stress (drought and salinity) conditions resulted in increased protection through maintenance of better photosynthetic efficiency, higher relative water content and less photooxidative damage. The accumulation of reactive oxygen species was remarkably decreased in the transgenic lines as compared with the wild type plants. This study of engineering multiple stress tolerance in blackgram, is the first report to date and this strategy for trait improvement is proposed to provide a novel germplasm for blackgram production on marginal lands.

  7. Novel oxime-bearing coumarin derivatives act as potent Nrf2/ARE activators in vitro and in mouse model.

    Science.gov (United States)

    Chang, Ken-Ming; Chen, Huang-Hui; Wang, Tai-Chi; Chen, I-Li; Chen, Yu-Tsen; Yang, Shyh-Chyun; Chen, Yeh-Long; Chang, Hsin-Huei; Huang, Chih-Hsiang; Chang, Jang-Yang; Shih, Chuan; Kuo, Ching-Chuan; Tzeng, Cherng-Chyi

    2015-12-01

    We have designed and synthesized certain novel oxime- and amide-bearing coumarin derivatives as nuclear factor erythroid 2 p45-related factor 2 (Nrf2) activators. The potency of these compounds was measured by antioxidant responsive element (ARE)-driven luciferase activity, level of Nrf2-related cytoprotective genes and proteins, and antioxidant activity. Among them, (Z)-3-(2-(hydroxyimino)-2-phenylethoxy)-2H-chromen-2-one (17a) was the most active, and more potent than the positive t-BHQ in the induction of ARE-driven luciferase activity. Exposure of HSC-3 cells to various concentrations of 17a strongly increased Nrf2 nuclear translocation and the expression level of Nrf2-mediated cytoprotective proteins in a concentration-dependent manner. HSC-3 cells pretreated with 17a significantly reduced t-BOOH-induced oxidative stress. In the animal experiment, Nrf2-mediated cytoprotective proteins, such as aldo-keto reductase 1 subunit C-1 (AKR1C1), glutathione reductase (GR), and heme oxygenase (HO-1), were obviously elevated in the liver of 17a-treated mice than that of control. These results suggested that novel oxime-bearing coumarin 17a is able to activate Nrf2/ARE pathway in vivo and are therefore seen as a promising candidate for further investigation.

  8. Sulforaphane promotes murine hair growth by accelerating the degradation of dihydrotestosterone.

    Science.gov (United States)

    Sasaki, Mari; Shinozaki, Shohei; Shimokado, Kentaro

    2016-03-25

    Dihydrotestosterone (DHT) causes the regression of human hair follicles in the parietal scalp, leading to androgenic alopecia (AGA). Sulforaphane (SFN) increases the expression of DHT degrading enzymes, such as 3α-hydroxysteroid dehydrogenases (3α-HSDs), and, therefore, SFN treatment may improve AGA. To determine the effects of SFN on hair growth, we administered SFN (10 mg/kg BW, IP) or vehicle (DMSO) to ob/ob mice for six weeks and examined hair regeneration and the plasma levels of testosterone and DHT. We also tested the effects of SFN on the expression of two forms of 3α-HSD, aldo-keto reductase 1c21 and dehydrogenase/reductase (SDR family) member 9, both in vitro and in vivo. SNF significantly enhanced hair regeneration in ob/ob mice. The mice treated with SFN showed lower plasma levels of testosterone and DHT than those treated with vehicle. SFN increased the mRNA and protein levels of the two forms of 3α-HSD in the liver of the mice and in cultured murine hepatocyte Hepa1c1c7 cells. These results suggest that SFN treatment increases the amount of 3α-HSDs in the liver, accelerates the degradation of blood DHT, and subsequently blocks the suppression of hair growth by DHT.

  9. 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.

  10. 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.

  11. 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...

  12. 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...

  13. 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.

  14. 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.

  15. 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.

  16. 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.

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

    Science.gov (United States)

    Ozturk, Levent; Yazici, Atilla; Eker, Selim; Gokmen, Ozgur; Römheld, Volker; Cakmak, Ismail

    2008-01-01

    Iron (Fe) deficiency is increasingly being observed in cropping systems with frequent glyphosate applications. A likely reason for this is that glyphosate interferes with root uptake of Fe by inhibiting ferric reductase in roots required for Fe acquisition by dicot and nongrass species. This study investigated the role of drift rates of glyphosate (0.32, 0.95 or 1.89 mm glyphosate corresponding to 1, 3 and 6% of the recommended herbicidal dose, respectively) on ferric reductase activity of sunflower (Helianthus annuus) roots grown under Fe deficiency conditions. Application of 1.89 mm glyphosate resulted in almost 50% inhibition of ferric reductase within 6 h and complete inhibition 24 h after the treatment. Even at lower rates of glyphosate (e.g. 0.32 mm and 0.95 mm), ferric reductase was inhibited. Soluble sugar concentration and the NAD(P)H oxidizing capacity of apical roots were not decreased by the glyphosate applications. To our knowledge, this is the first study reporting the effects of glyphosate on ferric reductase activity. The nature of the inhibitory effect of glyphosate on ferric reductase could not be identified. Impaired ferric reductase could be a major reason for the increasingly observed Fe deficiency in cropping systems associated with widespread glyphosate usage.

  18. Role of carbonyl reducing enzymes in the phase I biotransformation of the non-steroidal anti-inflammatory drug nabumetone in vitro.

    Science.gov (United States)

    Skarydova, Lucie; Nobilis, Milan; Wsól, Vladimir

    2013-04-01

    1. Nabumetone is a clinically used non-steroidal anti-inflammatory drug, its biotransformation includes major active metabolite 6-methoxy-2-naphtylacetic acid and another three phase I as well as corresponding phase II metabolites which are regarded as inactive. One important biotransformation pathway is carbonyl reduction, which leads to the phase I metabolite, reduced nabumetone. 2. The aim of this study is the determination of the role of a particular human liver subcellular fraction in the nabumetone reduction and the identification of participating carbonyl reducing enzymes along with their stereospecificities. 3. Both subcellular fractions take part in the carbonyl reduction of nabumetone and the reduction is at least in vitro the main biotransformation pathway. The activities of eight cytosolic carbonyl reducing enzymes--CBR1, CBR3, AKR1B1, AKR1B10, AKR1C1-4--toward nabumetone were tested. Except for CBR3, all tested reductases transform nabumetone to its reduced metabolite. AKR1C4 and AKR1C3 have the highest intrinsic clearances. 4. The stereospecificity of the majority of the tested enzymes is shifted to the production of an (+)-enantiomer of reduced nabumetone; only AKR1C1 and AKR1C4 produce predominantly an (-)-enantiomer. This project provides for the first time evidence that seven specific carbonyl reducing enzymes participate in nabumetone metabolism.

  19. UNC50 prompts G1/S transition and proliferation in HCC by regulation of epidermal growth factor receptor trafficking.

    Directory of Open Access Journals (Sweden)

    Zhou Fang

    Full Text Available UNC50 has long been recognized as a Golgi apparatus protein in yeast, and is involved in nicotinic receptor trafficking in Caenorhabditis elegans, but little is known about UNC50 gene function in human biology despite it being conserved from yeast to high eukaryotes.We investigated the relation between UNC50 and human hepatocellular carcinoma (HCC and the potential mechanisms underlying HCC development.UNC50 mRNA expression patterns in 12 HCC and adjacent non-cancerous tissues determined using northern blotting were confirmed by real-time PCR in another 44 paired tissues. Microarray experiments were used to screen for global effects of UNC50 knockdown in the Hep3B cell line, and were confirmed by real-time PCR, western blotting, flow cytometry, and tetrazolium assay in both UNC50 overexpression and knockdown Hep3B cells.UNC50 expression levels were upregulated in HCC tissues in comparison with the adjacent non-cancerous tissues. UNC50 knockdown reduced mRNA levels of the downstream targets of the epidermal growth factor receptor (EGFR pathway: cyclin D1 (CCND1, EGF, matrix metalloproteinase-7 (MMP7, aldose reductase-like 1 (AKR1B10, cell surface-associated mucin 1 (MUC1, and gastrin (GAST. Moreover, UNC50 influenced EGF, inducing cell cycle entry by affecting cell surface EGFR amounts.UNC50 may plays some roles in HCC progression by affecting the EGFR pathway.

  20. 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

  1. 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

  2. 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.

  3. 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...

  4. 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.

  5. 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...

  6. 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

  7. 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.

  8. 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.

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

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    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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. Identification and Validation of HCC-specific Gene Transcriptional Signature for Tumor Antigen Discovery.

    Science.gov (United States)

    Petrizzo, Annacarmen; Caruso, Francesca Pia; Tagliamonte, Maria; Tornesello, Maria Lina; Ceccarelli, Michele; Costa, Valerio; Aprile, Marianna; Esposito, Roberta; Ciliberto, Gennaro; Buonaguro, Franco M; Buonaguro, Luigi

    2016-07-08

    A novel two-step bioinformatics strategy was applied for identification of signatures with therapeutic implications in hepatitis-associated HCC. Transcriptional profiles from HBV- and HCV-associated HCC samples were compared with non-tumor liver controls. Resulting HCC modulated genes were subsequently compared with different non-tumor tissue samples. Two related signatures were identified, namely "HCC-associated" and "HCC-specific". Expression data were validated by RNA-Seq analysis carried out on unrelated HCC samples and protein expression was confirmed according to The Human Protein Atlas" (http://proteinatlas.org/), a public repository of immunohistochemistry data. Among all, aldo-keto reductase family 1 member B10, and IGF2 mRNA-binding protein 3 were found strictly HCC-specific with no expression in 18/20 normal tissues. Target peptides for vaccine design were predicted for both proteins associated with the most prevalent HLA-class I and II alleles. The described novel strategy showed to be feasible for identification of HCC-specific proteins as highly potential target for HCC immunotherapy.

  18. De novo Sequencing and Analysis of Lemongrass Transcriptome Provides First Insights into the Essential Oil Biosynthesis of Aromatic Grasses

    Directory of Open Access Journals (Sweden)

    Seema Meena

    2016-07-01

    Full Text Available Aromatic grasses of the genus Cymbopogon (Poaceae family represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavour, fragrance, cosmetic and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step towards understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases (TPS, pyrophosphatases (PPase, alcohol dehydrogenases (ADH, aldo-keto reductases (AKR, carotenoid cleavage dioxygenases (CCD, alcohol acetyltransferases (AAT and aldehyde dehydrogenases (ALDH, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes and acetates. Molecular modeling and docking further supported the role of identified enzymes in aroma formation in Cymbopogon. Also, simple sequence repeats (SSRs were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition.

  19. Oxidative Stress and Carbonyl Lesions in Ulcerative Colitis and Associated Colorectal Cancer

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    Zhiqi Wang

    2016-01-01

    Full Text Available Oxidative stress has long been known as a pathogenic factor of ulcerative colitis (UC and colitis-associated colorectal cancer (CAC, but the effects of secondary carbonyl lesions receive less emphasis. In inflammatory conditions, reactive oxygen species (ROS, such as superoxide anion free radical (O2∙-, hydrogen peroxide (H2O2, and hydroxyl radical (HO∙, are produced at high levels and accumulated to cause oxidative stress (OS. In oxidative status, accumulated ROS can cause protein dysfunction and DNA damage, leading to gene mutations and cell death. Accumulated ROS could also act as chemical messengers to activate signaling pathways, such as NF-κB and p38 MAPK, to affect cell proliferation, differentiation, and apoptosis. More importantly, electrophilic carbonyl compounds produced by lipid peroxidation may function as secondary pathogenic factors, causing further protein and membrane lesions. This may in turn exaggerate oxidative stress, forming a vicious cycle. Electrophilic carbonyls could also cause DNA mutations and breaks, driving malignant progression of UC. The secondary lesions caused by carbonyl compounds may be exceptionally important in the case of host carbonyl defensive system deficit, such as aldo-keto reductase 1B10 deficiency. This review article updates the current understanding of oxidative stress and carbonyl lesions in the development and progression of UC and CAC.

  20. Neurosteroid biosynthetic pathways changes in prefrontal cortex in Alzheimer's disease.

    Science.gov (United States)

    Luchetti, Sabina; Bossers, Koen; Van de Bilt, Saskia; Agrapart, Vincent; Morales, Rafael Ramirez; Frajese, Giovanni Vanni; Swaab, Dick F

    2011-11-01

    Expression of the genes for enzymes involved in neurosteroid biosynthesis was studied in human prefrontal cortex (PFC) in the course of Alzheimer's disease (AD) (n=49). Quantitative RT-PCR (qPCR) revealed that mRNA levels of diazepam binding inhibitor (DBI), which is involved in the first step of steroidogenesis and in GABAergic transmission, were increased, as were mRNA levels for several neurosteroid biosynthetic enzymes. Aromatase, 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) and aldo-keto reductase 1C2 (AKR1C2), were all increased in the late stages of AD. Several GABA-A subunits were significantly reduced in AD. Increased expression of aromatase in the PFC was confirmed by immunohistochemistry and was found to be localized predominantly in astrocytes. These data suggest a role for estrogens and allopregnanolone produced by astrocytes in the PFC in AD, possibly as part of a rescue program. The reduced gene expression of some synaptic and extra-synaptic GABA-A subunits may indicate a deficit of modulation of GABA-A receptors by neuroactive steroids, which may contribute to the neuropsychiatric characteristics of this disease.

  1. Involvement of an octose ketoreductase and two acyltransferases in the biosynthesis of paulomycins

    Science.gov (United States)

    Li, Jine; Wang, Min; Ding, Yong; Tang, Yue; Zhang, Zhiguo; Chen, Yihua

    2016-02-01

    C-4 hydroxyethyl branched octoses have been observed in polysaccharides of several genera of gram negative bacteria and in various antibiotics produced by gram positive bacteria. The C-4 hydroxyethyl branch was proposed to be converted from C-4 acetyl branch by an uncharacterized ketoreduction step. Paulomycins (PAUs) are glycosylated antibiotics with potent inhibitory activity against gram positive bacteria and are structurally defined by its unique C-4‧ hydroxyethyl branched paulomycose moiety. A novel aldo-keto-reductase, Pau7 was characterized as the enzyme catalyzing the stereospecific ketoreduction of 7‧-keto of PAU E (1) to give the C-4‧ hydroxyethyl branched paulomycose moiety of PAU F (2). An acyltransferase Pau6 further decorates the C-4‧ hydroxyethyl branch of paulomycose moiety of 2 by attaching various fatty acyl chains to 7‧-OH to generate diverse PAUs. In addition, another acyltransferase Pau24 was proposed to be responsible for the 13-O-acetylation of PAUs.

  2. Cooperative function of antioxidant and redox systems against oxidative stress in male reproductive tissues

    Institute of Scientific and Technical Information of China (English)

    JunichiFujii; YoshihitoIuchi; ShingoMatsuki; TatsuyaIshii

    2003-01-01

    Reactive oxygen species (ROS) are produced under oxidative stress, such as high oxygen concentration and during the metabolic consumption of oxygen molecules. Male reproductive tissues appear to be continuously exposed to ROS produced by active metabolism. In addition, spermatozoa must pass through a high oxygen environ-ment during the mating process. Thus, to maintain viable reproductive ability, a protective mechanism against oxida-tive stress is of importance. Here, we overview our current understanding of the cooperative function of antioxidative and redox systems that are involved in male fertility. Superoxide dismutase and glutathione peroxidase are major enzymes that scavenge harmful ROS in male reproductive organs. In turn, glutathione and thioredoxin systems constitute the main redox systems that repair oxidized and damaged molecules and also play a role in regulating a variety of cellular functions. While glutathione functions as an antioxidant by donating electrons to glutathione peroxidase and thioredoxin donates electrons to peroxiredoxin as a counterpart of glutathione peroxidase. In addition,aldo-keto reductases, which detoxify carbonyl compounds produced by oxidative stress, are present at high levels in the epithelia of the genital tract and Sertoli cells of the testis. Since these systems are involved in cross-talk, a comprehensive understanding will be required to maintain the physiological functions of male reproductive system.( Asian J Andro12003 Sep; 5: 231-242)

  3. Methyl Jasmonate: Putative Mechanisms of Action on Cancer Cells Cycle, Metabolism, and Apoptosis

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    Italo Mario Cesari

    2014-01-01

    Full Text Available Methyl jasmonate (MJ, an oxylipid that induces defense-related mechanisms in plants, has been shown to be active against cancer cells both in vitro and in vivo, without affecting normal cells. Here we review most of the described MJ activities in an attempt to get an integrated view and better understanding of its multifaceted modes of action. MJ (1 arrests cell cycle, inhibiting cell growth and proliferation, (2 causes cell death through the intrinsic/extrinsic proapoptotic, p53-independent apoptotic, and nonapoptotic (necrosis pathways, (3 detaches hexokinase from the voltage-dependent anion channel, dissociating glycolytic and mitochondrial functions, decreasing the mitochondrial membrane potential, favoring cytochrome c release and ATP depletion, activating pro-apoptotic, and inactivating antiapoptotic proteins, (4 induces reactive oxygen species mediated responses, (5 stimulates MAPK-stress signaling and redifferentiation in leukemia cells, (6 inhibits overexpressed proinflammatory enzymes in cancer cells such as aldo-keto reductase 1 and 5-lipoxygenase, and (7 inhibits cell migration and shows antiangiogenic and antimetastatic activities. Finally, MJ may act as a chemosensitizer to some chemotherapics helping to overcome drug resistant. The complete lack of toxicity to normal cells and the rapidity by which MJ causes damage to cancer cells turn MJ into a promising anticancer agent that can be used alone or in combination with other agents.

  4. Engineering Escherichia coli for renewable benzyl alcohol production

    Directory of Open Access Journals (Sweden)

    Shawn Pugh

    2015-12-01

    Full Text Available Benzyl alcohol is an aromatic hydrocarbon used as a solvent and an intermediate chemical in the pharmaceutical, cosmetics, and flavor/fragrance industries. The de novo biosynthesis of benzyl alcohol directly from renewable glucose was herein explored using a non-natural pathway engineered in Escherichia coli. Benzaldehyde was first produced from endogenous phenylpyruvate via three heterologous steps, including hydroxymandelate synthase (encoded by hmaS from Amycolatopsis orientalis, followed by (S-mandelate dehydrogenase (encoded by mdlB and phenylglyoxylate decarboxylase (encoded by mdlC from Pseudomonas putida ATCC 12633. The subsequent rapid and efficient reduction of benzaldehyde to benzyl alcohol occurred by the combined activity and native regulation of multiple endogenous alcohol dehydrogenases and/or aldo-keto reductases. Through systematic deletion of competing aromatic amino acid biosynthesis pathways to promote endogenous phenylpyruvate availability, final benzyl alcohol titers as high as 114±1 mg/L were realized, representing a yield of 7.6±0.1 mg/g on glucose and a ~5-fold improvement over initial strains.

  5. De Novo Sequencing and Analysis of Lemongrass Transcriptome Provide First Insights into the Essential Oil Biosynthesis of Aromatic Grasses

    Science.gov (United States)

    Meena, Seema; Kumar, Sarma R.; Venkata Rao, D. K.; Dwivedi, Varun; Shilpashree, H. B.; Rastogi, Shubhra; Shasany, Ajit K.; Nagegowda, Dinesh A.

    2016-01-01

    Aromatic grasses of the genus Cymbopogon (Poaceae family) represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavor, fragrance, cosmetic, and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step toward understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass) by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases, pyrophosphatases, alcohol dehydrogenases, aldo-keto reductases, carotenoid cleavage dioxygenases, alcohol acetyltransferases, and aldehyde dehydrogenases, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type) with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes, and acetates. Molecular modeling and docking further supported the role of identified protein sequences in aroma formation in Cymbopogon. Also, simple sequence repeats were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition. PMID:27516768

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. [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.

  20. 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.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

    We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest...... sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruzi HMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG...... 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...

  2. 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.

  3. 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.

  4. 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

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

    Science.gov (United States)

    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. 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.

  7. 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.

  8. 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

  9. 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...

  10. 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.

  11. 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

  12. 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...

  13. 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....

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

    Institute of Scientific and Technical Information of China (English)

    Russell A DeBose-Boyd

    2008-01-01

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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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...

  4. 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.

  5. 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...

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    We studied the electrochemical behavior of the redox metalloenzyme copper nitrite reductase (CNiR, Achromobacter xylosoxidans) immobilized on a Au(111)‐electrode surface modified by a self‐assembled cysteamine molecular monolayer (SAM) using a combination of cyclic voltammetry and electrochemical...

  7. 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.

  8. 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.

  9. 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.

  10. 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...

  11. 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

  12. 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...

  13. 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

  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. 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.

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    E.V. Pradedova

    2016-02-01

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

  18. 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.

  19. 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.

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    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...... 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...

  1. 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.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial......) of bacteria and archaea were 4.59 x 10(9) and 6.68 x 10(5), respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for the detection of sulfur...... oxygenase reductase (SOR) genes. Three sor-like genes, namely, sor (Fx), sor (SA), and sor (SB) were identified from metagenomic DNAs of the bioreactors. The sor (Fx) is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sor (SA) and sor (SB) showed...

  3. 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.

  4. 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

  5. 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...

  6. 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...

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Herpes simplex virus type 1 ribonucleotide reductase null mutants induce lesions in guinea pigs.

    Science.gov (United States)

    Turk, S R; Kik, N A; Birch, G M; Chiego, D J; Shipman, C

    1989-12-01

    Two herpes simplex virus type 1 ribonucleotide reductase null mutants, hrR3 and ICP6 delta, produced cutaneous lesions in guinea pigs as severe as those of wild-type strains. The lesions induced by hrR3 resulted from in vivo replication of the mutant virus, suggesting that this virus-encoded enzyme is nonessential for virus replication in guinea pigs.

  12. Identification of ubiquinol cytochrome c reductase hinge (UQCRH) as a potential diagnostic biomarker for lung adenocarcinoma

    OpenAIRE

    Gao, Feng; Liu, Qicai; Li, Guoping; Dong, Feng; Qiu, Minglian; Lv, Xiaoting; Zhang, Sheng; Guo, Zheng

    2016-01-01

    Ubiquinol cytochrome c reductase hinge (UQCRH) is a novel protein that localizes in the mitochondrial membrane and induces mitochondrial reactive oxygen species (ROS) generation. It had a high expression rate of 87.10% (108/124) in lung adenocarcinoma. Moreover, serum UQCRH level in patients with lung adenocarcinoma was significantly increased compared with that of pneumonia patients (p < 0.0001) and normal control subjects (p < 0.0001). Receiver operating characteristic curve analysis using ...

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

    Institute of Scientific and Technical Information of China (English)

    Mostafa Behpour-Oskooee; Abdollah Karimi; Shirin Sayyahfar

    2014-01-01

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

  14. Aminoglycoside-Resistant Mutation of Pseudomonas aeruginosa Defective in Cytochrome c552 and Nitrate Reductase

    OpenAIRE

    Bryan, L E; Nicas, Thalia; Holloway, B W; Crowther, Carol

    1980-01-01

    A gentamicin-resistant mutant of Pseudomonas aeruginosa PAO503 was selected after ethyl methane sulfonate mutagenesis. The strain, P. aeruginosa PAO2401 had increased resistance to all aminoglycosides tested but exhibited no change for other antibiotics. The mutation designated aglA (aminoglycoside resistance) was 50% cotransducible with the 8-min ilvB,C marker on the P. aeruginosa chromosome. It showed a marked reduction in cytochrome c552 and nitrate reductase (Nar) and a change in terminal...

  15. Purification of the Cytochrome c Reductase/Cytochrome c Oxidase Super Complex of Yeast Mitochondria

    OpenAIRE

    Braun, Hans-Peter; Sunderhaus, Stephanie; Boekema, Egbert J.; Kouřil, Roman

    2009-01-01

    The protein complexes of the respiratory chain interact by forming large protein particles called respiratory supercomplexes or ‘‘respirasomes’’. Biochemical characterization of these particles proved to be difficult because of their instability. Here we describe a strategy to isolate and characterize the cytochrome c reductase/cytochrome c oxidase supercomplex of yeast, also termed the III + IV supercomplex, which is based on lactate cultivation of yeast, gentle isolation of mitochondria, me...

  16. CLINICAL SIGNIFICANCE OF 5αα-REDUCTASE AND ANDROGEN RECEPTOR GENE POLYMORPHISMS IN PROSTATE CANCER

    Directory of Open Access Journals (Sweden)

    O. B. Loran

    2014-07-01

    Full Text Available The development of prostate cancer is inseparably linked with the effect of androgens on the fundamental prostatic intracellular processes,such as proliferation, apoptosis, which is realized through a number of second messengers. Major of them are the AR gene encoding androgenreceptors and the SRD5A2 gene encoding 5α-reductase enzyme. This paper deals with the study of the role of these genes in prostate cancer.  

  17. CLINICAL SIGNIFICANCE OF 5αα-REDUCTASE AND ANDROGEN RECEPTOR GENE POLYMORPHISMS IN PROSTATE CANCER

    Directory of Open Access Journals (Sweden)

    O. B. Loran

    2009-01-01

    Full Text Available The development of prostate cancer is inseparably linked with the effect of androgens on the fundamental prostatic intracellular processes,such as proliferation, apoptosis, which is realized through a number of second messengers. Major of them are the AR gene encoding androgenreceptors and the SRD5A2 gene encoding 5α-reductase enzyme. This paper deals with the study of the role of these genes in prostate cancer.  

  18. Smith-Lemli-Opitz syndrome is caused by mutations in the 7-dehydrocholesterol reductase gene.

    OpenAIRE

    Waterham, H. R.; Wijburg, F.A.; Hennekam, R. C.; Vreken, P; Poll-The, B T; Dorland, L.; Duran, M.; Jira, P.E.; Smeitink, J. A.; Wevers, R. A.; Wanders, R J

    1998-01-01

    Smith-Lemli-Opitz syndrome is a frequently occurring autosomal recessive developmental disorder characterized by facial dysmorphisms, mental retardation, and multiple congenital anomalies. Biochemically, the disorder is caused by deficient activity of 7-dehydrocholesterol reductase, which catalyzes the final step in the cholesterol-biosynthesis pathway-that is, the reduction of the Delta7 double bond of 7-dehydrocholesterol to produce cholesterol. We identified a partial transcript coding for...

  19. A Modified Method for Measuring Root Iron Reductase Activity Under Normal Laboratory Conditions

    Institute of Scientific and Technical Information of China (English)

    ZHENG Shao-Jian; HE Yun-Feng; TANG Cai-Xian; Y. MASAOKA

    2005-01-01

    Based on the strong chelating property of bathophenanthroline disulfonic acid (BPDS) with Fe(Ⅱ), root Fe(Ⅲ) chelate reductase activity is usually measured with a spectrophotometer using MES (2-morpholinoethanesulfonic acid) or HEPES (2-(4-(2-Hydroxyethyl)-1-piperazinyl) ethanesulfonic acid) buffer in the dark because of high autoreduction rate of Fe(Ⅲ)in the presence of light. However, the exclusion of light is inconvenient, especially when analyzing a large number of samples. The objective of this study was to develop a new method for determination of root reductase activity under normal laboratory conditions using a suitable buffer composition and Fe(Ⅲ) concentration to eliminate the autoreduction of Fe(Ⅲ). A modified method using a Tris (2-amino-2-hydroxymethyl-1,3-propanediol) buffer at pH 7.5 instead of MES or HEPES buffer and a decreased FeEDTA (Fe ethylene diamine tetraacetic acid) concentration of 50 μmol L-1 was developed. The autoreduction of Fe(Ⅲ) using the Tris buffer was undetectable for temperatures at 4 and 28 ℃ and was also much lower than that using the other buffers even with sunlight during measurement of Fe(Ⅲ) reduction.Furthermore, the differences in Fe(Ⅲ) reductase activity among 5 plant species and 14 red clover cultivars (Trifolium pratense L.) could be easily detected with the modified method. The method developed in this study to measure root Fe chelate reductase activity was not only effective and reliable but also easily managed under normal laboratory light conditions.

  20. Purification and characterization of a 15-ketoprostaglandin d-reductase from bovine lung

    DEFF Research Database (Denmark)

    Hansen, Harald S.

    1979-01-01

    . The turnover number of the enzyme was determined to be either 60 or 42 min. The low value of the turnover number is compensated by a high concentration (96.4 mU/g tissue) of the enzyme in lung tissue, resulting in a high metabolic capacity. Thus, 15-ketoprostaglandin d-reductase together with 15......-hydroxyprostaglandin dehydrogenase ensures an irreversible catabolism of prostaglandins. © 1979....

  1. Molecular modeling toward selective inhibitors of dihydrofolate reductase from the biological warfare agent Bacillus anthracis.

    Science.gov (United States)

    Giacoppo, Juliana O S; Mancini, Daiana T; Guimarães, Ana P; Gonçalves, Arlan S; da Cunha, Elaine F F; França, Tanos C C; Ramalho, Teodorico C

    2015-02-16

    In the present work, we applied docking and molecular dynamics techniques to study 11 compounds inside the enzymes dihydrofolate reductase (DHFR) from the biological warfare agent Bacillus anthracis (BaDHFR) and Homo sapiens sapiens (HssDHFR). Six of these compounds were selected for a study with the mutant BaF96IDHFR. Our results corroborated with experimental data and allowed the proposition of a new molecule with potential activity and better selectivity for BaDHFR.

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

    Science.gov (United States)

    Wu, Sangwook

    2016-04-01

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

  3. Tetrahydrobiopterin non-responsiveness in dihydropteridine reductase deficiency is associated with the presence of mutant protein.

    Science.gov (United States)

    Cotton, R G; Jennings, I; Bracco, G; Ponzone, A; Guardamagna, O

    1986-01-01

    Correlation of the response to a load of tetrahydrobiopterin (BH4) in dihydropterin reductase (DHPR) deficient patients to the type of mutation in these patients has led to the conclusion that 4 patients without mutant DHPR molecules in their cells respond to the BH4 load, whereas 3 patients with mutant DHPR in their cells do not respond. Intravenous injection of BH4 in 1 of the cases not responding to BH4 again showed no response.

  4. 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.

  5. 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.

  6. Screening for inhibitors of dihydrofolate reductase using pulsed ultrafiltration mass spectrometry.

    Science.gov (United States)

    Nikolic, D; van Breemen, R B

    1998-04-01

    A method of screening combinatorial libraries for inhibitors of eukaryotic dihydrofolate reductase has been developed using pulsed ultra-filtration electrospray mass spectrometry, which is a continuous-flow affinity separation system for extracting and identifying high affinity ligands in combinatorial libraries. In this application, pulsed ultrafiltration conditions were optimized for the isolation and identification of inhibitors of dihydrofolate reductase from a 22 compound library containing six known inhibitors of the enzyme including trimethoprim, aminopterin, methotrexate, pyrimethamine, folic acid, and folinic acid, and 16 compounds without known affinity. In order to optimize the screening method, sources of non-specific binding were identified and minimized. A significant source of non-specific binding for this set of library compounds was hydrophobic interaction with the surfaces of the ultrafiltration chamber. After affinity separation of bound (high affinity) versus free (low affinity) library compounds during pulsed ultrafiltration, receptor-bound ligands were released and eluted using either organic solvent or acidified mobile phase. Although 80% methanol easily disrupted the receptor-ligand complexes, organic solvent had the undesirable effect of releasing non-specifically bound compounds from the chamber and thereby increasing the background noise. Interference from non-specific binding was minimized by releasing bound ligands using a low pH mobile phase eluent instead of organic solvent. Under the conditions used, pulsed ultrafiltration mass spectrometry selectively identified the two library compounds with the highest affinity for dihydrofolate reductase, methotrexate and aminopterin.

  7. A preliminary study on estimating extra-cellular nitrate reductase activities in estuarine systems

    Directory of Open Access Journals (Sweden)

    Pant H. K.

    2009-07-01

    Full Text Available Enzymes catalyzing ammonium (NH4+/nitrate (NO3– into nitrous oxide (N2O/molecular nitrogen (N2, play critical roles in water quality management. The objective of this paper was to investigate the role of extra-cellular enzymes in cycling of nitrogen (N in aquatic systems. It appears that N in estuaries, salt marshes, etc., does not stay long enough to be available for uptake, thus, creating N limited conditions. This study showed that indigenous extra-cellular nitrate reductase along with others involved in N transformations in the waters/sediments of estuarine systems can cause complete removal of NH4+ and NO3– from the waters and available NH4+ and NO3– from the sediments. These results indicate that due to high extra-cellular nitrate reductase and other enzymes associated with N transformations in sediments/waters, substantial amounts of NH4+ and NO3– can be quickly lost from the systems as N2O and/or nitric oxide (NO, in turn, creating N limited conditions in estuarine systems. Such high activities of indigenous nitrate reductase and others are useful in removing readily bioavailable N from the systems, thereby avoidance of eutrophic conditions. However, they might contribute in increasing the N2O, a potent greenhouse gas with global warming potential (GWP of 296, in the atmosphere.

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

    Science.gov (United States)

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

    2015-01-01

    Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity.

  9. Purification and characterization of a novel carbonyl reductase with high stereo-selectivity

    Institute of Scientific and Technical Information of China (English)

    YANG Ming; XU Yan; MU Xiaoqing; XIAO Rong

    2007-01-01

    A novel NADPH-dependent carbonyl reductase was separated from Candida parapsilosis CCTCC 203011.The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),which was purified through ammonium sulfate,Diethylamino Ethanol (DEAE) sepharose Fast flow (FF),phenyl-sepharose FF and blue sepharose FF chromatography from cell-free extract.The molecular mass of the enzyme was about 30 kDa.The optimum pH and temperature for reduction were 4.5℃ and 35℃,respectively.The Cu2+ had strong restrictive effect on enzyme activity.In addition,the carbonyl reductase was an enzyme with high substrate specificity and stereo-selectivity,and showed high asymmetric reduction activity towards α-hydroxyacetophenone and ethyl 4-chloro acetoacetate.For the asymmetric reduction of α-hydroxyacetophenone and ethyl 4-chloro acetoacetate,(S)-1-phenyl-1,2-ethanediol and (R)-ethyl 4-chloro-3-hydroxybutanoate were produced by the purified enzyme,with the 100% and 94.3% e.e.value,respectively.Therefore,the enzyme could be one of the effective biocatalysts for asymmetric synthesis of chiral alcohols.The amino acid sequences of one peptide from the purified enzyme were analyzed by LC-MASS-MASS,and the carbonyl reductase showed some identity to the hypothetical protein CaO 19.10414 reported.

  10. Trichomonas vaginalis flavin reductase 1 and its role in metronidazole resistance.

    Science.gov (United States)

    Leitsch, David; Janssen, Brian D; Kolarich, Daniel; Johnson, Patricia J; Duchêne, Michael

    2014-01-01

    The enzyme flavin reductase 1 (FR1) from Trichomonas vaginalis, formerly known as NADPH oxidase, was isolated and identified. Flavin reductase is part of the antioxidative defence in T. vaginalis and indirectly reduces molecular oxygen to hydrogen peroxide via free flavins. Importantly, a reduced or absent flavin reductase activity has been reported in metronidazole-resistant T. vaginalis, resulting in elevated intracellular oxygen levels and futile cycling of metronidazole. Interestingly, FR1 has no close homologue in any other sequenced genome, but seven full-length and three truncated isoforms exist in the T. vaginalis genome. However, out of these, only FR1 has an affinity for flavins, i.e. FMN, FAD and riboflavin, which is high enough to be of physiological relevance. Although there are no relevant changes in the gene sequence or any alterations of the predicted FR1-mRNA structure in any of the strains studied, FR1 is not expressed in highly metronidazole-resistant strains. Transfection of a metronidazole-resistant clinical isolate (B7268), which does not express any detectable amounts of FR, with a plasmid bearing a functional FR1 gene nearly completely restored metronidazole sensitivity. Our results indicate that FR1 has a significant role in the emergence of metronidazole resistance in T. vaginalis.

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

    Directory of Open Access Journals (Sweden)

    Vanitha Reddy Palvai

    2014-01-01

    Full Text Available Phytochemicals are the bioactive components that contribute to the prevention of cardiovascular and other degenerative diseases. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA reductase would be an effective means of lowering plasma cholesterol in humans. The present study explores the HMG CoA reductase inhibitory effect of extracts from leaves of Morus indica varieties, M5, V1, and S36, compared with the statin, using an ex vivo method. The assay is based on the stoichiometric formation of coenzyme A during the reduction of microsomal HMG CoA to mevalonate. Dechlorophyllised extract of three varieties was studied at 300 µg. The coenzyme A released at the end of assay in control (100.31 nmoles and statins (94.46 nm was higher than the dechlorphyllised extracts of the samples. The coenzyme A released during the reduction of HMG CoA to mevalonate in dechlorophyllised extracts of the samples was as follows: S36 < M5 < V1. The results indicated that the samples were highly effective in inhibiting the enzyme compared to statins (standard drug. The results indicate the role of Morus varieties extracts in modulating the cholesterol metabolism by inhibiting the activity of HMG CoA reductase. These results provide scope for designing in vivo animal studies to confirm their effect.

  12. Comparative azo reductase activity of red azo dyes through caecal and hepatic microsomal fraction in rats.

    Science.gov (United States)

    Singh, S; Das, M; Khanna, S K

    1997-09-01

    In order to study the rate of formation of toxic aromatic amines, anaerobic reduction of four red azo dyes viz. amaranth, carmoisine, fast Red E and ponceau 4R was investigated by incubating caecal content and hepatic microsomal fraction of rats with 37.5 microM concentration of dyes in sodium phosphate buffer pH 7.4 using NADPH generating system, glucose oxidase system and nitrogen as the gaseous phase. Caecal suspension exhibited higher azo reductase activity than that of hepatic microsomal fraction using any of the 4 azo dyes. Caecal microbes showed maximal azo reductase activity when ponceau 4R was used as a substrate followed by fast Red E and carmoisine, while with amaranth the activity was minimum. Similarly ponceau 4 R exhibited maximum hepatic microsomal azo reductase activity followed by fast Red E and carmoisine whereas, amaranth had minimum activity. Caecal flora possessed almost 17 fold higher degradative capability of ponceau 4 R and fast Red E colourants than the hepatic microsomal fraction. The higher reductive ability through caecal flora for ponceau 4R and fast Red E signifies the formation of more aromatic amines which may be re-absorbed through the intestine to be either eliminated through urine as conjugates or retained in the target tissues to elicit toxic effects.

  13. Nitrosative Stress Response in Vibrio cholerae: Role of S-Nitrosoglutathione Reductase.

    Science.gov (United States)

    Patra, Sourav Kumar; Bag, Prasanta Kumar; Ghosh, Sanjay

    2016-12-20

    Vibrio cholerae, the causative agent of cholera, poses serious threats to humans worldwide. V. cholerae faces host inflammatory response and encounters nitrosative stress before establishing successful colonization. It is not clear how V. cholerae combats nitric oxide and reactive nitrogen species. In the present study, we used three clinical strains of V. cholerae and tested their nitrosative stress response pattern towards sodium nitroprusside (SNP) and S-Nitrosoglutathione (GSNO). Among them, V. cholerae, belonging to both O1 and O139 serotypes, showed moderate resistance to SNP and GSNO. However, a V. cholerae strain belonging to non O1 and non O139 showed sensitivity to SNP but resistance towards GSNO. Reduced glutathione and glutathione reductase play a significant role to combat nitrosative stress in V. cholerae. This is the first report where we show the presence of GSNO reductase activity in V. cholerae and that it plays an important role to detoxify S-Nitrosoglutathione. GSNO reductase activity of V. cholerae was regulated by posttranslational modification through S-nitrosylation under in vitro conditions which could be reversed by dithiothreitol (DTT). In addition, we show that biofilm formation remained unaffected under nitrosative stress in V. cholerae.

  14. Molecular dissection of a putative iron reductase from Desulfotomaculum reducens MI-1

    Science.gov (United States)

    Li, Zhi; Kim, David D.; Nelson, Ornella D.; Otwell, Anne E.; Richardson, Ruth E.; Callister, Stephen J.; Lin, Hening

    2015-01-01

    Desulfotomaculum reducens MI-1 is a Firmicute strain capable of reducing a variety of heavy metal ions and has a great potential in heavy metal bioremediation. We recently identified Dred_2421 as a potential iron reductase through proteomic study of D. reducens. The current study examines its iron-reduction mechanism. Dred_2421, like its close homolog from Escherichia coli (2, 4-dienoyl-CoA reductase), has an FMN-binding N-terminal domain (NTD), an FAD-binding C-terminal domain (CTD), and a 4Fe-4S cluster between the two domains. To understand the mechanism of the iron-reduction activity and the role of each domain, we generated a series of variants for each domain and investigated their iron-reduction activity. Our results suggest that CTD is the main contributor of the iron-reduction activity, and that NTD and the 4Fe-4S cluster are not directly involved in such activity. This study provides a mechanistic understanding of the iron-reductase activity of Dred_2421 and may also help to elucidate other physiological activities this enzyme may have. PMID:26454174

  15. Evolution of the ferric reductase domain (FRD) superfamily: modularity, functional diversification, and signature motifs.

    Science.gov (United States)

    Zhang, Xuezhi; Krause, Karl-Heinz; Xenarios, Ioannis; Soldati, Thierry; Boeckmann, Brigitte

    2013-01-01

    A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria.

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

    Directory of Open Access Journals (Sweden)

    Rosana Aparecida Manólio Soares

    2015-02-01

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

  17. Stereospecific micellar electrokinetic chromatography assay of methionine sulfoxide reductase activity employing a multiple layer coated capillary.

    Science.gov (United States)

    Zhu, Qingfu; El-Mergawy, Rabab G; Heinemann, Stefan H; Schönherr, Roland; Jáč, Pavel; Scriba, Gerhard K E

    2013-09-01

    A micellar electrokinetic chromatography method for the analysis of the l-methionine sulfoxide diastereomers employing a successive multiple ionic-polymer layer coated fused-silica capillary was developed and validated in order to investigate the stereospecificity of methionine sulfoxide reductases. The capillary coating consisted of a first layer of hexadimethrine and a second layer of dextran sulfate providing a stable strong cathodic EOF and consequently highly repeatable analyte migration times. The methionine sulfoxide diastereomers, methionine as product as well as β-alanine as internal standard were derivatized by dabsyl chloride and separated using a 35 mM sodium phosphate buffer, pH 8.0, containing 25 mM SDS as BGE and a separation voltage of 25 kV. The method was validated in the range of 0.15-2.0 mM with respect to linearity and precision. The LODs of the analytes ranged between 0.04 and 0.10 mM. The assay was subsequently applied to determine the stereospecificity of methionine sulfoxide reductases as well as the enzyme kinetics of human methionine sulfoxide reductase A. Monitoring the decrease of the l-methionine-(S)-sulfoxide Km = 411.8 ± 33.8 μM and Vmax = 307.5 ± 10.8 μM/min were determined.

  18. Molecular dissection of a putative iron reductase from Desulfotomaculum reducens MI-1.

    Science.gov (United States)

    Li, Zhi; Kim, David D; Nelson, Ornella D; Otwell, Anne E; Richardson, Ruth E; Callister, Stephen J; Lin, Hening

    2015-11-20

    Desulfotomaculum reducens MI-1 is a Firmicute strain capable of reducing a variety of heavy metal ions and has a great potential in heavy metal bioremediation. We recently identified Dred_2421 as a potential iron reductase through proteomic study of D. reducens. The current study examines its iron-reduction mechanism. Dred_2421, like its close homolog from Escherichia coli (2, 4-dienoyl-CoA reductase), has an FMN-binding N-terminal domain (NTD), an FAD-binding C-terminal domain (CTD), and a 4Fe-4S cluster between the two domains. To understand the mechanism of the iron-reduction activity and the role of each domain, we generated a series of variants for each domain and investigated their iron-reduction activity. Our results suggest that CTD is the main contributor of the iron-reduction activity, and that NTD and the 4Fe-4S cluster are not directly involved in such activity. This study provides a mechanistic understanding of the iron-reductase activity of Dred_2421 and may also help to elucidate other physiological activities this enzyme may have.

  19. Molecular cloning and catalytic characterization of a recombinant tropine biosynthetic tropinone reductase from Withania coagulans leaf.

    Science.gov (United States)

    Kushwaha, Amit K; Sangwan, Neelam S; Tripathi, Sandhya; Sangwan, Rajender S

    2013-03-10

    Tropinone reductases (TRs) are small proteins belonging to the SDR (short chain dehydrogenase/reductase) family of enzymes. TR-I and TR-II catalyze the conversion of tropinone into tropane alcohols (tropine and pseudotropine, respectively). The steps are intermediary enroute to biosynthesis of tropane esters of medicinal importance, hyoscyamine/scopolamine, and calystegins, respectively. Biosynthesis of tropane alkaloids has been proposed to occur in roots. However, in the present report, a tropine forming tropinone reductase (TR-I) cDNA was isolated from the aerial tissue (leaf) of a medicinal plant, Withania coagulans. The ORF was deduced to encode a polypeptide of 29.34 kDa. The complete cDNA (WcTRI) was expressed in E. coli and the recombinant His-tagged protein was purified for functional characterization. The enzyme had a narrow pH range of substantial activity with maxima at 6.6. Relatively superior thermostability of the enzyme (30% retention of activity at 60 °C) was catalytic novelty in consonance with the desert area restricted habitat of the plant. The in vitro reaction kinetics predominantly favoured the forward reaction. The enzyme had wide substrate specificity but did not cover the substrates of other well-known plant SDR related to menthol metabolism. To our knowledge, this pertains to be the first report on any gene and enzyme of secondary metabolism from the commercially and medicinally important vegetable rennet species.

  20. Reductive metabolism of nabumetone by human liver microsomal and cytosolic fractions: exploratory prediction using inhibitors and substrates as marker probes.

    Science.gov (United States)

    Matsumoto, Kaori; Hasegawa, Tetsuya; Koyanagi, Junichi; Takahashi, Tamiko; Akimoto, Masayuki; Sugibayashi, Kenji

    2015-06-01

    The metabolic reduction of nabumetone was examined by inhibition and correlation studies using human liver microsomes and cytosol. This reduction was observed in both fractions, with the V(max) values for reduction activity being approximately fourfold higher, and the V(max)/K(m) values approximately three-fold higher, in the microsomes than in the cytosol. The reduction of nabumetone was inhibited by 18β-glycyrrhetinic acid, an 11β-hydroxysteroid dehydrogenase (11β-HSD) inhibitor, in the microsomal fraction. The reduction activity was also inhibited by quercetin and menadione [carbonyl reductase (CBR) inhibitors], and by phenolphthalein and medroxyprogesterone acetate [potent inhibitors of aldo-keto reductase (AKR) 1C1, 1C2 and 1C4] in the cytosol. A good correlation (r² = 0.93) was observed between the reduction of nabumetone and of cortisone, as a marker of 11β-HSD activity, in the microsomal fractions. There was also an excellent relationship between reduction of nabumetone and of the AKR1C substrates, acetohexamide, and ethacrynic acid (r 2 = 0.92 and 0.93, respectively), in the cytosol fractions. However, a poor correlation was observed between the formation of 4-(6-methoxy-2-naphthyl)-butan-2-ol (MNBO) from nabumetone and CBR activity (with 4-benzoyl pyridine reduction as a CBR substrate) in the cytosol fractions (r² = 0.24). These findings indicate that nabumetone may be metabolized by 11β-HSD in human liver microsomes, and primarily by AKR1C4 in human liver cytosol, although multiple enzymes in the AKR1C subfamily may be involved. It cannot be completely denied that CBR is involved to some extent in the formation of MNBO from nabumetone in the cytosol fraction.

  1. Hydroxysteroid dehydrogenases (HSDs) in bacteria: a bioinformatic perspective.

    Science.gov (United States)

    Kisiela, Michael; Skarka, Adam; Ebert, Bettina; Maser, Edmund

    2012-03-01

    Steroidal compounds including cholesterol, bile acids and steroid hormones play a central role in various physiological processes such as cell signaling, growth, reproduction, and energy homeostasis. Hydroxysteroid dehydrogenases (HSDs), which belong to the superfamily of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR), are important enzymes involved in the steroid hormone metabolism. HSDs function as an enzymatic switch that controls the access of receptor-active steroids to nuclear hormone receptors and thereby mediate a fine-tuning of the steroid response. The aim of this study was the identification of classified functional HSDs and the bioinformatic annotation of these proteins in all complete sequenced bacterial genomes followed by a phylogenetic analysis. For the bioinformatic annotation we constructed specific hidden Markov models in an iterative approach to provide a reliable identification for the specific catalytic groups of HSDs. Here, we show a detailed phylogenetic analysis of 3α-, 7α-, 12α-HSDs and two further functional related enzymes (3-ketosteroid-Δ(1)-dehydrogenase, 3-ketosteroid-Δ(4)(5α)-dehydrogenase) from the superfamily of SDRs. For some bacteria that have been previously reported to posses a specific HSD activity, we could annotate the corresponding HSD protein. The dominating phyla that were identified to express HSDs were that of Actinobacteria, Proteobacteria, and Firmicutes. Moreover, some evolutionarily more ancient microorganisms (e.g., Cyanobacteria and Euryachaeota) were found as well. A large number of HSD-expressing bacteria constitute the normal human gastro-intestinal flora. Another group of bacteria were originally isolated from natural habitats like seawater, soil, marine and permafrost sediments. These bacteria include polycyclic aromatic hydrocarbons-degrading species such as Pseudomonas, Burkholderia and Rhodococcus. In conclusion, HSDs are found in a wide variety of microorganisms including

  2. Single-molecule enzymology of steroid transforming enzymes: Transient kinetic studies and what they tell us.

    Science.gov (United States)

    Penning, Trevor M

    2016-07-01

    Structure-function studies on steroid transforming enzymes often use site-directed mutagenesis to inform mechanisms of catalysis and effects on steroid binding, and data are reported in terms of changes in steady state kinetic parameters kcat, Km and kcat/Km. However, this dissection of function is limited since kcat is governed by the rate-determining step and Km is a complex macroscopic kinetic constant. Often site-directed mutagenesis can lead to a change in the rate-determining step which cannot be revealed by just reporting a decrease in kcat alone. These issues are made more complex when it is considered that many steroid transforming enzymes have more than one substrate and product. We present the case for using transient-kinetics performed with stopped-flow spectrometry to assign rate constants to discrete steps in these multi-substrate reactions and their use to interpret enzyme mechanism and the effects of disease and engineered mutations. We demonstrate that fluorescence kinetic transients can be used to measure ligand binding that may be accompanied by isomerization steps, revealing the existence of new enzyme intermediates. We also demonstrate that single-turnover reactions can provide a klim for the chemical step and Ks for steroid-substrate binding and that when coupled with kinetic isotope effect measurements can provide information on transition state intermediates. We also demonstrate how multiple turnover experiments can provide evidence for either "burst-phase" kinetics, which can reveal a slow product release step, or linear-phase kinetics, in which the chemical step can be rate-determining. With these assignments it becomes more straightforward to analyze the effects of mutations. We use examples from the hydroxysteroid dehydrogenases (AKR1Cs) and human steroid 5β-reductase (AKR1D1) to illustrate the utility of the approach, which are members of the aldo-keto reductase (AKR) superfamily.

  3. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders.

    Science.gov (United States)

    Miller, Walter L; Auchus, Richard J

    2011-02-01

    Steroidogenesis entails processes by which cholesterol is converted to biologically active steroid hormones. Whereas most endocrine texts discuss adrenal, ovarian, testicular, placental, and other steroidogenic processes in a gland-specific fashion, steroidogenesis is better understood as a single process that is repeated in each gland with cell-type-specific variations on a single theme. Thus, understanding steroidogenesis is rooted in an understanding of the biochemistry of the various steroidogenic enzymes and cofactors and the genes that encode them. The first and rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone by a single enzyme, P450scc (CYP11A1), but this enzymatically complex step is subject to multiple regulatory mechanisms, yielding finely tuned quantitative regulation. Qualitative regulation determining the type of steroid to be produced is mediated by many enzymes and cofactors. Steroidogenic enzymes fall into two groups: cytochrome P450 enzymes and hydroxysteroid dehydrogenases. A cytochrome P450 may be either type 1 (in mitochondria) or type 2 (in endoplasmic reticulum), and a hydroxysteroid dehydrogenase may belong to either the aldo-keto reductase or short-chain dehydrogenase/reductase families. The activities of these enzymes are modulated by posttranslational modifications and by cofactors, especially electron-donating redox partners. The elucidation of the precise roles of these various enzymes and cofactors has been greatly facilitated by identifying the genetic bases of rare disorders of steroidogenesis. Some enzymes not principally involved in steroidogenesis may also catalyze extraglandular steroidogenesis, modulating the phenotype expected to result from some mutations. Understanding steroidogenesis is of fundamental importance to understanding disorders of sexual differentiation, reproduction, fertility, hypertension, obesity, and physiological homeostasis.

  4. Effect of graded Nrf2 activation on phase-I and -II drug metabolizing enzymes and transporters in mouse liver.

    Directory of Open Access Journals (Sweden)

    Kai Connie Wu

    Full Text Available Nuclear factor erythroid 2-related factor 2 (Nrf2 is a transcription factor that induces a battery of cytoprotective genes in response to oxidative/electrophilic stress. Kelch-like ECH associating protein 1 (Keap1 sequesters Nrf2 in the cytosol. The purpose of this study was to investigate the role of Nrf2 in regulating the mRNA of genes encoding drug metabolizing enzymes and xenobiotic transporters. Microarray analysis was performed in livers of Nrf2-null, wild-type, Keap1-knockdown mice with increased Nrf2 activation, and Keap1-hepatocyte knockout mice with maximum Nrf2 activation. In general, Nrf2 did not have a marked effect on uptake transporters, but the mRNAs of organic anion transporting polypeptide 1a1, sodium taurocholate cotransporting polypeptide, and organic anion transporter 2 were decreased with Nrf2 activation. The effect of Nrf2 on cytochrome P450 (Cyp genes was minimal, with only Cyp2a5, Cyp2c50, Cyp2c54, and Cyp2g1 increased, and Cyp2u1 decreased with enhanced Nrf2 activation. However, Nrf2 increased mRNA of many other phase-I enzymes, such as aldo-keto reductases, carbonyl reductases, and aldehyde dehydrogenase 1. Many genes involved in phase-II drug metabolism were induced by Nrf2, including glutathione S-transferases, UDP- glucuronosyltransferases, and UDP-glucuronic acid synthesis enzymes. Efflux transporters, such as multidrug resistance-associated proteins, breast cancer resistant protein, as well as ATP-binding cassette g5 and g8 were induced by Nrf2. In conclusion, Nrf2 markedly alters hepatic mRNA of a large number of drug metabolizing enzymes and xenobiotic transporters, and thus Nrf2 plays a central role in xenobiotic metabolism and detoxification.

  5. Identification, purification and characterization of furfural transforming enzymes from Clostridium beijerinckii NCIMB 8052.

    Science.gov (United States)

    Zhang, Yan; Ujor, Victor; Wick, Macdonald; Ezeji, Thaddeus Chukwuemeka

    2015-06-01

    Generation of microbial inhibitory compounds such as furfural and 5-hydroxymethylfurfural (HMF) is a formidable roadblock to fermentation of lignocellulose-derived sugars to butanol. Bioabatement offers a cost effective strategy to circumvent this challenge. Although Clostridium beijerinckii NCIMB 8052 can transform 2-3 g/L of furfural and HMF to their less toxic alcohols, higher concentrations present in biomass hydrolysates are intractable to microbial transformation. To delineate the mechanism by which C. beijerinckii detoxifies furfural and HMF, an aldo/keto reductase (AKR) and a short-chain dehydrogenase/reductase (SDR) found to be over-expressed in furfural-challenged cultures of C. beijerinckii were cloned and over-expressed in Escherichia coli Rosetta-gami™ B(DE3)pLysS, and purified by histidine tag-assisted immobilized metal affinity chromatography. Protein gel analysis showed that the molecular weights of purified AKR and SDR are close to the predicted values of 37 kDa and 27 kDa, respectively. While AKR has apparent Km and Vmax values of 32.4 mM and 254.2 mM s(-1) respectively, using furfural as substrate, SDR showed lower Km (26.4 mM) and Vmax (22.6 mM s(-1)) values on the same substrate. However, AKR showed 7.1-fold higher specific activity on furfural than SDR. Further, both AKR and SDR were found to be active on HMF, benzaldehyde, and butyraldehyde. Both enzymes require NADPH as a cofactor for aldehydes reduction. Based on these results, it is proposed that AKR and SDR are involved in the biotransformation of furfural and HMF by C. beijerinckii.

  6. 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...

  7. Light-regulated expression of the nitrate-reductase and nitrite-reductase genes in tomato and in the phytochrome-deficient aurea mutant of tomato.

    Science.gov (United States)

    Becker, T W; Foyer, C; Caboche, M

    1992-08-01

    The phytochrome-deficient aurea mutant of tomato (Lycopersicon esculentum (L.) Mill) was used to investigate if phytochrome plays a role in the regulation of nitrate-reductase (NR, EC 1.6.6.1) and nitrite-reductase (NiR, EC 1.7.7.1) gene expression. We show that the expression of the tomato NR and NiR genes is stimulated by light and that this light response is mediated by the photoreceptor phytochrome. The red-light response of the NR and NiR genes was reduced in etiolated aurea seedlings when compared to isogenic wild-type cotyledons. The relative levels of NR mRNA and NiR transcripts and their diurnal fluctuations were identical in mature white-light-grown leaves of the wild-type and of the aurea mutant. The transcript levels for cab and RbcS (genes for the chlorophyll-a/b-binding protein of PSII and the small subunit of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively) in aurea leaves grown in white light were indistinguishable from the respective transcript levels in the leaves of the wildtype grown under the same conditions. Despite a severe reduction in the chlorophyll content, the rate of net CO2 uptake by leaves of the aurea mutant was only slightly reduced when compared to the rate of net photosynthesis of wild-type leaves. This difference in the photosynthetic performances of wild-type and aurea mutant plants disappeared during aging of the plants. The increase in zeaxanthin and the concomitant decrease in violaxanthin in leaves of the aurea mutant compared with the same pigment levels in leaves of the wild-type indicate that the activity of the xanthophyll cycle is increased in aurea leaves as a consequence of the reduced CO2-fixation capacity of the mutant leaves.

  8. Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJI operon.

    Science.gov (United States)

    Blasco, F; Iobbi, C; Ratouchniak, J; Bonnefoy, V; Chippaux, M

    1990-06-01

    The structural genes for NRZ, the second nitrate reductase of Escherichia coli, have been sequenced. They are organized in a transcription unit, narZYWV, encoding four subunits, NarZ, NarY, NarW and NarV. The transcription unit is homologous (73% identity) to the narGHJI operon which encodes the genes for NRA, the better characterized nitrate reductase of this organism. The level of homology between the corresponding polypeptides ranges from 69% for the NarW/NarJ pair to 86% for the NarV/NarI pair. The NarZ polypeptide contains the five conserved regions present in all other known molybdoproteins of E. coli and their relative order is the same. The NarY polypeptide, which contains the same four cysteine clusters in the same order as NarH, is probably an electron transfer unit of the complex. Upstream of narZ, an open reading frame, ORFA, is present which could encode a product which has homology (73% identity) with the COOH-terminal end of NarK. The ORFA-narZ intergenic region, however, is about 80 nucleotides long and does not contain the cis-acting elements, NarL and Fnr boxes, nor the terC4 terminator sequence present in the 500 nucleotide narK-narG intergenic region. This might explain why the narZYWV and the narGHJI operons are regulated differently. Our results tend to support the hypothesis that a DNA fragment larger than that encompassing the narGHJI genes has been duplicated.

  9. Aromatic substitution of the FAD-shielding tryptophan reveals its differential role in regulating electron flux in methionine synthase reductase and cytochrome P450 reductase.

    Science.gov (United States)

    Meints, Carla E; Simtchouk, Svetlana; Wolthers, Kirsten R

    2013-03-01

    Methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR) transfer reducing equivalents from NADPH via an FAD and FMN cofactor to a redox partner protein. In both enzymes, hydride transfer from NADPH to FAD requires displacement of a conserved tryptophan that lies coplanar to the FAD isoalloxazine ring. Swapping the tryptophan for a smaller aromatic side chain revealed a distinct role for the residue in regulating MSR and CPR catalysis. MSR W697F and W697Y showed enhanced catalysis, noted by increases in kcat and k(cat)/K(m)(NADPH) for steady-state cytochrome c(3+) reduction and a 10-fold increase in the rate constant (k(obs1)) associated with hydride transfer. Elevated primary kinetic isotope effects on k(obs1) for W697F and W697Y suggest that preceding isotopically insensitive steps like displacement of W697 are less rate determining. MSR W697Y, but not MSR W697F, showed detectable formation of the disemiquinone intermediate, indicating that the polarity of the aromatic side chain influences the rate of interflavin electron transfer. By contrast, the CPR variants (W676F and W676Y) displayed modest decreases in cytochrome c(3+) reduction, a 30- and 3.5-fold decrease in the rate of FAD reduction, accumulation of a FADH2 -NADP(+) charge-transfer complex and dramatically suppressed rates of interflavin electron transfer. We conclude for MSR that hydride transfer is 'gated' by the free energy required to disrupt dispersion forces between the FAD isoalloxazine ring and W697. By contrast, the bulky indole ring of W676 accelerates catalysis in CPR by lowering the energy barrier for displacement of the oxidized nicotinamide ring coplanar with the FAD.

  10. 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.

  11. Downregulation of thioredoxin reductase 1 expression in the substantia nigra pars compacta of Parkinson’s disease mice

    Institute of Scientific and Technical Information of China (English)

    Zihua Liu; Yuhong Jing; Jie Yin; Jiying Mu; Tingting Yao; Liping Gao

    2013-01-01

    Because neurons are susceptible to oxidative damage and thioredoxin reductase 1 is extensively distributed in the central nervous system and has antioxidant properties, we speculated that the enzyme may be involved in the pathogenesis of Parkinson’s disease. A Parkinson’s disease model was produced by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine into C57BL/6 mice. Real-time reverse transcription-PCR, western blot analysis and colorimetric assay showed that the levels of thioredoxin reductase 1 mRNA and protein were decreased, along with a significant reduction in thioredoxin reductase activity, in the midbrain of Parkinson’s disease mice compared with normal mice. Immunohistochemical staining revealed that the number of thioredoxin reductase 1-positive neurons in the substantia nigra pars compacta of Parkinson’s disease mice was significantly decreased compared with normal mice. These experimental findings suggest that the expression of thioredoxin reductase 1 in the substantia nigra pars compacta of Parkinson’s disease mice is significantly decreased, and that the enzyme may be associated with disease onset.

  12. The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans.

    Science.gov (United States)

    Zhang, Yuru; Wang, Haizhen; Zhang, Jingjing; Hu, Ying; Zhang, Linqiang; Wu, Xiaoyun; Su, Xiong; Li, Tingting; Zou, Xiaoju; Liang, Bin

    2016-04-01

    Polyunsaturated fatty acids (PUFAs) are fatty acids with backbones containing more than one double bond, which are introduced by a series of desaturases that insert double bonds at specific carbon atoms in the fatty acid chain. It has been established that desaturases need flavoprotein-NADH-dependent cytochrome b5 reductase (simplified as cytochrome b5 reductase) and cytochrome b5 to pass through electrons for activation. However, it has remained unclear how this multi-enzyme system works for distinct desaturases. The model organism Caenorhabditis elegans contains seven desaturases (FAT-1, -2, -3, -4, -5, -6, -7) for the biosynthesis of PUFAS, providing an excellent model in which to characterize different desaturation reactions. Here, we show that RNAi inactivation of predicted cytochrome b5 reductases hpo-19 and T05H4.4 led to increased levels of C18:1n-9 but decreased levels of PUFAs, small lipid droplets, decreased fat accumulation, reduced brood size and impaired development. Dietary supplementation with different fatty acids showed that HPO-19 and T05H4.4 likely affect the activity of FAT-1, FAT-2, FAT-3, and FAT-4 desaturases, suggesting that these four desaturases use the same cytochrome b5 reductase to function. Collectively, these findings indicate that cytochrome b5 reductase HPO-19/T05H4.4 is required for desaturation to biosynthesize PUFAs in C. elegans.

  13. Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase.

    Science.gov (United States)

    Klein, B; Pawlowski, K; Höricke-Grandpierre, C; Schell, J; Töpfer, R

    1992-05-01

    A cDNA encoding beta-ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned from Cuphea lanceolata. This cDNA of 1276 bp codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp beta-ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the beta-ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, beta-ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced in Escherichia coli, was isolated and proved to possess beta-ketoacyl-ACP reductase activity. Hybridization studies revealed that in C. lanceolata beta-ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.

  14. New roles of flavoproteins in molecular cell biology: an unexpected role for quinone reductases as regulators of proteasomal degradation.

    Science.gov (United States)

    Sollner, Sonja; Macheroux, Peter

    2009-08-01

    Quinone reductases are ubiquitous soluble enzymes found in bacteria, fungi, plants and animals. These enzymes utilize a reduced nicotinamide such as NADH or NADPH to reduce the flavin cofactor (either FMN or FAD), which then affords two-electron reduction of cellular quinones. Although the chemical nature of the quinone substrate is still a matter of debate, the reaction appears to play a pivotal role in quinone detoxification by preventing the generation of potentially harmful semiquinones. In recent years, an additional role of quinone reductases as regulators of proteasomal degradation of transcription factors and possibly intrinsically unstructured protein has emerged. To fulfil this role, quinone reductase binds to the core particle of the proteasome and recruits certain transcription factors such as p53 and p73alpha to the complex. The latter process appears to be governed by the redox state of the flavin cofactor of the quinone reductase, thus linking the stability of transcription factors to cellular events such as oxidative stress. Here, we review the current evidence for protein complex formation between quinone reductase and the 20S proteasome in eukaryotic cells and describe the regulatory role of this complex in stabilizing transcription factors by acting as inhibitors of their proteasomal degradation.

  15. 5α—reductase type 2 gene expression in human testis,epididymis and vas deferens

    Institute of Scientific and Technical Information of China (English)

    LiuDY; WuYW

    2002-01-01

    Objective:To study the expression patterm of 5-α-reductase type 2 gene in human male reproductive organs.Methods:The expression level of 5α-reductase type 2 gene in human testis,epididymis and vas deferens tissues was determined by in situ hybridization using a digoxin-labeled 5α-reductase type 2 cRNA probe.Results:The brown granules of hybridizing signals distributed in the cytoplasm of the Sertoli and Leydig cells of the testis,the principle cells of epididymis and the epithelial cells of vas deferens,but there was no positive signal in the nuclei of these cellsNo positive signal was observed in the germ cells,basement of the testis, interstium of the epididymis and basement and the smooth muscle cells of vas deferens.conclusion:This study confirmed that the 5α-reductase type 2 gene expressed in the Sertoli and Leydig cells of the testis and the principle cells of the epididymis.The expression pattern of the gene in these cells in the human was similar to that in the rat and monkey.The presence of 5α-reductase type 2 gene in the epithelial cells of the vas deferens suggests that it may play a physiological role in human reproduction.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

  18. Evidence that the intra-amoebal Legionella drancourtii acquired a sterol reductase gene from eukaryotes

    Directory of Open Access Journals (Sweden)

    Fournier Pierre-Edouard

    2009-03-01

    Full Text Available Abstract Background Free-living amoebae serve as a natural reservoir for some bacteria that have evolved into «amoeba-resistant» bacteria. Among these, some are strictly intra-amoebal, such as Candidatus "Protochlamydia amoebophila" (Candidatus "P. amoebophila", whose genomic sequence is available. We sequenced the genome of Legionella drancourtii (L. drancourtii, another recently described intra-amoebal bacterium. By comparing these two genomes with those of their closely related species, we were able to study the genetic characteristics specific to their amoebal lifestyle. Findings We identified a sterol delta-7 reductase-encoding gene common to these two bacteria and absent in their relatives. This gene encodes an enzyme which catalyses the last step of cholesterol biosynthesis in eukaryotes, and is probably functional within L. drancourtii since it is transcribed. The phylogenetic analysis of this protein suggests that it was acquired horizontally by a few bacteria from viridiplantae. This gene was also found in the Acanthamoeba polyphaga Mimivirus genome, a virus that grows in amoebae and possesses the largest viral genome known to date. Conclusion L. drancourtii acquired a sterol delta-7 reductase-encoding gene of viridiplantae origin. The most parsimonious hypothesis is that this gene was initially acquired by a Chlamydiales ancestor parasite of plants. Subsequently, its descendents transmitted this gene in amoebae to other intra-amoebal microorganisms, including L. drancourtii and Coxiella burnetii. The role of the sterol delta-7 reductase in prokaryotes is as yet unknown but we speculate that it is involved in host cholesterol parasitism.

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Yvonne D Trigoso

    Full Text Available The enzyme dihydrodipicolinate reductase (DHDPR is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(PH dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3. The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

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

    Science.gov (United States)

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

    2016-01-01

    The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

  2. Direct antioxidant properties of bilirubin andbiliverdin. Is there a role for biliverdin reductase?

    Directory of Open Access Journals (Sweden)

    Thomas eJansen

    2012-03-01

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

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

    Science.gov (United States)

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

    2010-03-24

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

  4. 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.

  5. 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.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  7. Biological evaluation of some uracil derivatives as potent glutathione reductase inhibitors

    Science.gov (United States)

    Güney, Murat; Ekinci, Deniz; Ćavdar, Huseyin; Şentürk, Murat; Zilbeyaz, Kani

    2016-04-01

    Discovery of glutathione reductase (GR) inhibitors has become very popular recently due to antimalarial and anticancer activities. In this study, GR inhibitory capacities of some uracil derivatives (UDCs) (1-4) were reported. Some commercially available molecules (5-6) were also tested for comparison reasons. The novel UDCs were obtained in high yields using simple chemical procedures and exhibited much potent inhibitory activities against GR at low nanomolar concentrations with IC50 values ranging from 2.68 to 166.6 nM as compared with well-known agents.

  8. Structural basis for target protein recognition by the protein disulfide reductase thioredoxin

    DEFF Research Database (Denmark)

    Maeda, Kenji; Hägglund, Per; Finnie, Christine;

    2006-01-01

    Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley alpha-amylase/subtilisin inhibitor (BASI). The crystal structure...... a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares...

  9. STUDY OF THE RELATIONSHIP BETWEEN PSORIASIS AND THE POLYMORPHIC SITE C677T OF METHYLENETETRAHYDROFOLATE REDUCTASE

    Institute of Scientific and Technical Information of China (English)

    王柏秋; 傅松滨; 张贵寅; 李严璞

    2000-01-01

    Objective. In order to investigate 5, l0-methylenetetrahydrofolate reductase (MTHFR)'s polymorphic changes in psoriasis vulgaris. Methods. We detected mutation of site C677V of MTHFR in 39 psoriasfics by PCR-RFLP. Results. Genotype frequencies of the psoriasfics were C/C= 20.15%,C/T= 48.72% and T/T= 30.77%; the allelic frequencies were C = 0.4487 and T=0.5513. Homozygous mutant (TT) of the psoriastics was significantly different from the normal control goup by X2 test. Conclusion. C677V mutant of MTHFR might be related with psoriasis.

  10. STUDY OF THE RELATIONSHIP BETWEEN PSORIASIS AND THE POLYMORPHIC SITE C677T OF METHYLENETETRAHYDROFOLATE REDUCTASE

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective. In order to investigate 5,10-methylenetetrahydrofolate reductase (MTHFR)s polymorphic changes in psoriasis vulgaris.Methods.We detected mutation of site C677V of MTHFR in 39 psoriastics by PCR-RFLP.Results.Genotype frequencies of the psoriastics were C/C=20.15%,C/T=48.72% and T/T=30.77%; the allelic frequencies were C=0.4487 and T=0.5513. Homozygous mutant (TT) of the psoriastics was significantly different from the normal control group by X2 test.Conclusion.C677V mutant of MTHFR might be related with psoriasis.

  11. Aspects of Antithrombotic Effect of HMG-CoA Reductase Inhibitors

    Institute of Scientific and Technical Information of China (English)

    贺石林

    2005-01-01

    @@ Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors are widely used for the treatment of hypercholesteremia and have showed remarkable activity in preventing cardiovascular morbidity and mortality. Recent studies demonstrated that statins have significant antithrombotic effect in addition to cholesterollowering action. Although the efficacy of statins for reducing cardiovascular events has historically been ascribed to their inhibitory activity on cholesterol synthesis, the degree of low-density lipoprotein cholesterol reduction by statins generally does not correlate with the magnitude of coronary risk reduction.

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

    Science.gov (United States)

    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.

  13. Quantum chemical study of the mechanism of action of vitamin K epoxide reductase (VKOR)

    Science.gov (United States)

    Deerfield, David, II; Davis, Charles H.; Wymore, Troy; Stafford, Darrel W.; Pedersen, Lee G.

    Possible model, but simplistic, mechanisms for the action of vitamin K epoxide reductase (VKOR) are investigated with quantum mechanical methods (B3LYP/6-311G**). The geometries of proposed model intermediates in the mechanisms are energy optimized. Finally, the energetics of the proposed (pseudo-enzymatic) pathways are compared. We find that the several pathways are all energetically feasible. These results will be useful for designing quantum mechanical/molecular mechanical method (QM/MM) studies of the enzymatic pathway once three-dimensional structural data are determined and available for VKOR.

  14. Crystal structure of the electron transfer complex rubredoxin rubredoxin reductase of Pseudomonas aeruginosa.

    OpenAIRE

    2007-01-01

    Crude oil spills represent a major ecological threat because of the chemical inertness of the constituent n-alkanes. The Gram-negative bacterium Pseudomonas aeruginosa is one of the few bacterial species able to metabolize such compounds. Three chromosomal genes, rubB, rubA1, and rubA2 coding for an NAD(P)H:rubredoxin reductase (RdxR) and two rubredoxins (Rdxs) are indispensable for this ability. They constitute an electron transport (ET) pathway that shuttles reducing equivalents from carbon...

  15. Crystal structure of the electron transfer complex rubredoxin–rubredoxin reductase of Pseudomonas aeruginosa

    OpenAIRE

    2007-01-01

    Crude oil spills represent a major ecological threat because of the chemical inertness of the constituent n-alkanes. The Gram-negative bacterium Pseudomonas aeruginosa is one of the few bacterial species able to metabolize such compounds. Three chromosomal genes, rubB, rubA1, and rubA2 coding for an NAD(P)H:rubredoxin reductase (RdxR) and two rubredoxins (Rdxs) are indispensable for this ability. They constitute an electron transport (ET) pathway that shuttles reducing equivalents from carbon...

  16. A structural account of substrate and inhibitor specificity differences between two Naphthol reductases

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Thompson, J.E.; Fahnestock, S.; Valent, B.; Jordan, D.B. (DuPont)

    2010-03-08

    Two short chain dehydrogenase/reductases mediate naphthol reduction reactions in fungal melanin biosynthesis. An X-ray structure of 1,3,6,8-tetrahydroxynaphthalene reductase (4HNR) complexed with NADPH and pyroquilon was determined for examining substrate and inhibitor specificities that differ from those of 1,3,8-trihydroxynaphthalene reductase (3HNR). The 1.5 {angstrom} resolution structure allows for comparisons with the 1.7 {angstrom} resolution structure of 3HNR complexed with the same ligands. The sequences of the two proteins are 46% identical, and they have the same fold. The 30-fold lower affinity of the 4HNR-NADPH complex for pyroquilon (a commercial fungicide that targets 3HNR) in comparison to that of the 3HNR-NADPH complex can be explained by unfavorable interactions between the anionic carboxyl group of the C-terminal Ile282 of 4HNR and CH and CH{sub 2} groups of the inhibitor that are countered by favorable inhibitor interactions with 3HNR. 1,3,8-Trihydroxynaphthalene (3HN) and 1,3,6,8-tetrahydroxynaphthalene (4HN) were modeled onto the cyclic structure of pyroquilon in the 4HNR-NADPH-pyroquilon complex to examine the 300-fold preference of the enzyme for 4HN over 3HN. The models suggest that the C-terminal carboxyl group of Ile282 has a favorable hydrogen bonding interaction with the C6 hydroxyl group of 4HN and an unfavorable interaction with the C6 CH group of 3HN. Models of 3HN and 4HN in the 3HNR active site suggest a favorable interaction of the sulfur atom of the C-terminal Met283 with the C6 CH group of 3HN and an unfavorable one with the C6 hydroxyl group of 4HN, accounting for the 4-fold difference in substrate specificities. Thus, the C-terminal residues of the two naphthol reductase are determinants of inhibitor and substrate specificities.

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

    Science.gov (United States)

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

    2009-06-08

    Posterior circulation vascular occlusive disease in children is a rare and uncommonly reported event. Among the numerous risk factors, the methylenetetrahydrofolate reductase (MTHFR) mutation is considered to be a common genetic cause of thrombosis in adults and children. Recently, a link between the MTHFR mutation and cerebrovascular disorders was reported in children. Diffusion tensor imaging (DTI) is a great improvement on magnetic resonance imaging (MRI), making the in vivo anatomical and pathological study of the brain and its fibers possible. In our patient cerebellar infarction was associated with MTHFR mutation and, in a standard neurological examination, DTI revealed normal white matter tracts.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Kai Wang

    2014-04-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1983-12-01

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

  1. Sulfur globule oxidation in green sulfur bacteria is dependent on the dissimilatory sulfite reductase system

    DEFF Research Database (Denmark)

    Holkenbrink, Carina; Ocón Barbas, Santiago; Mellerup, Anders;

    2011-01-01

    Green sulfur bacteria oxidize sulfide and thiosulfate to sulfate with extracellular globules of elemental sulfur as intermediate. Here we investigated which genes are involved in the formation and consumption of these sulfur globules in the green sulfur bacterium Chlorobaculum tepidum. We show...... that sulfur globule oxidation is strictly dependent on the dissimilatory sulfite reductase (DSR) system. Deletion of dsrM/CT2244 or dsrT/CT2245 or the two dsrCABL clusters (CT0851-CT0854, CT2247-2250) abolished sulfur globule oxidation and prevented formation of sulfate from sulfide, whereas deletion of dsr...

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    We have performed a systematic study of chemically possible peroxo-type intermediates occurring in the non-heme di-iron enzyme class la ribonucleotide reductase, using spectroscopically calibrated computational chemistry. Density functional computations of equilibrium structures, Fe-O and O...... in carboxylate conformations occurring during the O-2 reaction of this class of non-heme iron enzymes. Our procedure identifies and characterizes various possible candidates for peroxo intermediates experimentally observed along the ribonucleotide reductase dioxygen activation reaction. The study explores how...... water or a proton can bind to the di-iron site of ribonucleotide reductase and facilitate changes that affect the electronic structure of the iron sites and activate the site for further reaction. Two potential reaction pathways are presented: one where water adds to Fe1 of the cis-mu-1,2 peroxo...

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

    Science.gov (United States)

    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.

  4. 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.

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

    Science.gov (United States)

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

    2008-02-01

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

  6. 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.

  7. A recombinant thioredoxin-glutathione reductase from Fasciola hepatica induces a protective response in rabbits.

    Science.gov (United States)

    Maggioli, Gabriela; Silveira, Fernando; Martín-Alonso, José M; Salinas, Gustavo; Carmona, Carlos; Parra, Francisco

    2011-12-01

    Antioxidant systems are fundamental components of host-parasite interactions, and often play a key role in parasite survival. Here, we report the cloning, heterologous expression, and characterization of a thioredoxin glutathione reductase (TGR) from Fasciola hepatica. The deduced polypeptide sequence of the cloned open reading frame (ORF) confirmed the experimental N-terminus previously determined for a native F. hepatica TGR showing thioredoxin reductase (TR) activity. The sequence revealed the presence of a fusion between a glutaredoxin (Grx) and a TR domain, similar to that previously reported in Schistosoma mansoni and Echinococcus granulosus. The F. hepatica TGR sequence included an additional redox active center (ACUG; U being selenocysteine) located at the C-terminus. The addition of a recombinant selenocysteine insertion sequence (SECIS) element in the Escherichia coli expression vector, or the substitution of the native selenocysteine by a cysteine, indicated the relevance of this unusual amino acid residue for the activity of F. hepatica TGR. Rabbit vaccination with recombinant F. hepatica TGR reduced the worm burden by 96.7% following experimental infection, further supporting the relevance of TGR as a promising target for anti Fasciola treatments.

  8. 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.

  9. Molecular cloning, functional characterization, and subcellular localization of soybean nodule dihydrolipoamide reductase.

    Science.gov (United States)

    Moran, Jose F; Sun, Zhaohui; Sarath, Gautam; Arredondo-Peter, Raúl; James, Euan K; Becana, Manuel; Klucas, Robert V

    2002-01-01

    Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed Km and kcat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH.

  10. Corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique mycothiol redox relay mechanism.

    Science.gov (United States)

    Tossounian, Maria-Armineh; Pedre, Brandán; Wahni, Khadija; Erdogan, Huriye; Vertommen, Didier; Van Molle, Inge; Messens, Joris

    2015-05-01

    Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.

  11. Protective Role of Aldose Reductase Deletion in an Animal Model of Oxygen-Induced Retinopathy

    Directory of Open Access Journals (Sweden)

    Zhongjie Fu

    2011-05-01

    Full Text Available Retinopathy of prematurity (ROP is a common disease occurred in premature babies. Both vascular abnormality and neural dysfunction of the retina were reported, and oxidative stress was involved. Previously, it has been showed that deficiency of aldose reductase (AR, the rate-limiting enzyme in polyol pathway, lowered oxidative stress. Here, the effect of AR deletion on neonatal retinal injury was investigated by using a mouse model of ROP (oxygen-induced retinopathy, OIR. Seven-day-old pups were exposed to 75% oxygen for 5 days and then returned to room air. The vascular changes and neuronal/glial responses were examined and compared between wild-type and AR-deficient OIR mice. Significantly reduced vaso-obliterated area, blood vessel leakage, and early revascularization were observed in AR-deficient OIR mice. Moreover, reduced amacrine cells and less distorted strata were observed in AR-deficient OIR mice. Less astrocytic immunoreactivity and reduced Müller cell gliosis were also observed in AR-deficient mice. After OIR, nitrotyrosine immunoreactivity and poly (ADP-ribose (PAR translocation, which are two oxidative stress markers, were decreased in AR-deficient mice. Significant decrease in VEGF, pho-Erk1/2, pho-Akt, and pho-I?B expression was found in AR-deficient OIR retinae. Thus, these observations suggest that the deficiency of aldose reductase may protect the retina in the OIR model.

  12. Reduction of azo dyes by flavin reductase from Citrobacter freundii A1

    Directory of Open Access Journals (Sweden)

    Mohd Firdaus Abdul-Wahab

    2012-12-01

    Full Text Available Citrobacter freundii A1 isolated from a sewage treatment facility was demonstrated to be able to effectively decolorize azo dyes as pure and mixed culture. This study reports on the investigation on the enzymatic systems involved. An assay performed suggested the possible involvement of flavin reductase (Fre as an azo reductase. A heterologouslyexpressed recombinant Fre from C. freundii A1 was used to investigate its involvement in the azo reduction process. Three model dyes were used, namely Acid Red 27 (AR27, Direct Blue 15 (DB15 and Reactive Black 5 (RB5. AR27 was found to be reduced the fastest by Fre, followed by RB5, and lastly DB15. Redox mediators nicotinamide adenine dinucleotide (NADH and riboflavin enhance the reduction, suggesting the redox activity of the enzyme. The rate and extent of reduction of the model dyes correlate well with the reduction potentials (Ep. The data presented here strongly suggest that Fre is one of the enzymes responsible for azo reduction in C. freundii A1, acting via an oxidation-reduction reaction.

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

    Science.gov (United States)

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

    2007-12-01

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

  14. Different effects of two aldose reductase inhibitors on nociception and prostaglandin E.

    Science.gov (United States)

    Calcutt, N A; Li, L; Yaksh, T L; Malmberg, A B

    1995-10-16

    This study examined the effect of two structurally dissimilar aldose reductase inhibitors, N-[[5-(trifluoromethyl)-6-methoxy-1- napthalenyl]thioxomethyl]-N-methlyglycine (tolrestat) and 4-amino-2,6-dimethylphenyl-sulphonyl nitromethane (ICI 222155), on formalin-evoked behavioural responses in control and diabetic rats and on capsaicin-evoked release of prostaglandin E from spinal cord slices in vitro. Both compounds, given orally for 4 weeks, prevented hyperalgesia in diabetic rats 5-20 min after hindpaw formalin injection. ICI 222155 also prevented hyperalgesia in diabetic rats 21-60 min after formalin, whereas tolrestat suppressed activity in diabetic rats below controls and also suppressed activity in controls when given orally or intrathecally. Capsaicin-evoked release of prostaglandin E from spinal cord slices of control rats was significantly reduced by tolrestat, but not ICI 222155. These data suggest that hyperalgesia in diabetic rats is related to glucose metabolism by aldose reductase, whereas tolrestat has specific effects on formalin-evoked nociception associated with an ability to reduce spinal prostaglandin release.

  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. Kinetic characterization of an oxidative, cooperative HMG-CoA reductase from Burkholderia cenocepacia.

    Science.gov (United States)

    Schwarz, Benjamin H; Driver, Joseph; Peacock, Riley B; Dembinski, Holly E; Corson, Melissa H; Gordon, Samuel S; Watson, Jeffrey M

    2014-02-01

    3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme in endogenous cholesterol biosynthesis in mammals and isoprenoid biosynthesis via the mevalonate pathway in other eukaryotes, archaea and some eubacteria. In most organisms that express this enzyme, it catalyzes the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have cloned and characterized the 6x-His-tagged HMGR from the opportunistic lung pathogen Burkholderia cenocepacia. Kinetic characterization shows that the enzyme prefers NAD(H) over NADP(H) as a cofactor, suggesting an oxidative physiological role for the enzyme. This hypothesis is supported by the fact that the Burkholderia cenocepacia genome lacks the genes for the downstream enzymes of the mevalonate pathway. The enzyme exhibits positive cooperativity toward the substrates of the reductive reaction, but the oxidative reaction exhibits unusual double-saturation kinetics, distinctive among characterized HMG-CoA reductases. The unusual kinetics may arise from the presence of multiple active oligomeric states, each with different Vmax values.

  17. 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.

  18. Conditional gene expression and promoter replacement in Zymoseptoria tritici using fungal nitrate reductase promoters.

    Science.gov (United States)

    Marchegiani, Elisabetta; Sidhu, Yaadwinder; Haynes, Ken; Lebrun, Marc-Henri

    2015-06-01

    Studying essential genes in haploid fungi requires specific tools. Conditional promoter replacement (CPR) is an efficient method for testing gene essentiality. However, this tool requires promoters that can be strongly down-regulated. To this end, we tested the nitrate reductase promoters of Magnaporthe oryzae (pMoNIA1) and Zymoseptoria tritici (pZtNIA1) for their conditional expression in Z. tritici. Expression of EGFP driven by pMoNIA1 or pZtNIA1 was induced on nitrate and down-regulated on glutamate (10-fold less than nitrate). Levels of differential expression were similar for both promoters, demonstrating that the Z. tritici nitrogen regulatory network functions with a heterologous promoter similarly to a native promoter. To establish CPR, the promoter of Z. tritici BGS1, encoding a β-1,3-glucan synthase, was replaced by pZtNIA1 using targeted sequence replacement. Growth of pZtNIA1::BGS1 CPR transformants was strongly reduced in conditions repressing pZtNIA1, while their growth was similar to wild type in conditions inducing pZtNIA1. This differential phenotype demonstrates that BGS1 is important for growth in Z. tritici. In addition, in inducing conditions, pZtNIA1::BGS1 CPR transformants were hyper-sensitive to Calcofluor white, a cell wall disorganizing agent. Nitrate reductase promoters are therefore suitable for conditional promoter replacement in Z. tritici. This tool is a major step toward identifying novel fungicide targets.

  19. A New Type of YumC-Like Ferredoxin (Flavodoxin) Reductase Is Involved in Ribonucleotide Reduction

    DEFF Research Database (Denmark)

    Chen, Jun; Shen, Jing; Solem, Christian;

    2015-01-01

    The trxB2 gene, which is annotated as a thioredoxin reductase, was found to be essential for growth of Lactococcus lactis in the presence of oxygen. The corresponding protein (TrxB2) showed a high similarity with Bacillus subtilis YumC (E value = 4.0E-88), and YumC was able to fully complement....... Genome sequencing of two independent isolates, which were able to grow as well as the wild-type strain under aerated conditions, revealed the importance of mutations in nrdI, encoding a flavodoxin involved in aerobic ribonucleotide reduction. We suggest a role for TrxB2 in nucleotide metabolism, where....... subtilis but that the addition of deoxynucleosides cannot compensate for the lethal phenotype displayed by the B. subtilis yumC knockout mutant. Ferredoxin (flavodoxin) reductase (FdR) is involved in many important reactions in both eukaryotes and prokaryotes, such as photosynthesis, nitrate reduction, etc. The recently...

  20. Purification and characterization of virginiamycin M1 reductase from Streptomyces virginiae.

    Science.gov (United States)

    Suzuki, N; Lee, C K; Nihira, T; Yamada, Y

    1998-11-01

    Virginiamycin M1 (VM1), produced by Streptomyces virginiae, is a polyunsaturated macrocyclic lactone antibiotic belonging to the virginiamycin A group. S. virginiae possesses an activity which stereospecifically reduces a 16-carbonyl group of VM1, resulting in antibiotically inactive 16R-dihydroVM1. The corresponding VM1 reductase was purified to homogeneity from crude extracts of S. virginiae in five steps, with 5,650-fold purification and 23% overall yield. The N-terminal amino acid sequence was determined to be MAIKLVIA. The purified enzyme showed an apparent Mr of 73,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Mr of 280,000 by native molecular sieve high-performance liquid chromatography, indicating the tetrameric nature of the native enzyme. NADPH served as a coenzyme for the reduction, with a Km value of 0.13 mM, but NADH did not support the reaction, even at a concentration of 5 mM, indicating the NADPH-specific nature of the enzyme. The Km for VM1 was determined to be 1.5 mM in the presence of 2 mM NADPH. In the reverse reaction, only 16R-dihydroVM1, not the 16S-epimer, served as a substrate, with a less than 0.1% overall reaction rate compared to that of the forward reaction, confirming that the VM1 reductase participates solely in VM1 inactivation in vivo.

  1. The Quaternary Structure of NADPH Thioredoxin Reductase C Is Redox-Sensitive

    Institute of Scientific and Technical Information of China (English)

    Juan Manuel Pérez-Ruiz; Maricruz González; Maria Cristina Spinola; Luisa Maria Sandali; Francisco Javier Cejudo

    2009-01-01

    NADPH thioredoxin reductase C (NTRC) is a chloroplast enzyme able to conjugate NADPH thioredoxin reduc-tase (NTR) and thioredoxin (TRX) activities for the efficient reduction of 2-Cys peroxiredoxin (2-Cys PRX).Because NADPH can be produced in chloroplasts during darkness,NTRC plays a key role for plant peroxide detoxification during the night.Here,it is shown that the quaternary structure of NTRC is highly dependent on its redox status.In vitro,most of the enzyme adopted an oligomeric state that disaggregated in dimers upon addition of NADPH,NADH,or DTr.Gel filtration and West-ern blot analysis of protein extracts from Arabidopsis chloroplast stroma showed that native NTRC forms aggregates,which are sensitive to NADPH and DTT,suggesting that the aggregation state might be a significant aspect of NTRC activity in vivo.Moreover,the enzyme is localized in clusters in Arabidopsis chloroplasts.NTRC triple and double mutants,A164G-V182E-R183F and A164G-R183F,replacing key residues of NADPH binding site,showed reduced activity but were still able to dimerize though with an increase in intermediary forms.Based on these results,we propose that the catalytically active form of NTRC is the dimer,which formation is induced by NADPH.

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

    Science.gov (United States)

    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.

  3. Overexpression of soybean isoflavone reductase (GmIFR enhances resistance to Phytophthora sojae in soybean

    Directory of Open Access Journals (Sweden)

    Qun eCheng

    2015-11-01

    Full Text Available Isoflavone reductase (IFR is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. The cDNA of GmIFR was 1199 bp containing a 939 bp open reading frame encoding a polypeptide of 312 amino acids. Sequence analysis suggested that GmIFR contained a NAD(P domain of 107 amino acids. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET, abscisic acid (ABA, salicylic acid (SA. It is located in the cytoplasmic when transiently expressed in Arabidopsis protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while levels of genistein and glycitein had little change compared to that of control plants. Furthermore, we also found that the reactive oxygen species (ROS content of transgenic soybean plants was significantly lower than that of control plants, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean.

  4. Mechanostability of the Single-Electron-Transfer Complexes of Anabaena Ferredoxin-NADP(+) Reductase.

    Science.gov (United States)

    Marcuello, Carlos; de Miguel, Rocío; Martínez-Júlvez, Marta; Gómez-Moreno, Carlos; Lostao, Anabel

    2015-10-26

    The complexes formed between the flavoenzyme ferredoxin-NADP(+) reductase (FNR; NADP(+) =nicotinamide adenine dinucleotide phosphate) and its redox protein partners, ferredoxin (Fd) and flavodoxin (Fld), have been analysed by using dynamic force spectroscopy through AFM. A strategy is developed to immobilise proteins on a substrate and AFM tip to optimise the recognition ability. The differences in the recognition efficiency regarding a random attachment procedure, together with nanomechanical results, show two binding models for these systems. The interaction of the reductase with the natural electron donor, Fd, is threefold stronger and its lifetime is longer and more specific than that with the substitute under iron-deficient conditions, Fld. The higher bond probability and two possible dissociation pathways in Fld binding to FNR are probably due to the nature of this complex, which is closer to a dynamic ensemble model. This is in contrast with the one-step dissociation kinetics that has been observed and a specific interaction described for the FNR:Fd complex.

  5. Synthesis of organic nitrates of luteolin as a novel class of potent aldose reductase inhibitors.

    Science.gov (United States)

    Wang, Qi-Qin; Cheng, Ning; Zheng, Xiao-Wei; Peng, Sheng-Ming; Zou, Xiao-Qing

    2013-07-15

    Aldose reductase (AR) plays an important role in the design of drugs that prevent and treat diabetic complications. Aldose reductase inhibitors (ARIs) have received significant attentions as potent therapeutic drugs. Based on combination principles, three series of luteolin derivatives were synthesised and evaluated for their AR inhibitory activity and nitric oxide (NO)-releasing capacity in vitro. Eighteen compounds were found to be potent ARIs with IC50 values ranging from (0.099±0.008) μM to (2.833±0.102) μM. O(7)-Nitrooxyethyl-O(3'),O(4')-ethylidene luteolin (La1) showed the most potent AR inhibitory activity [IC50=(0.099±0.008) μM]. All organic nitrate derivatives released low concentrations of NO in the presence of l-cysteine. Structure-activity relationship studies suggested that introduction of an NO donor, protection of the catechol structure, and the ether chain of a 2-carbon spacer as a coupling chain on the luteolin scaffold all help increase the AR inhibitory activity of the resulting compound. This class of NO-donor luteolin derivatives as efficient ARIs offer a new concept for the development and design of new drug for preventive and therapeutic drugs for diabetic complications.

  6. 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).

  7. Deletion of thioredoxin reductase and effects of selenite and selenate toxicity in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Christopher J Boehler

    Full Text Available Thioredoxin reductase-1 (TRXR-1 is the sole selenoprotein in C. elegans, and selenite is a substrate for thioredoxin reductase, so TRXR-1 may play a role in metabolism of selenium (Se to toxic forms. To study the role of TRXR in Se toxicity, we cultured C. elegans with deletions of trxr-1, trxr-2, and both in axenic media with increasing concentrations of inorganic Se. Wild-type C. elegans cultured for 12 days in Se-deficient axenic media grow and reproduce equivalent to Se-supplemented media. Supplementation with 0-2 mM Se as selenite results in inverse, sigmoidal response curves with an LC50 of 0.20 mM Se, due to impaired growth rather than reproduction. Deletion of trxr-1, trxr-2 or both does not modulate growth or Se toxicity in C. elegans grown axenically, and (75Se labeling showed that TRXR-1 arises from the trxr-1 gene and not from bacterial genes. Se response curves for selenide (LC50 0.23 mM Se were identical to selenite, but selenate was 1/4(th as toxic (LC50 0.95 mM Se as selenite and not modulated by TRXR deletion. These nutritional and genetic studies in axenic media show that Se and TRXR are not essential for C. elegans, and that TRXR alone is not essential for metabolism of inorganic Se to toxic species.

  8. Methionine sulfoxide reductase A expression is regulated by the DAF-16/FOXO pathway in Caenorhabditis elegans.

    Science.gov (United States)

    Minniti, Alicia N; Cataldo, Romina; Trigo, Carla; Vasquez, Luis; Mujica, Patricio; Leighton, Federico; Inestrosa, Nibaldo C; Aldunate, Rebeca

    2009-12-01

    The methionine sulfoxide reductase system has been implicated in aging and protection against oxidative stress. This conserved system reverses the oxidation of methionine residues within proteins. We analyzed one of the components of this system, the methionine sulfoxide reductase A gene, in Caenorhabditis elegans. We found that the msra-1 gene is expressed in most tissues, particularly in the intestine and the nervous system. Worms carrying a deletion of the msra-1 gene are more sensitive to oxidative stress, show chemotaxis and locomotory defects, and a 30% decrease in median survival. We established that msra-1 expression decreases during aging and is regulated by the DAF-16/FOXO3a transcription factor. The absence of this enzyme decreases median survival and affects oxidative stress resistance of long lived daf-2 worms. A similar effect of MSRA-1 absence in wild-type and daf-2 (where most antioxidant enzymes are activated) backgrounds, suggests that the lack of this member of the methionine repair system cannot be compensated by the general antioxidant response. Moreover, FOXO3a directly activates the human MsrA promoter in a cell culture system, implying that this could be a conserved mechanism of MsrA regulation. Our results suggest that repair of oxidative damage in proteins influences the rate at which tissues age. This repair mechanism, rather than the general decreased of radical oxygen species levels, could be one of the main determinants of organisms' lifespan.

  9. Molecular dissection of a putative iron reductase from Desulfotomaculum reducens MI-1

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhi; Kim, David D.; Nelson, Ornella D.; Otwell, Annie E.; Richardson, Ruth E.; Callister, Stephen J.; Lin, Hening

    2015-10-08

    Desulfotomaculum reducens MI-1 is a Firmicute strain capable of reducing a variety of heavy metal ions and has a great potential in heavy metal bioremediation.We recently identified Dred_2421 as a potential iron reductase through proteomic study of D. reducens. The current study examines its iron-reduction mechanism. Dred_2421, like its close homolog from Escherichia coli (2, 4-dienoyl-CoA reductase), has an FMN-binding N-terminal domain (NTD), an FAD-binding C-terminal domain (CTD), and a 4Fee4S cluster between the two domains. To understand the mechanism of the iron-reduction activity and the role of each domain, we generated a series of variants for each domain and investigated their iron reduction activity. Our results suggest that CTD is the main contributor of the iron-reduction activity, and that NTD and the 4Fee4S cluster are not directly involved in such activity. This study provides a mechanistic understanding of the ironereductase activity of Dred_2421 and may also help to elucidate other physiological activities this enzyme may have.

  10. Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple

    Directory of Open Access Journals (Sweden)

    Yuepeng eHan

    2015-04-01

    Full Text Available Proanthocyanidins (PAs are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR and anthocyanidin reductase (ANR was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis.

  11. Calmodulin-mediated suppression of 2-ketoisovalerate reductase in Beauveria bassiana beauvericin biosynthetic pathway.

    Science.gov (United States)

    Kim, Jiyoung; Yoon, Deok-Hyo; Oh, Junsang; Hyun, Min-Woo; Han, Jae-Gu; Sung, Gi-Ho

    2016-11-01

    Ketoisovalerate reductase (KIVR, E.C. 1.2.7.7) mediates the specific reduction of 2-ketoisovalerate (2-Kiv) to d-hydroxyisovalerate (d-Hiv), a precursor for beauvericin biosynthesis. Beauvericin, a famous mycotoxin produced by many fungi, is a cyclooligomer depsipeptide, which has insecticidal, antimicrobial, antiviral, and cytotoxic activities. In this report, we demonstrated that Beauveria bassiana 2-ketoisovalerate reductase (BbKIVR) acts as a typical KIVR enzyme in the entomopathogenic fungus B. bassiana. In addition, we found that BbKIVR interacts with calmodulin (CaM) in vitro and in vivo. The functional role of CaM-binding to BbKIVR was to negatively regulate the BbKIVR activity in B. bassiana. Environmental stimuli such as light and salt stress suppressed BbKIVR activity in B. bassiana. Interestingly, this negative effect of BbKIVR activity by light and salt stress was recovered by CaM inhibitors, suggesting that the inhibitory mechanism is mediated through stimulation of CaM activity. Therefore, this work suggests that BbKIVR plays an important role in the beauvericin biosynthetic pathway mediated by environmental stimuli such as light and salt stress via the CaM signaling pathway.

  12. 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) (-).

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Schormann, Norbert; Velu, Sadanandan E.; Murugesan, Srinivasan; Senkovich, Olga; Walker, Kiera; Chenna, Bala C.; Shinkre, Bidhan; Desai, Amar; Chattopadhyay, Debasish (UAB)

    2010-09-17

    Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas disease. We have undertaken a detailed structure-activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme-ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60-70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.

  15. One statin, two statins, three statins, more: similarities and differences of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

    Science.gov (United States)

    Turkoski, Beatrice B

    2011-01-01

    Statin drugs (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) are one of the most widely prescribed drugs today. They are considered first-line therapy to lower blood serum cholesterol levels in conjunction with therapeutic lifestyle changes for both primary and secondary prevention of cardiovascular events. In the following discussion, a brief explanation of the background of statins will explain why they are deemed so important today. The similarities and differences between the different statins will be addressed, including a look at dosage, side effects, and cautions for the seven 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors currently available.

  16. Evaluation of the food grade expression systems NICE and pSIP for the production of 2,5-diketo-D-gluconic acid reductase from Corynebacterium glutamicum.

    Science.gov (United States)

    Kaswurm, Vanja; Nguyen, Tien-Thanh; Maischberger, Thomas; Kulbe, Klaus D; Michlmayr, Herbert

    2013-01-28

    2,5-diketo-D-gluconic acid reductase (2,5-DKG reductase) catalyses the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-keto-L-gulonic acid (2-KLG), a direct precursor (lactone) of L-ascorbic acid (vitamin C). This reaction is an essential step in the biocatalytic production of the food supplement vitamin C from D-glucose or D-gluconic acid. As 2,5-DKG reductase is usually produced recombinantly, it is of interest to establish an efficient process for 2,5-DKG reductase production that also satisfies food safety requirements. In the present study, three recently described food grade variants of the Lactobacillales based expression systems pSIP (Lactobacillus plantarum) and NICE (Lactococcus lactis) were evaluated with regard to their effictiveness to produce 2,5-DKG reductase from Corynebacterium glutamicum. Our results indicate that both systems are suitable for 2,5-DKG reductase expression. Maximum production yields were obtained with Lb. plantarum/pSIP609 by pH control at 6.5. With 262 U per litre of broth, this represents the highest heterologous expression level so far reported for 2,5-DKG reductase from C. glutamicum. Accordingly, Lb. plantarum/pSIP609 might be an interesting alternative to Escherichia coli expression systems for industrial 2,5-DKG reductase production.

  17. Polycyclic aromatic hydrocarbons: from metabolism to lung cancer.

    Science.gov (United States)

    Moorthy, Bhagavatula; Chu, Chun; Carlin, Danielle J

    2015-05-01

    Excessive exposure to polycyclic aromatic hydrocarbons (PAHs) often results in lung cancer, a disease with the highest cancer mortality in the United States. After entry into the lung, PAHs induce phase I metabolic enzymes such as cytochrome P450 (CYP) monooxygenases, i.e. CYP1A1/2 and 1B1, and phase II enzymes such as glutathione S-transferases, UDP glucuronyl transferases, NADPH quinone oxidoreductases (NQOs), aldo-keto reductases (AKRs), and epoxide hydrolases (EHs), via the aryl hydrocarbon receptor (AhR)-dependent and independent pathways. Humans can also be exposed to PAHs through diet, via consumption of charcoal broiled foods. Metabolism of PAHs through the CYP1A1/1B1/EH pathway, CYP peroxidase pathway, and AKR pathway leads to the formation of the active carcinogens diol-epoxides, radical cations, and o-quinones. These reactive metabolites produce DNA adducts, resulting in DNA mutations, alteration of gene expression profiles, and tumorigenesis. Mutations in xenobiotic metabolic enzymes, as well as polymorphisms of tumor suppressor genes (e.g. p53) and/or genes involved in gene expression (e.g. X-ray repair cross-complementing proteins), are associated with lung cancer susceptibility in human populations from different ethnicities, gender, and age groups. Although various metabolic activation/inactivation pathways, AhR signaling, and genetic susceptibilities contribute to lung cancer, the precise points at which PAHs induce tumor initiation remain unknown. The goal of this review is to provide a current state-of-the-science of the mechanisms of human lung carcinogenesis mediated by PAHs, the experimental approaches used to study this complex class of compounds, and future directions for research of these compounds.

  18. Correlation between sequence, structure and function for trisporoid processing proteins in the model zygomycete Mucor mucedo.

    Science.gov (United States)

    Ellenberger, Sabrina; Schuster, Stefan; Wöstemeyer, Johannes

    2013-03-01

    Terpenoids, steroids, carotenoids, phytoenes and other chemically related substance groups fulfill multiple functions in all realms of the organismic world. This analysis focuses on trisporoids that operate as pheromones in the phylogenetically ancient fungal group of mucoralean zygomycetes. Trisporoids serve as pheromones for recognizing complementary mating partners and for inducing the differentiation program towards sexual spore formation. Trisporoids are synthesized by oxidative degradation of β-carotene. Structurally, they are related to retinoids in mammals and abscisic acid in vascular plants. In order to evaluate evolutionary relationships between proteins involved in trisporoid binding and also for checking possibilities to recognize functionally related proteins by sequence and structure comparisons, we compared representative proteins of different origins. Towards this goal, we calculated three-dimensional structures for 4-dihydromethyltrisporate dehydrogenase (TSP1) and 4-dihydrotrisporin dehydrogenase (TSP2), the two proteins involved in trisporic acid synthesis that have unequivocally been correlated with their catalytic function for the model zygomycete Mucor mucedo. TSP1 is an aldo-keto reductase with a TIM-barrel structure, TSP2 belongs to short-chain dehydrogenases, characterized by a Rossmann fold. Evidently, functional conservation, even implying very similar substrates and identical cosubstrates of enzymes in a single organism, turns out to be essentially independent of basic protein structure. The binding sites for NADP and trisporoid ligands in the proteins were determined by docking studies, revealing those regions affecting substrate specificity. Despite the pronounced differences in amino acid sequence and tertiary structure, the surfaces around the active sites are comparable between TSP1 and TSP2. Two binding regions were identified, one sterically open and a second closed one. In contrast to TSP1, all docking models for TSP2 place the

  19. 4-Dihydromethyltrisporate dehydrogenase from Mucor mucedo, an enzyme of the sexual hormone pathway: purification, and cloning of the corresponding gene.

    Science.gov (United States)

    Czempinski, K; Kruft, V; Wöstemeyer, J; Burmester, A

    1996-09-01

    We have purified the NADP-dependent 4-dihydromethyltrisporate dehydrogenase from the zygomycete Mucor mucedo. The enzyme is involved in the biosynthesis of trisporic acid, the sexual hormone of zygomycetes, which induces the first steps of zygophore development. Protein was obtained from the (-) mating type of M. mucedo after induction with trisporic acid, and purified by gel filtration and affinity chromatography steps. On SDS-PAGE a band with an apparent molecular mass of 33 kDa was ascribed to the enzyme. After transferring onto PVDF membranes the protein was digested with endoprotease Lys-C, and several peptides were sequenced. Oligonucleotides derived from protein sequence data were used for PCR amplification of genomic M. mucedo DNA. The PCR fragment was used as probe for isolation of the corresponding cDNA and complete genomic DNA clones. Comparison of protein and DNA sequence data showed that the cloned fragment corresponded to the purified protein. Search for similarity with protein sequences of the Swiss-Prot database revealed a relationship to enzymes belonging to the aldo/keto reductase superfamily. Southern-blot analysis of genomic DNA with the labelled cloned fragment detected a single-copy gene in both mating types of M. mucedo. PCR with genomic DNA from other zygomycetes gave rise to several fragments. Hybridization analysis with the cloned M. mucedo fragment showed that a fragment of similar length cross-hybridized in Blakeslea trispora (Choanephoraceae) as well as in Parasitella parasitica and Absidia glauca (Mucoraceae). The promoter region of the gene contains DNA elements with similarity to a cAMP-regulated gene of Dictyostelium discoideum.

  20. 4-dihydrotrisporin-dehydrogenase, an enzyme of the sex hormone pathway of Mucor mucedo: purification, cloning of the corresponding gene, and developmental expression.

    Science.gov (United States)

    Wetzel, Jana; Scheibner, Olaf; Burmester, Anke; Schimek, Christine; Wöstemeyer, Johannes

    2009-01-01

    The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (-) mating-type-specific enzyme in the pathway from beta-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (-) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (-) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation.

  1. 4-Dihydrotrisporin-Dehydrogenase, an Enzyme of the Sex Hormone Pathway of Mucor mucedo: Purification, Cloning of the Corresponding Gene, and Developmental Expression▿

    Science.gov (United States)

    Wetzel, Jana; Scheibner, Olaf; Burmester, Anke; Schimek, Christine; Wöstemeyer, Johannes

    2009-01-01

    The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (−) mating-type-specific enzyme in the pathway from β-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (−) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (−) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation. PMID:18931040

  2. AKRs expression in peripheral blood lymphocytes from smokers: the role of body mass index.

    Science.gov (United States)

    Barrón-Vivanco, B S; Rothenberg, S J; Medina-Díaz, I M; Robledo-Marenco, L; Rojas-García, A E; Hernández-Cadena, L; Poblete-Naredo, I; Elizondo, G; Albores, A

    2013-04-01

    Aldo-keto reductases (AKRs) metabolize a wide range of substrates, including polycyclic aromatic hydrocarbons (PAHs), generating metabolites (o-quinones) and reactive oxygen species (ROS), which are capable of initiating and promoting carcinogenesis. Exposure to PAHs, their metabolites, and ROS further increase AKRs isoform expression that may amplify oxidative damage. Human AKR enzymes are highly polymorphic, and allelic variants may contribute to different AKRs expression in individuals. Despite the importance of AKRs in PAHs metabolism, there are no studies that evaluate, in general human populations, the effect of PAHs on AKRs expression in peripheral blood lymphocytes (PBLs). The aim of this study was to determine the effect of tobacco smoke exposure, and AKR1A1*2 and AKR1C3*2 polymorphisms, on AKR1A1 and AKR1C1-AKR1C3 messenger RNA (mRNA) levels in PBLs from smokers. In the smoker group, there is a statistically significant positive association between AKR1A1, AKR1C1, and AKR1C3 mRNA induction and urine cotinine levels in individuals with a body mass index (BMI) less than 25. However, AKR1A1*2 and AKR1C3*2 alleles did not influence AKR1A1 and AKR1C1-AKR1C3 mRNA levels. These results suggest that AKRs induction by PAHs in smokers' PBLs is associated with BMI; therefore, the role of adipose tissue accumulation in PAHs' effects needs further investigation.

  3. Rational design of an AKR1C3-resistant analog of PR-104 for enzyme-prodrug therapy.

    Science.gov (United States)

    Mowday, Alexandra M; Ashoorzadeh, Amir; Williams, Elsie M; Copp, Janine N; Silva, Shevan; Bull, Matthew R; Abbattista, Maria R; Anderson, Robert F; Flanagan, Jack U; Guise, Christopher P; Ackerley, David F; Smaill, Jeff B; Patterson, Adam V

    2016-09-15

    The clinical stage anti-cancer agent PR-104 has potential utility as a cytotoxic prodrug for exogenous bacterial nitroreductases expressed from replicating vector platforms. However substrate selectivity is compromised due to metabolism by the human one- and two-electron oxidoreductases cytochrome P450 oxidoreductase (POR) and aldo-keto reductase 1C3 (AKR1C3). Using rational drug design we developed a novel mono-nitro analog of PR-104A that is essentially free of this off-target activity in vitro and in vivo. Unlike PR-104A, there was no biologically relevant cytotoxicity in cells engineered to express AKR1C3 or POR, under aerobic or anoxic conditions, respectively. We screened this inert prodrug analog, SN34507, against a type I bacterial nitroreductase library and identified E. coli NfsA as an efficient bioactivator using a DNA damage response assay and recombinant enzyme kinetics. Expression of E. coli NfsA in human colorectal cancer cells led to selective cytotoxicity to SN34507 that was associated with cell cycle arrest and generated a robust 'bystander effect' at tissue-like cell densities when only 3% of cells were NfsA positive. Anti-tumor activity of SN35539, the phosphate pre-prodrug of SN34507, was established in 'mixed' tumors harboring a minority of NfsA-positive cells and demonstrated marked tumor control following heterogeneous suicide gene expression. These experiments demonstrate that off-target metabolism of PR-104 can be avoided and identify the suicide gene/prodrug partnership of E. coli NfsA/SN35539 as a promising combination for development in armed vectors.

  4. Integration of HPV6 and downregulation of AKR1C3 expression mark malignant transformation in a patient with juvenile-onset laryngeal papillomatosis.

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    Christian Ulrich Huebbers

    Full Text Available Juvenile-onset recurrent respiratory papillomatosis (RRP is associated with low risk human papillomavirus (HPV types 6 and 11. Malignant transformation has been reported solely for HPV11-associated RRP in 2-4% of all RRP-cases, but not for HPV6. The molecular mechanisms in the carcinogenesis of low risk HPV-associated cancers are to date unknown. We report of a female patient, who presented with a laryngeal carcinoma at the age of 24 years. She had a history of juvenile-onset RRP with an onset at the age of three and subsequently several hundred surgical interventions due to multiple recurrences of RRP. Polymerase chain reaction (PCR or bead-based hybridization followed by direct sequencing identified HPV6 in tissue sections of previous papilloma and the carcinoma. P16(INK4A, p53 and pRb immunostainings were negative in all lesions. HPV6 specific fluorescence in situ hybridization (FISH revealed nuclear staining suggesting episomal virus in the papilloma and a single integration site in the carcinoma. Integration-specific amplification of papillomavirus oncogene transcripts PCR (APOT-PCR showed integration in the aldo-keto reductase 1C3 gene (AKR1C3 on chromosome 10p15.1. ArrayCGH detected loss of the other gene copy as part of a deletion at 10p14-p15.2. Western blot analysis and immunohistochemistry of the protein AKR1C3 showed a marked reduction of its expression in the carcinoma. In conclusion, we identified a novel molecular mechanism underlying a first case of HPV6-associated laryngeal carcinoma in juvenile-onset RRP, i.e. that HPV6 integration in the AKR1C3 gene resulted in loss of its expression. Alterations of AKR1C gene expression have previously been implicated in the tumorigenesis of other (HPV-related malignancies.

  5. Proteome analysis of early lineage specification in bovine embryos.

    Science.gov (United States)

    Demant, Myriam; Deutsch, Daniela R; Fröhlich, Thomas; Wolf, Eckhard; Arnold, Georg J

    2015-02-01

    During mammalian embryo development, the zygote undergoes embryonic cleavage in the oviduct and reaches the uterus at the morula stage, when compaction and early lineage specification take place. To increase knowledge about the associated changes of the embryonic protein repertoire, we performed a comprehensive proteomic analysis of in vitro produced bovine morulae and blastocysts (six biological replicates), using an iTRAQ-based approach. A total of 560 proteins were identified of which 502 were quantified. The abundance of 140 proteins was significantly different between morulae and blastocysts, among them nucleophosmin (NPM1), eukaryotic translation initiation factor 5A-1 (EIF5A), receptor of activated protein kinase C 1 (GNB2L1/RACK1), and annexin A6 (ANXA6) with increased, and glutathione S-transferase mu 3 (GSTM3), peroxiredoxin 2 (PRDX2), and aldo-keto reductase family 1 member B1 (AKR1B1) with decreased abundance in blastocysts. Seventy-three percent of abundance altered proteins increased, reflecting an increase of translation activity in this period. This is further supported by an increase in the abundance of proteins involved in the translation machinery and the synthesis of ATP. Additionally, a complementary 2D saturation DIGE analysis led to the detection of protein isoforms, e.g. of GSTM3 and PRDX2, relevant for this period of mammalian development, and exemplarily verified the results of the iTRAQ approach. In summary, our systematic differential proteome analysis of bovine morulae and blastocysts revealed new molecular correlates of early lineage specification and differentiation events during bovine embryogenesis.

  6. Sulforaphane Preconditioning Sensitizes Human Colon Cancer Cells towards the Bioreductive Anticancer Prodrug PR-104A.

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    Melanie M Erzinger

    Full Text Available The chemoprotective properties of sulforaphane (SF, derived from cruciferous vegetables, are widely acknowledged to arise from its potent induction of xenobiotic-metabolizing and antioxidant enzymes. However, much less is known about the impact of SF on the efficacy of cancer therapy through the modulation of drug-metabolizing enzymes. To identify proteins modulated by a low concentration of SF, we treated HT29 colon cancer cells with 2.5 μM SF. Protein abundance changes were detected by stable isotope labeling of amino acids in cell culture. Among 18 proteins found to be significantly up-regulated, aldo-keto reductase 1C3 (AKR1C3, bioactivating the DNA cross-linking prodrug PR-104A, was further characterized. Preconditioning HT29 cells with SF reduced the EC50 of PR-104A 3.6-fold. The increase in PR-104A cytotoxicity was linked to AKR1C3 abundance and activity, both induced by SF in a dose-dependent manner. This effect was reproducible in a second colon cancer cell line, SW620, but not in other colon cancer cell lines where AKR1C3 abundance and activity were absent or barely detectable and could not be induced by SF. Interestingly, SF had no significant influence on PR-104A cytotoxicity in non-cancerous, immortalized human colonic epithelial cell lines expressing either low or high levels of AKR1C3. In conclusion, the enhanced response of PR-104A after preconditioning with SF was apparent only in cancer cells provided that AKR1C3 is expressed, while its expression in non-cancerous cells did not elicit such a response. Therefore, a subset of cancers may be susceptible to combined food-derived component and prodrug treatments with no harm to normal tissues.

  7. Changes in gene expression induced by aromatic amine drugs: testing the danger hypothesis.

    Science.gov (United States)

    Ng, Winnie; Uetrecht, Jack

    2013-01-01

    Virtually all drugs that contain a primary aromatic amine are associated with a high incidence of idiosyncratic drug reactions (IDRs), suggesting that this functional group has biological effects that may be used as biomarkers to predict IDR risk. Most IDRs exhibit evidence of immune involvement and the ability of aromatic amines to form reactive metabolites and redox cycle may be responsible for initiation of an immune response through induction of cell stress, as postulated by the Danger Hypothesis. If true, danger signals could be biomarkers of IDR risk. A previous attempt to test the Danger Hypothesis found that sulfamethoxazole (SMX), the only aromatic amine tested, was also the only drug not associated with an increase of cell stress genes in mice. To ensure that these observations were not species-specific, and to determine biomarkers of IDR risk common to aromatic amines, rats were treated with SMX and two other aromatic amine drugs, dapsone (DDS) and aminoglutethimide (AMG), and hepatic gene expression was determined using microarrays. As in mice, SMX induced minimal gene changes in the rat, and none indicated cell stress, whereas DDS and AMG induced several changes including up-regulation of enzymes such as aldo-keto reductase, glutathione-S-transferase, and aldehyde dehydrogenase, which may represent danger signals. Early insulin-induced hepatic gene (Eiih) was up-regulated by all three drugs. Some mRNA changes were observed in the Keap-1-Nrf2-ARE pathway; however, the pattern was significantly different for each drug. Overall, the most salient finding was that the changes in the liver were minimal, even though aromatic amines cause a high incidence of IDRs. The liver generates a large number of reactive species; however, the ability of aromatic amines to be bioactivated by cells of the immune system may be why they cause a high incidence of IDRs.

  8. Comparative genomics of aldehyde dehydrogenase 5a1 (succinate semialdehyde dehydrogenase and accumulation of gamma-hydroxybutyrate associated with its deficiency

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    Malaspina Patrizia

    2009-01-01

    Full Text Available Abstract Succinic semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5A1 [ALDH5A1]; locus 6p22 occupies a central position in central nervous system (CNS neurotransmitter metabolism as one of two enzymes necessary for γ-aminobutyric acid (GABA recycling from the synaptic cleft. Its importance is highlighted by the neurometabolic disease associated with its inherited deficiency in humans, as well as the severe epileptic phenotype observed in Aldh5a1-/- knockout mice. Expanding evidence now suggests, however, that even subtle decreases in human SSADH activity, associated with rare and common single nucleotide polymorphisms, may produce subclinical pathological effects. SSADH, in conjunction with aldo-keto reductase 7A2 (AKR7A2, represent two neural enzymes responsible for further catabolism of succinic semialdehyde, producing either succinate (SSADH or γ-hydroxybutyrate (GHB; AKR7A2. A GABA analogue, GHB is a short-chain fatty alcohol with unusual properties in the CNS and a long pharmacological history. Moreover, SSADH occupies a further role in the CNS as the enzyme responsible for further metabolism of the lipid peroxidation aldehyde 4-hydroxy-2-nonenal (4-HNE, an intermediate known to induce oxidant stress. Accordingly, subtle decreases in SSADH activity may have the capacity to lead to regional accumulation of neurotoxic intermediates (GHB, 4-HNE. Polymorphisms in SSADH gene structure may also associate with quantitative traits, including intelligence quotient and life expectancy. Further population-based studies of human SSADH activity promise to reveal additional properties of its function and additional roles in CNS tissue.

  9. Green tea consumption, genetic susceptibility, PAH-rich smoky coal, and the risk of lung cancer.

    Science.gov (United States)

    Bonner, Matthew R; Rothman, Nathaniel; Mumford, Judy L; He, Xingzhou; Shen, Min; Welch, Robert; Yeager, Meredith; Chanock, Stephen; Caporaso, Neil; Lan, Qing

    2005-04-04

    Experimental evidence suggests that green tea (Camellia sinesis) may reduce the risk of lung cancer through several hypothesized mechanisms including scavenging oxidative radicals, inhibition of tumor initiation, and modulation of detoxification enzymes. However, epidemiologic results have not been consistent as to the relationship between green tea consumption and lung caner prevention. We employed a population-based case-control study of 122 cases and 122 controls to investigate the effect that green tea consumption may have on the risk of lung cancer and whether polymorphisms in 8-oxoguanine-DNA glycosylase (OGG1), glutathione-S-transferase M1 (GSTM1), and aldo-keto reductase 1C3 (AKR1C3) modify such an association. Daily green tea consumption was associated with a non-significant reduction in lung cancer risk. However, the effect of smoky coal exposure was higher for non-drinkers (odds ratio (OR)=4.93; 95% confidence interval (95% CI)=1.27-19.13) than for drinkers (OR=1.88; 95% CI=1.01-3.48). Further, among individuals with the OGG1 Cys(326) allele, daily consumption was associated with a 72% reduction (95% CI=0.09-0.94). Among GSTM1 null homozygotes, those who consumed green tea daily had a non-significant reduction in risk compared with non-consumers. Green tea consumption had no effect among OGG1 Ser(326) homozygotes or GSTM1 carriers. In addition, AKR1C3 genotype did not modulate the effect of green tea consumption. The chemopreventive effects of green tea in this population may be restricted to individuals who are particularly susceptible to oxidative stress and oxidative DNA damage.

  10. GENOMIC APPROACHES FOR IMPROVEMENT OF DROUGHT ADAPTATION IN WHEAT

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    Dénes Dudits

    2008-09-01

    Full Text Available Breeding for yield stability under water limited conditions plays an essential role in the reduction of economic and social consequences of global climate changes. We show that two exotic drought resistant genotypes (Kobomughi and Plainsmann differ in root growth rate, root/shoot ratio, and adaptation to low soil water content. These genotypes exhibit characteristic transcript profiles as shown by barley macroarray studies using 10500 unigenes. Reprogramming of gene expression primarily occurred during the 1-2 weeks of water stress, and 6,1% of tested genes were up-regulated in roots of the more adaptive Plainsmann plants. The time course for expression of gene clusters from Kobomughi genotype revealed a prompt and transient gene activation that can help the survival of plants through function of various defense mechanisms. The aldo-keto reductases (AKRs can detoxify lipid peroxidation products (4-hydroxynon-2-enal and glycolysis-derived reactive aldehydes (metylglyoxal that contribute significantly to cellular damages caused by variety of environmental stresses such as drought, high light intensity, UV-B irradiation, cold. Overproduction of AKRs in transgenic tobacco or wheat plants provides considerable stress tolerance and resistance to methylglyoxal. Several transgenic wheat genotypes have been produced with production of elevated level of AKR enzyme. The drought tolerance of these materials was tested by a complex stress diagnostic system, that integrates imaging of plants and monitoring the leaf temperature and fluorescence induction. Based on these parameters, we can conclude that this transgenic strategy that is based on detoxification of lipid aldehyde can result in improved stress adaptation and reduced yield loss.

  11. Diversity of assimilatory nitrate reductase genes from plankton and epiphytes associated with a seagrass bed.

    Science.gov (United States)

    Adhitya, Anita; Thomas, Florence I M; Ward, Bess B

    2007-11-01

    Assimilatory nitrate reductase gene fragments were isolated from epiphytes and plankton associated with seagrass blades collected from Tampa Bay, Florida, USA. Nitrate reductase genes from diatoms (NR) and heterotrophic bacteria (nasA) were amplified by polymerase chain reaction (PCR) using two sets of degenerate primers. A total of 129 NR and 75 nasA clones from four clone libraries, two from each of epiphytic and planktonic components, were sequenced and aligned. In addition, genomic DNA sequences for the NR fragment were obtained from Skeletonema costatum and Thalassiosira weissflogii diatom cultures. Rarefaction analysis with an operational taxonomic unit cut-off of 6% indicated that diversity of the NR and nasA clone libraries were similar, and that sequencing of the clone libraries was not yet saturated. Phylogenetic analysis indicated that 121 of the 129 NR clones sequenced were similar to diatom sequences. Of the eight non-diatom sequences, four were most closely related to the sequence of Chlorella vulgaris. Introns were found in 8% of the Tampa Bay NR sequences; introns were also observed in S. costatum, but not T. weissflogii. Introns from within the same clone library exhibited close similarity in nucleotide sequence, position and length; the corresponding exon sequences were unique. Introns from within the same component were similar in position and length, but not in nucleotide sequence. These findings raise questions about the function of introns, and mechanisms or time evolution of intron formation. A large cluster of 14 of the 75 nasA sequences was similar to sequences from Vibrio species; other sequences were closely related to sequences from Alteromonas, alpha-proteobacteria and Marinomonas-like species. Biogeographically consistent patterns were observed for the nasA Tampa Bay sequences compared with sequences from other locations: for example, Tampa Bay sequences were similar to those from the South Atlantic Bight, but not the Barents Sea. The

  12. Hamster SRD5A3 lacks steroid 5α-reductase activity in vitro.

    Science.gov (United States)

    Chávez, B; Ramos, L; García-Becerra, R; Vilchis, F

    2015-02-01

    According to current knowledge, two steroid 5α-reductases, designated type 1 (SRD5A1) and type 2 (SRD5A2), are present in all species examined to date. These isozymes play a central role in steroid hormone physiology by catalyzing the reduction of 3-keto-4-ene-steroids into more active 5α-reduced derivatives, including the conversion of testosterone (T) to dihydrotestosterone (DHT). A third 5α-reductase (SRD5A3, -type 3), which is overexpressed in hormone-refractory prostate cancer cells, has been identified; however, its enzymatic characteristics are practically unknown. Here, we isolated a cDNA encoding hamster Srd5a3 (hSrd5a3) and performed functional metabolic assays to investigate its biochemical properties. The cloned cDNA encodes a 330 amino acid protein that is 87% identical to the homologous protein in mice and 78% to that in humans. However, hSrd5a3 exhibits low sequence homology with its counterparts hSrd5a1 (19%) and hSrd5a2 (17%). A fusion protein consisting of hSrd5a3 and green fluorescent protein provided evidence for cytoplasmic localization in transfected mammalian cells. Real-time PCR analysis revealed that, Srd5a3 mRNA was present in nearly all hamster tissues, with high expression in the cerebellum, Harderian gland and testis. Functional assays expressing hSrd5a3 cDNA in HEK-293 cells revealed that this isozyme is unable to reduce T into DHT. Further expression assays confirmed that similar to testosterone, progesterone, androstenedione and corticosterone are not reduced by hSrd5a3 or human SRD5A3. Together, these results indicate that hSrd5a3 lacks the catalytic activity to transform 3-keto-4-ene-compounds; therefore 5α-reductase type 3 may not be involved in 5α-reduction of steroids.

  13. Comparative molecular modeling study of Arabidopsis NADPH-dependent thioredoxin reductase and its hybrid protein.

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    Yuno Lee

    Full Text Available 2-Cys peroxiredoxins (Prxs play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C of thioredoxin reductase (TrxR in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D, which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD simulations on AtNTRC and AtNTRA-(Trx-D proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D because of following reasons: i unstable and unfavorable interaction of the linker region, ii shifted Trx domain, and iii different or weak interface interaction of Trx domains. This study is one of the good examples for understanding the relationship between structure formation and reaction activity in hybrid protein. In addition, this study would be helpful for further study on the mechanism of electron transfer reaction in NADPH-dependent thioredoxin reductase proteins.

  14. 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...

  15. 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

  16. Genotypic variation in sulfur assimilation and metabolism of onion (Allium cepa L.) III. Characterization of sulfite reductase

    Science.gov (United States)

    Genomic and cDNA sequences corresponding to a ferredoxin-sulfite reductase (SiR) have been cloned from bulb onion (Allium cepa L.) and the expression of the gene and activity of the enzyme characterised with respect to sulfur (S) supply. Cloning, mapping and expression studies revealed that onion ha...

  17. Impacts of Elevated CO2 Concentration on Biochemical Composition,Carbonic Anhydrase, and Nitrate Reductase Activity of Freshwater Green Algae

    Institute of Scientific and Technical Information of China (English)

    Jian-Rong XIA; Kun-Shan GAO

    2005-01-01

    To investigate the biochemical response of freshwater green algae to elevated CO2 concentrations,Chlorella pyrenoidosa Chick and Chlamydomonas reinhardtii Dang cells were cultured at different CO2concentrations within the range 3-186 μmol/L and the biochemical composition, carbonic anhydrase (CA),and nitrate reductase activities of the cells were investigated. Chlorophylls (Chl), carotenoids, carbonhydrate,and protein contents were enhanced to varying extents with increasing CO2 concentration from 3-186μmol/L. The CO2 enrichment significantly increased the Chl a/Chl b ratio in Chlorella pyrenoidosa, but not in Chlamydomonas reinhardtii. The CO2 concentration had significant effects on CA and nitrate reductase activity. Elevating CO2 concentration to 186 μmol/L caused a decline in intracellular and extracellullar CA activity. Nitrate reductase activity, under either light or dark conditions, in C. reinhardtii and C. pyrenoidosa was also significantly decreased with CO2 enrichment. From this study, it can be concluded that CO2enrichment can affect biochemical composition, CA, and nitrate reductase activity, and that the biochemical response was species dependent.

  18. Long-term Use of 5α-Reductase Inhibitors and the Risk of Male Breast Cancer.

    NARCIS (Netherlands)

    Duijnhoven, R.G.; Straus, S.M.J.M.; Souverein, P.C.; de Boer, A.; Bosch, J.L.H.R.; Hoes, A.W.; De Bruin, M.L.; Sub Pharmacoepidemiology; Dep Farmaceutische wetenschappen; Sub Pharmacotherapy, Theoretical

    2014-01-01

    Background The 5α-reductase inhibitors (5-ARI) finasteride and dutasteride are indicated for the treatment of lower urinary tract symptoms caused by benign prostatic hyperplasia. Case reports have suggested that 5-ARIs increase the risk for male breast cancer, with no conclusive evidence. The object

  19. Quantification of dissimilatory (bi)sulphite reductase gene expression in Desulfobacterium autotrophicum using real-time RT-PCR

    DEFF Research Database (Denmark)

    Neretin, LN; Schippers, A.; Pernthaler, A.;

    2003-01-01

    We developed a real-time RT-PCR method for the quantification of dissimilatory (bi)sulphite reductase (DSR) mRNA in Desulfobacterium autotrophicum cells. The amount of DSR mRNA was determined relative to the amount of 16S rRNA at different growth conditions during transition from exponential...

  20. Production of (R)-Ethyl-4-Chloro-3-Hydroxybutanoate Using Saccharomyces cerevisiae YOL151W Reductase Immobilized onto Magnetic Microparticles.

    Science.gov (United States)

    Choo, Jin Woo; Kim, Hyung Kwoun

    2015-11-01

    For the synthesis of various pharmaceuticals, chiral alcohols are useful intermediates. Among them, (R)-ethyl-4-chloro-3-hydroxybutanoate ((R)-ECHB) is an important building block for the synthesis of L-carnitine. (R)-ECHB is produced from ethyl-4-chloro-3-oxobutanoate (ECOB) by a reductase-mediated, enantioselective reduction reaction. The Saccharomyces cerevisiae YOL151W reductase that is expressed in Escherichia coli cells exhibited an enantioselective reduction reaction toward ECOB. By virtue of the C-terminal His-tag, the YOL151W reductase was purified from the cell-free extract using Ni(2+)-NTA column chromatography and immobilized onto Ni(2+)-magnetic microparticles. The physical properties of the immobilized reductase (Imm-Red) were measured using electron microscopy, a magnetic property measurement system, and a zeta potential system; the average size of the particles was approximately 1 μm and the saturated magnetic value was 31.76 emu/g. A neodymium magnet was used to recover the immobilized enzyme within 2 min. The Imm-Red showed an optimum temperature at 45°C and an optimum pH at 6.0. In addition, Bacillus megaterium glucose dehydrogenase (GDH) was produced in the E. coli cells and was used in the coupling reaction to regenerate the NADPH cofactor. The reduction/oxidation coupling reaction composed of the Imm-Red and GDH converted 20 mM ECOB exclusively into (R)- ECHB with an e.e.p value of 98%.

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

    Science.gov (United States)

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

    2006-09-06

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

  2. The anaerobic (Class III) ribonucleotide reductase from Lactococcus lactis : Catalytic properties and allosteric regulation of the pure enzyme system

    NARCIS (Netherlands)

    Torrents, Eduard; Buist, Girbe; Liu, Aimin; Eliasson, Rolf; Kok, Jan; Gibert, Isidre; Gräslund, Astrid; Reichard, Peter

    2000-01-01

    Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase, Strict anaerobic growth depends on the activity of these genes. Both were sequenced, cloned, and overproduced in Escherichia coli, The corresponding proteins, NrdD and NrdG, were purified c

  3. 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.

  4. DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE

    Science.gov (United States)

    Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

  5. [Intensity of cardiac free-radicals processes and expression of glutathione peroxidase and glutathione reductase genes in rats with adrenaline].

    Science.gov (United States)

    Iskusnykh, I Iu; Popova, T N; Musharova, O S

    2012-01-01

    The correlation between changes in activities of glutathione peroxidase and glutathione reductase in heart of rats during development of adrenaline myocarditis and intensity of free radical processes estimated by biochemiluminesce parameters and the content of lipoperoxidation products was demonstrated. The maximal increase of glutathione peroxidase and glutathione reductase activities (in 1.8 and 1.4 times accordingly) was observed t 24 h after the development of the pathological process; this coincided with the maximum intensity of prosesses of free radical oxidation. Using combination of reverse transcriptions with real-time polymerase chain reaction the cardiac mRNA levels of glutathione peroxidase and glutathione reductase genes were determined during the development of adrenaline myocarditis in rats. Analysis of expression of glutathione peroxidase and glutathione reductase genes showed, that the level of this transcripts demonstrated 2,8- and 7,3- increase in rats with adrenaline myocarditis, respectively. Obviously, overexpression of these enzymes can increase the resistance of cardiomyocites to oxidative stress.

  6. HMG-CoA reductase inhibitors, other lipid-lowering medication, antiplatelet therapy, and the risk of venous thrombosis

    NARCIS (Netherlands)

    Ramcharan, A.S.; Stralen, van K.J.; Snoep, J.D.; Mantel-Teeuwisse, A.K.; Doggen, C.J.M.

    2009-01-01

    Background: Statins [3-hydroxymethyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors] and antiplatelet therapy reduce the risk of atherosclerotic disease. Besides a reduction of lipid levels, statins might also have antithrombotic and anti-inflammatory properties, and anti-platelet therap

  7. Mitochondrial localization of the mevalonate pathway enzyme 3-Hydroxy-3-methyl-glutaryl-CoA reductase in the Trypanosomatidae

    DEFF Research Database (Denmark)

    Pena Diaz, Javier; Montalvetti, Andrea; Flores, Carmen-Lisset

    2004-01-01

    3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is a key enzyme in the sterol biosynthesis pathway, but its subcellular distribution in the Trypanosomatidae family is somewhat controversial. Trypanosoma cruzi and Leishmania HMGRs are closely related in their catalytic domains to bacterial and eu...... that the parasite enzyme exhibits a mitochondrial localization, suggesting a conservation between the targeting signals of both organisms....

  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. 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

  10. 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

  11. 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

  12. Long-term use of 5α-reductase inhibitors and the risk of male breast cancer

    DEFF Research Database (Denmark)

    Duijnhoven, Ruben G; Straus, Sabine M J M; Souverein, Patrick C;

    2014-01-01

    BACKGROUND: The 5α-reductase inhibitors (5-ARI) finasteride and dutasteride are indicated for the treatment of lower urinary tract symptoms caused by benign prostatic hyperplasia. Case reports have suggested that 5-ARIs increase the risk for male breast cancer, with no conclusive evidence...

  13. Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural

    Science.gov (United States)

    An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerev...

  14. Met144Ala mutation of the copper-containing nitrite reductase from Alcaligenes xylosoxidans reverses the intramolecular electron transfer

    DEFF Research Database (Denmark)

    Farver, Ole; Eady, Robert R; Sawers, Gary

    2004-01-01

    Pulse radiolysis has been employed to investigate the intramolecular electron transfer (ET) between the type 1 (T1) and type 2 (T2) copper sites in the Met144Ala Alcaligenes xylosoxidans nitrite reductase (AxCuNiR) mutant. This mutation increases the reduction potential of the T1 copper center...

  15. Identification of a functional antioxidant responsive element in the promoter of the Chinese hamster carbonyl reductase 3 (Chcr3) gene.

    Science.gov (United States)

    Miura, Takeshi; Taketomi, Ayako; Nakabayashi, Toshikatsu; Nishinaka, Toru; Terada, Tomoyuki

    2015-07-01

    CHCR3, a member of the short-chain dehydrogenase/reductase superfamily, is a carbonyl reductase 3 enzyme in Chinese hamsters. Carbonyl reductase 3 in humans has been believed to involve the metabolism and/or pharmacokinetics of anthracycline drugs, and the mechanism underlying the gene regulation has been investigated. In this study, the nucleotide sequence of the Chcr3 promoter was originally determined, and its promoter activity was characterised. The proximal promoter region is TATA-less and GC-rich, similar to the promoter region of human carbonyl reductase 3. Cobalt stimulated the transcriptional activity of the Chcr3 gene. The results of a luciferase gene reporter assay demonstrated that cobalt-induced stimulation required an antioxidant responsive element. Forced expression of Nrf2, the transcription factor that binds to antioxidant responsive elements, enhanced the transcriptional activity of the Chcr3 gene. These results suggest that cobalt induces the expression of the Chcr3 gene via the Nrf2-antioxidant responsive element pathway.

  16. Enzymatic reduction of complex redox dyes using NADH-dependent reductase from Bacillus subtilis coupled with cofactor regeneration.

    Science.gov (United States)

    Bozic, Mojca; Pricelius, Sina; Guebitz, Georg M; Kokol, Vanja

    2010-01-01

    Conventional vat dyeing involves chemical reduction of dyes into their water-soluble leuco form generating considerable amounts of toxic chemicals in effluents. In the present study, a new beta-nicotinamide adenine dinucleotide disodium salt (NADH)-dependent reductase isolated from Bacillus subtilis was used to reduce the redox dyes CI Acid Blue 74, CI Natural Orange 6, and CI Vat Blue 1 into their water-soluble leuco form. Enzymatic reduction was optimized in relation to pH and temperature conditions. The reductase was able to reduce Acid Blue 74 and Natural Orange 6 in the presence of the stoichiometrically consumed cofactor NADH; meanwhile, Vat Blue 1 required the presence of mediator 1,8-dihydroxyanthraquinone. Oxygen from air was used to reoxidize the dyes into their initial forms. The enzymatic reduction of the dyes was studied and the kinetic constants determined, and these were compared to the chemically-reduced leuco form. The enzyme responsible for the reduction showed homology to a NADH-dependent reductase from B. subtilis based on results from the MS/MS peptide mass mapping of the tryptically digested protein. Additionally, the reduction of Acid Blue 74 to its leuco form by reductase from B. subtilis was confirmed using NADH regenerated by the oxidation of formic acid with formate dehydrogenase from Candida boidinii in the same solution.

  17. Methylenetetrahydrofolate reductase mutations, a genetic cause for familial recurrent neural tube defects

    Directory of Open Access Journals (Sweden)

    Laxmi V Yaliwal

    2012-01-01

    Full Text Available Methylenetetrahydrofolate reductase (MTHFR gene mutations have been implicated as risk factors for neural tube defects (NTDs. The best-characterized MTHFR genetic mutation 677C→T is associated with a 2-4 fold increased risk of NTD if patient is homozygous for this mutation. This risk factor is modulated by folate levels in the body. A second mutation in the MTHFR gene is an A→C transition at position 1298. The 1298A→C mutation is also a risk factor for NTD, but with a smaller relative risk than 677C→T mutation. Under conditions of low folate intake or high folate requirements, such as pregnancy, this mutation could become of clinical importance. We present a case report with MTHFR genetic mutation, who presented with recurrent familial pregnancy losses due to anencephaly/NTDs.

  18. 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.

  19. The radical mechanism of biological methane synthesis by methyl-coenzyme M reductase

    Energy Technology Data Exchange (ETDEWEB)

    Wongnate, T.; Sliwa, D.; Ginovska, B.; Smith, D.; Wolf, M. W.; Lehnert, N.; Raugei, S.; Ragsdale, S. W.

    2016-05-19

    Methyl-coenzyme M reductase (MCR), the rate-limiting enzyme in methanogenesis and anaerobic methane oxidation, is responsible for the production of over one billion tons of methane per year. The mechanism of methane synthesis is unknown, with the two leading proposals involving either a methyl-nickel(III) (Mechanism I) or methyl radical/Ni(II)-thiolate (Mechanism II) intermediate(s). When the reaction between the active Ni(I) enzyme with substrates was studied by transient kinetic, spectroscopic and computational methods, formation of an EPR-silent Ni(II)-thiolate intermediate was positively identified by magnetic circular dichroism spectroscopy. There was no evidence for an EPR-active methyl-Ni(III) species. Temperature-dependent transient kinetic studies revealed that the activation energy for the initial catalytic step closely matched the value computed by density functional theory for Mechanism II. Thus, our results demonstrate that biological methane synthesis occurs by generation of a methyl radical.

  20. Comparative stability of dihydrofolate reductase mutants in vitro and in vivo.

    Science.gov (United States)

    Leontiev, V V; Uversky, V N; Gudkov, A T

    1993-01-01

    Dihydrofolate reductase mutants with amino acid replacements in the active center (Thr35-->Asp mutant, Arg57-->His mutant and the mutant with triple replacement Thr35-->Asp, Asn37-->Ser, Arg57-->His) were obtained by site-directed mutagenesis. The stabilization effect of trimethoprim and NADP.H on the protein tertiary structure in vitro has been investigated. In the case of mutants with a 'weak' tertiary structure (Thr35-->Asp35 and the triple mutant) the separate addition of ligands does not affect their stability. The simultaneous addition of these ligands to Thr35-->Asp35 and the triple mutant leads to the large increase in their stability. A distinct correlation was found between the in vitro studied stability of the mutant proteins to the urea- or heat-induced denaturation and the level of proteolytic degradation of these mutants previously observed in vivo.

  1. Anti-inflammatory and Quinone Reductase Inducing Compounds from Fermented Noni (Morinda citrifolia) Juice Exudates.

    Science.gov (United States)

    Youn, Ui Joung; Park, Eun-Jung; Kondratyuk, Tamara P; Sang-Ngern, Mayuramas; Wall, Marisa M; Wei, Yanzhang; Pezzuto, John M; Chang, Leng Chee

    2016-06-24

    A new fatty acid ester disaccharide, 2-O-(β-d-glucopyranosyl)-1-O-(2E,4Z,7Z)-deca-2,4,7-trienoyl-β-d-glucopyranose (1), a new ascorbic acid derivative, 2-caffeoyl-3-ketohexulofuranosonic acid γ-lactone (2), and a new iridoid glycoside, 10-dimethoxyfermiloside (3), were isolated along with 13 known compounds (4-16) from fermented noni fruit juice (Morinda citrifolia). The structures of the new compounds, together with 4 and 5, were determined by 1D and 2D NMR experiments, as well as comparison with published values. Compounds 2 and 7 showed moderate inhibitory activities in a TNF-α-induced NF-κB assay, and compounds 4 and 6 exhibited considerable quinone reductase-1 (QR1) inducing effects.

  2. Synergistic effect of reductase and keratinase for facile synthesis of protein-coated gold nanoparticles.

    Science.gov (United States)

    Gupta, Sonali; Singh, Surinder P; Singh, Rajni

    2015-05-01

    We have synthesized gold nanoparticles (GNPs) using chicken feathers (poultry waste) and Bacillus subtilis RSE163. Disulfide reductase and keratinase produced by Bacillus subtilis during the degradation of chicken feather has been used to reduce Au(3+) from HAuCl4 precursor to produce gold nanoparticles. The synthesized biogenic GNPs were characterized by UV-visible spectroscopy, transmission electron microscopy (TEM), and zeta potential measurements. Fourier transform infrared (FTIR) spectroscopy indicated the presence of protein capping on synthesized GNPs, imparting multifunctionality to the GNP surface. Furthermore, the nontoxic nature of biogenic GNPs was insured by interaction with Escherichia coli (ATCC11103), where TEM images and enhancement of growth rate of E. coli in log phase signified their nontoxic nature. The results indicate that the synthesis of biocompatible GNPs using poultry waste may find potential applications in drug delivery and sensing.

  3. In silico screening for Plasmodium falciparum enoyl-ACP reductase inhibitors

    Science.gov (United States)

    Lindert, Steffen; Tallorin, Lorillee; Nguyen, Quynh G.; Burkart, Michael D.; McCammon, J. Andrew

    2015-01-01

    The need for novel therapeutics against Plasmodium falciparum is urgent due to recent emergence of multi-drug resistant malaria parasites. Since fatty acids are essential for both the liver and blood stages of the malarial parasite, targeting fatty acid biosynthesis is a promising strategy for combatting P. falciparum. We present a combined computational and experimental study to identify novel inhibitors of enoyl-acyl carrier protein reductase ( PfENR) in the fatty acid biosynthesis pathway. A small-molecule database from ChemBridge was docked into three distinct PfENR crystal structures that provide multiple receptor conformations. Two different docking algorithms were used to generate a consensus score in order to rank possible small molecule hits. Our studies led to the identification of five low-micromolar pyrimidine dione inhibitors of PfENR.

  4. 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.

  5. Evaluation of Enoyl-Acyl Carrier Protein Reductase Inhibitors as Pseudomonas aeruginosa Quorum-Quenching Reagents

    Directory of Open Access Journals (Sweden)

    Søren Molin

    2010-02-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic pathogen which is responsible for a wide range of infections. Production of virulence factors and biofilm formation by P. aeruginosa are partly regulated by cell-to-cell communication quorum-sensing systems. Identification of quorum-quenching reagents which block the quorum-sensing process can facilitate development of novel treatment strategies for P. aeruginosa infections. We have used molecular dynamics simulation and experimental studies to elucidate the efficiencies of two potential quorum-quenching reagents, triclosan and green tea epigallocatechin gallate (EGCG, which both function as inhibitors of the enoyl-acyl carrier protein (ACP reductase (ENR from the bacterial type II fatty acid synthesis pathway. Our studies suggest that EGCG has a higher binding affinity towards ENR of P. aeruginosa and is an efficient quorum-quenching reagent. EGCG treatment was further shown to be able to attenuate the production of virulence factors and biofilm formation of P. aeruginosa.

  6. 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

    gene, rs17238484 (for the main analysis) and rs12916 (for a subsidiary analysis) as proxies for HMGCR inhibition by statins. We examined associations of these variants with plasma lipid, glucose, and insulin concentrations; bodyweight; waist circumference; and prevalent and incident type 2 diabetes......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....... Study-specific effect estimates per copy of each LDL-lowering allele were pooled by meta-analysis. These findings were compared with a meta-analysis of new-onset type 2 diabetes and bodyweight change data from randomised trials of statin drugs. The effects of statins in each randomised trial were...

  7. Enantioselective bioconversion using Escherichia coli cells expressing Saccharomyces cerevisiae reductase and Bacillus subtilis glucose dehydrogenase.

    Science.gov (United States)

    Park, Hyun Joo; Jung, Jihye; Choi, Hyejeong; Uhm, Ki-Nam; Kim, Hyung Kwoun

    2010-09-01

    Ethyl (R, S)-4-chloro-3-hydroxybutanoate (ECHB) is a useful chiral building block for the synthesis of L-carnitine and hypercholesterolemia drugs. The yeast reductase, YOL151W (GenBank locus tag), exhibits an enantioselective reduction activity, converting ethyl-4-chlorooxobutanoate (ECOB) exclusively into (R)-ECHB. YOL151W was generated in Escherichia coli cells and purified via Ni- NTA and desalting column chromatography. It evidenced an optimum temperature of 45 degrees C and an optimum pH of 6.5-7.5. Bacillus subtilis glucose dehydrogenase (GDH) was also expressed in Escherichia coli, and was used for the recycling of NADPH, required for the reduction reaction. Thereafter, Escherichia coli cells co-expressing YOL151W and GDH were constructed. After permeablization treatment, the Escherichia coli whole cells were utilized for ECHB synthesis. Through the use of this system, the 30 mM ECOB substrate could be converted to (R)-ECHB.

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

    Science.gov (United States)

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

    2015-08-01

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

  9. Characterizing and predicting carboxylic acid reductase activity for diversifying bioaldehyde production.

    Science.gov (United States)

    Moura, Matthew; Pertusi, Dante; Lenzini, Stephen; Bhan, Namita; Broadbelt, Linda J; Tyo, Keith E J

    2016-05-01

    Chemicals with aldehyde moieties are useful in the synthesis of polymerization reagents, pharmaceuticals, pesticides, flavors, and fragrances because of their high reactivity. However, chemical synthesis of aldehydes from carboxylic acids has unfavorable thermodynamics and limited specificity. Enzymatically catalyzed reductive bioaldehyde synthesis is an attractive route that overcomes unfavorable thermodynamics by ATP hydrolysis in ambient, aqueous conditions. Carboxylic acid reductases (Cars) are particularly attractive, as only one enzyme is required. We sought to increase the knowledge base of permitted substrates for four Cars. Additionally, the Lys2 enzyme family was found to be mechanistically the same as Cars and two isozymes were also tested. Our results show that Cars prefer molecules where the carboxylic acid is the only polar/charged group. Using this data and other published data, we develop a support vector classifier (SVC) for predicting Car reactivity and make predictions on all carboxylic acid metabolites in iAF1260 and Model SEED.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    Different lines of evidence indicate that methylenetetrahydrofolate reductase (MTHFR) functional gene polymorphisms, causative in aberrant folate-homocysteine metabolism, are associated with increased vulnerability to several heritable developmental disorders. Opposing views are expressed conside...... 677T allele showed earlier age at onset than siblings being homozygous for the wild-type allele (P = 0.008). The MTHFR C677T polymorphism may play a role as a modifying factor for age of onset in schizophrenia....... considering the possible association between MTHFR and susceptibility for schizophrenia. In order to evaluate if age of onset could explain some of this discrepancy we investigated the relationship between two functional MTHFR gene polymorphisms and age at onset in this disorder. Scandinavian patients (n...... = 820) diagnosed with schizophrenia, schizoaffective disorder, and schizophreniform disorder were investigated. Two functional MTHFR single nucleotide polymorphisms (SNPs; rs1801131 and rs1801133) were genotyped and the effect of MTHFR polymorphisms on the age of onset was examined with survival...

  11. (+)-Pinoresinol/(-)-lariciresinol reductase from Linum perenne Himmelszelt involved in the biosynthesis of justicidin B.

    Science.gov (United States)

    Hemmati, Shiva; Schmidt, Thomas J; Fuss, Elisabeth

    2007-02-20

    A cDNA encoding a pinoresinol-lariciresinol reductase PLR (PLR-Lp1) was isolated from a cell culture of Linum perenne Himmelszelt accumulating the arylnaphthalene lignan justicidin B. The recombinant PLR-Lp1 prefers (+)-pinoresinol in the first reaction step, but (-)-lariciresinol in the second step. Therefore, it is the first PLR described with opposite enantiospecificity within the two reaction steps catalysed by PLRs. Hairy root lines transformed with an ihpRNAi construct to suppress plr gene expression show less mRNA accumulation for the plr-Lp1 gene and PLR enzyme activity. Justicidin B accumulation was reduced down to 24% in comparison to control lines showing the involvement of PLR-Lp1 in the biosynthesis of justicidin B.

  12. Pinoresinol-lariciresinol reductase gene expression and secoisolariciresinol diglucoside accumulation in developing flax (Linum usitatissimum) seeds.

    Science.gov (United States)

    Hano, C; Martin, I; Fliniaux, O; Legrand, B; Gutierrez, L; Arroo, R R J; Mesnard, F; Lamblin, F; Lainé, E

    2006-11-01

    The transcription activity of the pinoresinol-lariciresinol reductase (PLR) gene of Linum usitatissimum (so-called LuPLR), a key gene in lignan synthesis, was studied by RT-PCR and promoter-reporter transgenesis. The promoter was found to drive transcription of a GUSint reporter gene in the seed coats during the flax seed development. This fitted well with the tissue localization monitored by semi-quantitative RT-PCR of LuPLR expression. Accumulation of the main flax lignan secoisolariciresinol diglucoside was coherent with LuPLR expression during seed development. This three-way approach demonstrated that the LuPLR gene is expressed in the seed coat of flax seeds, and that the synthesis of PLR enzyme occurs where flax main lignan is found stored in mature seeds, confirming its involvement in SDG synthesis.

  13. Direct and reversible hydrogenation of CO2 to formate by a bacterial carbon dioxide reductase.

    Science.gov (United States)

    Schuchmann, K; Müller, V

    2013-12-13

    Storage and transportation of hydrogen is a major obstacle for its use as a fuel. An increasingly considered alternative for the direct handling of hydrogen is to use carbon dioxide (CO2) as an intermediate storage material. However, CO2 is thermodynamically stable, and developed chemical catalysts often require high temperatures, pressures, and/or additives for high catalytic rates. Here, we present the discovery of a bacterial hydrogen-dependent carbon dioxide reductase from Acetobacterium woodii directly catalyzing the hydrogenation of CO2. We also demonstrate a whole-cell system able to produce formate as the sole end product from dihydrogen (H2) and CO2 as well as syngas. This discovery opens biotechnological alternatives for efficient CO2 hydrogenation either by using the isolated enzyme or by employing whole-cell catalysis.

  14. 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.

  15. Sneddon Syndrome with Factor V Leiden, Methylene Tetrahydrofolate Reductase and FMF Gene Mutations

    Directory of Open Access Journals (Sweden)

    Murat Terzi

    2010-03-01

    Full Text Available Sneddon syndrome (SNS, characterized by livedo racemosa and stroke, is a rare disease, especially in young adults. Livedo racemosa are large lesions, widespread on the extremities and the body, that are violet-colored and have a good appearance and ambiguous limits. A 33-years-old female presented to our clinic for headache. She had a two-year history of blue-purple skin marks on her body and legs. The skin lesions were consistent with livedo racemosa. She had experienced right hemiparesis according to her medical history. Factor V Leiden (G1691A mutation was heterozygote-positive. Methylenetetrahydrofolate reductase (MTHFR C677T and FMF gene (MEFV V726A mutations were determined. SNS is the cause of stroke, rarely seen in young adults. We considered this case to be of value since it is the first SNS case having factor V Leiden, MTHFR and MEFV mutations concomitantly.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    considering the possible association between MTHFR and susceptibility for schizophrenia. In order to evaluate if age of onset could explain some of this discrepancy we investigated the relationship between two functional MTHFR gene polymorphisms and age at onset in this disorder. Scandinavian patients (n......Different lines of evidence indicate that methylenetetrahydrofolate reductase (MTHFR) functional gene polymorphisms, causative in aberrant folate-homocysteine metabolism, are associated with increased vulnerability to several heritable developmental disorders. Opposing views are expressed...... = 820) diagnosed with schizophrenia, schizoaffective disorder, and schizophreniform disorder were investigated. Two functional MTHFR single nucleotide polymorphisms (SNPs; rs1801131 and rs1801133) were genotyped and the effect of MTHFR polymorphisms on the age of onset was examined with survival...

  17. Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and age at onset of schizophrenia

    DEFF Research Database (Denmark)

    Saetre, Peter; Grove, Jakob; Børglum, Anders;

    2012-01-01

    Methylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in metabolic pathways of importance for nucleotide synthesis and methylation of DNA, membranes, proteins and lipids. The MTHFR gene includes a common polymorphism (rs1801133 or C677T), which is associated with enzyme activity. The T......-allele of the C677T polymorphism has been associated with earlier age at onset of schizophrenia in a Scandinavian population, although no association was found in replication attempts in other populations. Extending the study to five Nordic samples consisting of 2,198 patients with schizophrenia, including...... the original Scandinavian samples, there was no significant association between MTHFR C677T polymorphism and age at onset in schizophrenia. The present results do not suggest that the investigated MTHFR polymorphism has any significant influence on age at onset of schizophrenia in the Nordic population. © 2012...

  18. Isolation and characterization of thioredoxin and NADPH-dependent thioredoxin reductase from tomato (Solanum lycopersicum).

    Science.gov (United States)

    Dai, Changbo; Wang, Myeong-Hyeon

    2011-10-01

    To investigate the pathways of oxidoreductases in plants, 2 key components in thioredox systems i.e. thioredoxin h (Trx h) and NADPH-dependent thioredoxin reductase (NTR) genes were first isolated from tomatoes (Solanum lycopersicum). Subsequently, the coding sequences of Trx h and NTR were inserted into pET expression vectors, and overexpressed in Escherichia coli. In the UV-Visible spectra of the purified proteins, tomato Trx h was shown to have a characteristic 'shoulder' at -290 nm, while the NTR protein had the 3 typical peaks unique to flavoenzymes. The activities of both proteins were demonstrated by following insulin reduction, as well as DTNB reduction. Moreover, both NADPH and NADH could serve as substrates in the NTR reduction system, but the catalytic efficiency of NTR with NADPH was 2500-fold higher than with NADH. Additionally, our results reveal that the tomato Trx system might be involved in oxidative stress, but not in cold damage.

  19. Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase

    Directory of Open Access Journals (Sweden)

    Margit Winkler

    2013-08-01

    Full Text Available Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S-selectivity and together with a highly (R-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

  20. 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.

  1. Combined choroidal neovascularization and hypopituitarism in a patient with homozygous mutation in methylenetetrahydrofolate reductase gene

    Directory of Open Access Journals (Sweden)

    Aydogan Aydogdu

    2014-01-01

    Full Text Available We report a case of choroidal neovascularization (CNV secondary to methylenetetrahydrofolate reductase (MTHFR gene mutation in a 20-year-old male patient with hypopituitarism. Treatment with three consecutive injections of intravitreal ranibizumab (anti-vascular endothelial growth factor resulted in significant improvement of the patient′s vision and the appearance of the macula. A search of the literature produced no previously reported case of MTHFR gene mutation associated both CNV and possibly hypopituitarism. With hormone replacement therapy of hypopituitarism, acetyl salicylic acid 100 mg/day also was started. The patient was clinically stable both for CNV and other thromboembolic disorders over a 6-month follow-up and also 1-year follow-up period.

  2. Response to arsenate treatment in Schizosaccharomyces pombe and the role of its arsenate reductase activity.

    Directory of Open Access Journals (Sweden)

    Alejandro Salgado

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

  3. Study on the violet LED-induced fluorescence spectra of thioredoxin reductase from human brain

    Institute of Scientific and Technical Information of China (English)

    Xiufeng Lan(兰秀风); Tao Yang(杨涛); Shumei Gao(高淑梅); Xiaosen Luo(骆晓森); Zhonghua Shen(沈中华); Jian Lu(陆建); Xiaowu Ni(倪晓武); Lin Xu(许琳)

    2003-01-01

    The technique of fluorescence spectroscopy is applied to study thioredoxin reductase (TrxR) in the ce;lsof human brain. Experimental results show that, by the violet light emitting diode (LED, λmax=407 nm)light irradiation, TrxR is able to emit three striking spectral bands (528 - 582 nm; 588 - 660 nm; 683 - 700nm). The fluorescence intensity is linear to the concentration of TrxR. The spectrum of denatured TrxR israther different from that of organized TrxR, which reflects the structure change between denatured TrxRand organized TrxR. Furthermore, physical and biochemical mechanisms of fluorescence production forLED light-induced TrxR spectra and its characteristics are analyzed. This paper may be useful to betterunderstand the structure of TrxR, and to provide new spectroscopic information to improve the resolutionfor this kind of biology structure.

  4. Localization of Methyl-Coenzyme M Reductase as Metabolic Marker for Diverse Methanogenic Archaea

    Directory of Open Access Journals (Sweden)

    Christoph Wrede

    2013-01-01

    Full Text Available Methyl-Coenzyme M reductase (MCR as key enzyme for methanogenesis as well as for anaerobic oxidation of methane represents an important metabolic marker for both processes in microbial biofilms. Here, the potential of MCR-specific polyclonal antibodies as metabolic marker in various methanogenic Archaea is shown. For standard growth conditions in laboratory culture, the cytoplasmic localization of the enzyme in Methanothermobacter marburgensis, Methanothermobacter wolfei, Methanococcus maripaludis, Methanosarcina mazei, and in anaerobically methane-oxidizing biofilms is demonstrated. Under growth limiting conditions on nickel-depleted media, at low linear growth of cultures, a fraction of 50–70% of the enzyme was localized close to the cytoplasmic membrane, which implies “facultative” membrane association of the enzyme. This feature may be also useful for assessment of growth-limiting conditions in microbial biofilms.

  5. The Impact of dUTPase on Ribonucleotide Reductase-Induced Genome Instability in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Chih-Wei Chen

    2016-08-01

    Full Text Available The appropriate supply of dNTPs is critical for cell growth and genome integrity. Here, we investigated the interrelationship between dUTP pyrophosphatase (dUTPase and ribonucleotide reductase (RNR in the regulation of genome stability. Our results demonstrate that reducing the expression of dUTPase increases genome stress in cancer. Analysis of clinical samples reveals a significant correlation between the combination of low dUTPase and high R2, a subunit of RNR, and a poor prognosis in colorectal and breast cancer patients. Furthermore, overexpression of R2 in non-tumorigenic cells progressively increases genome stress, promoting transformation. These cells display alterations in replication fork progression, elevated genomic uracil, and breaks at AT-rich common fragile sites. Consistently, overexpression of dUTPase abolishes R2-induced genome instability. Thus, the expression level of dUTPase determines the role of high R2 in driving genome instability in cancer cells.

  6. THE IMPACT OF THE ENZYME STEROID 5α-REDUCTASE 2 DEFICIENCY ON UROGENITAL BIRTH DEFECT IN MALES

    Directory of Open Access Journals (Sweden)

    Livia-Irina Olaru

    2014-07-01

    Full Text Available This research work on 5α-reductase 2 is aiming to put into evidence how a mutation of the gene codifying for this enzyme can affect the phenotype of different males, leading at a partial female phenotype.  This type of research focuses on offering a scientific explanation on different health problems affecting the normal lives of some people, even from early stages of their evolution.The experiment is focused on studying the impact of an enzyme called “steroid 5α-reductase” on the development of male urogenital components during the embryological life.  In order to study the steroid 5α-reductase 2 activity, the following procedures were used: isolation of a 5α-reductase 2 gene, genomicDNAisolation, and polymerase chain reactions.Biochemical analysis put into evidence two mutations representing opposite poles of disease manifestation, i.e. feminization of external genitalia versus predominantly male development. It suggested a correlation between clinical expression and severity of the impairment of enzyme function.  As mentioned by Imperato-McGinley, Guerrero, Gautier, & Peterson (1974, one of the mutations is called “G196S mutation.”  This mutation consists of a serine substitution for a glycine at position 196.  In this case, the level of 5α-reductase activity was sufficient to induce partial virilization.  The second type of mutation is called “G34R mutation.”  This mutation consists of an arginine substitution for glycine at position 34.  The G34R enzyme is essentially inactive, thus giving rise to the female phenotype.Mutations in the type 2 gene are responsible for autosomal recessive genetic disease of 5α-reductase deficiency.

  7. Evaluation of constitutive iron reductase (AtFRO2) expression on mineral accumulation and distribution in soybean (Glycine max. L).

    Science.gov (United States)

    Vasconcelos, Marta W; Clemente, Thomas E; Grusak, Michael A

    2014-01-01

    Iron is an important micronutrient in human and plant nutrition. Adequate iron nutrition during crop production is central for assuring appropriate iron concentrations in the harvestable organs, for human food or animal feed. The whole-plant movement of iron involves several processes, including the reduction of ferric to ferrous iron at several locations throughout the plant, prior to transmembrane trafficking of ferrous iron. In this study, soybean plants that constitutively expressed the AtFRO2 iron reductase gene were analyzed for leaf iron reductase activity, as well as the effect of this transgene's expression on root, leaf, pod wall, and seed mineral concentrations. High Fe supply, in combination with the constitutive expression of AtFRO2, resulted in significantly higher concentrations of different minerals in roots (K, P, Zn, Ca, Ni, Mg, and Mo), pod walls (Fe, K, P, Cu, and Ni), leaves (Fe, P, Cu, Ca, Ni, and Mg) and seeds (Fe, Zn, Cu, and Ni). Leaf and pod wall iron concentrations increased as much as 500% in transgenic plants, while seed iron concentrations only increased by 10%, suggesting that factors other than leaf and pod wall reductase activity were limiting the translocation of iron to seeds. Protoplasts isolated from transgenic leaves had three-fold higher reductase activity than controls. Expression levels of the iron storage protein, ferritin, were higher in the transgenic leaves than in wild-type, suggesting that the excess iron may be stored as ferritin in the leaves and therefore unavailable for phloem loading and delivery to the seeds. Also, citrate and malate levels in the roots and leaves of transgenic plants were significantly higher than in wild-type, suggesting that organic acid production could be related to the increased accumulation of minerals in roots, leaves, and pod walls, but not in the seeds. All together, these results suggest a more ubiquitous role for the iron reductase in whole-plant mineral accumulation and distribution.

  8. The retinaldehyde reductase activity of DHRS3 is reciprocally activated by retinol dehydrogenase 10 to control retinoid homeostasis.

    Science.gov (United States)

    Adams, Mark K; Belyaeva, Olga V; Wu, Lizhi; Kedishvili, Natalia Y

    2014-05-23

    The retinoic acid-inducible dehydrogenase reductase 3 (DHRS3) is thought to function as a retinaldehyde reductase that controls the levels of all-trans-retinaldehyde, the immediate precursor for bioactive all-trans-retinoic acid. However, the weak catalytic activity of DHRS3 and the lack of changes in retinaldehyde conversion to retinol and retinoic acid in the cells overexpressing DHRS3 undermine its role as a physiologically important all-trans-retinaldehyde reductase. This study demonstrates that DHRS3 requires the presence of retinol dehydrogenase 10 (RDH10) to display its full catalytic activity. The RDH10-activated DHRS3 acts as a robust high affinity all-trans-retinaldehyde-specific reductase that effectively converts retinaldehyde back to retinol, decreasing the rate of retinoic acid biosynthesis. In turn, the retinol dehydrogenase activity of RDH10 is reciprocally activated by DHRS3. At E13.5, DHRS3-null embryos have ∼4-fold lower levels of retinol and retinyl esters, but only slightly elevated levels of retinoic acid. The membrane-associated retinaldehyde reductase and retinol dehydrogenase activities are decreased by ∼4- and ∼2-fold, respectively, in Dhrs3(-/-) embryos, and Dhrs3(-/-) mouse embryonic fibroblasts exhibit reduced metabolism of both retinaldehyde and retinol. Neither RDH10 nor DHRS3 has to be itself catalytically active to activate each other. The transcripts encoding DHRS3 and RDH10 are co-localized at least in some tissues during development. The mutually activating interaction between the two related proteins may represent a highly sensitive and conserved mechanism for precise control over the rate of retinoic acid biosynthesis.

  9. Characterisation of PduS, the pdu metabolosome corrin reductase, and evidence of substructural organisation within the bacterial microcompartment.

    Directory of Open Access Journals (Sweden)

    Joshua B Parsons

    Full Text Available PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(IIIalamin to cob(IIalamin and cob(IIalamin to cob(Ialamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment.

  10. Evaluation of constitutive iron reductase (AtFRO2 expression on mineral accumulation and distribution in soybean (Glycine max. L

    Directory of Open Access Journals (Sweden)

    Marta Wilton Vasconcelos

    2014-04-01

    Full Text Available Iron is an important micronutrient in human and plant nutrition. Adequate iron nutrition during crop production is central for assuring appropriate iron concentrations in the harvestable organs, for human food or animal feed. The whole-plant movement of iron involves several processes, including the reduction of ferric to ferrous iron at several locations throughout the plant, prior to transmembrane trafficking of ferrous iron. In this study, soybean plants that constitutively expressed the AtFRO2 iron reductase gene were analyzed for leaf iron reductase activity, as well as the effect of this transgene's expression on root, leaf, pod wall, and seed mineral concentrations. High Fe supply, in combination with the constitutive expression of AtFRO2, resulted in significantly higher concentrations of different minerals in roots (K, P, Zn, Ca, Ni, Mg and Mo, pod walls (Fe, K, P, Cu and Ni, leaves (Fe, P, Cu, Ca, Ni and Mg and seeds (Fe, Zn, Cu and Ni. Leaf and pod wall iron concentrations increased as much as 500% in transgenic plants, while seed iron concentrations only increased by 10%, suggesting that factors other than leaf and pod wall reductase activity were limiting the translocation of iron to seeds. Protoplasts isolated from transgenic leaves had three-fold higher reductase activity than controls. Expression levels of the iron storage protein, ferritin, were higher in the transgenic leaves than in wild-type, suggesting that the excess iron may be stored as ferritin in the leaves and therefore unavailable for phloem loading and delivery to the seeds. Also, citrate and malate levels in the roots and leaves of transgenic plants were significantly higher than in wild-type, suggesting that organic acid production could be related to the increased accumulation of minerals in roots, leaves and pod walls, but not in the seeds. All together, these results suggest a more ubiquitous role for the iron reductase in whole-plant mineral accumulation and

  11. 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.

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

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    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.

  13. Lantibiotic Reductase LtnJ Substrate Selectivity Assessed with a Collection of Nisin Derivatives as Substrates.

    Science.gov (United States)

    Mu, Dongdong; Montalbán-López, Manuel; Deng, Jingjing; Kuipers, Oscar P

    2015-06-01

    Lantibiotics are potent antimicrobial peptides characterized by the presence of dehydrated amino acids, dehydroalanine and dehydrobutyrine, and (methyl)lanthionine rings. In addition to these posttranslational modifications, some lantibiotics exhibit additional modifications that usually confer increased biological activity or stability on the peptide. LtnJ is a reductase responsible for the introduction of D-alanine in the lantibiotic lacticin 3147. The conversion of L-serine into D-alanine requires dehydroalanine as the substrate, which is produced in vivo by the dehydration of serine by a lantibiotic dehydratase, i.e., LanB or LanM. In this work, we probe the substrate specificity of LtnJ using a system that combines the nisin modification machinery (dehydratase, cyclase, and transporter) and the stereospecific reductase LtnJ in Lactococcus lactis. We also describe an improvement in the production yield of this system by inserting a putative attenuator from the nisin biosynthesis gene cluster in front of the ltnJ gene. In order to clarify the sequence selectivity of LtnJ, peptides composed of truncated nisin and different mutated C-terminal tails were designed and coexpressed with LtnJ and the nisin biosynthetic machinery. In these tails, serine was flanked by diverse amino acids to determine the influence of the surrounding residues in the reaction. LtnJ successfully hydrogenated peptides when hydrophobic residues (Leu, Ile, Phe, and Ala) were flanking the intermediate dehydroalanine, while those in which dehydroalanine was flanked by one or two polar residues (Ser, Thr, Glu, Lys, and Asn) or Gly were either less prone to be modified by LtnJ or not modified at all. Moreover, our results showed that dehydrobutyrine cannot serve as a substrate for LtnJ.

  14. Methyl-coenzyme M reductase A as an indicator to estimate methane production from dairy cows.

    Science.gov (United States)

    Aguinaga Casañas, M A; Rangkasenee, N; Krattenmacher, N; Thaller, G; Metges, C C; Kuhla, B

    2015-06-01

    The evaluation of greenhouse gas mitigation strategies requires the quantitative assessment of individual methane production. Because methane measurement in respiration chambers is highly accurate, but also comprises various disadvantages such as limited capacity and high costs, the establishment of an indicator for estimating methane production of individual ruminants would provide an alternative to direct methane measurement. Methyl-coenzyme M reductase is involved in methanogenesis and the subunit α of methyl-coenzyme M reductase is encoded by the mcrA gene of rumen archaea. We therefore examined the relationship between methane emissions of Holstein dairy cows measured in respiration chambers with 2 different diets (high- and medium-concentrate diet) and the mcrA DNA and mcrA cDNA abundance determined from corresponding rumen fluid samples. Whole-body methane production per kilogram of dry matter intake and mcrA DNA normalized to the abundance of the rrs gene coding for 16S rRNA correlated significantly when using qmcrA primers. Use of qmcrA primers also revealed linear correlation between mcrA DNA copy number and methane yield. Regression analyses based on normalized mcrA cDNA abundances revealed no significant linear correlation with methane production per kilogram of dry matter intake. Furthermore, the correlations between normalized mcrA DNA abundance and the rumen fluid concentration of acetic and isobutyric acid were positive, whereas the correlations with propionic and lactic acid were negative. These data suggest that the mcrA DNA approach based on qmcrA primers could potentially be a molecular proxy for methane yield after further refinement.

  15. Functional profiling of mercuric reductase (mer A genes in biofilm communities of a technical scale biocatalyzer

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    von Canstein Harald

    2003-10-01

    Full Text Available Abstract Background Bacterial mercury resistance is based on enzymatic reduction of ionic mercury to elemental mercury and has recently been demonstrated to be applicable for industrial wastewater clean-up. The long-term monitoring of such biocatalyser systems requires a cultivation independent functional community profiling method targeting the key enzyme of the process, the merA gene coding for the mercuric reductase. We report on the development of a profiling method for merA and its application to monitor changes in the functional diversity of the biofilm community of a technical scale biocatalyzer over 8 months of on-site operation. Results Based on an alignment of 30 merA sequences from Gram negative bacteria, conserved primers were designed for amplification of merA fragments with an optimized PCR protocol. The resulting amplicons of approximately 280 bp were separated by thermogradient gelelectrophoresis (TGGE, resulting in strain specific fingerprints for mercury resistant Gram negative isolates with different merA sequences. The merA profiling of the biofilm community from a technical biocatalyzer showed persistence of some and loss of other inoculum strains as well as the appearance of new bands, resulting in an overall increase of the functional diversity of the biofilm community. One predominant new band of the merA community profile was also detected in a biocatalyzer effluent isolate, which was identified as Pseudomonas aeruginosa. The isolated strain showed lower mercury reduction rates in liquid culture than the inoculum strains but was apparently highly competitive in the biofilm environment of the biocatalyzer where moderate mercury levels were prevailing. Conclusions The merA profiling technique allowed to monitor the ongoing selection for better adapted strains during the operation of a biocatalyzer and to direct their subsequent isolation. In such a way, a predominant mercury reducing Ps. aeruginosa strain was identified by

  16. Characterization of Arabidopsis thaliana pinoresinol reductase, a new type of enzyme involved in lignan biosynthesis.

    Science.gov (United States)

    Nakatsubo, Tomoyuki; Mizutani, Masaharu; Suzuki, Shiro; Hattori, Takefumi; Umezawa, Toshiaki

    2008-06-01

    A lignan, lariciresinol, was isolated from Arabidopsis thaliana, the most widely used model plant in plant bioscience sectors, for the first time. In the A. thaliana genome database, there are two genes (At1g32100 and At4g13660) that are annotated as pinoresinol/lariciresinol reductase (PLR). The recombinant AtPLRs showed strict substrate preference toward pinoresinol but only weak or no activity toward lariciresinol, which is in sharp contrast to conventional PLRs of other plants that can reduce both pinoresinol and lariciresinol efficiently to lariciresinol and secoisolariciresinol, respectively. Therefore, we renamed AtPLRs as A. thaliana pinoresinol reductases (AtPrRs). The recombinant AtPrR2 encoded by At4g13660 reduced only (-)-pinoresinol to (-)-lariciresinol and not (+)-pinoresinol in the presence of NADPH. This enantiomeric selectivity accords with that of other PLRs of other plants so far reported, which can reduce one of the enantiomers selectively, whatever the preferential enantiomer. In sharp contrast, AtPrR1 encoded by At1g32100 reduced both (+)- and (-)-pinoresinols to (+)- and (-)-lariciresinols efficiently with comparative k(cat)/K(m) values. Analysis of lignans and spatiotemporal expression of AtPrR1 and AtPrR2 in their functionally deficient A. thaliana mutants and wild type indicated that both genes are involved in lariciresinol biosynthesis. In addition, the analysis of the enantiomeric compositions of lariciresinol isolated from the mutants and wild type showed that PrRs together with a dirigent protein(s) are involved in the enantiomeric control in lignan biosynthesis. Furthermore, it was demonstrated conclusively for the first time that differential expression of PrR isoforms that have distinct selectivities of substrate enantiomers can determine enantiomeric compositions of the product, lariciresinol.

  17. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

    Energy Technology Data Exchange (ETDEWEB)

    Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E. (UIC)

    2012-10-25

    The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.

  18. 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.)

  19. S-glutathionyl-(chloro)hydroquinone reductases: a new class of glutathione transferases functioning as oxidoreductases.

    Science.gov (United States)

    Belchik, Sara M; Xun, Luying

    2011-05-01

    Glutathione transferases (GSTs) are best known for transferring glutathione (GSH) to hydrophobic organic compounds, making the conjugates more soluble. However, the omega-class GSTs of animals and the lambda-class GSTs and dehydroascorbate reductases (DHARs) of plants have little or no activity for GSH transfer. Instead, they catalyze GSH-dependent oxidoreductions. The lambda-class GSTs reduce disulfide bonds, the DHARs reduce the disulfide bonds and dehydroascorbate, and the omega-class GSTs can reduce more substrates, including disulfide bonds, dehydroascorbate, and dimethylarsinate. Glutathionyl-(chloro)hydroquinone reductases (GS-HQRs) are the newest class of GSTs that mainly catalyze oxidoreductions. Besides the activities of the other three classes, GS-HQRs also reduce GS-hydroquinones, including GS-trichloro-p-hydroquinone, GS-dichloro-p-hydroquinone, GS-2-hydroxy-p-hydroquinone, and GS-p-hydroquinone. They are conserved and widely distributed in bacteria, fungi, protozoa, and plants, but not in animals. The four classes are phylogenetically more related to each other than to other GSTs, and they share a Cys-Pro motif at the GSH-binding site. Hydroquinones are metabolic intermediates of certain aromatic compounds. They can be auto-oxidized by O(2) to benzoquinones, which spontaneously react with GSH to form GS-hydroquinones via Michael's addition. GS-HQRs are expected to channel GS-hydroquinones, formed spontaneously or enzymatically, back to hydroquinones. When the released hydroquinones are intermediates of metabolic pathways, GS-HQRs play a maintenance role for the pathways. Further, the common presence of GS-HQRs in plants, green algae, cyanobacteria, and halobacteria suggest a beneficial role in the light-using organisms.

  20. The nitric-oxide reductase from Paracoccus denitrificans uses a single specific proton pathway.

    Science.gov (United States)

    ter Beek, Josy; Krause, Nils; Reimann, Joachim; Lachmann, Peter; Ädelroth, Pia

    2013-10-18

    The NO reductase from Paracoccus denitrificans reduces NO to N2O (2NO + 2H(+) + 2e(-) → N2O + H2O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O2-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O2 reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O2. We further show that electron transfer during single-turnover reduction of O2 is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.

  1. A transgenic Neospora caninum strain based on mutations of the dihydrofolate reductase-thymidylate synthase gene.

    Science.gov (United States)

    Pereira, Luiz Miguel; Baroni, Luciana; Yatsuda, Ana Patrícia

    2014-03-01

    Neospora caninum is an Apicomplexa parasite related to abortion and losses of fertility in cattle. The amenability of Toxoplasma gondii and Plasmodium to genetic manipulation offers several tools to determine the invasion and replication processes, which support posterior strategies related to the combat of these diseases. For Plasmodium the use of pyrimethamine as an auxiliary drug on malaria treatment has been affected by the rise of resistant strains and the analyses on Dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene indicated several point mutations. In this work we developed a method for stable insertion of genes based on resistance to pyrimethamine. For that, the coding sequence of NcDHFR-TS (Dihydrofolate reductase-thymidylate synthase) was point mutated in two amino acids, generating DHFRM2M3. The DHFRM2M3 flanked by the promoter and 3'UTR of Ncdhfr-ts (Ncdhfr-DHFRM2M3) conferred resistance to pyrimethamine after transfection. For illustration of stability and expression, the cassette Ncdhfr-DHFRM2M3 was ligated to the reporter gene Lac-Z (β-galactosidase enzyme) controlled by the N. caninum tubulin promoter and was transfected and selected in N. caninum. The cassette was integrated into the genome and the selected tachyzoites expressed Lac-Z, allowing the detection of tachyzoites by the CPRG reaction and X-gal precipitation. The obtainment of transgenic N. caninum resistant to pyrimethamine confirms the effects on DHFR-TS among the Apicomplexa members and will support future approaches on pholate inhibitors for N. caninum prophylaxis. The construction of stable tachyzoites based on vectors with N. caninum promoters initiates the molecular manipulation of this parasite independently of T. gondii.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Swapping FAD binding motifs between plastidic and bacterial ferredoxin-NADP(H) reductases.

    Science.gov (United States)

    Musumeci, Matías A; Botti, Horacio; Buschiazzo, Alejandro; Ceccarelli, Eduardo A

    2011-03-29

    Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 β-hairpin from a plastidic FNR and the carboxy-terminal tryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the β-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme with decreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the β-hairpin into the corresponding position of the bacterial FNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxy-terminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased k(cat) and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme's ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 β-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs.

  4. Temperature dependence of methyl-coenzyme M reductase activity and of the formation of the methyl-coenzyme M reductase red2 state induced by coenzyme B.

    Science.gov (United States)

    Goenrich, Meike; Duin, Evert C; Mahlert, Felix; Thauer, Rudolf K

    2005-06-01

    Methyl-coenzyme M reductase (MCR) catalyses the formation of methane from methyl-coenzyme M (CH(3)-S-CoM) and coenzyme B (HS-CoB) in methanogenic archaea. The enzyme has an alpha(2)beta(2)gamma(2) subunit structure forming two structurally interlinked active sites each with a molecule F(430) as a prosthetic group. The nickel porphinoid must be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-based electron paramagnetic resonance (EPR) signal and a UV-vis spectrum with an absorption maximum at 385 nm. This state is called the MCR-red1 state. In the presence of coenzyme M (HS-CoM) and coenzyme B the MCR-red1 state is in part converted reversibly into the MCR-red2 state, which shows a rhombic Ni(I)-based EPR signal and a UV-vis spectrum with an absorption maximum at 420 nm. We report here for MCR from Methanothermobacter marburgensis that the MCR-red2 state is also induced by several coenzyme B analogues and that the degree of induction by coenzyme B is temperature-dependent. When the temperature was lowered below 20 degrees C the percentage of MCR in the red2 state decreased and that in the red1 state increased. These changes with temperature were fully reversible. It was found that at most 50% of the enzyme was converted to the MCR-red2 state under all experimental conditions. These findings indicate that in the presence of both coenzyme M and coenzyme B only one of the two active sites of MCR can be in the red2 state (half-of-the-sites reactivity). On the basis of this interpretation a two-stroke engine mechanism for MCR is proposed.

  5. Diversity in mechanisms of substrate oxidation by cytochrome P450 2D6. Lack of an allosteric role of NADPH-cytochrome P450 reductase in catalytic regioselectivity.

    Science.gov (United States)

    Hanna, I H; Krauser, J A; Cai, H; Kim, M S; Guengerich, F P

    2001-10-26

    Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Differences in the regioselectivity of oxidation products formed in systems containing NADPH-P450 reductase/NADPH and the model oxidant cumene hydroperoxide have been proposed by others to be due to an allosteric influence of the reductase on P450 2D6 (Modi, S., Gilham, D. E., Sutcliffe, M. J., Lian, L.-Y., Primrose, W. U., Wolf, C. R., and Roberts, G. C. K. (1997) Biochemistry 36, 4461-4470). We examined the differences in the formation of oxidation products of N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, metoprolol, and bufuralol between reductase-, cumene hydroperoxide-, and iodosylbenzene-supported systems. Catalytic regioselectivity was not influenced by the presence of the reductase in any of the systems supported by model oxidants, ruling out allosteric influences. The presence of the reductase had little effect on the affinity of P450 2D6 for any of these three substrates. The addition of the reaction remnants of the model oxidants (cumyl alcohol and iodobenzene) to the reductase-supported system did not affect reaction patterns, arguing against steric influences of these products on catalytic regioselectivity. Label from H(2)18O was quantitatively incorporated into 1'-hydroxybufuralol in the iodosylbenzene- but not in the reductase- or cumene hydroperoxide-supported reactions. We conclude that the P450 systems utilizing NADPH-P450 reductase, cumene hydroperoxide, and iodosylbenzene use similar but distinct chemical mechanisms. These differences are the basis for the variable product distributions, not an allosteric influence of the reductase.

  6. 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.

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

    Digital Repository Service at National Institute of Oceanography (India)

    Puttananjaiah, M.H.; Dhale, M.A.; Gaonkar, V.; Keni, S.

    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...

  8. 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.

  9. In vivo induction of phase II detoxifying enzymes, glutathione transferase and quinone reductase by citrus triterpenoids

    Directory of Open Access Journals (Sweden)

    Ahmad Hassan

    2010-09-01

    Full Text Available Abstract Background Several cell culture and animal studies demonstrated that citrus bioactive compounds have protective effects against certain types of cancer. Among several classes of citrus bioactive compounds, limonoids were reported to prevent different types of cancer. Furthermore, the structures of citrus limonoids were reported to influence the activity of phase II detoxifying enzymes. The purpose of the study was to evaluate how variations in the structures of citrus limonoids (namely nomilin, deacetyl nomilin, and isoobacunoic acid and a mixture of limonoids would influence phase II enzyme activity in excised tissues from a mouse model. Methods In the current study, defatted sour orange seed powder was extracted with ethyl acetate and subjected to silica gel chromatography. The HPLC, NMR and mass spectra were used to elucidate the purity and structure of compounds. Female A/J mice were treated with three limonoids and a mixture in order to evaluate their effect on phase II enzymes in four different tissues. Assays for glutathione S-transferase and NAD(PH: quinone reductase (QR were used to evaluate induction of phase II enzymatic activity. Results The highest induction of GST against 1-chloro-2,4-dinitrobenzene (CDNB was observed in stomach (whole, 58% by nomilin, followed by 25% isoobacunoic acid and 19% deacetyl nomilin. Deacetyl nomilin in intestine (small as well as liver significantly reduced GST activity against CDNB. Additionally isoobacunoic acid and the limonoid mixture in liver demonstrated a significant reduction of GST activity against CDNB. Nomilin significantly induced GST activity against 4-nitroquinoline 1-oxide (4NQO, intestine (280% and stomach (75% while deacetyl nomilin showed significant induction only in intestine (73%. Induction of GST activity was also observed in intestine (93% and stomach (45% treated with the limonoid mixture. Finally, a significant induction of NAD(PH: quinone reductase (QR activity was

  10. Structure and mechanism of styrene monooxygenase reductase: new insight into the FAD-transfer reaction.

    Science.gov (United States)

    Morrison, Eliot; Kantz, Auric; Gassner, George T; Sazinsky, Matthew H

    2013-09-03

    The two-component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH- and FAD-dependent epoxidation of styrene to styrene oxide. In this study, we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to the epoxidase (NSMOA) component and report our findings in light of the 2.2 Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH → FADox charge-transfer intermediate and catalyzes a hydride-transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s(-1), in a step that is coupled to the rapid dissociation of NAD(+). Electrochemical and equilibrium-binding studies indicate that NSMOA binds FADhq ∼13-times more tightly than SMOB, which supports a vectoral transfer of FADhq from the reductase to the epoxidase. After binding to NSMOA, FADhq rapidly reacts with molecular oxygen to form a stable C(4a)-hydroperoxide intermediate. The half-life of apoSMOB generated in the FAD-transfer reaction is increased ∼21-fold, supporting a protein-protein interaction between apoSMOB and the peroxide intermediate of NSMOA. The mechanisms of FAD dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. On the basis of these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S state) and more reactive, transfer state (T state). The dissociation of NAD(+) after the hydride-transfer reaction transiently populates the T state, promoting the transfer of FADhq to NSMOA. The binding of pyridine nucleotides to SMOB-FADhq shifts the FADhq-binding equilibrium from the T state to the S state. Additionally, the 2.2 Å crystal structure of SMOB-FADox reported in this work is discussed in light of the pyridine nucleotide-gated flavin-transfer and electron-transfer reactions.

  11. Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase.

    Science.gov (United States)

    Turner, Glenn W; Croteau, Rodney

    2004-12-01

    We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.

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

    Science.gov (United States)

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

  13. Immunochemical and immunoelectron microscope studies on localization of NADPH-cytochrome c reductase on rat liver microsomes

    Science.gov (United States)

    1976-01-01

    By the use of ferritin-conjugated antibody (conjugate) indirect immunoelectron microscopy, NADPH-cytochrome c reductase was localized on rat liver microsomes. Most microsomes in the sections had from 1 to 12 conjugates on their outer surfaces. Among the conjugates, 83% was estimated to bind to NADPH-cytochrome c reductase at a molecular ratio of 1:1, 12% at the ratio of 2:1, and 5% at the ratio of 3 or 4:1. The correlation between immunochemical and morphological data confirmed that most of the NADPH-cytochrome c reducatase reacted with the conjugates. Subsequent morphological analyses have revealed that the enzyme is distributed homogeneously on the outer surfaces of microsomes but heterogeneously within microsomes in groups of three to five enzyme molecules. PMID:812876

  14. Functional assembly of camphor converting two-component Baeyer-Villiger monooxygenases with a flavin reductase from E. coli.

    Science.gov (United States)

    Kadow, Maria; Balke, Kathleen; Willetts, Andrew; Bornscheuer, Uwe T; Bäckvall, J-E

    2014-05-01

    The major limitation in the synthetic application of two-component Baeyer-Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.

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

    Energy Technology Data Exchange (ETDEWEB)

    Fujii, Tomomi; Goda, Yuko [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Yoshida, Masahiro; Oikawa, Tadao [Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka 564-8680 (Japan); Hata, Yasuo, E-mail: hata@scl.kyoto-u.ac.jp [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2008-08-01

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

  16. 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.

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

    DEFF Research Database (Denmark)

    Gøtterup, Jacob; Olsen, Karsten; Knøchel, Susanne

    2008-01-01

    Three Staphylococcus strains, S. carnosus, S. simulans and S. saprophyticus, selected due to their varying nitrite and/or nitrate-reductase activities, were used to initiate colour formation during sausage fermentation. During fermentation of sausages with either nitrite or nitrate added, colour...... nitrate depended on the specific Staphylococcus strain. Strains with high nitrate-reductase activity showed a significantly faster rate of pigment formation, but other factors were of influence as well. Product stability for the sliced, packaged sausage was evaluated as surface colour and oxidation...... by autofluorescence and hexanal content, respectively. No significant direct effect of the Staphylococcus addition was observed, however, there was a clear correspondence between high initial amount of MbFeIINO in the different sausages and the colour stability during storage. Autofluorescence data correlated well...

  18. Synthesis and activity of 8-substituted benzo[c]quinolizin-3-ones as dual inhibitors of human 5alpha-reductases 1 and 2.

    Science.gov (United States)

    Ferrali, Alessandro; Menchi, Gloria; Occhiato, Ernesto G; Danza, Giovanna; Mancina, Rosa; Serio, Mario; Guarna, Antonio

    2005-01-03

    Some potent dual inhibitors of 5alpha-reductases 1 and 2, based on the benzo[c]quinolizin-3-one structure and with IC(50) values ranging between 93 and 166nM for both isozymes, were found. The presence of the F atom on the ester moiety at the position 8 was crucial. This result can help in the design of other potent, dual inhibitors to be developed as drugs in the treatment of 5alpha-reductase related diseases.

  19. Assessment of Folic Acid Supplementation in Pregnant Women by Estimation of Serum Levels of Tetrahydrofolic Acid, Dihydrofolate Reductase, and Homocysteine

    OpenAIRE

    Manisha Naithani; Vartika Saxena; Anissa Atif Mirza; Ranjeeta Kumari; Kapil Sharma; Jyoti Bharadwaj

    2016-01-01

    Background. Status of folic acid use in pregnant women of the hilly regions in North India was little known. This study was carried out to assess the folic acid use and estimate folate metabolites in pregnant women of this region. Materials and Methods. This cross-sectional study is comprised of 76 pregnant women, whose folic acid supplementation was assessed by a questionnaire and serum levels of homocysteine, tetrahydrofolic acid (THFA), and dihydrofolate reductase (DHFR) were estimated usi...

  20. The development of the 2, 4-dienoyl CoA reductase 1 gene (DECR 1) in pig

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    2,4-dienoyl CoA reductase gene (DECR 1) is mapped on pig 4 q1.2, includes ten exons and nine introns of variable sizethat span 30 kb. DECR 1 gene participates in the β-oxidation pathway, affects the content of intramuscular fatty acid, especially thepercentage of linoleic acid. The expression of DECR 1 gene has important influence on IMF, the pH, and the meat colour of pork,further affects the meat quality.

  1. First Synthesis of 3'- Geranyl-2', 3,4,4'-tetrahydroxychalcone with 5 α- Reductase Inhibitory Properties

    Institute of Scientific and Technical Information of China (English)

    LIU Hong-Xing; HUANG Chu-Sheng; CHEN Xi-Hui; ZHU Yan-Yun

    2003-01-01

    @@ The geranylated chalcone 1,3′-geranyl-2′, 3,4,4′-tetvahydroxychalcone with high 5α-reductase inhibitory prop erties, was isolated from Artocarpus incisus by Shimizu in 2000. [1] Herein, we describe the first synthesis of the natural product 1 by four steps starting from compounds 2 and 7. The synthetic route was shown in Scheme 1:

  2. 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....

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

    Science.gov (United States)

    Moon, Jaewoong; Liu, Z Lewis

    2015-04-01

    The aldehyde reductase gene ARI1 is a recently characterized member of an intermediate subfamily within the short-chain dehydrogenase/reductase (SDR) superfamily that clarified mechanisms of in situ detoxification of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde by Saccharomyces cerevisiae. Uncharacterized open reading frames (ORFs) are common among tolerant candidate genes identified for lignocellulose-to-advanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehyde-inhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates, with high levels of reduction activity for Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co-factor NADPH, Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr-X-X-X-Lys; and a cofactor-binding sequence motif, Gly-X-X-Gly-X-X-Ala, near the N-terminus that are shared by Ydr541cp, Ygl039wp, Yol151wp/GRE2 and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next-generation biocatalyst for advanced biofuels production.

  4. New functional sulfide oxidase-oxygen reductase supercomplex in the membrane of the hyperthermophilic bacterium Aquifex aeolicus.

    Science.gov (United States)

    Prunetti, Laurence; Infossi, Pascale; Brugna, Myriam; Ebel, Christine; Giudici-Orticoni, Marie-Thérèse; Guiral, Marianne

    2010-12-31

    Aquifex aeolicus, a hyperthermophilic and microaerophilic bacterium, obtains energy for growth from inorganic compounds alone. It was previously proposed that one of the respiratory pathways in this organism consists of the electron transfer from hydrogen sulfide (H(2)S) to molecular oxygen. H(2)S is oxidized by the sulfide quinone reductase, a membrane-bound flavoenzyme, which reduces the quinone pool. We have purified and characterized a novel membrane-bound multienzyme supercomplex that brings together all the molecular components involved in this bioenergetic chain. Our results indicate that this purified structure consists of one dimeric bc(1) complex (complex III), one cytochrome c oxidase (complex IV), and one or two sulfide quinone reductases as well as traces of the monoheme cytochrome c(555) and quinone molecules. In addition, this work strongly suggests that the cytochrome c oxidase in the supercomplex is a ba(3)-type enzyme. The supercomplex has a molecular mass of about 350 kDa and is enzymatically functional, reducing O(2) in the presence of the electron donor, H(2)S. This is the first demonstration of the existence of such a respirasome carrying a sulfide oxidase-oxygen reductase activity. Moreover, the kinetic properties of the sulfide quinone reductase change slightly when integrated in the supercomplex, compared with the free enzyme. We previously purified a complete respirasome involved in hydrogen oxidation and sulfur reduction from Aquifex aeolicus. Thus, two different bioenergetic pathways (sulfur reduction and sulfur oxidation) are organized in this bacterium as supramolecular structures in the membrane. A model for the energetic sulfur metabolism of Aquifex aeolicus is proposed.

  5. Single dose testosterone increases total cholesterol levels and induces the expression of HMG CoA Reductase

    OpenAIRE

    2012-01-01

    Abstract Background Cholesterol is mainly synthesised in liver and the rate-limiting step is the reduction of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) to mevalonate, a reaction catalysed by HMG-CoA reductase (HMGCR). There is a comprehensive body of evidence documenting that anabolic-androgenic steroids are associated with deleterious alterations of lipid profile. In this study we investigated whether a single dose of testosterone enanthate affects the cholesterol biosynthesis and the e...

  6. Anthranoyl-CoA monooxygenase/reductase from Azoarcus evansii possesses both FMN and FAD in two distinct and independent active sites.

    Science.gov (United States)

    Bergner, Thomas; Pavkov-Keller, Tea; Kreuzer, Katharina; Kowaliuk, Jakob; Plank, Markus; Runggatscher, Kathrin; Turrini, Nikolaus G; Zucol, Benjamin; Wallner, Silvia; Faber, Kurt; Gruber, Karl; Macheroux, Peter

    2015-08-01

    Anthranoyl-CoA monooxygenase/reductase (ACMR) participates in an unusual pathway for the degradation of aromatic compounds in Azoarcus evansii. It catalyzes the monooxygenation of anthranoyl-CoA to 5-hydroxyl-2-aminobenzoyl-CoA and the subsequent reduction to the dearomatized product 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. The two reactions occur in separate domains, termed the monooxygenase and reductase domain. Both domains were reported to utilize FAD as a cofactor for hydroxylation and reduction, respectively. We have heterologously expressed ACMR in Escherichia coli BL21 and found that the monooxygenase domain contains FAD. However, the reductase domain utilizes FMN and not FAD for the reduction of the intermediate 5-hydroxyl-2-aminobenzoyl-CoA. A homology model for the reductase domain predicted a topology similar to the Old Yellow Enzyme family, which exclusively bind FMN, in accordance with our results. Binding studies with 2-aminobenzoyl-CoA (AbCoA) and p-hydroxybenzaldehyde (pHB) as probes for the monooxygenase and reductase domain, respectively, indicated that two functionally distinct and independent active sites exist. Given the homodimeric quartenary structure of ACMR and the compact shape of the dimer as determined by small-angle X-ray scattering experiments we propose that the monooxygenase and reductase domain of opposite peptide chains are involved in the transformation of anthranoyl-CoA to 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA.

  7. 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.

  8. A 1,536-well-based kinetic HTS assay for inhibitors of Schistosoma mansoni thioredoxin glutathione reductase.

    Science.gov (United States)

    Lea, Wendy A; Jadhav, Ajit; Rai, Ganesha; Sayed, Ahmed A; Cass, Cynthia L; Inglese, James; Williams, David L; Austin, Christopher P; Simeonov, Anton

    2008-08-01

    Abstract: Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, glutathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of a highly miniaturized and robust screening assay for TGR. The 5-mul final volume assay is based on the Ellman reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes that utilize DTNB as a model substrate.

  9. The effect of oxidative stress on human red cells glutathione peroxidase, glutathione reductase level, and prevalence of anemia among diabetics

    Directory of Open Access Journals (Sweden)

    Hisham Waggiallah

    2011-01-01

    Full Text Available Background: The oxidative stress is considered as major consequence of diabetes mellitus affecting red cell antioxidant enzymes. Aim: The present study was conducted to assess the impact of oxidative stress (reduced glutathione on glutathione peroxidase, and glutathione reductse and prevalence of anemia among diabetic patients. Materials and Methods: The study involved 100 adult patients attending Buraidah Central Hospital and 30 healthy controls. Blood samples were collected and analyzed for glutathione (GSH concentration, glutathione peroxidase (GPO, glutathione reductase (GR, fasting blood sugar (RBS, hemoglobin (HGB, red cell count (RBCs hematocrit (HCT mean cell volume (MCV mean cell hemoglobin (MCH and mean cell hemoglobin concentration (MCHC and hemoglobin A1c. Blood urea, serum creatinine, and microalbuminuria were measured to exclude diabetes mellitus nephropathy. Results : were obtained showed significant correlation between deficiency of glutathione peroxidase, glutathione reductase and deficient of glutathione among diabetics, which has significant correlation between low hemoglobin concentration (females <120 g/L, males <130 g/L, also there is low concentration of red cell count and red cell indices (MCV, MCH and MCHC. The prevalence of anemia was 22% in diabetes patients. Conclusion: It can be concluded that there is strong significant effect of oxidative stress (reduced glutathione on glutathione peroxidase, glutathione reductase level these may reduce hemoglobin concentration in diabetic patients. This means oxidative stress of diabetes mellitus is the possible cause of anemia in diabetics without nephropathy.

  10. 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.

  11. Crystallization and preliminary X-ray studies of ferredoxin-NAD(P){sup +} reductase from Chlorobium tepidum

    Energy Technology Data Exchange (ETDEWEB)

    Muraki, Norifumi [Department of Life Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Seo, Daisuke [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan); Shiba, Tomoo [Department of Life Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Sakurai, Takeshi [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan); Kurisu, Genji, E-mail: gkurisu@xtal.c.u-tokyo.ac.jp [Department of Life Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902 (Japan)

    2008-03-01

    Ferredoxin-NAD(P){sup +} reductase from C. tepidum has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. Ferredoxin-NAD(P){sup +} reductase (FNR) is a key enzyme that catalyzes the photoreduction of NAD(P){sup +} to generate NAD(P)H during the final step of the photosynthetic electron-transport chain. FNR from the green sulfur bacterium Chlorobium tepidum is a homodimeric enzyme with a molecular weight of 90 kDa; it shares a high level of amino-acid sequence identity to thioredoxin reductase rather than to conventional plant-type FNRs. In order to understand the structural basis of the ferredoxin-dependency of this unique photosynthetic FNR, C. tepidum FNR has been heterologously expressed, purified and crystallized in two forms. Form I crystals belong to space group C222{sub 1} and contain one dimer in the asymmetric unit, while form II crystals belong to space group P4{sub 1}22 or P4{sub 3}22. Diffraction data were collected from a form I crystal to 2.4 Å resolution on the synchrotron-radiation beamline NW12 at the Photon Factory.

  12. Isolation and preliminary characterization of a respiratory nitrate reductase from hydrocarbon-degrading bacterium Gordonia alkanivorans S7.

    Science.gov (United States)

    Romanowska, Irena; Kwapisz, Ewa; Mitka, Magdalena; Bielecki, Stanisław

    2010-06-01

    Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors. The respiratory nitrate reductase (Nar) from this organism has been isolated using ion exchange chromatography and gel filtration, and then preliminarily characterized. PAGE, SDS-PAGE and gel filtration chromatography revealed that Nar consisted of three subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40 degrees C. K(m) values for NO(3)(-) (110 microM) and for ClO(3)(-) (138 microM) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from Gordonia species.

  13. Linear array of conserved sequence motifs to discriminate protein subfamilies: study on pyridine nucleotide-disulfide reductases

    Directory of Open Access Journals (Sweden)

    De Las Rivas Javier

    2007-03-01

    Full Text Available Abstract Background The pyridine nucleotide disulfide reductase (PNDR is a large and heterogeneous protein family divided into two classes (I and II, which reflect the divergent evolution of its characteristic disulfide redox active site. However, not all the PNDR members fit into these categories and this suggests the need of further studies to achieve a more comprehensive classification of this complex family. Results A workflow to improve the clusterization of protein families based on the array of linear conserved motifs is designed. The method is applied to the PNDR large family finding two main groups, which correspond to PNDR classes I and II. However, two other separate protein clusters, previously classified as class I in most databases, are outgrouped: the peroxide reductases (NAOX, NAPE and the type II NADH dehydrogenases (NDH-2. In this way, two novel PNDR classes III and IV for NAOX/NAPE and NDH-2 respectively are proposed. By knowledge-driven biochemical and functional data analyses done on the new class IV, a linear array of motifs putatively related to Cu(II-reductase activity is detected in a specific subset of NDH-2. Conclusion The results presented are a novel contribution to the classification of the complex and large PNDR protein family, supporting its reclusterization into four classes. The linear array of motifs detected within the class IV PNDR subfamily could be useful as a signature for a particular subgroup of NDH-2.

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

    Energy Technology Data Exchange (ETDEWEB)

    Saito, Jun, E-mail: jun-saito@meiji.co.jp; Yamada, Mototsugu; Watanabe, Takashi; Kitagawa, Hideo; Takeuchi, Yasuo [Pharmaceutical Research Center, Meiji Seika Kaisha Ltd, 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567 (Japan)

    2006-06-01

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

  15. 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.

  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. Structural and mutational studies of an electron transfer complex of maize sulfite reductase and ferredoxin.

    Science.gov (United States)

    Kim, Ju Yaen; Nakayama, Masato; Toyota, Hiroshi; Kurisu, Genji; Hase, Toshiharu

    2016-08-01

    The structure of the complex of maize sulfite reductase (SiR) and ferredoxin (Fd) has been determined by X-ray crystallography. Co-crystals of the two proteins prepared under different conditions were subjected to the diffraction analysis and three possible structures of the complex were solved. Although topological relationship of SiR and Fd varied in each of the structures, two characteristics common to all structures were found in the pattern of protein-protein interactions and positional arrangements of redox centres; (i) a few negative residues of Fd contact with a narrow area of SiR with positive electrostatic surface potential and (ii) [2Fe-2S] cluster of Fd and [4Fe-4S] cluster of SiR are in a close proximity with the shortest distance around 12 Å. Mutational analysis of a total of seven basic residues of SiR distributed widely at the interface of the complex showed their importance for supporting an efficient Fd-dependent activity and a strong physical binding to Fd. These combined results suggest that the productive electron transfer complex of SiR and Fd could be formed through multiple processes of the electrostatic intermolecular interaction and this implication is discussed in terms of the multi-functionality of Fd in various redox metabolisms.

  18. 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

  19. Inhibitory effect of two Indian medicinal plants on aldose reductase of rat lensin vitro

    Institute of Scientific and Technical Information of China (English)

    Rajesh Kumar; Dinesh Kumar Patel; Damiki Laloo; Krishnamurthy Sairam; Siva Hemalatha

    2011-01-01

    Objective:To assesse the inhibitory effect of alcoholic extract of two Indian medicinal plants namelyCeasalpinia digyna Rottler and, Alangium lamarckii Thwaits on aldose reductase(AR) of rat lens.Methods: Rats lens were enucleated through posterior approach and their homogenate was prepared and centrifuged to obtain a clear supernatant for the determination ofAR activity and protein content.Results:The alcoholic extract of Ceasalpinia digyna andAlangium lamarckii had a potent inhibitory effect on the lensAR enzyme. TheIC50 values of alcoholic extract of the selected plants were calculated and were (46.29±11.17)and(106.00±5.11) μg/mL, respectively. Quercetin was used as a positive control and itsIC50value was (2.95±1.53)μg/mL.Conclusions:Thus, it is concluded that alcoholic extracts of the selected plant exhibit significant inhibitory effects on AR in the rat lensin vitro.

  20. Generation of an attenuated Tiantan vaccinia virus by deletion of the ribonucleotide reductase large subunit.

    Science.gov (United States)

    Kan, Shifu; Jia, Peng; Sun, Lili; Hu, Ningning; Li, Chang; Lu, Huijun; Tian, Mingyao; Qi, Yanxin; Jin, Ningyi; Li, Xiao

    2014-09-01

    Attenuation of the virulence of vaccinia Tiantan virus (VTT) underlies the strategy adopted for mass vaccination campaigns. This strategy provides advantages of safety and efficacy over traditional vaccines and is aimed at minimization of adverse health effects. In this study, a mutant form of the virus, MVTT was derived from VTT by deletion of the ribonucleotide reductase large subunit (R1) (TI4L). Compared to wild-type parental (VTT) and revertant (VTT-rev) viruses, virulence of the mutant MVTT was reduced by 100-fold based on body weight reduction and by 3,200-fold based on determination of the intracranial 50% lethal infectious dose. However, the immunogenicity of MVTT was equivalent to that of the parental VTT. We also demonstrated that the TI4L gene is not required for efficient replication. These data support the conclusion that MVTT can be used as a replicating virus vector or as a platform for the development of vaccines against infectious diseases and for cancer therapy.

  1. Alkyl hydroperoxide reductase enhances the growth of Leuconostoc mesenteroides lactic acid bacteria at low temperatures.

    Science.gov (United States)

    Goto, Seitaro; Kawamoto, Jun; Sato, Satoshi B; Iki, Takashi; Watanabe, Itaru; Kudo, Kazuyuki; Esaki, Nobuyoshi; Kurihara, Tatsuo

    2015-01-01

    Lactic acid bacteria (LAB) can cause deterioration of food quality even at low temperatures. In this study, we investigated the cold-adaptation mechanism of a novel food spoilage LAB, Leuconostoc mesenteroides NH04 (NH04). L. mesenteroides was isolated from several spoiled cooked meat products at a high frequency in our factories. NH04 grew rapidly at low temperatures within the shelf-life period and resulted in heavy financial losses. NH04 grew more rapidly than related strains such as Leuconostoc mesenteroides NBRC3832 (NBRC3832) at 10°C. Proteome analysis of NH04 demonstrated that this strain produces a homolog of alkyl hydroperoxide reductase--AhpC--the expression of which can be induced at low temperatures. The expression level of AhpC in NH04 was approximately 6-fold higher than that in NBRC3832, which was grown under the same conditions. Although AhpC is known to have an anti-oxidative role in various bacteria by catalyzing the reduction of alkyl hydroperoxide and hydrogen peroxide, the involvement of AhpC in cold adaptation of food spoilage bacteria was unclear. We introduced an expression plasmid containing ahpC into NBRC3832, which grows slower than NH04 at 10°C, and found that expression of AhpC enhanced growth. These results demonstrated that AhpC, which likely increases anti-oxidative capacity of LAB, plays an important role in their rapid growth at low temperatures.

  2. Functional thioredoxin reductase from pathogenic and free-living Leptospira spp.

    Science.gov (United States)

    Sasoni, Natalia; Iglesias, Alberto A; Guerrero, Sergio A; Arias, Diego G

    2016-08-01

    Low molecular mass thiols and antioxidant enzymes have essential functions to detoxify reactive oxygen and nitrogen species maintaining cellular redox balance. The metabolic pathways for redox homeostasis in pathogenic (Leptospira interrogans) and free-living (Leptospira biflexa) leptospires species were not functionally characterized. We performed biochemical studies on recombinantly produced proteins to in depth analyze kinetic and structural properties of thioredoxin reductase (LinTrxR) and thioredoxin (LinTrx) from L. interrogans, and two TrxRs (LbiTrxR1 and LbiTrxR2) from L. biflexa. All the TrxRs were characterized as homodimeric flavoproteins, with LinTrxR and LbiTrxR1 catalyzing the NADPH dependent reduction of LinTrx and DTNB. The thioredoxin system from L. interrogans was able to use glutathione disulfide, lipoamide disulfide, cystine and bis-γ-glutamyl cysteine and homologous peroxiredoxin as substrates. Classic TrxR activity of LinTrxR2 had not been evidenced in vitro, but recombinant Escherichia coli cells overexpressing LbiTrxR2 showed high tolerance to oxidative stress. The enzymatic systems herein characterized could play a key role for the maintenance of redox homeostasis and the function of defense mechanisms against reactive oxidant species in Leptospira spp. Our results contribute to the general knowledge about redox biochemistry in these bacteria, positioning TrxR as a critical molecular target for the development of new anti-leptospiral drugs.

  3. 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.

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

    Institute of Scientific and Technical Information of China (English)

    Safinaz S Ibrahim; Noha N Nassar

    2008-01-01

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

  5. Association between methylenetetrahydrofolate reductase C677T polymorphism and psoriasis: A meta-analysis.

    Science.gov (United States)

    Wu, Dongze; Shi, Deshun; Yang, Li; Zhu, Xiaoliang

    2016-02-01

    Several studies have evaluated the associations between methylenetetrahydrofolate reductase (MTHFR) C677T and psoriasis. However, the results remain inconclusive. The objective of the present study was to conduct a qualitative and quantitative meta-analysis investigating the associations between MTHFR C677T and psoriasis. A published work search of PubMed, Embase, Web of Science and Chinese National Knowledge Infrastructure database were conducted to identify all publications concerning MTHFR C677T polymorphism and psoriasis on 1 October 2014. The principal outcome measure for evaluating the strength of the association was crude odds ratios along with their corresponding 95% confidence intervals. Data were extracted and statistical analyses were implemented using STATA version 12.0 software. A total of 1179 psoriatic cases and 937 controls from five case-control studies concentrating on the association between MTHFR C677T polymorphism and psoriasis were included in this qualitative meta-analysis. Pooled analysis revealed that there is no association between this polymorphism and susceptibility to psoriasis in dominant, recessive, allele and additive models under a random-effect model. However, a marginal significant association was found in the overdominant model under fixed-effect model. Subgroup analysis of ethnicity demonstrated that there is no association between MTHFR C677T polymorphism and either Asian or European psoriatic patients. In conclusion, MTHFR C677T polymorphism, qualitatively, is not a genetic factor for the pathogenesis of psoriasis but could quantitatively reflect the severity of psoriasis to some extent.

  6. Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in psoriasis in southern Turkey*

    Science.gov (United States)

    Izmirli, Muzeyyen; Sen, Bilge Bulbul; Rifaioglu, Eminenur; Gogebakan, Bulent; Aldemir, Ozgur; Sen, Tuba; Ekiz, Ozlem; Alptekin, Davut

    2016-01-01

    Background Psoriasis is a multigenic and multifactorial dermatological disease linked to cardiovascular diseases. Increased levels of homocysteine in patients with psoriasis have been demonstrated in many studies. The most frequently investigated genetic defect that plays a role in homocysteine metabolism is single point substitution (C to T) located on the 677th nucleotide of the methylenetetrahydrofolate reductase gene (MTHFR). Objective In this study, we aimed to investigate methylenetetrahydrofolate C677T polymorphism in psoriasis patients in Turkey. Methods The study included 96 patients with psoriasis and 77 controls from southern Turkey. Methylenetetrahydrofolate C677T polymorphism was analysed using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism methods. Results In the psoriasis group, 34 CC (35.4%), 46 CT (47.9%) and 16 TT (16.7%) genotypes were found, respectively; while in the control group, the figures were 39 (50.6%), 35 (45.5%), 3 (3.9%). Homozygote and heterozygote T alleles of methylenetetrahydrofolate C677T polymorphism were significantly higher in the psoriasis than in the control group (p=0.013). Conclusion We firstly found a correlation between methylenetetrahydrofolate C677T polymorphism and psoriasis among the southern Turkish population. PMID:27828634

  7. Identification of Novel Aldose Reductase Inhibitors from Spices: A Molecular Docking and Simulation Study.

    Science.gov (United States)

    Antony, Priya; Vijayan, Ranjit

    2015-01-01

    Hyperglycemia in diabetic patients results in a diverse range of complications such as diabetic retinopathy, neuropathy, nephropathy and cardiovascular diseases. The role of aldose reductase (AR), the key enzyme in the polyol pathway, in these complications is well established. Due to notable side-effects of several drugs, phytochemicals as an alternative has gained considerable importance for the treatment of several ailments. In order to evaluate the inhibitory effects of dietary spices on AR, a collection of phytochemicals were identified from Zingiber officinale (ginger), Curcuma longa (turmeric) Allium sativum (garlic) and Trigonella foenum graecum (fenugreek). Molecular docking was performed for lead identification and molecular dynamics simulations were performed to study the dynamic behaviour of these protein-ligand interactions. Gingerenones A, B and C, lariciresinol, quercetin and calebin A from these spices exhibited high docking score, binding affinity and sustained protein-ligand interactions. Rescoring of protein ligand interactions at the end of MD simulations produced binding scores that were better than the initially docked conformations. Docking results, ligand interactions and ADMET properties of these molecules were significantly better than commercially available AR inhibitors like epalrestat, sorbinil and ranirestat. Thus, these natural molecules could be potent AR inhibitors.

  8. Nitrate reductase activity and its diurnal variation rhythm for Camptotheca acuminata seedlings

    Institute of Scientific and Technical Information of China (English)

    SUNShi-qin; YANXiu-feng

    2004-01-01

    Nitrate reductase activity (NRA) in different plant organs and leaves in different positions of Camptotheca acuminata seedlings was determined by an In vivo assay, the diurnal variation rhythm of NRA in leaves of different positions was observed,and the correlations between leaf NRA, leaf area and lamina mass per unit area (LMA) were also examined. The results showed that NRA in the leaf was significantly highest, compared with that in other organs such as roots, stems and leaves. In this experiment, the 10 leaves were selected from the apex to the base of the seedlings in order. The different NRA occurred obviously in leaves of different positions of C. acuminata seedlings from the apex to the base, and NRA was higher in the 4th-6th leaves.The diurnal change rhythm of leaf NRA showed a one peak curve, and maximum NRA value appeared at about midday (at 12:30 or so). No obvious correlations between NRA and leaf area or lamina mass per unit area were observed. This study offered scientific foundation for the further research on nitrogen metabolism of C. acuminata.

  9. Decreased glutathione reductase2 leads to early leaf senescence in Arabidopsis.

    Science.gov (United States)

    Ding, Shunhua; Wang, Liang; Yang, Zhipan; Lu, Qingtao; Wen, Xiaogang; Lu, Congming

    2016-01-01

    Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) and participates in the ascorbate-glutathione cycle, which scavenges H2 O2 . Here, we report that chloroplastic/mitochondrial GR2 is an important regulator of leaf senescence. Seed development of the homozygous gr2 knockout mutant was blocked at the globular stage. Therefore, to investigate the function of GR2 in leaf senescence, we generated transgenic Arabidopsis plants with decreased GR2 using RNAi. The GR2 RNAi plants displayed early onset of age-dependent and dark- and H2 O2 -induced leaf senescence, which was accompanied by the induction of the senescence-related marker genes SAG12 and SAG13. Furthermore, transcriptome analysis revealed that genes related to leaf senescence, oxidative stress, and phytohormone pathways were upregulated directly before senescence in RNAi plants. In addition, H2 O2 accumulated to higher levels in RNAi plants than in wild-type plants and the levels of H2 O2 peaked in RNAi plants directly before the early onset of leaf senescence. RNAi plants showed a greater decrease in GSH/GSSG levels than wild-type plants during leaf development. Our results suggest that GR2 plays an important role in leaf senescence by modulating H2 O2 and glutathione signaling in Arabidopsis.

  10. Design of Deinococcus radiodurans thioredoxin reductase with altered thioredoxin specificity using computational alanine mutagenesis.

    Science.gov (United States)

    Obiero, Josiah; Sanders, David A R

    2011-06-01

    In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface suggested that only four residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent residues in DR TrxR (M84, K137, F148, and F149) with alanine resulted in drastic changes in binding affinity, confirming that the four residues account for most of TrxR-Trx interface stability. When M84 and K137 were changed to match equivalent EC TrxR residues (K137R and M84F), the DR TrxR substrate specificity was altered from its own Trx to that of EC Trx. The results suggest that a small subset of the TrxR-Trx interface residues is responsible for the majority of Trx binding affinity and species-specific recognition.

  11. Homologous recombination and gene replacement at the dihydrofolate reductase-thymidylate synthase locus in Toxoplasma gondii.

    Science.gov (United States)

    Donald, R G; Roos, D S

    1994-02-01

    To investigate the feasibility of genomic transgene expression and gene targeting in Toxoplasma gondii, parasites have been transfected with constructs differing in the length of contiguous genomic sequence spanning the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. We have previously reported that vectors derived from a DHFR-TS cDNA 'minigene' containing mutations in the DHFR coding sequence confer pyrimethamine resistance to transfected parasites (Donald and Roos, 1993). Stably resistant parasite clones arise at high frequency, generally by virtue of transgene integration into parasite chromosomes at locations scattered throughout the genome. In contrast, using a vector which contains 8 kb of contiguous genomic sequence (vs. homologous recombination. Homologous recombination appears to occur at even higher frequency when a 16 kb genomic clone is used. Circular plasmids were more efficient than linearized molecules at producing homologous recombination in this system, integrating by reciprocal crossing-over to produce a duplication of the DHFR-TS locus. Double crossing-over (or gene conversion) was also observed at low frequency, resulting in complete allelic replacement in this haploid stage of the parasite. The ability to produce either homologous or non-homologous recombinants, by the selection of appropriate transformation constructs, has considerable genetic potential.

  12. Expression and Enzyme Activity Detection of a Sepiapterin Reductase Gene from Musca domestica Larva.

    Science.gov (United States)

    Tang, Yan; Pei, Zhihua; Liu, Lei; Wang, Dongfang; Kong, Lingcong; Liu, Shuming; Jiang, Xiuyun; Gao, Yunhang; Ma, Hongxia

    2017-02-01

    Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases and nitric oxide synthase. Sepiapterin reductase (SPR) catalyzes the final steps of BH4 biosynthesis. Studies on SPR from several insects and other organisms have been reported. However, thus far, enzyme activity of SPR in Musca domestica is kept unknown. In this study, 186 differentially expressed genes including SPR gene from Musca domestica (MDSPR) were screened in subtractive cDNA library. The MDSPR gene was cloned, and the recombinant MDSPI16 protein was expressed as a 51-kDa protein in soluble form. The MDSPR exhibited strong activity to the substrate sepiapterin (SP). The values of Vmax and Km of the MDSPR for SP were 6.83 μM/min and 23.48 μM, and the optimum temperature and pH of MDSPR were 50 °C and 4.0, respectively. This study provides new hypotheses and methods for the production of BH4 using insect-derived SPR.

  13. Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Y.W.; Robinson, H.; Yeung, N.; Gao, Y.-G.; Miner, K. D.; Tian, S.; Lu, Y.

    2010-05-11

    A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative FeB site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by {approx}110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a {approx}100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the FeB site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.

  14. Role of alkyl hydroperoxide reductase (AhpC) in the biofilm formation of Campylobacter jejuni.

    Science.gov (United States)

    Oh, Euna; Jeon, Byeonghwa

    2014-01-01

    Biofilm formation of Campylobacter jejuni, a major cause of human gastroenteritis, contributes to the survival of this pathogenic bacterium in different environmental niches; however, molecular mechanisms for its biofilm formation have not been fully understood yet. In this study, the role of oxidative stress resistance in biofilm formation was investigated using mutants defective in catalase (KatA), superoxide dismutase (SodB), and alkyl hydroperoxide reductase (AhpC). Biofilm formation was substantially increased in an ahpC mutant compared to the wild type, and katA and sodB mutants. In contrast to the augmented biofilm formation of the ahpC mutant, a strain overexpressing ahpC exhibited reduced biofilm formation. A perR mutant and a CosR-overexpression strain, both of which upregulate ahpC, also displayed decreased biofilms. However, the introduction of the ahpC mutation to the perR mutant and the CosR-overexpression strain substantially enhanced biofilm formation. The ahpC mutant accumulated more total reactive oxygen species and lipid hydroperoxides than the wild type, and the treatment of the ahpC mutant with antioxidants reduced biofilm formation to the wild-type level. Confocal microscopy analysis showed more microcolonies were developed in the ahpC mutant than the wild type. These results successfully demonstrate that AhpC plays an important role in the biofilm formation of C. jejuni.

  15. 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.

  16. Cloning, expression, crystallization and preliminary structural studies of dihydrodipicolinate reductase from Acinetobacter baumannii

    Science.gov (United States)

    Kaushik, Sanket; Singh, Avinash; Sinha, Mau; Kaur, Punit; Sharma, Sujata; Singh, Tej P.

    2013-01-01

    Acinetobacter baumannii is a virulent pathogenic bacterium that is resistant to most currently available antibiotics. Therefore, the design of drugs for the treatment of infections caused by A. baumannii is urgently required. Dihydrodipicolinate reductase (DHDPR) is an important enzyme which is involved in the biosynthetic pathway that leads to the production of l-lysine in bacteria. In order to design potent inhibitors against this enzyme, its detailed three-dimensional structure is required. DHDPR from A. baumannii (AbDHDPR) has been cloned, expressed, purified and crystallized. Here, the preliminary X-ray crystallographic data of AbDHDPR are reported. The crystals were grown using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitating agent The crystals belonged to the orthorhombic space group P222, with unit-cell parameters a = 80.0, b = 100.8, c = 147.6 Å, and contained four molecules in the asymmetric unit. The complete structure determination of AbDHDPR is in progress. PMID:23722845

  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. 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.

  19. 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

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

    Science.gov (United States)

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

    2016-03-15

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

  1. Genome-wide analysis of glutathione reductase (GR) genes from rice and Arabidopsis.

    Science.gov (United States)

    Trivedi, Dipesh Kumar; Gill, Sarvajeet Singh; Yadav, Sandep; Tuteja, Narendra

    2013-02-01

    Plant cells and tissues remain always on risk under abiotic and biotic stresses due to increased production of reactive oxygen species (ROS). Plants protect themselves against ROS induced oxidative damage by the upregulation of antioxidant machinery. Out of many components of antioxidant machinery, glutathione reductase (GR, EC 1.6.4.2) and glutathione (GSH, γ-Glu-Cys-Gly) play important role in the protection of cell against oxidative damage. In stress condition, the GR helps in maintaining the reduced glutathione pool for strengthening the antioxidative processes in plants. Present study investigates genome wide analysis of GR from rice and Arabidopsis. We were able to identify 3 rice GR genes (LOC_Os02 g56850, LOC_Os03 g06740, LOC_Os10 g28000) and 2 Arabidopsis GR genes (AT3G54660, AT3G24170) from their respective genomes on the basis of their annotation as well as the presence of pyridine nucleotide-disulphide oxidoreductases class-I active site. The evolutionary relationship of the GR genes from rice and Arabidopsis genomes was analyzed using the multiple sequence alignment and phylogenetic tree. This revealed evolutionary conserved pyridine nucleotide-disulphide oxidoreductases class-I active site among the GR protein in rice and Arabidopsis. This study should make an important contribution to our better understanding of the GR under normal and stress condition in plants.

  2. Expression and purification of growth hormone-releasing factor with the aid of dihydrofolate reductase handle.

    Science.gov (United States)

    Iwakura, M; Obara, K; Kokubu, T; Ohashi, S; Izutsu, H

    1992-07-01

    Expression of a fusion protein composed of dihydrofolate reductase and a derivative of growth hormone-releasing factor resulted in the formation of inclusion bodies in Escherichia coli at 37 degrees C. Among various chemicals, such as detergents, protein denaturants, and acetic acid, tested for the ability to dissolve the inclusion bodies, acetic acid, Brij-35, deoxycholic acid sodium salts, guanidine-HCl, and urea showed a strong solubilizing effect without damaging the DHFR activity. Acetic acid was useful in terms of preparing GRF derivatives, since it could be easily removed by lyophilization, and this made it easy to perform the succeeding BrCN treatment for cutting out the GRF derivative from the fusion protein. The GRF derivative was purified by reversed phase HPLC from the BrCN digest of the acetic acid extract, and its growth hormone-releasing activity was demonstrated. However, for obtaining a highly purified fusion protein itself, solubilization of inclusion bodies by urea was preferred because urea was the only agent which did not cause serious precipitation of the regenerated fusion protein after 10-fold dilution of the extracted inclusion bodies with buffer. The fusion protein was highly purified by means of a methotrexate affinity chromatography.

  3. Folate metabolism gene 5,10-methylenetetrahydrofolate reductase (MTHFR is associated with ADHD in myelomeningocele patients.

    Directory of Open Access Journals (Sweden)

    Catherine J Spellicy

    Full Text Available The objective of this study was to examine the relation between the 5, 10-methylenetetrahydrofolate reductase (MTHFR gene and behaviors related to attention- deficit/hyperactivity disorder (ADHD in individuals with myelomeningocele. The rationale for the study was twofold: folate metabolizing genes, (e.g. MTHFR, are important not only in the etiology of neural tube defects but are also critical to cognitive function; and individuals with myelomeningocele have an elevated incidence of ADHD. Here, we tested 478 individuals with myelomeningocele for attention-deficit hyperactivity disorder behavior using the Swanson Nolan Achenbach Pelham-IV ADHD rating scale. Myelomeningocele participants in this group for whom DNAs were available were genotyped for seven single nucleotide polymorphisms (SNPs in the MTHFR gene. The SNPs were evaluated for an association with manifestation of the ADHD phenotype in children with myelomeningocele. The data show that 28.7% of myelomeningocele participants exhibit rating scale elevations consistent with ADHD; of these 70.1% had scores consistent with the predominantly inattentive subtype. In addition, we also show a positive association between the SNP rs4846049 in the 3'-untranslated region of the MTHFR gene and the attention-deficit hyperactivity disorder phenotype in myelomeningocele participants. These results lend further support to the finding that behavior related to ADHD is more prevalent in patients with myelomeningocele than in the general population. These data also indicate the potential importance of the MTHFR gene in the etiology of the ADHD phenotype.

  4. New insights into the mechanism of substrates trafficking in Glyoxylate/Hydroxypyruvate reductases.

    Science.gov (United States)

    Lassalle, Louise; Engilberge, Sylvain; Madern, Dominique; Vauclare, Pierre; Franzetti, Bruno; Girard, Eric

    2016-02-11

    Glyoxylate accumulation within cells is highly toxic. In humans, it is associated with hyperoxaluria type 2 (PH2) leading to renal failure. The glyoxylate content within cells is regulated by the NADPH/NADH dependent glyoxylate/hydroxypyruvate reductases (GRHPR). These are highly conserved enzymes with a dual activity as they are able to reduce glyoxylate to glycolate and to convert hydroxypyruvate into D-glycerate. Despite the determination of high-resolution X-ray structures, the substrate recognition mode of this class of enzymes remains unclear. We determined the structure at 2.0 Å resolution of a thermostable GRHPR from Archaea as a ternary complex in the presence of D-glycerate and NADPH. This shows a binding mode conserved between human and archeal enzymes. We also determined the first structure of GRHPR in presence of glyoxylate at 1.40 Å resolution. This revealed the pivotal role of Leu53 and Trp138 in substrate trafficking. These residues act as gatekeepers at the entrance of a tunnel connecting the active site to protein surface. Taken together, these results allowed us to propose a general model for GRHPR mode of action.

  5. 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.

  6. Premature and ectopic anthocyanin formation by silencing of anthocyanidin reductase in strawberry (Fragaria × ananassa).

    Science.gov (United States)

    Fischer, Thilo C; Mirbeth, Beate; Rentsch, Judith; Sutter, Corina; Ring, Ludwig; Flachowsky, Henryk; Habegger, Ruth; Hoffmann, Thomas; Hanke, Magda-Viola; Schwab, Wilfried

    2014-01-01

    Strawberry (Fragaria × ananassa) is a fruit crop with a distinct biphasic flavonoid biosynthesis. Whereas, in the immature receptacle, high levels of proanthocyanidins accumulate, which are associated with herbivore deterrence and pathogen defense, the prominent color-giving anthocyanins are primarily produced in ripe 'fruits' helping to attract herbivores for seed dispersal. Here, constitutive experimental down-regulation of one branch of proanthocyanidin biosynthesis was performed. As a result, the proportion of epicatechin monomeric units within the proanthocyanidin polymer chains was reduced, but this was not the case for the epicatechin starter unit. Shortened chain lengths of proanthocyanidins were also observed. All enzymatic activities for the production of color-giving anthocyanins were already present in unripe fruits at levels allowing a striking red anthocyanin phenotype in unripe fruits of the RNAi silencing lines. An immediately recognizable phenotype was also observed for the stigmata of flowers, which is another epicatechin-forming tissue. Thus, the down-regulation of anthocyanidin reductase (ANR) induced a redirection of the proanthocyanidin pathway, leading to premature and ectopic anthocyanin biosynthesis via enzymatic glycosylation as the alternative pathway. This redirection is also seen in flavonol biosynthesis, which is paralleled by higher pollen viability in silencing lines. ANRi transgenic lines of strawberry provide a versatile tool for the study of the biological functions of proanthocyanidins.

  7. Isolation and characterization of an enoyl-acyl carrier protein reductase gene from microalga Isochrysis galbana

    Institute of Scientific and Technical Information of China (English)

    ZHENG Minggang; LIANG Kepeng; WANG Bo; SUN Xiuqin; YUE Yanyan; WAN Wenwen; ZHENG Li

    2013-01-01

    In most bacteria,plants and algae,fatty acid biosynthesis is catalyzed by a group of freely dissociable proteins known as the type Ⅱ fatty acid synthase (FAS Ⅱ) system.In the FAS Ⅱ system,enoylacyl carrier protein reductase (ENR) acts as a determinant for completing the cycles of fatty acid elongation.In this study,the cDNA sequence of ENR,designated as IgENR,was isolated from the microalga Isochrysis galbana CCMM5001.RACE (rapid amplification of cDNA ends) was used to isolate the full-length cDNA ofIgENR (1 503 bp),which contains an open reading frame (ORF) of 1 044 bp and encodes a protein of 347 amino acids.The genomic DNA sequence ofIgENR is interrupted by four introns.The putative amino acid sequence is homologous to the ENRs of seed plants and algae,and they contain common coenzymebinding sites and active site motifs.Under different stress conditions,real-time quantitative polymerase chain reaction (RT-qPCR) showed the expression ofIgENR was upregulated by high temperature (35℃),and downregulated by depleted nitrogen (0 mol/L).To clarify the mechanism of lipids accumulating lipids,other genes involved in lipids accumulation should be studied.

  8. 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.

  9. Folate levels in mucosal tissue but not methylenetetrahydrofolate reductase polymorphisms are associated with gastric carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Yu-Rong Weng; Dan-Feng Sun; Jing-Yuan Fang; Wei-Qi Gu; Hong-Yin Zhu

    2006-01-01

    AIM: To evaluate whether folate levels in mucosal tissue and some common methylenetetrahydrofolate reductase (MTHFR) variants are associated with the risk of gastric cancer through DNA methylation.METHODS: Real-time PCR was used to study the expression of tumor related genes in 76 mucosal tissue samples from 38 patients with gastric cancer. Samples from the gastroscopic biopsy tissues of 34 patients with chronic superficial gastritis (CSG) were used as controls.Folate concentrations in these tissues were detected by the FOL ACS: 180 automated chemiluminescence system.MTHFR polymorphisms were analyzed by PCR-RFLP, and the promoter methylation of tumor-related genes was determined by methylation-specific PCR (MSP).RESULTS: Folate concentrations were significantly higher in CSG than in cancerous tissues. Decreased expression and methylation of c-myc accompanied higher folate concentrations. Promoter hypermethylation and loss of p16INK4A in samples with MTHFR 677CC were more frequent than in samples with the 677TT or 677CT genotype. And the promoter hypermethylation and loss of p21WAF1 in samples with MTHFR 677CT were more frequent than when 677CC or 677TT was present. The 677CT genotype showed a non-significant higher risk for gastric cancer as compared with the 677CC genotype.CONCLUSION: Lower folate levels in gastric mucosal tissue may confer a higher risk of gastric carcinogenesis through hypomethylation and overexpression of c-myc.

  10. Regulation of Expression of Oxacillin-Inducible Methionine Sulfoxide Reductases in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Kyle R. Baum

    2015-01-01

    Full Text Available Cell wall-active antibiotics cause induction of a locus that leads to elevated synthesis of two methionine sulfoxide reductases (MsrA1 and MsrB in Staphylococcus aureus. To understand the regulation of this locus, reporter strains were constructed by integrating a DNA fragment consisting of the msrA1/msrB promoter in front of a promoterless lacZ gene in the chromosome of wild-type and MsrA1-, MsrB-, MsrA1/MsrB-, and SigB-deficient methicillin-sensitive S. aureus strain SH1000 and methicillin-resistant S. aureus strain COL. These reporter strains were cultured in TSB and the cellular levels of β-galactosidase activity in these cultures were assayed during different growth phases. β-galactosidase activity assays demonstrated that the lack of MsrA1, MsrB, and SigB upregulated the msrA1/msrB promoter in S. aureus strain SH1000. In S. aureus strain COL, the highest level of β-galactosidase activity was observed under the conditions when both MsrA1 and MsrB proteins were absent. The data suggest that the msrA1/msrB locus, in part, is negatively regulated by MsrA1, MsrB, and SigB in S. aureus.

  11. Conformational transitions of cinnamoyl CoA reductase 1 from Leucaena leucocephala.

    Science.gov (United States)

    Sonawane, Prashant D; Khan, Bashir M; Gaikwad, Sushama M

    2014-03-01

    Conformational transitions of cinnamoyl CoA reductase, a key regulatory enzyme in lignin biosynthesis, from Leucaena leucocephala (Ll-CCRH1) were studied using fluorescence and circular dichroism spectroscopy. The native protein possesses four trp residues exposed on the surface and 66% of helical structure, undergoes rapid structural transitions at and above 45 °C and starts forming aggregates at 55 °C. Ll-CCRH1 was transformed into acid induced (pH 2.0) molten globule like structure, exhibiting altered secondary structure, diminished tertiary structure and exposed hydrophobic residues. The molten globule like structure was examined for the thermal and chemical stability. The altered secondary structure of L1-CCRH1 at pH 2.0 was stable up to 90 °C. Also, in presence of 0.25 M guanidine hydrochloride (GdnHCl), it got transformed into different structure which was stable in the vicinity of 2M GdnHCl (as compared to drastic loss of native structure in 2M GdnHCl) as seen in far UV-CD spectra. The structural transition of Ll-CCRH1 at pH 2.0 followed another transition after readjusting the pH to 8.0, forming a structure with hardly any similarity to that of native protein.

  12. Isolation and characterization of an enoyl-acyl carrier protein reductase gene from microalga Isochrysis galbana

    Science.gov (United States)

    Zheng, Minggang; Liang, Kepeng; Wang, Bo; Sun, Xiuqin; Yue, Yanyan; Wan, Wenwen; Zheng, Li

    2013-03-01

    In most bacteria, plants and algae, fatty acid biosynthesis is catalyzed by a group of freely dissociable proteins known as the type II fatty acid synthase (FAS II) system. In the FAS II system, enoylacyl carrier protein reductase (ENR) acts as a determinant for completing the cycles of fatty acid elongation. In this study, the cDNA sequence of ENR, designated as IgENR, was isolated from the microalga Isochrysis galbana CCMM5001. RACE (rapid amplification of cDNA ends) was used to isolate the full-length cDNA of IgENR (1 503 bp), which contains an open reading frame (ORF) of 1 044 bp and encodes a protein of 347 amino acids. The genomic DNA sequence of IgENR is interrupted by four introns. The putative amino acid sequence is homologous to the ENRs of seed plants and algae, and they contain common coenzymebinding sites and active site motifs. Under different stress conditions, real-time quantitative polymerase chain reaction (RT-qPCR) showed the expression of IgENR was upregulated by high temperature (35°C), and downregulated by depleted nitrogen (0 mol/L). To clarify the mechanism of lipids accumulating lipids, other genes involved in lipids accumulation should be studied.

  13. Identification of Novel Aldose Reductase Inhibitors from Spices: A Molecular Docking and Simulation Study.

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    Priya Antony

    Full Text Available Hyperglycemia in diabetic patients results in a diverse range of complications such as diabetic retinopathy, neuropathy, nephropathy and cardiovascular diseases. The role of aldose reductase (AR, the key enzyme in the polyol pathway, in these complications is well established. Due to notable side-effects of several drugs, phytochemicals as an alternative has gained considerable importance for the treatment of several ailments. In order to evaluate the inhibitory effects of dietary spices on AR, a collection of phytochemicals were identified from Zingiber officinale (ginger, Curcuma longa (turmeric Allium sativum (garlic and Trigonella foenum graecum (fenugreek. Molecular docking was performed for lead identification and molecular dynamics simulations were performed to study the dynamic behaviour of these protein-ligand interactions. Gingerenones A, B and C, lariciresinol, quercetin and calebin A from these spices exhibited high docking score, binding affinity and sustained protein-ligand interactions. Rescoring of protein ligand interactions at the end of MD simulations produced binding scores that were better than the initially docked conformations. Docking results, ligand interactions and ADMET properties of these molecules were significantly better than commercially available AR inhibitors like epalrestat, sorbinil and ranirestat. Thus, these natural molecules could be potent AR inhibitors.

  14. A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

    Science.gov (United States)

    Fu, Yuan; Lin, Hong-Yu; Wisitpitthaya, Somsinee; Blessing, William A; Aye, Yimon

    2014-11-24

    Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger α-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation.

  15. Preparative SDS-PAGE Electroelution for Rapid Purification of Alkyl Hydroperoxide Reductase from Helicobacter pylori

    Directory of Open Access Journals (Sweden)

    T Mohammadian

    2010-03-01

    Full Text Available "nBackground: Alkyl hydroperoxide reductase (AhpC of Helicobacter pylori is considered as a diagnostic antigen. There­fore, this antigen can be used to detect H. pylori infection by stool immunoassays such as ELISA. The aim of this study was to simplify the AhpC protein purification procedures."nMethods: For whole cell protein extraction, the bacterial cells were ruptured by octly-β-D glucopyranoside. The isolation and purification of  AhpC protein were attempted by various techniques including ammonium sulfate precipitation, dialysis, preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE and electroelution."nResults:A simple method was used for protein purification AhpC protein. One-dimensional preparative gel electrophoresis allows a single and short purification step; the high resolution capacity of this technique leads to a high level of purity of the protein. Moreover, it avoids contamination by other non-specific proteins which often appear during protein purification by column chromatography."nConclusion: The present method is simple, rapid and makes it possible to preparate AhpC from H. pylori

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

    Science.gov (United States)

    Rodriguez, Gabriel M; Atsumi, Shota

    2014-09-01

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

  17. Folate Intake and Methylenetetrahydrofolate Reductase Gene Polymorphisms as Predictive and Prognostic Biomarkers for Ovarian Cancer Risk

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    Ke Wang

    2012-03-01

    Full Text Available Folic acid and methylenetetrahydrofolate reductase (MTHFR may affect the development of human cancer. However, few studies have evaluated folate intake and MTHFR in susceptibility to and prognosis of patients with ovarian cancer. We conducted a prospective case-control study in 215 ovarian cancer patients and 218 controls (all Chinese between Jan. 2004 and Jan. 2007. MTHFR C677T genotyping was done by PCR-RFLP. All patients were followed up until Dec. 2010. We found a 2.43-fold increased risk of ovarian cancer among MTHFR 677TT carriers, and a decreased risk of ovarian cancer in individuals with high folate intake (OR = 0.54, 95% CI = 0.32–0.94. Cox regression survival analysis showed that among the ovarian cancer patients, those carrying the 677TT genotype had a higher risk of death (HR = 2.17, 95% CI = 1.20–4.79, while high folate intake was associated with a lower risk of death (HR = 0.43, 95% CI = 0.33–0.88. Moreover, MTHFR 677CC carriers with higher folate intake showed a lower risk of death from ovarian cancer (HR = 0.32, 95% CI = 0.27–0.82. In summary, high folate intake may lessen susceptibility and improve the prognosis of ovarian cancer patients, while the MTHFR 677TT genotype appears to increase ovarian cancer risk and worsen its prognosis in a Chinese population.

  18. Molecular Properties and Functional Divergence of the Dehydroascorbate Reductase Gene Family in Lower and Higher Plants.

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    Yuan-Jie Zhang

    Full Text Available Dehydroascorbate reductase (DHAR, which reduces oxidized ascorbate, is important for maintaining an appropriate ascorbate redox state in plant cells. To date, genome-wide molecular characterization of DHARs has only been conducted in bryophytes (Physcomitrella patens and eudicots (e.g. Arabidopsis thaliana. In this study, to gain a general understanding of the molecular properties and functional divergence of the DHARs in land plants, we further conducted a comprehensive analysis of DHARs from the lycophyte Selaginella moellendorffii, gymnosperm Picea abies and monocot Zea mays. DHARs were present as a small gene family in all of the land plants we examined, with gene numbers ranging from two to four. All the plants contained cytosolic and chloroplastic DHARs, indicating dehydroascorbate (DHA can be directly reduced in the cytoplasm and chloroplast by DHARs in all the plants. A novel vacuolar DHAR was found in Z. mays, indicating DHA may also be reduced in the vacuole by DHARs in Z. mays. The DHARs within each species showed extensive functional divergence in their gene structures, subcellular localizations, and enzymatic characteristics. This study provides new insights into the molecular characteristics and functional divergence of DHARs in land plants.

  19. Roles of Glutamates and Metal ions in a Rationally Designed Nitric Oxide Reductase Based on Myoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Y Lin; N Yeung; Y Gao; K Miner; S Tian; H Robinson; Y Lu

    2011-12-31

    A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative FeB site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by {approx}110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a {approx}100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the FeB site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.

  20. Hydrogen-Bonded Networks Along and Bifurcation of the E-Pathway in Quinol: Fumarate Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Herzog, Elena; Gu, Wei; Juhnke, Hanno D.; Haas, Alexander H.; Mantele, Werner; Simon, Jorg; Helms, Volkhard H.; Lancaster , C. Roy D.

    2012-09-19

    The E-pathway of transmembrane proton transfer has been demonstrated previously to be essential for catalysis by the diheme-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes. Two constituents of this pathway, Glu- C180 and heme bD ring C (bD-C-) propionate, have been validated experimentally. Here, we identify further constituents of the E-pathway by analysis of molecular dynamics simulations. The redox state of heme groups has a crucial effect on the connectivity patterns of mobile internal water molecules that can transiently support proton transfer from the bD-C-propionate to Glu-C180. The short H-bonding paths formed in the reduced states can lead to high proton conduction rates and thus provide a plausible explanation for the required opening of the E-pathway in reduced QFR. We found evidence that the bD-C-propionate group is the previously postulated branching point connecting proton transfer to the E-pathway from the quinol-oxidation site via interactions with the heme bD ligand His-C44. An essential functional role of His-C44 is supported experimentally by site-directed mutagenesis resulting in its replacement with Glu. Although the H44E variant enzyme retains both heme groups, it is unable to catalyze quinol oxidation. All results obtained are relevant to the QFR enzymes from the human pathogens Campylobacter jejuni and Helicobacter pylori.