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Sample records for aldo keto reductase

  1. The aldo-keto reductase superfamily homepage.

    Science.gov (United States)

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

    2003-02-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  3. 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.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

    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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

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

    Science.gov (United States)

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

    2012-02-01

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

  11. 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

  12. 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...

  13. 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.

  14. 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.

  15. 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-...

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

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    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

  17. 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

  18. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

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    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

  1. 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.

  2. 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.

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

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    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.

  4. 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

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

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    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

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

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    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

  7. 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.

  8. 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

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    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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. Stereoselective reduction of carbonyl compounds with Actinomycete: purification and characterization of three alpha-keto ester reductases from Streptomyces avermitilis.

    Science.gov (United States)

    Ishihara, Kohji; Kato, Chiaki; Yamaguchi, Hitomi; Iwai, Rieko; Yoshida, Momoko; Ikeda, Natsumi; Hamada, Hiroki; Masuoka, Noriyoshi; Nakajima, Nobuyoshi

    2008-12-01

    We achieved the purification of three alpha-keto ester reductases (Streptomyces avermitilis keto ester reductase, SAKERs-I, -II, and -III) from Streptomyces avermitilis NBRC14893 whole cells. The molecular masses of the native SAKERs-I, -II, and -III were estimated to be 72, 38, and 36 kDa, respectively, by gel filtration chromatography. The subunit molecular masses of SAKERs-I, -II, and -III were also estimated to be 32, 32, and 34 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The purified SAKERs-II and -III showed a reducing activity for alpha-keto esters (in particular, for ethyl pyruvate). SAKER-I showed a high reducing activity not only toward the alpha- and beta-keto esters, but also toward alpha-keto acid. The N-terminal region amino acid sequences of SAKERs-I, -II, and -III were identical to that of a putative oxidoreductase, SAV2750, a putative oxidoreductase, SAV1849, and a putative oxidoreductase, SAV4117, respectively, hypothetical proteins coded on the S. avermitilis genome.

  15. 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

  16. Clustered Genes Encoding 2-Keto-l-Gulonate Reductase and l-Idonate 5-Dehydrogenase in the Novel Fungal d-Glucuronic Acid Pathway.

    Science.gov (United States)

    Kuivanen, Joosu; Arvas, Mikko; Richard, Peter

    2017-01-01

    D-Glucuronic acid is a biomass component that occurs in plant cell wall polysaccharides and is catabolized by saprotrophic microorganisms including fungi. A pathway for D-glucuronic acid catabolism in fungal microorganisms is only partly known. In the filamentous fungus Aspergillus niger, the enzymes that are known to be part of the pathway are the NADPH requiring D-glucuronic acid reductase forming L-gulonate and the NADH requiring 2-keto-L-gulonate reductase that forms L-idonate. With the aid of RNA sequencing we identified two more enzymes of the pathway. The first is a NADPH requiring 2-keto-L-gulonate reductase that forms L-idonate, GluD. The second is a NAD(+) requiring L-idonate 5-dehydrogenase forming 5-keto-gluconate, GluE. The genes coding for these two enzymes are clustered and share the same bidirectional promoter. The GluD is an enzyme with a strict requirement for NADP(+)/NADPH as cofactors. The kcat for 2-keto-L-gulonate and L-idonate is 21.4 and 1.1 s(-1), and the Km 25.3 and 12.6 mM, respectively, when using the purified protein. In contrast, the GluE has a strict requirement for NAD(+)/NADH. The kcat for L-idonate and 5-keto-D-gluconate is 5.5 and 7.2 s(-1), and the Km 30.9 and 8.4 mM, respectively. These values also refer to the purified protein. The gluD deletion resulted in accumulation of 2-keto-L-gulonate in the liquid cultivation while the gluE deletion resulted in reduced growth and cessation of the D-glucuronic acid catabolism.

  17. Clustered Genes Encoding 2-Keto-l-Gulonate Reductase and l-Idonate 5-Dehydrogenase in the Novel Fungal d-Glucuronic Acid Pathway

    Science.gov (United States)

    Kuivanen, Joosu; Arvas, Mikko; Richard, Peter

    2017-01-01

    D-Glucuronic acid is a biomass component that occurs in plant cell wall polysaccharides and is catabolized by saprotrophic microorganisms including fungi. A pathway for D-glucuronic acid catabolism in fungal microorganisms is only partly known. In the filamentous fungus Aspergillus niger, the enzymes that are known to be part of the pathway are the NADPH requiring D-glucuronic acid reductase forming L-gulonate and the NADH requiring 2-keto-L-gulonate reductase that forms L-idonate. With the aid of RNA sequencing we identified two more enzymes of the pathway. The first is a NADPH requiring 2-keto-L-gulonate reductase that forms L-idonate, GluD. The second is a NAD+ requiring L-idonate 5-dehydrogenase forming 5-keto-gluconate, GluE. The genes coding for these two enzymes are clustered and share the same bidirectional promoter. The GluD is an enzyme with a strict requirement for NADP+/NADPH as cofactors. The kcat for 2-keto-L-gulonate and L-idonate is 21.4 and 1.1 s-1, and the Km 25.3 and 12.6 mM, respectively, when using the purified protein. In contrast, the GluE has a strict requirement for NAD+/NADH. The kcat for L-idonate and 5-keto-D-gluconate is 5.5 and 7.2 s-1, and the Km 30.9 and 8.4 mM, respectively. These values also refer to the purified protein. The gluD deletion resulted in accumulation of 2-keto-L-gulonate in the liquid cultivation while the gluE deletion resulted in reduced growth and cessation of the D-glucuronic acid catabolism. PMID:28261181

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

    Directory of Open Access Journals (Sweden)

    Emily Yun-Chia Chang

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

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

    Science.gov (United States)

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

    2014-09-01

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

  20. 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.

  1. ALDO-DHF & PARAMOUNT

    Directory of Open Access Journals (Sweden)

    Ahmed M ElGuindy

    2012-12-01

    Full Text Available Results of the ALDOsterone receptor blockade in Diastolic Heart Failure (ALDO-­-DHF were recently presented at the European Society of Cardiology (ESC meeting in Munich.1 The multicenter, double-­-blind randomized, placebo-­-controlled phase IIb mechanistic study was conducted to test the hypothesis that 12 months treatment with spironolactone would improve cardiac function and structure as well as exercise capacity and quality of life in patients with heart failure with preserved ejection fraction (HFpEF. HFpEF continues to be a challenging form of heart failure – one in which no therapy has yet been proven to improve outcome and with a prevalence that continues to rise at an alarming rate. An extensive review on HFpEF – including various drugs in current use as well as those under trial – was published in the last issue of the journal.

  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. 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.

  4. 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.

  5. 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.

  6. Polifilo e o sonho de Aldo Manuzio/Polifilo and the dream of Aldo Manuzio

    Directory of Open Access Journals (Sweden)

    Ubirajara Alencar Rodrigues

    2007-12-01

    Full Text Available Estudo sobre a tipologia gráfica e ilustrações da obra Hypnerotomachia Poliphili, de Aldo Manuzio, através da Arte da Memória e da rede de imagens que a envolve. Study on the graphical typology and illustrations of the work Hypnerotomachia Poliphili, of Aldo Manuzio, through the Art of Memory and the network of images that involves it.

  7. 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.

  8. 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.

  9. 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

  10. Avalik küsimus peaminister Juhan Partsile / Aldo Vinkel

    Index Scriptorium Estoniae

    Vinkel, Aldo

    2003-01-01

    Eesti Kristliku Rahvapartei esimees Aldo Vinkel soovib peaministrilt selgitust, kas tema vihjed Jeruusalemmale kui Euroopa ühisele usuruumile ning kirikualtari ees antud valimisvanne on kantud siirast soovist arendada Eestit kui kristlikku maad.

  11. 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.

  12. An Ecological Probe into Land Ethics by Aldo Leopold

    Institute of Scientific and Technical Information of China (English)

    白虎跃

    2015-01-01

    Aldo Leopold has gained considerable repute as the initiator of eco-conservationism and been globally known as the father of American wilderness governance."Land ethics",which possesses paramount importance to eco-systems,is brought forth in his Sand Country Almanac.This thesis mainly touches upon the engendering,contents and significance of the thoughts to nowadays society by means of which the contemporary values of ecological civilization embodied in"land ethics"can be concluded provokingly.

  13. An Ecological Probe into Land Ethics by Aldo Leopold

    Institute of Scientific and Technical Information of China (English)

    白虎跃

    2015-01-01

    Aldo Leopold has gained considerable repute as the initiator of eco-conservationism and been globally known as the father of American wilderness governance.“Land ethics”,which possesses paramount importance to eco-systems,is brought forth in his Sand Country Almanac.This thesis mainly touches upon the engendering,contents and significance of the thoughts to nowadays society by means of which the contemporary values of ecological civilization embodied in“land ethics”can be concluded provokingly.

  14. 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

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

    Science.gov (United States)

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

    2014-03-01

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

  16. The facile insertion of β-keto sulfones to arynes: The direct preparation of polysubstituted ortho-keto benzyl sulfones

    Institute of Scientific and Technical Information of China (English)

    Jian Xue; Lu Ling Wu; Xian Huang

    2008-01-01

    One novel carbon-carbon bond insertion reaction of arynes has been developed. By this reaction β-keto sulfones can insert the triple bond of arynes to prepare polysubstituted ortho-keto benzyl sulfones.

  17. Oskari juht loovutab peagi vastvõidetud aktsiapaki / Aldo Parik ; interv. Hans Väre

    Index Scriptorium Estoniae

    Parik, Aldo

    2007-01-01

    Saarepeedil tegutseva lihatööstuse Oskar tegevjuht ja omanik Aldo Parik suurendas oma osalust 100%-ni, kuid kavatseb lähiajal aktsiate kontrollpakist loobuda. Lisa: Taust. Arvamust avaldab Wõro Kommertsi juhatuse esimees Kaido Kaare

  18. 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.

  19. Keto-enol tautomerism in estrogen hormone. A theoretical study

    Science.gov (United States)

    Jameh-Bozorghi, Saeed; Shirani, Hossein; Ghaempanah, Aram; Ghapanvari, Hamed

    2015-01-01

    The HF/6-311+G** calculation was used to investigate Keto-Enol tautomerism of Estrogen Hormone. Molecular geometries of keto, enol and transition state of this reaction were optimized and NBO calculations were performed. These calculation results showed that activation energy (Ea) of Keto-Enol tautomerization of Estrogen is 118.65 Kcal mol-1. Energetic study at B3LYP/6-311+G** level of theory revealed that keto tautomer is more stable structure. NBO analysis results have a good agreements with optimized geometries and experimental data.

  20. Synthesis of Keto-RDX and its Characterizations Calculation%Keto-RDX的合成及性能计算

    Institute of Scientific and Technical Information of China (English)

    Arash Shokrollahi; Abbas Zali; Hamid Reza Pouretedal; Mohammad Hossein Keshavarz

    2008-01-01

    Keto-RDX was obtained by one-step method with a certain amount of RDX as by-product. The effects of various parameters on high yield were studied. A simple analytical method was also introduced to determine simultaneously Keto-RDX/RDX mole ratio. Some important theoretical characterizations of Keto-RDX such as detonation performance at maximum nominal density and shock sensitivity were determined by new methods and compared with RDX.

  1. Keto-Enol Thermodynamics of Breslow Intermediates.

    Science.gov (United States)

    Paul, Mathias; Breugst, Martin; Neudörfl, Jörg-Martin; Sunoj, Raghavan B; Berkessel, Albrecht

    2016-04-20

    Breslow intermediates, first postulated in 1958, are pivotal intermediates in carbene-catalyzed umpolung. Attempts to isolate and characterize these fleeting amino enol species first met with success in 2012 when we found that saturated bis-Dipp/Mes imidazolidinylidenes readily form isolable, though reactive diamino enols with aldehydes and enals. In contrast, triazolylidenes, upon stoichiometric reaction with aldehydes, gave exclusively the keto tautomer, and no isolable enol. Herein, we present the synthesis of the "missing" keto tautomers of imidazolidinylidene-derived diamino enols, and computational thermodynamic data for 15 enol-ketone pairs derived from various carbenes/aldehydes. Electron-withdrawing substituents on the aldehyde favor enol formation, the same holds for N,N'-Dipp [2,6-di(2-propyl)phenyl] and N,N'-Mes [2,4,6-trimethylphenyl] substitution on the carbene component. The latter effect rests on stabilization of the diamino enol tautomer by Dipp substitution, and could be attributed to dispersive interaction of the 2-propyl groups with the enol moiety. For three enol-ketone pairs, equilibration of the thermodynamically disfavored tautomer was attempted with acids and bases but could not be effected, indicating kinetic inhibition of proton transfer.

  2. Site specific incorporation of keto amino acids into proteins

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  3. Site specific incorporation of keto amino acids into proteins

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, Peter G. (La Jolla, CA); Wang, Lei (San Diego, CA)

    2011-03-22

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  4. Site specific incorporation of keto amino acids into proteins

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    2011-12-06

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  5. Aldo Järvsoo: kirivööd ma ei lõigu / Tiiu Laks

    Index Scriptorium Estoniae

    Laks, Tiiu, 1984-

    2009-01-01

    Eesti 2008. aasta parim moekunstnik ja auhinna Kuldnõel omanik on Aldo Järvsoo, tema kollektsioonist "Virve". Lühidalt auhinna teiste nominentide Liisi Eesmaa, Tiina Talumehe ja Liivia Leškini kollektsioonidest. Võidukollektsiooni saab 15. märtsini vaadata Tallinna Kaubamaja Naistemaailmas paikneval näitusepinnal

  6. Pärandihoidja 2009 / kommenteerivad Helgi Põllo, Aldo Järvsoo

    Index Scriptorium Estoniae

    2010-01-01

    13. veebruaril toimus käsitööliidu Pärandihoidja auhinna üleandmine ning pidulik vastuvõtt ja moeetendus Estonia talveaias. Auhinna pälvisid Eevi Astel, Helgi Põllo, Aldo Järvsoo. Sisaldab lühiintervjuusid A. Järvsoo ja H. Põlloga

  7. Synthesis of Indole Derivatives from 2-Keto Glycoside

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@ 2-Oxo derivatives of glycosides, called 2-keto glycosides or glycoside 2-uloses, are biologically important in carbohydrate metabolism and are very useful in synthesis of branched-chain sugars and amino sugars. Very little is known of their chemistry because of the high susceptibility of these compounds to degradation in solution, and in particular their instability to base. Thus it is important to study the reactivities of 2-keto glycosides. In a previous paper, we reported the transformation of 2-keto glycosides in pyridine solution. During the transformation of 2-and 3-keto glycosides, a demethoxylation reaction of the enol intermediate was shown to take place simultaneously with elimination to give hex-1-enopyran-3-ulose.

  8. Synthesis of Indole Derivatives from 2-Keto Glycoside

    Institute of Scientific and Technical Information of China (English)

    LIU; HongMin

    2001-01-01

    2-Oxo derivatives of glycosides, called 2-keto glycosides or glycoside 2-uloses, are biologically important in carbohydrate metabolism and are very useful in synthesis of branched-chain sugars and amino sugars. Very little is known of their chemistry because of the high susceptibility of these compounds to degradation in solution, and in particular their instability to base. Thus it is important to study the reactivities of 2-keto glycosides. In a previous paper, we reported the transformation of 2-keto glycosides in pyridine solution. During the transformation of 2-and 3-keto glycosides, a demethoxylation reaction of the enol intermediate was shown to take place simultaneously with elimination to give hex-1-enopyran-3-ulose.……

  9. Citrate-Linked Keto- and Aldo-Hexose Monosaccharide Cellulose Conjugates Demonstrate Selective Human Neutrophil Elastase-Lowering Activity in Cotton Dressings

    Directory of Open Access Journals (Sweden)

    Sonya Caston-Pierre

    2013-05-01

    Full Text Available Sequestration of harmful proteases as human neutrophil elastase (HNE from the chronic wound environment is an important goal of wound dressing design and function. Monosaccharides attached to cellulose conjugates as ester-appended aldohexoses and ketohexoses were prepared on cotton gauze as monosccharide-citrate-cellulose-esters for HNE sequestration. The monosaccharide-cellulose analogs demonstrated selective binding when the derivatized cotton dressings were measured for sequestration of HNE. Each monosaccharide-cellulose conjugate was prepared as a cellulose citrate-linked monosaccharide ester on the cotton wound dressing, and assayed under wound exudate-mimicked conditions for elastase sequestration activity. A series of three aldohexose and four ketohexose ester cellulose conjugates were prepared on cotton gauze through citric acid-cellulose cross linking esterification. The monosaccharide portion of the conjugate was characterized by hydrolysis of the citrate-monosaccharide ester bond, and subsequent analysis of the free monosaccharide with high performance anion exchange chromatography. The ketohexose and aldohexose conjugate levels on cotton were quantified on cotton using chromatography and found to be present in milligram/gram amounts. The citrate-cellulose ester bonds were characterized with FTIR. Ketohexose-citrate-cellulose conjugates sequestered more elastase activity than aldohexose-citrate-cellulose conjugates. The monosaccharide cellulose conjugate families each gave distinctive profiles in elastase-lowering effects. Possible mechanisms of elastase binding to the monosaccharide-cellulose conjugates are discussed.

  10. The Reduction of a Natural Diterpene Containing o, β-Unsaturated Keto Group

    Institute of Scientific and Technical Information of China (English)

    Qing ZHAO; Xin HONG; Cheng ZOU; Yue Mao SHEN; Xiao Jiang HAO

    2003-01-01

    Hedychenone (1), a diterpene containing α, β-unsaturated keto group, was reduced byaluminum-mercury alloy, and a dimerized product (2) was obtained as the major product. Thecoupling occurred at β position of the keto group.

  11. PENSAMIENTOS COMPARTIDOS. ALDO VAN EYCK, EL GRUPO COBRA Y EL ARTE / Shared thoughts. Aldo van Eyck, the COBRA group, and art

    Directory of Open Access Journals (Sweden)

    Esther Mayoral Campa

    2014-11-01

    Full Text Available RESUMEN El periodo inmediatamente posterior a la II Guerra Mundial es uno de los episodios más interesantes desde el punto de vista cultural del siglo XX, un momento vivido por muchos de los intelectuales europeos coetáneos a esta época como un punto de inflexión, una oportunidad para repensar el mundo, para comenzar de nuevo tras el cataclismo bélico. En ese contexto comienza su andadura como arquitecto Aldo van Eyck, así como su colaboración con el breve, pero intenso, movimiento Cobra, grupo esencial para comprender el panorama cultural europeo de posguerra y una de las últimas vanguardias del siglo XX. Este artículo explora la vinculación del arquitecto holandés Aldo van Eyck con el mundo del arte. Una relación poliédrica, parte esencial de su discurso, que engloba su formación cultural, sus relaciones de amistad, su pensamiento crítico y su obra. En esa correlación entre la arquitectura y las artes será determinante la vinculación del arquitecto con Cobra, con el que compartirá una mirada común sobre la realidad, una relación compleja con líneas de investigación comunes, escritos, exposiciones y trabajos compartidos. A todo ello se suma la aportación fundamental que supone un trasvase de valores constantes entre la arquitectura y el mundo del arte, que caracterizó la relación entre el arquitecto y los miembros del grupo.

  12. Lactate Dehydrogenase Catalysis: Roles of Keto, Hydrated, and Enol Pyruvate

    Science.gov (United States)

    Meany, J. E.

    2007-01-01

    Many carbonyl substrates of oxidoreductase enzymes undergo hydration and enolization so that these substrate systems are partitioned between keto, hydrated (gem-diol), and enol forms in aqueous solution. Some oxidoreductase enzymes are subject to inhibition by high concentrations of substrate. For such enzymes, two questions arise pertaining to…

  13. The enantioselective b-keto ester reductions by Saccharomyces cerevisiae

    OpenAIRE

    HASSAN TAJIK; KHALIL TABATABAEIAN; MAHMOOD SHAHBAZI

    2006-01-01

    The enantioselective yeast reduction of aromatic b-keto esters, by use of potassium dihydrogen phosphate, calcium phosphate (monobasic), magnesium sulfate and ammonium tartrate (diammonium salt) (10:1:1:50) in water at pH 7 as a buffer for 72–120 h with 45–90 % conversion to the corresponding aromatic -hydroxy esters was achieved by means of Saccharomyces cerevisiae.

  14. Destrutturare le maiuscole. Pensiero debole, Italian Theory e politica. Conversazione con Pier Aldo Rovatti

    Directory of Open Access Journals (Sweden)

    Pier Aldo Rovatti

    2014-06-01

    Full Text Available The conversation focuses on the social and political role of the philosopher nowadays. Pier Aldo Rovatti discusses about the growing philosophical movement called “Italian Theory” while revisiting his own recent intellectual path. The italian philosopher retraces the cultural experience of the “pensiero debole”, whereof he has been one of the two promoters, and underlines the intellectual and political fight, against all the so-called universal truths (and ideological violences, inspired by this philosophical trend at the beginning of the Eighties. The interview ends with a discussion about the dawning perspectives of the political-philosophical action in the post-modern age.

  15. 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.

  16. La contestación al desarrollo moderno en Europa: el pensamiento y la obra de Aldo Rossi

    OpenAIRE

    González Capitel, Antón

    1996-01-01

    3. La contestación al desarrollo moderno en Europa: el pensamiento y la obra de Aldo Rossi 3. l. La teoría rossiana: L'architettura della citta 3. 2. La obra de Aldo Rossi 3. 2. l. La arquitectura entendida corno la razón formal 3. 2. 2. La razón al servicio del sentimiento: la arnplia~ión del cementerio de Módena. Otras obras de esta primera época 3. 2. 3. De lo teórico a lo personal: la arquitectura de Rossi en su segunda etapa 3. 3. La arquitectura del racionalism...

  17. Quantum chemical investigation of the electronic spectra of the keto, enol, and keto-imine tautomers of cytosine.

    Science.gov (United States)

    Tomić, Katarina; Tatchen, Jörg; Marian, Christel M

    2005-09-22

    The low-lying excited singlet states of the keto, enol, and keto-imine tautomers of cytosine have been investigated employing a combined density functional/multireference configuration interaction (DFT/MRCI) method. Unconstrained geometry optimizations have yielded out-of-plain distorted structures of the pi --> pi and n --> pi excited states of all cytosine forms. For the keto tautomer, the DFT/MRCI adiabatic excitation energy of the pi --> pi state (4.06 eV including zero-point vibrational energy corrections) supports the resonant two-photon ionization (R2PI) spectrum (Nir et al. Phys. Chem. Chem. Phys. 2002, 5, 4780). On its S1 potential energy surface, a conical intersection between the 1pipi state and the electronic ground state has been identified. The barrier height of the reaction along a constrained minimum energy path amounts to merely 0.2 eV above the origin and explains the break-off of the R2PI spectrum. The 1pipi minimum of the enol tautomer is found at considerably higher excitation energies (4.50 eV). Because of significant geometry shifts with respect to the ground state, long vibrational progressions are expected, in accord with experimental observations. For the keto-imine tautomer, a crossing of the 1pipi potential energy surface with the ground-state surface has been found, too. Its n --> pi minimum (3.27 eV) is located well below the conical intersection between the pi --> pi and S0 states, but it will be difficult to observe because of its small transition moment. The identified conical intersections of the pi --> pi excited states of the keto cytosine tautomers are made responsible for the ultrafast decay to the electronic ground states and thus may explain their subpicoseconds lifetimes.

  18. The Architecture of a Lifetime: Structures of Remembrance and Invention in Walter Benjamin and Aldo Rossi

    Directory of Open Access Journals (Sweden)

    Jolien Paeleman

    2016-05-01

    Full Text Available This article presents the result of research on the influence of Walter Benjamin’s thinking in the work of Italian architect Aldo Rossi (1931–1997. In present-day architectural criticism, Aldo Rossi’s oeuvre still constitutes a rich subject for discussion because of its resistance to easy pinpointing, even if Rossi himself explained his theories and methods of design on numerous occasions. In his writings, among these A Scientific Autobiography, Rossi quotes from a collection of Benjamin’s memoirs: Berlin Childhood around 1900. The architect believes that these short prose pieces express better than anything else what he himself had not been able to explain in his writing. In this paper I intend to show the poignancy of the words Rossi referred to and the implications they had on his architecture by offering close comparisons of Benjamin’s and Rossi’s autobiographical writings. In addition, this study examines how one of Rossi’s most famous architectural artefacts, the ossuary of San Cataldo cemetery at Modena, can be viewed as a coalescence of a Benjaminian thought-image, thereby fortifying the philosopher’s presence in modern architecture.

  19. Biotechnological production of alpha-keto acids: Current status and perspectives.

    Science.gov (United States)

    Song, Yang; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng; Chen, Jian

    2016-11-01

    Alpha-keto (α-keto) acids are used widely in feeds, food additives, pharmaceuticals, and in chemical synthesis processes. Although most α-keto acids are currently produced by chemical synthesis, their biotechnological production from renewable carbohydrates is a promising new approach. In this mini-review, we first present the different types of α-keto acids as well as their applications; next, we summarize the recent progresses in the biotechnological production of some important α-keto acids; namely, pyruvate, α-ketoglutarate, α-ketoisovalerate, α-ketoisocaproate, phenylpyruvate, α-keto-γ-methylthiobutyrate, and 2,5-diketo-d-gluconate. Finally, we discuss the future prospects as well as favorable directions for the biotechnological production of keto acids that ultimately would be more environment-friendly and simpler compared with the production by chemical synthesis.

  20. 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...

  1. 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...

  2. I2-Catalyzed Oxidative Condensation of Aldoses with Diamines: Synthesis of Aldo-Naphthimidazoles for Carbohydrate Analysis

    Directory of Open Access Journals (Sweden)

    Chunchi Lin

    2010-03-01

    Full Text Available A novel method for the conversion of unprotected and unmodified aldoses to aldo-imidazoles has been developed. Using iodine as a catalyst in acetic acid solution, a series of mono- and oligosaccharides, including those containing carboxyl and acetamido groups, undergo an oxidative condensation reaction with aromatic vicinal diamines at room temperature to give the corresponding aldo-imidazole products in high yields. No cleavage of the glycosidic bond occurs under the mild reaction conditions. The compositional analysis of saccharides is commonly realized by capillary electropheresis of the corresponding aldo-imidazole derivatives, which are easily synthesized by the reported iodine-promoted oxidative condensation. In addition, a series of aldo-imidazoles were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS to analyze molecular weight and ion intensity. The diamine-labeled saccharides showed enhanced signals in MALDI–TOF MS. The combined use of aldoimidazole derivatization and mass spectrometric analysis thus provides a rapid method for identification of saccharides, even when less than 1 pmol of saccharide is present in the sample. These results can be further applied to facilitate the isolation and analysis of novel saccharides.

  3. The enantioselective b-keto ester reductions by Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    HASSAN TAJIK

    2006-09-01

    Full Text Available The enantioselective yeast reduction of aromatic b-keto esters, by use of potassium dihydrogen phosphate, calcium phosphate (monobasic, magnesium sulfate and ammonium tartrate (diammonium salt (10:1:1:50 in water at pH 7 as a buffer for 72–120 h with 45–90 % conversion to the corresponding aromatic -hydroxy esters was achieved by means of Saccharomyces cerevisiae.

  4. Aldo-X Bifunctional Building Blocks for the Synthesis of Heterocycles.

    Science.gov (United States)

    Ravichandiran, Palanisamy; Lai, Bingbing; Gu, Yanlong

    2017-02-01

    Compounds containing oxygen, nitrogen, or sulfur atoms inside the rings are attracting much attention and interest due to their biological importance. In recent years, several methods for the synthesis of such molecules have been reported by using aldo-X bifunctional building blocks (AXB3 s) as substrates; these are a wide class of organic molecules that contain at least two reactive sites, among them, one aldehyde, acetal, or semiacetal group was involved. Because of the multiple reactivities, AXB3 s are widely used in the one-pot synthesis of biologically important heterocycles. This review summarizes the synthesis of important heterocycles by using AXB3 s as pivotal components in establishing multicomponent reactions, tandem reactions, and so forth. In many cases, the established reaction systems with AXB3 s were characterized by some green properties, such as easy access to the substrate, mild and environmentally benign conditions, and wide scope of the substrate.

  5. Synthesis, spectral studies, antimicrobial and insect antifeedant potent keto oxiranes

    Directory of Open Access Journals (Sweden)

    Ganesamoorthy Thirunarayanan

    2016-09-01

    Full Text Available A series of ee (αS, βR biphenyl keto oxiranes (biphenyl-4-yl[3-(substituted phenyloxiran-2-yl]methanones have been synthesized by phase transfer catalysed epoxidation of biphenyl 2E-chalcones. The yields of oxiranes are more than 95%. The synthesized oxiranes have been characterized by IR, 1H, 13C and GC–MS spectra. The spectral data are correlated with Hammett substituent constants and Swain–Lupton parameters. From the regression analyses, the effect of substituent on the group frequencies has been predicted. The antimicrobial and insect antifeedant activities of all synthesized oxiranes have been evaluated.

  6. La Huella del montaje Aby Warburg; Aldo van Eyck, Jerzy Grotowski : recorridos a partir del Atlas Mnemosyne

    OpenAIRE

    Konstantopoulou, Dimitra

    2011-01-01

    Premi extraordinari doctorat curs 2010-2011, àmbit d’Arquitectura, Urbanisme i Edificació The thesis consists in constructing an itinerary through three fields "culture of science, architecture, theatre" and three authors Aby Warburg, Aldo van Eyck, Jerzy Grotowski, on the basis of the idea of the montage. Its methodological reference in spite of the inadequacy of any literal reference to the term "method" in this case' is Warburg's Atlas Mnemosyne; on the one hand, since it constitutes it...

  7. Identification of 2-keto-3-deoxy-d-Gluconate Kinase and 2-keto-3-deoxy-d-Phosphogluconate Aldolase in an Alginate-Assimilating Bacterium, Flavobacterium sp. Strain UMI-01

    Science.gov (United States)

    Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

    2017-01-01

    Recently, we identified an alginate-assimilating gene cluster in the genome of Flavobacterium sp. strain UMI-01, a member of Bacteroidetes. Alginate lyase genes and a 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH) reductase gene in the cluster have already been characterized; however, 2-keto-3-deoxy-d-gluconate (KDG) kinase and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase genes, i.e., flkin and flald, still remained uncharacterized. The amino acid sequences deduced from flkin and flald showed low identities with those of corresponding enzymes of Saccharophagus degradans 2-40T, a member of Proteobacteria (Kim et al., Process Biochem., 2016). This led us to consider that the DEH-assimilating enzymes of Bacteroidetes species are somewhat deviated from those of Proteobacteria species. Thus, in the present study, we first assessed the characteristics in the primary structures of KDG kinase and KDG aldolase of the strain UMI-01, and then investigated the enzymatic properties of recombinant enzymes, recFlKin and recFlAld, expressed by an Escherichia coli expression system. Multiple-sequence alignment among KDG kinases and KDG aldolases from several Proteobacteria and Bacteroidetes species indicated that the strain UMI-01 enzymes showed considerably low sequence identities (15%–25%) with the Proteobacteria enzymes, while they showed relatively high identities (47%–68%) with the Bacteroidetes enzymes. Phylogenetic analyses for these enzymes indicated the distant relationship between the Proteobacteria enzymes and the Bacteroidetes enzymes, i.e., they formed distinct clusters in the phylogenetic tree. recFlKin and recFlAld produced with the genes flkin and flald, respectively, were confirmed to show KDG kinase and KDPG aldolase activities. Namely, recFlKin produced 1.7 mM KDPG in a reaction mixture containing 2.5 mM KDG and 2.5 mM ATP in a 90-min reaction, while recFlAld produced 1.2 mM pyruvate in the reaction mixture containing 5 mM KDPG at the equilibrium

  8. Synthesis of α-amino-β-keto-esters (β-oxodipeptides)

    Institute of Scientific and Technical Information of China (English)

    Yanjie XU; Ligong CHEN

    2008-01-01

    The synthesis of α-amino-β-keto-esters (β-oxo dipeptides) was studied. Corresponding α-amino-β-keto-esters were prepared from BOC-(L)-Valine and BOC-(L)-isoleucine by coupling with (D,L)-threonine hydro-chloride and oxidation with Dess-Martin periodinane (DMP) with a total yield of 48% and 38%, respectively.

  9. 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.

  10. Theoretical Study on Enol-keto Tautomerism of α-Fluorine-β-diketones

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Density Functional Theory method is applied to investigate the enol-keto tautome- rism of both acyclic and cyclic α-fluorine-β-diketones. It is shown that, for acyclic cases, α-fluorine could improve the relative stability of keto tautomer by lessening intramolecular hydrogen bond of enol form, whereas the relative stability of cyclic enol could be attributed to two factors: destabi- lization of keto and stabilization of enol. Furthermore, the relative stabilities of all enol tautomers are improved in THF to different extents.

  11. Purification and characterization of 2-keto-3-deoxy-6-phosphogluconate aldolase from Azotobacter vinelandii: evidence that the enzyme is bifunctional towards 2-keto-4-hydroxy glutarate cleavage.

    Science.gov (United States)

    Taha, T S; Deits, T L

    1994-04-15

    2-keto-3-deoxy-6-phosphogluconate aldolase (E.C. 4.1.2.14) has been purified in two chromatographic steps to 99% purity in 73% overall yield from Azotobacter vinelandii. The pure enzyme is a 70 kD trimeric Class I aldolase, inhibitable by bromopyruvate or pyruvate plus sodium borohydride, with a specific activity of 625 mumol per min per mg protein and a Km of 38 microM for 2-keto-3-deoxy-6-phosphogluconate. The enzyme also has 2-keto-4-hydroxy glutarate aldolase (E.C. 4.1.3.16) activity, with a specific activity of 4.8 mumol per min per mg protein and a Km of 39 microM. 2-keto-4-hydroxy glutarate inhibits the 2-keto-3-deoxy-6-phosphogluconate aldolase activity of the enzyme with an apparent Ki of 0.17 mM. Slow steps following formation of the Schiff base intermediate between KHG and the enzyme are responsible for both the slower turnover of this substrate and for its inhibitory effect.

  12. Preliminary Results of the NASA Beacon Receiver for Alphasat Aldo Paraboni TDP5 Propagation Experiment

    Science.gov (United States)

    Nessel, James; Morse, Jacquelynne; Zemba, Michael; Riva, Carlo; Luini, Lorenzo

    2014-01-01

    NASA Glenn Research Center (GRC) and the Politecnico di Milano (POLIMI) have initiated a joint propagation campaign within the framework of the Alphasat propagation experiment to characterize rain attenuation, scintillation, and gaseous absorption effects of the atmosphere in the 40 GHz band. NASA GRC has developed and installed a K/Q-band (20/40 GHz) beacon receiver at the POLIMI campus in Milan, Italy, which receives the 20/40 GHz signals broadcast from the Alphasat Aldo Paraboni TDP#5 beacon payload. The primary goal of these measurements is to develop a physical model to improve predictions of communications systems performance within the Q-band. Herein, we describe the design and preliminary performance of the NASA propagation terminal, which has been installed and operating in Milan since May 2014. The receiver is based upon a validated Fast Fourier Transform (FFT) I/Q digital design approach utilized in other operational NASA propagation terminals, but has been modified to employ power measurement via a frequency estimation technique and to coherently track and measure the amplitude of the 20/40 GHz beacon signals. The system consists of a 1.2-m K-band and a 0.6-m Qband Cassegrain reflector employing synchronous open-loop tracking to track the inclined orbit of the Alphasat satellite. An 8 Hz sampling rate is implemented to characterize scintillation effects, with a 1-Hz measurement bandwidth dynamic range of 45 dB. A weather station with an optical disdrometer is also installed to characterize rain drop size distribution for correlation with physical based models.

  13. Performance of the NASA Beacon Receiver for the Alphasat Aldo Paraboni TDP5 Propagation Experiment

    Science.gov (United States)

    Nessel, James; Morse, Jacquelynne; Zemba, Michael; Riva, Carlo; Luini, Lorenzo

    2015-01-01

    NASA Glenn Research Center (GRC) and the Politecnico di Milano (POLIMI) have initiated a joint propagation campaign within the framework of the Alphasat propagation experiment to characterize rain attenuation, scintillation, and gaseous absorption effects of the atmosphere in the 40 gigahertz band. NASA GRC has developed and installed a K/Q-band (20/40 gigahertz) beacon receiver at the POLIMI campus in Milan, Italy, which receives the 20/40 gigahertz signals broadcast from the Alphasat Aldo Paraboni Technology Demonstration Payload (TDP) no. 5 beacon payload. The primary goal of these measurements is to develop a physical model to improve predictions of communications systems performance within the Q-band. Herein, we describe the design and preliminary performance of the NASA propagation terminal, which has been installed and operating in Milan since June 2014. The receiver is based upon a validated Fast Fourier Transform (FFT) I/Q digital design approach utilized in other operational NASA propagation terminals, but has been modified to employ power measurement via a frequency estimation technique and to coherently track and measure the amplitude of the 20/40 gigahertz beacon signals. The system consists of a 1.2-meter K-band and a 0.6-meter Q-band Cassegrain reflector employing synchronous open-loop tracking to track the inclined orbit of the Alphasat satellite. An 8 hertz sampling rate is implemented to characterize scintillation effects, with a 1-hertz measurement bandwidth dynamic range of 45 decibels. A weather station with an optical disdrometer is also installed to characterize rain drop size distribution for correlation with physical based models.

  14. Aldo Bernardini, Cinema italiano delle origini. Gli ambulanti, Gemona, La Cineteca del Friuli, 2001, 196 p.

    OpenAIRE

    Gili, Jean Antoine

    2007-01-01

    Les recherches poursuivies en Italie sur le cinéma des origines continuent sur bien des points à demeurer exemplaires. Le travail de l’infatigable Aldo Bernardini sur les forains entre dans une perspective qui devra comporter deux autres volumes consacrés à une filmographie des documentaires, le premier sur l’ensemble des films « dal vero » tournés en Italie de 1896 à 1914, le second sur les quelques centaines de documentaires commandités par des sociétés étrangères de 1896 à 1908. Dans le pr...

  15. Efficient Syntheses of 2-Substituted Benzimidazoles and Benzoxazoles from β-Keto Esters

    Institute of Scientific and Technical Information of China (English)

    XIAO Li-wei; ZHANG Min; SUN Wen-hua

    2011-01-01

    An efficient synthetic method was developed to synthesize 2-substituted benzimidazoles and benzoxazoles withβ-keto esters as starting materials under mild reaction conditions, during which other functional groups are bearable from reactants to products.

  16. Substrate selectivity of Gluconobacter oxydans for production of 2,5-diketo-D-gluconic acid and synthesis of 2-keto-L-gulonic acid in a multienzyme system.

    Science.gov (United States)

    Ji, A; Gao, P

    2001-06-01

    Substrate selectivity of Gluconobacter oxydans (ATCC 9937) for 2,5-diketo-D-gluconic acid (2,5-DKG) production was investigated with glucose, gluconic acid, and gluconolactone in different concentrations using a resting-cell system. The results show that gluconic acid was utilized favorably by G. oxydans as substrate to produce 2,5-DKG. The strain was coupled with glucose dehydrogenase (GDH) and 2,5-DKG reductase for synthesis of 2-keto-L-gulonic acid (2-KLG), a direct precursor of L-ascorbic acid, from glucose. NADP and NADPH were regenerated between GDH and 2,5-DKG reductase. The mole yield of 2-KLG of this multienzyme system was 16.8%. There are three advantages for using the resting cells of G. oxydans to connect GDH with 2,5-DKG reductase for production of 2-KLG: gluconate produced by GDH may immediately be transformed into 2,5-DKG so that a series of problems generally caused by the accumulation of gluconate would be avoided; 2,5-DKG is supplied directly and continuously for 2,5-DKG reductase, so it is unnecessary to take special measures to deal with this unstable substrate as it was in Sonoyama's tandem fermentation process; and NADP(H) was regenerated within the system without any other components or systems.

  17. 5-Lipoxygenase inhibition by acetyl-11-keto-β-boswellic acid (AKBA) by a novel mechanism.

    Science.gov (United States)

    Safayhi, H; Sailer, E R; Ammon, H P

    1996-05-01

    Acetyl-11-keto-β-boswellic acid (AKAB) from Boswellia serrata and B. carterii acts directly on purified 5-lipoxygenase of human blood leukocytes at a selective site for pentacyclic triterpenes that is different from the arachidonate substrate binding site. The pentacyclic triterpene ring is crucial for binding to the enzyme, whereas functional groups (11-keto function in addition to a hydrophilic group on C 4 of ring A) are essential for the 5-lipoxygenase activity.

  18. Natural polyprenylated benzophenones: keto-enol tautomerism and stereochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Martins, Felipe T.; Cruz Junior, Jose W.; Doriguetto, Antonio C. [Universidade Federal de Alfenas, MG (Brazil). Dept. de Ciencias Exatas. Lab. de Cristalografia]. E-mail: doriguetto@unifal-mg.edu.br; Derogis, Priscilla B.M.C.; Santos, Marcelo H. dos; Veloso, Marcia P. [Universidade Federal de Alfenas, MG (Brazil). Dept. de Ciencias Exatas. Lab. de Fitoquimica e Quimica Medicinal; Ellena, Javier [Universidade de Sao Paulo, Sao Carlos, SP (Brazil). Inst. de Fisica

    2007-07-01

    The keto-enol tautomerism and stereochemistry study of a HIV-inhibitory natural benzophenone, (1R,5R,7R,8S)-(+)-3-(10-(3,4-dihydroxyphenyl)-10-hydroxymethylene) -8-methyl-1,5,7-tris(3-methyl-2-butenyl)-8-(4-methyl-3-pentenyl)-bicyclo [3.3.1]nonane-2,4,9-trione (a), isolated from Garcinia brasiliensis seeds is presented. The crystal structure of (a), which is also know as guttiferona A, was determined by X-ray diffraction and its intra and inter-molecular geometries discussed and compared with two analogue natural benzophenones: clusianone and epiclusianone. In (a), the hydroxyl H atom from enolizable 2,4,10-trione moiety is linked in the oxygen atom bonded to 10-(3,4-dihydroxyphenyl)methylene group, in opposition to the related natural benzophenones, where this analogue H-atom is placed in different O-atoms from bicyclo[3.3.1]nonane ring system. Such behaviour can be explained by the presence of aromatic OH6 group in (a) that origins a further delocalized resonance path along of 3,4-dihydroxyphenyl- C10-OH2 group. In addition, the (a) stereochemistry around C7 atom is compared with known structures of clusianone and epiclusianone and the influence from configuration in this chiral Catom to structural features found in the enolizable system is proposed. (author)

  19. Louis I. Kahn ja Aldo van Eyck : paralleelid moodsa arhitektuuri teises traditsioonis / Robert McCarter ; tõlk. Tiina Randus

    Index Scriptorium Estoniae

    McCarter, Robert

    2007-01-01

    Vaadeldakse Louis I. Kahni ja Aldo van Eycki esinemist 1959. a. CIAMi (Congres Internatinaux d'Architecture Moderne) XI kongressil Otterlos, ajaloolistes paikades saadud kogemuste mõju nende loomingule, suhteid kaasaja kunstnikega ja kunstnike loomingu mõju neile, hoonete kavandamist, linnaarhitektuuri ja -planeerimist. Bibliograafia lk.101-102

  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. 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

  2. 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

  3. 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

  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. Quorum Sensing Inhibition and Structure–Activity Relationships of β-Keto Esters

    Directory of Open Access Journals (Sweden)

    Stephanie Forschner-Dancause

    2016-07-01

    Full Text Available Traditional therapeutics to treat bacterial infections have given rise to multi-drug resistant pathogens, which pose a major threat to human and animal health. In several pathogens, quorum sensing (QS—a cell-cell communication system in bacteria—controls the expression of genes responsible for pathogenesis, thus representing a novel target in the fight against bacterial infections. Based on the structure of the autoinducers responsible for QS activity and other QS inhibitors, we hypothesize that β-keto esters with aryl functionality could possess anti-QS activity. A panel of nineteen β-keto ester analogs was tested for the inhibition of bioluminescence (a QS-controlled phenotype in the marine pathogen Vibrio harveyi. Initial screening demonstrated the need of a phenyl ring at the C-3 position for antagonistic activity. Further additions to the phenyl ring with 4-substituted halo groups or a 3- or 4-substituted methoxy group resulted in the most active compounds with IC50 values ranging from 23 µM to 53 µM. The compounds additionally inhibit green fluorescent protein production by E. coli JB525. Evidence is presented that aryl β-keto esters may act as antagonists of bacterial quorum sensing by competing with N-acyl homoserine lactones for receptor binding. Expansion of the β-keto ester panel will enable us to obtain more insight into the structure–activity relationships needed to allow for the development of novel anti-virulence agents.

  6. Design and synthesis of selective keto-1,2,4-oxadiazole-based tryptase inhibitors.

    Science.gov (United States)

    Palmer, James T; Rydzewski, Robert M; Mendonca, Rohan V; Sperandio, David; Spencer, Jeffrey R; Hirschbein, Bernard L; Lohman, Julia; Beltman, Jeri; Nguyen, Margaret; Liu, Liang

    2006-07-01

    Using a scaleable, directed library approach based on orthogonally protected advanced intermediates, we have prepared a series of potent keto-1,2,4-oxadiazoles designed to explore the P(2) binding pocket of human mast cell tryptase, while building in a high degree of selectivity over human trypsin and other serine proteases.

  7. Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase

    NARCIS (Netherlands)

    Wolterink-van Loo, S.; Siemerink, M.A.J.; Perrakis, G.; Kaper, T.; Kengen, S.W.M.; Oost, van der J.

    2009-01-01

    Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 degrees C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthe

  8. Enzymatic production of microthecin by aldos-2-ulose dehydratase from 1,5-anhydro-D-fructose and stability studies of microthecin

    DEFF Research Database (Denmark)

    Yu, Shukun; Andreassen, Mikkel; Lundt, Inge

    2008-01-01

    .2.2.13) from glycogen and starch by aldos-2-ulose dehydratase (AUDH; EC 4. 2.1.110). In the current study, the yield and purity of microthecin was examined with respect to pH and buffers using AUDH purified from the fungus Phanerochaete chrysosporium. It was found that AUDH had a Km of 5.4 and 4.9 mM towards...

  9. ALDO: A radiation-tolerant, low-noise, adjustable low drop-out linear regulator in 0.35 μm CMOS technology

    Energy Technology Data Exchange (ETDEWEB)

    Carniti, P., E-mail: paolo.carniti@mib.infn.it [INFN, Sezione di Milano Bicocca, I-20126 Milano (Italy); Dipartimento di Fisica, Università di Milano Bicocca, I-20126 Milano (Italy); Cassina, L.; Gotti, C.; Maino, M.; Pessina, G. [INFN, Sezione di Milano Bicocca, I-20126 Milano (Italy); Dipartimento di Fisica, Università di Milano Bicocca, I-20126 Milano (Italy)

    2016-07-11

    In this work we present ALDO, an adjustable low drop-out linear regulator designed in AMS 0.35 μm CMOS technology. It is specifically tailored for use in the upgraded LHCb RICH detector in order to improve the power supply noise for the front end readout chip (CLARO). ALDO is designed with radiation-tolerant solutions such as an all-MOS band-gap voltage reference and layout techniques aiming to make it able to operate in harsh environments like High Energy Physics accelerators. It is capable of driving up to 200 mA while keeping an adequate power supply filtering capability in a very wide frequency range from 10 Hz up to 100 MHz. This property allows us to suppress the noise and high frequency spikes that could be generated by a DC/DC regulator, for example. ALDO also shows a very low noise of 11.6 μV RMS in the same frequency range. Its output is protected with over-current and short detection circuits for a safe integration in tightly packed environments. Design solutions and measurements of the first prototype are presented.

  10. ALDO: A radiation-tolerant, low-noise, adjustable low drop-out linear regulator in 0.35 μm CMOS technology

    Science.gov (United States)

    Carniti, P.; Cassina, L.; Gotti, C.; Maino, M.; Pessina, G.

    2016-07-01

    In this work we present ALDO, an adjustable low drop-out linear regulator designed in AMS 0.35 μm CMOS technology. It is specifically tailored for use in the upgraded LHCb RICH detector in order to improve the power supply noise for the front end readout chip (CLARO). ALDO is designed with radiation-tolerant solutions such as an all-MOS band-gap voltage reference and layout techniques aiming to make it able to operate in harsh environments like High Energy Physics accelerators. It is capable of driving up to 200 mA while keeping an adequate power supply filtering capability in a very wide frequency range from 10 Hz up to 100 MHz. This property allows us to suppress the noise and high frequency spikes that could be generated by a DC/DC regulator, for example. ALDO also shows a very low noise of 11.6 μV RMS in the same frequency range. Its output is protected with over-current and short detection circuits for a safe integration in tightly packed environments. Design solutions and measurements of the first prototype are presented.

  11. 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.

  12. Enantioselective α-Hydroxylation by Modified Salen-Zirconium(IV)-Catalyzed Oxidation of β-Keto Esters.

    Science.gov (United States)

    Yang, Fan; Zhao, Jingnan; Tang, Xiaofei; Zhou, Guangli; Song, Wangze; Meng, Qingwei

    2017-02-03

    The highly enantioselective α-hydroxylation of β-keto esters using cumene hydroperoxide (CHP) as the oxidant was realized by a chiral (1S,2S)-cyclohexanediamine backbone salen-zirconium(IV) complex as the catalyst. A variety of corresponding chiral α-hydroxy β-keto esters were obtained in excellent yields (up to 99%) and enantioselectivities (up to 98% ee). The zirconium-catalyzed enantioselective α-hydroxylation of β-keto esters was scalable, and the zirconium catalyst was recyclable. The reaction can be performed in gram scale, and corresponding chiral products were acquired in 95% yield and 99% ee.

  13. Effect of cholic acid and its keto derivatives on the analgesic action of lidocaine and associated biochemical parameters in rats.

    Science.gov (United States)

    Posa, Mihalj; Kevresan, Slavko; Mikov, Momir; Cirin-Novta, Vera; Kuhajda, Ksenija

    2007-01-01

    This study examined the effect of the structure and concentration of cholic acid and its keto derivatives on the local analgesic action of lidocaine in rats, measured by an analgesimetric method. The increase in bile acid concentrations in the administered lidocaine solution increased the duration of local anesthesia. It was found that the introduction of keto groups into the cholic acid molecule yielded derivatives with lower promotory action, i.e. decreased the duration of local anesthesia. The biochemical parameters investigated indicated that the keto derivatives of cholic acid exhibited no toxicity compared to that of cholic acid itself.

  14. 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.

  15. Aldo Leopold's land health from a resilience point of view: self-renewal capacity of social-ecological systems.

    Science.gov (United States)

    Berkes, Fikret; Doubleday, Nancy C; Cumming, Graeme S

    2012-09-01

    Health approaches to ecology have a strong basis in Aldo Leopold's thinking, and contemporary ecohealth in turn has a strong philosophical basis in Leopold. To commemorate the 125th anniversary of Leopold's birth (1887-1948), we revisit his ideas, specifically the notions of stewardship (land ethic), productive use of ecosystems (land), and ecosystem renewal. We focus on Leopold's perspective on the self-renewal capacity of the land, as understood in terms of integrity and land health, from the contemporary perspective of resilience theory and ecological theory more generally. Using a broad range of literature, we explore insights and implications of Leopold's work for today's human-environment relationships (integrated social-ecological systems), concerns for biodiversity, the development of agency with respect to stewardship, and key challenges of his time and of ours. Leopold's seminal concept of land health can be seen as a triangulation of productive use, self-renewal, and stewardship, and it can be reinterpreted through the resilience lens as the health of social-ecological systems. In contemporary language, this involves the maintenance of biodiversity and ecosystem services, and the ability to exercise agency both for conservation and for environmental justice.

  16. Spectroscopic Evidence of Keto-enol Tautomerism in Deliquesced Malonic Acid Particles

    Energy Technology Data Exchange (ETDEWEB)

    Ghorai, Suman; Laskin, Alexander; Tivanski, Alexei V.

    2011-04-11

    Scanning Transmission X-ray Microscopy combined with Near Edge X-ray Absorption Fine Structure Spectroscopy (STXM/NEXAFS), and optical microscopy coupled with Fourier Transform Infrared Spectroscopy (micro-FTIR) have been applied to observe hygroscopic growth and chemical changes in malonic acid particles deposited on substrates. Extent of the hygroscopic growth of particles has been quantified in terms of the corresponding water-to-solute ratios (WSR) based on STXM/NEXAFS and micro-FTIR data sets. WSR values derived separately from two applied methods displayed a remarkable agreement with previous data reported in the literature. Comparison of NEXAFS and FTIR spectra acquired at different relative humidity (RH) shows efficient keto-enol tautomerization of malonic acid, with the enol form dominated at higher RH. The keto-enol equilibrium constants were calculated using relevant peak intensities in the carbon and oxygen K-edge NEXAFS spectra as a function of RH.

  17. Density functional study on enantioselective reduction of keto oxime ether with borane catalyzed by oxazaborolidine

    Institute of Scientific and Technical Information of China (English)

    LI; Ming; ZHENG; Wenxu

    2006-01-01

    The enantioselective reduction of keto oxime ether with borane catalyzed by oxazaborolidine is discussed by the density functional theory (DFT) method. The main intermediates and transition states for this reaction are optimized completely at the B3LYP/6-31g(d) level, and the transition states are verified by vibrational modes. As shown, the chirality-controlled steps for this reaction are the hydride transfer from borane to carbonyl carbon and oxime carbon of keto oxime ether, and the chirality for the reduced products is determined in these two reaction steps. In all examined reaction paths, the first hydride is transferred via a six-membered ring and the second hydride via a five-membered ring or a four-membered ring.

  18. Catalytic and regioselective oxidation of carbohydrates to synthesize keto-sugars under mild conditions.

    Science.gov (United States)

    Muramatsu, Wataru

    2014-09-19

    A new catalytic and regioselective approach for the synthesis of keto-sugars is described. An organotin catalyst, Oc2SnCl2, in the presence of trimethylphenylammonium tribromide ([TMPhA](+)Br3(-)) accelerates the regioselective oxidation at the "axial"-OH group of 1,2-diol moieties in galactopyranosides. The reaction conditions can also be used for the regioselective oxidation of various carbohydrates.

  19. Keto amphetamine toxicity-focus on the redox reactivity of the cathinone designer drug mephedrone.

    Science.gov (United States)

    den Hollander, Bjørnar; Sundström, Mira; Pelander, Anna; Ojanperä, Ilkka; Mervaala, Eero; Korpi, Esa Risto; Kankuri, Esko

    2014-09-01

    The β-keto amphetamine (cathinone, β-KA) designer drugs such as mephedrone (4-methylmethcathinone, 4-MMC) show a large degree of structural similarity to amphetamines like methamphetamine (METH). However, little is currently known about whether these substances also share the potential neurotoxic properties of their non-keto amphetamine counterparts, or what mechanisms could be involved. Here, we evaluate the cytotoxicity of β-KAs in SH-SY5Y cells using lactate dehydrogenase (LDH) assays, assess the redox potential of a range of β-KAs and non-keto amphetamines using the sensitive redox indicator 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1), and explore the effect of 4-MMC on the formation of protein adducts using ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) and on the mitochondrial respiratory chain using high-resolution respirometry. We show that treatment with β-KAs increases LDH release. Further, we demonstrate that even under physiological pH, β-KAs are effective and selective-as compared with their non-keto analogues-reductants in the presence of electron acceptors. Increased pH (range 7.6-8.0) greatly enhanced the reactivity up to sixfold. We found no evidence of protein adduct formation, suggesting the reactivity is due to direct electron transfer by the β-KAs. Finally, we show that 4-MMC and METH produce dissimilar effects on the respiratory chain. Our results indicate that β-KAs such as 4-MMC possess cytotoxic properties in vitro. Furthermore, in the presence of an electron-accepting redox partner, the ketone moiety of β-KAs is vital for pH-dependent redox reactivity. Further work is needed to establish the importance of β-KA redox properties and its potential toxicological importance in vivo.

  20. Iron-regulated excretion of alpha-keto acids by Salmonella typhimurium.

    OpenAIRE

    Reissbrodt, R.; Kingsley, R; Rabsch, W.; Beer, W.; Roberts, M.; Williams, P H

    1997-01-01

    Excretion of alpha-keto acids by clinical isolates and laboratory strains of Salmonella typhimurium was determined by high-performance liquid chromatography analysis of culture supernatants. The levels of excretion increased markedly with increasing iron stress imposed by the presence of alpha,alpha'-dipyridyl or conalbumin in the medium. The major product was pyruvic acid, but significant concentrations of alpha-ketoglutaric acid, alpha-ketoisovaleric acid, and alpha-ketoisocaproic acid were...

  1. Antioxidative activities of algal keto carotenoids acting as antioxidative protectants in the chloroplast.

    Science.gov (United States)

    Dambeck, Michael; Sandmann, Gerhard

    2014-01-01

    Very diverse carotenoid structures exist in the photosynthesis apparatus of different algae. Among them, the keto derivatives are regarded the most antioxidative. Therefore, four different keto carotenoids, peridinin, fucoxanthin, siphonaxanthin and astaxanthin fatty acid monoesters, were isolated and purified from Amphidinium carterae, Phaeodactylum tricornutum, Caulerpa taxifolia and Haematococcus pluvialis, respectively. The carotenoids were assayed as inhibitors of photosensitizer initiated reactions or scavengers of radicals in the early events generating reactive oxygen species as starters for peroxidation and as protectants against the whole reaction chain finally leading to lipid peroxidation. These in vitro studies demonstrated the substantial antioxidative properties as indicated by the IC(50) values of all four keto carotenoids with superior protection by astaxanthin fatty acid monoesters which were as effective as free astaxanthin and of peridinin against radicals. As an example, the in vivo relevance of fucoxanthin for protection of photosynthesis from excess light and from peroxidative agents was evaluated with intact cells. Cultures of P. tricornutum with decreased fucoxanthin content generated by inhibitor treatment were exposed to strong light or cumene hydroperoxyde. In each case, oxidation of chlorophyll as marker for damaging of the photosynthesis apparatus was less severe when the fucoxanthin was at maximum level.

  2. Organocatalytic Asymmetric Michael Addition of 4-Hydroxycoumarin to β,γ-Unsaturated α-Keto Esters

    Energy Technology Data Exchange (ETDEWEB)

    Suh, Chang Won; Han, Tae Hyun; Kim, Dae Young [Soonchunhyang Univ., Asan (Korea, Republic of)

    2013-06-15

    In conclusion, we have developed organocatalytic enantioselective conjugate addition reaction of 4-hydroxycoumarin (1) to β,γ-unsaturated α-keto esters 2 to afford biologically valuable warfarin derivatives 3. The process is efficiently catalyzed by a binaphthyl-modified thiourea organocatalyst. The coumarin core is present as a characteristic structural motif in a large number of natural products and biologically active molecules.1 Particularly, many of these naturally occurring 4-hydroxycoumarin and their synthetic analogues are important precursors for the synthesis of natural products and pharmaceuticals. Enantioselective organocatalytic conjugate addition of 4-hydroxycoumarin to α,β-unsaturated ketones is a straightforward method to access warfarin which is an effective anticoagulants. Although a number of reactions of α,β-unsaturated ketones as Michael acceptors have been reported, the corresponding β,γ-unsaturated α-keto esters have received relatively little attention as Michael acceptors. Recently, several groups have reported the asymmetric Michael addition of 4-hydroxycoumarin to β,γ-unsaturated α-keto esters catalyzed by Cu(II)-bisoxazoline, N,N'-dioxide-Ni(II) complexes, thiourea catalysts. Although several efficient methods have been achieved by these systems, an effective method for the synthesis of warfarin analogues is still a challenge.

  3. Supramolecular Influence on Keto-Enol Tautomerism and Thermochromic Properties of o-Hydroxy Schiff Bases

    Directory of Open Access Journals (Sweden)

    Marija Zbačnik

    2016-06-01

    Full Text Available This work presents a study on thermo-optical properties of three Schiff bases (imines in the solid state. The Schiff bases were obtained by means of mechanochemical synthesis using monosubstituted o-hydroxy aromatic aldehydes and monosubstituted aromatic amines. The keto-enol tautomerism and proton transfer via intramolecular O∙∙∙N hydrogen bond of the reported compounds was found to be influenced more by supramolecular interactions than by a temperature change. All products were characterised by powder X-ray diffraction (PXRD, FT-IR spectroscopy, thermogravimetric (TG analysis and differential scanning calorimetry (DSC. Molecular and crystal structures of compounds 1, 2 and 3 were determined by single crystal X-ray diffraction (SCXRD. The molecules of 1 appear to be present as the enol-imine, the molecules of 2 as the keto-amine tautomer and the molecules of 3 exhibit keto-enol tautomeric equilibrium in the solid state. An analysis of Cambridge structural database (CSD data on similar imines has been used for structural comparison. This work is licensed under a Creative Commons Attribution 4.0 International License.

  4. Identification of a 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid Reductase, FlRed, in an Alginolytic Bacterium Flavobacterium sp. Strain UMI-01

    Directory of Open Access Journals (Sweden)

    Akira Inoue

    2015-01-01

    Full Text Available In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11. The monosaccharide is non-enzymatically converted to 4-deoxy-l-ery thro-5-hexoseulose uronic acid (DEH, then reduced to 2-keto-3-deoxy-d-gluconate (KDG by a specific reductase, and metabolized through the Entner–Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%–88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.

  5. La dimensión humana de la arquitectura de Aldo Van Eyck. Escrita y Construida: Reconocimiento de sus ideas y Estudio de su iglesia en La Haya

    OpenAIRE

    Fernández-Llébrez Muñoz, José Ángel

    2013-01-01

    El protagonista fundamental de esta tesis doctoral es Aldo van Eyck. Mención aparte del interés suscitado por el conjunto de su trayectoria profesional y por su propia figura, la principal motivación del presente trabajo consiste en aproximarse a la visión que el maestro holandés tenía de la relación que se establece entre 'arquitectura' y 'sociedad' o, más específicamente, de cómo los arquitectos pueden (y deben) introducir mejoras significativas en los modos de vida de las personas (usuario...

  6. Antistaphylococcal and biofilm inhibitory activities of acetyl-11-keto-β-boswellic acid from Boswellia serrata

    Directory of Open Access Journals (Sweden)

    Arora Daljit S

    2011-03-01

    Full Text Available Abstract Background Boswellic acids are pentacyclic triterpenes, which are produced in plants belonging to the genus Boswellia. Boswellic acids appear in the resin exudates of the plant and it makes up 25-35% of the resin. β-boswellic acid, 11-keto-β-boswellic acid and acetyl-11-keto-β-boswellic acid have been implicated in apoptosis of cancer cells, particularly that of brain tumors and cells affected by leukemia or colon cancer. These molecules are also associated with potent antimicrobial activities. The present study describes the antimicrobial activities of boswellic acid molecules against 112 pathogenic bacterial isolates including ATCC strains. Acetyl-11-keto-β-boswellic acid (AKBA, which exhibited the most potent antibacterial activity, was further evaluated in time kill studies, postantibiotic effect (PAE and biofilm susceptibility assay. The mechanism of action of AKBA was investigated by propidium iodide uptake, leakage of 260 and 280 nm absorbing material assays. Results AKBA was found to be the most active compound showing an MIC range of 2-8 μg/ml against the entire gram positive bacterial pathogens tested. It exhibited concentration dependent killing of Staphylococcus aureus ATCC 29213 up to 8 × MIC and also demonstrated postantibiotic effect (PAE of 4.8 h at 2 × MIC. Furthermore, AKBA inhibited the formation of biofilms generated by S. aureus and Staphylococcus epidermidis and also reduced the preformed biofilms by these bacteria. Increased uptake of propidium iodide and leakage of 260 and 280 nm absorbing material by AKBA treated cells of S aureus indicating that the antibacterial mode of action of AKBA probably occurred via disruption of microbial membrane structure. Conclusions This study supported the potential use of AKBA in treating S. aureus infections. AKBA can be further exploited to evolve potential lead compounds in the discovery of new anti-Gram-positive and anti-biofilm agents.

  7. Spectral correlation, antimicrobial and insect antifeedant activities of some 1-naphthyl keto-oxiranes

    Directory of Open Access Journals (Sweden)

    G. Thirunarayanan

    2014-12-01

    Full Text Available Thirteen optically active (αS,βR 1-naphthyl keto-oxiranes (1-naphthyl-4-yl[3-(substituted phenyloxiran-2-yl]methanones have been synthesised by phase transfer catalysed epoxidation of 1-naphthyl chalcones. The yields of oxiranes are more than 95%. The synthesized oxiranes have been characterized by IR, 1H, 13C and MS spectra. The spectral data are correlated with Hammett substituent constants and Swain–Lupton parameters. From the regression analysis the effect of substituent on the group frequencies has been predicted. The antimicrobial and insect antifeedant activities of all synthesised oxiranes have been evaluated.

  8. Bioconversion of D-galacturonate to keto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate using filamentous fungi

    Directory of Open Access Journals (Sweden)

    Wiebe Marilyn G

    2010-08-01

    Full Text Available Abstract Background The D-galacturonic acid derived from plant pectin can be converted into a variety of other chemicals which have potential use as chelators, clarifiers, preservatives and plastic precursors. Among these is the deoxy-keto acid derived from L-galactonic acid, keto-deoxy-L-galactonic acid or 3-deoxy-L-threo-hex-2-ulosonic acid. The keto-deoxy sugars have been found to be useful precursors for producing further derivatives. Keto-deoxy-L-galactonate is a natural intermediate in the fungal D-galacturonate metabolic pathway, and thus keto-deoxy-L-galactonate can be produced in a simple biological conversion. Results Keto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate accumulated in the culture supernatant when Trichoderma reesei Δlga1 and Aspergillus niger ΔgaaC were grown in the presence of D-galacturonate. Keto-deoxy-L-galactonate accumulated even if no metabolisable carbon source was present in the culture supernatant, but was enhanced when D-xylose was provided as a carbon and energy source. Up to 10.5 g keto-deoxy-L-galactonate l-1 was produced from 20 g D-galacturonate l-1 and A. niger ΔgaaC produced 15.0 g keto-deoxy-L-galactonate l-1 from 20 g polygalacturonate l-1, at yields of 0.4 to 1.0 g keto-deoxy-L-galactonate [g D-galacturonate consumed]-1. Keto-deoxy-L-galactonate accumulated to concentrations of 12 to 16 g l-1 intracellularly in both producing organisms. This intracellular concentration was sustained throughout production in A. niger ΔgaaC, but decreased in T. reesei. Conclusions Bioconversion of D-galacturonate to keto-deoxy-L-galactonate was achieved with both A. niger ΔgaaC and T. reesei Δlga1, although production (titre, volumetric and specific rates was better with A. niger than T. reesei. A. niger was also able to produce keto-deoxy-L-galactonate directly from pectin or polygalacturonate demonstrating the feasibility of simultaneous hydrolysis and bioconversion. Although keto

  9. Amino acids allosterically regulate the thiamine diphosphate-dependent alpha-keto acid decarboxylase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Werther, Tobias; Spinka, Michael; Tittmann, Kai; Schütz, Anja; Golbik, Ralph; Mrestani-Klaus, Carmen; Hübner, Gerhard; König, Stephan

    2008-02-29

    The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.

  10. Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase

    Directory of Open Access Journals (Sweden)

    Suzanne Wolterink-van Loo

    2009-01-01

    Full Text Available Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA displays optimal activity at 95°C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 °C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40–60 °C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.

  11. Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase.

    Science.gov (United States)

    Wolterink-van Loo, Suzanne; Siemerink, Marco A J; Perrakis, Georgios; Kaper, Thijs; Kengen, Servé W M; van der Oost, John

    2009-03-02

    Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 degrees C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 degrees C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40-60 degrees C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.

  12. Statistical Evaluation of HTS Assays for Enzymatic Hydrolysis of β-Keto Esters.

    Directory of Open Access Journals (Sweden)

    O Buß

    Full Text Available β-keto esters are used as precursors for the synthesis of β-amino acids, which are building blocks for some classes of pharmaceuticals. Here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of β-keto esters, the first step in the preparation of β-amino acids. Two of the tested high throughput screening (HTS assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. The third assay detects the pH shift due to acid formation using an indicator dye. To choose the most efficient approach for screening, we assessed these assays with different statistical methods-namely, the classical Z'-factor, standardized mean difference (SSMD, the Kolmogorov-Smirnov-test, and t-statistics. This revealed that all three assays are suitable for HTS, the pH assay performing best. Based on our data we discuss the explanatory power of different statistical measures. Finally, we successfully employed the pH assay to identify a very fast hydrolase in an enzyme-substrate screening.

  13. Investigation of Keto-enol Tautomers during the Synthesis of Aryl-bis (2-hydroxy-1-naphthyl)Methanes

    Indian Academy of Sciences (India)

    Papia Dutta; Mrinal Saikia; Rashmi Jyoti Das; Ruli Borah

    2014-11-01

    This study investigated the existence of keto-enol tautomers for the first time during the synthesis of aryl-bis(2-hydroxy-1-naphthyl)methane from 2-naphthol and -tolualdehyde or 4-chlorobenzaldehyde in methanol using CuSO4.5H2O as catalyst under reflux condition. The exclusive formation of aryl-bis(2-hydroxy-1-naphthyl)methaneswas observed in dichloromethane at room temperature in the presence of BF3.OEt2/AcOH as catalyst. The keto productswere isolated and characterized by 1HNMR, 13C NMR, COSY and DEPTspectra.

  14. Asymmetric synthesis of aromatic β-amino acids using ω-transaminase: Optimizing the lipase concentration to obtain thermodynamically unstable β-keto acids.

    Science.gov (United States)

    Mathew, Sam; Jeong, Seong-Su; Chung, Taeowan; Lee, Sang-Hyeup; Yun, Hyungdon

    2016-01-01

    Synthesized aromatic β-amino acids have recently attracted considerable attention for their application as precursors in many pharmacologically relevant compounds. Previous studies on asymmetric synthesis of aromatic β-amino acids using ω-transaminases could not be done efficiently due to the instability of β-keto acids. In this study, a strategy to circumvent the instability problem of β-keto acids was utilized to generate β-amino acids efficiently via asymmetric synthesis. In this work, thermodynamically stable β-ketoesters were initially converted to β-keto acids using lipase, and the β-keto acids were subsequently aminated using ω-transaminase. By optimizing the lipase concentration, we successfully overcame the instability problem of β-keto acids and enhanced the production of β-amino acids. This strategy can be used as a general approach to efficiently generate β-amino acids from β-ketoesters.

  15. Respiratory arsenate reductase as a bidirectional enzyme

    Science.gov (United States)

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

    2009-01-01

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

  16. 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.

  17. Powstanie i rozwój filozofii środowiskowej w USA na podstawie poglądów Johna Muira, Aldo Leopolda i J. Bairda Callicota

    Directory of Open Access Journals (Sweden)

    Leszek Pyra

    2013-06-01

    Full Text Available The Origin and Development of Environmental Philosophy in the US according to John Muir, Aldo Leopold and J. Baird Callicot. The publication refers to environmental philosophy, which is also called ecological philosophy or ecophilosophy. It shows in what way philosophical reflection on the environment has been shaped in the American tradition. In this context, the views of the thinkers listed below have been presented, analysed and evaluated. John Muir, an astute observer of wild nature, has been presented as an enthusiast and a prophet persuading a return to nature. Muir was a forester under the strong influence of the representatives of transcendentalism. As an activist, he founded national parks that served to preserve virgin areas, and as a writer he popularized the concept of the preservation of wild areas. For many representatives of ecophilosophy, the views of Aldo Leopold, as expressed in his well-known work, A Sand County Almanac, constituted a new pattern of thinking about wild nature. The book itself is almost generally regarded as a bible of environmental philosophy. Leopold, a forester interested in philosophy, created the fundamentals of holistic environmental philosophy, but the theory he developed is not without defects, to which some attention has been paid in this paper. The theory of J. Baird Callicot presents the attitude of an academic philosopher with regard to wild nature. His attitude is fully professional as regards the applied method, thus allowing the author to avoid committing a naturalistic fallacy. Callicot reinterprets the issues of facts and values, because he thinks that the discoveries of the dynamically developing ecology make possible the reformulation of the traditional approach to such issues by utilising a spirit of acceptance, and lead to a shift from is to ought to be

  18. Mitochondrial complex II participates in normoxic and hypoxic regulation of alpha-keto acids in the murine heart.

    NARCIS (Netherlands)

    Muhling, J.; Tiefenbach, M.; Lopez-Barneo, J.; Piruat, J.I.; Garcia-Flores, P.; Pfeil, U.; Gries, B.; Muhlfeld, C.; Weigand, M.A.; Kummer, W.; Weissmann, N.; Paddenberg, R.

    2010-01-01

    alpha-Keto acids (alpha-KAs) are not just metabolic intermediates but are also powerful modulators of different cellular pathways. Here, we tested the hypothesis that alpha-KA concentrations are regulated by complex II (succinate dehydrogenase=SDH), which represents an intersection between the mitoc

  19. Pyruvate: immunonutritional effects on neutrophil intracellular amino or alpha-keto acid profiles and reactive oxygen species production

    NARCIS (Netherlands)

    Mathioudakis, D.; Engel, J.; Welters, I.D.; Dehne, M.G.; Matejec, R.; Harbach, H.; Henrich, M.; Schwandner, T.; Fuchs, M.; Weismuller, K.; Scheffer, G.J.; Muhling, J.

    2011-01-01

    For the first time the immunonutritional role of pyruvate on neutrophils (PMN), free alpha-keto and amino acid profiles, important reactive oxygen species (ROS) produced [superoxide anion (O(2) (-)), hydrogen peroxide (H(2)O(2))] as well as released myeloperoxidase (MPO) acitivity has been investiga

  20. Adiponectin,leptin: focus on low-protein diet supplemented with keto acids in chronic glomerulonephritis with hbv patients

    Directory of Open Access Journals (Sweden)

    Shan Mou

    2012-06-01

    In conclusion: Short-term restriction of DPI 0.6–0.8 g of protein/ kg IBW/day is safe, when combined with keto acids, is associated with decreased of urinary protein and improvement of lipid metabolism

  1. Regulation of hepatic branched-chain alpha-keto acid dehydrogenase complex in rats fed a high-fat diet

    Science.gov (United States)

    Objective: Branched-chain alpha-keto acid dehydrogenase complex (BCKDC) regulates branched-chain amino acid (BCAA) metabolism at the level of branched chain alpha-ketoacid (BCKA) catabolism. It has been demonstrated that the activity of hepatic BCKDC is markedly decreased in type 2 diabetic animal...

  2. The diterpenoid 7-keto-sempervirol, derived from Lycium chinense, displays anthelmintic activity against both Schistosoma mansoni and Fasciola hepatica.

    Directory of Open Access Journals (Sweden)

    Jennifer Edwards

    2015-03-01

    Full Text Available BACKGROUND: Two platyhelminths of biomedical and commercial significance are Schistosoma mansoni (blood fluke and Fasciola hepatica (liver fluke. These related trematodes are responsible for the chronic neglected tropical diseases schistosomiasis and fascioliasis, respectively. As no vaccine is currently available for anti-flukicidal immunoprophylaxis, current treatment is mediated by mono-chemical chemotherapy in the form of mass drug administration (MDA (praziquantel for schistosomiasis or drenching (triclabendazole for fascioliasis programmes. This overreliance on single chemotherapeutic classes has dramatically limited the number of novel chemical entities entering anthelmintic drug discovery pipelines, raising significant concerns for the future of sustainable blood and liver fluke control. METHODOLOGY/ PRINCIPLE FINDINGS: Here we demonstrate that 7-keto-sempervirol, a diterpenoid isolated from Lycium chinense, has dual anthelmintic activity against related S. mansoni and F. hepatica trematodes. Using a microtiter plate-based helminth fluorescent bioassay (HFB, this activity is specific (Therapeutic index = 4.2, when compared to HepG2 cell lines and moderately potent (LD50 = 19.1 μM against S. mansoni schistosomula cultured in vitro. This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening, oviposition rates and egg morphology. As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD50 = 17.7 μM. Scanning electron microscopy (SEM evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental

  3. [Effects of carbon and nitrogen sources on 5-keto-gluconic acid production].

    Science.gov (United States)

    Tan, Zhilei; Wang, Hongcui; Wei, Yuqiao; Li, Yanyan; Zhong, Cheng; Jia, Shiru

    2014-01-01

    Gluconobacter oxydans is known to oxidize glucose to gluconic acid (GA), and subsequently, to 2-keto-gluconic acid (2KGA) and 5-keto-gluconic acid (5KGA), while 5KGA can be converted to L-(+)-tartaric acid. In order to increase the production of 5KGA, Gluconobacter oxydans HGI-1 that converts GA to 5KGA exclusively was chosen in this study, and effects of carbon sources (lactose, maltose, sucrose, amylum and glucose) and nitrogen sources (yeast extract, fish meal, corn steep liquor, soybean meal and cotton-seed meal) on 5KGA production were investigated. Results of experiment in 500 mL shake-flask show that the highest yield of 5KGA (98.20 g/L) was obtained using 100 g/L glucose as carbon source. 5KGA reached 100.20 g/L, 109.10 g/L, 99.83 g/L with yeast extract, fish meal and corn steep liquor as nitrogen source respectively, among which the optimal nitrogen source was fish meal. The yield of 5KGA by corn steep liquor is slightly lower than that by yeast extract. For the economic reason, corn steep liquor was selected as nitrogen source and scaled up to 5 L stirred-tank fermentor, and the final concentration of 5KGA reached 93.80 g/L, with its maximum volumetric productivity of 3.48 g/(L x h) and average volumetric productivity of 1.56 g/(L x h). The result obtained in this study showed that carbon and nitrogen sourses for large-scale production of 5KGA by Gluconobacter oxydans HGI-1 were glucose and corn steep liquor, respectively, and the available glucose almost completely (85.93%) into 5KGA.

  4. Pen and Pal are nucleotide-sugar dehydratases that convert UDP-GlcNAc to UDP-6-deoxy-D-GlcNAc-5,6-ene and then to UDP-4-keto-6-deoxy-L-AltNAc for CMP-pseudaminic acid synthesis in Bacillus thuringiensis.

    Science.gov (United States)

    Li, Zi; Hwang, Soyoun; Ericson, Jaime; Bowler, Kyle; Bar-Peled, Maor

    2015-01-09

    CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection. However, little is known about flagellin glycosylation in Gram-positive bacteria. Here, we identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. In contrast, Gram-negative bacteria complete this reaction with six enzymes. The first enzyme, which we named Pen, converts UDP-d-GlcNAc to an uncommon UDP-sugar, UDP-6-deoxy-D-GlcNAc-5,6-ene. Pen contains strongly bound NADP(+) and has distinct UDP-GlcNAc 4-oxidase, 5,6-dehydratase, and 4-reductase activities. The second enzyme, which we named Pal, converts UDP-6-deoxy-D-GlcNAc-5,6-ene to UDP-4-keto-6-deoxy-L-AltNAc. Pal is NAD(+)-dependent and has distinct UDP-6-deoxy-d-GlcNAc-5,6-ene 4-oxidase, 5,6-reductase, and 5-epimerase activities. We also show here using NMR spectroscopy and mass spectrometry that in B. thuringiensis, the enzymatic product of Pen and Pal, UDP-4-keto-6-deoxy-L-AltNAc, is converted to CMP-pseudaminic acid by the sequential activities of a C4″-transaminase (Pam), a 4-N-acetyltransferase (Pdi), a UDP-hydrolase (Phy), an enzyme (Ppa) that adds phosphoenolpyruvate to form pseudaminic acid, and finally a cytidylyltransferase that condenses CTP to generate CMP-pseudaminic acid. Knowledge of the distinct dehydratase-like enzymes Pen and Pal and their role in CMP-pseudaminic acid biosynthesis in Gram-positive bacteria provides a foundation to investigate the role of pseudaminic acid and flagellin glycosylation in Bacillus and their involvement in bacterial motility and pathogenicity.

  5. 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.

  6. Utilization of acidic α-amino acids as acyl donors: an effective stereo-controllable synthesis of aryl-keto α-amino acids and their derivatives.

    Science.gov (United States)

    Wang, Lei; Murai, Yuta; Yoshida, Takuma; Okamoto, Masashi; Tachrim, Zetryana Puteri; Hashidoko, Yasuyuki; Hashimoto, Makoto

    2014-05-16

    Aryl-keto-containing α-amino acids are of great importance in organic chemistry and biochemistry. They are valuable intermediates for the construction of hydroxyl α-amino acids, nonproteinogenic α-amino acids, as well as other biofunctional components. Friedel-Crafts acylation is an effective method to prepare aryl-keto derivatives. In this review, we summarize the preparation of aryl-keto containing α-amino acids by Friedel-Crafts acylation using acidic α-amino acids as acyl-donors and Lewis acids or Brönsted acids as catalysts.

  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. 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.

  9. Calcium salts of keto-amino acids, a phosphate binder alternative for patients on CAPD.

    Science.gov (United States)

    Macia, M; Coronel, F; Navarro, J F; Gallego, E; Herrero, J A; Méndez, M L; Chahin, J; García, J

    1997-09-01

    Control of hyperphosphoremia is crucial to the prevention of secondary hyperparathyroidism. Calcium salts of keto-amino acids (KAA) were employed as phosphate binders in hemodialysis patients. We wanted to assess the efficacy of these substances as quelating agents in patients under continuous ambulatory peritoneal dialysis (CAPD). Also, as an amino acid supplement, we determined their possible effect on some parameters related to nutritional status. We studied 13 patients (7 M; 6 F) with a mean age of 45.2 +/- 17 years and a mean time on CAPD of 18.4 +/- 11.4 months. None had severe secondary hyperparathyroidism and/or clinically relevant aluminium intoxication. They were not receiving calcitriol and none were using low-calcium peritoneal dialysis fluids. All were under aluminum hydroxide (AlOH3) treatment and 8 patients also received calcium carbonate. These quelating agents were withdrawn and after 21 days (wash-out period) KAA were initiated. We analyzed serum levels of bone metabolism parameters (calcium, phosphate, osteocalcin [OC], intact parathyroid hormone [iPTH], alkaline phosphatase [AP]) and nutritional parameters (total protein, albumin, pre-albumin, transferrin) in four periods: (A) during AlOH3; (B) immediately after the washout period; (C) after 1.5 months; and (D) after 3 months of KAA therapy. In 5 patients serum aluminum level was also measured in periods (A) and (D). The serum phosphate level at period (B) was significantly higher than in other periods. After 3 months of treatment phosphate levels decreased significantly (A = 1.77 +/- 0.3 mmol/l vs D = 1.48 +/- 0.2; p < 0.05). Serum calcium levels increased, while iPTH and OC decreased (p = ns). AP remained stable during the study. All nutritional parameters increased at the end of the study (p = ns). Calcium salts of keto-amino acids showed to be an effective alternative to aluminum-containing phosphate binders. They were well tolerated, without relevant side-effects. These compounds could also

  10. Synthesis and characterization of two new hydroxamic acids derivatives and their metal complexes. An investigation on the keto/enol, E/Z and hydroxamate/hydroximate forms

    Science.gov (United States)

    Adiguzel, Ekrem; Yilmaz, Fatih; Emirik, Mustafa; Ozil, Musa

    2017-01-01

    2-phenylbenzimidazole-N-acetohydroxamic acid (HL1), 2-phenylbenzimidazole-N-butanohydroxamic acid (HL2) and Ni(II), Cu(II), Zn(II) and Cd(II) metal complexes have been synthesized and characterized by elemental analyses, 1H NMR, 13C NMR, FT-IR spectrometry, LC-MS (ESI+) and thermal analyses. The results of NMR spectra and theoretical calculations showed that the hydroxamic acids were in the keto-E and keto-Z conformations. The elemental analysis and thermal analysis indicated that M:L ratio of the complexes are 1:1 and the spectral analysis confirmed that hydroxamate groups are keto form in the Ni(II) and Zn(II) complexes of 2-phenylbenzimidazole-N-butanohydroxamic acid and enol form in the other complexes.

  11. Reduction of Aromatic α-Keto Esters by Commercially Available Zinc Dust and Ammonium Formate:Formation of Aromatic a-Hydroxy Esters

    Institute of Scientific and Technical Information of China (English)

    CHEN Gang; YAO Guo-xin; SONG Guang-wei; ZHU Jin-tao

    2011-01-01

    Various aromatic α-keto esters were rapidly and selectively reduced to aromatic α-hydroxy esters by commercially available zinc dust and ammonium formate in the presence of other functional groups such as halogens,methoxy and esters.

  12. N-Heterocyclic Carbene-Catalyzed Vinylogous Mukaiyama Aldol Reaction of α-Keto Esters and α-Trifluoromethyl Ketones

    KAUST Repository

    Du, Guang-Fen

    2015-11-05

    © Georg Thieme Verlag Stuttgart · New York · Synthesis 2016. N-Heterocyclic carbene (NHC)-catalyzed vinylogous Mukaiyama aldol reaction of ketones was developed. Under the catalysis of 5 mol% NHC, α-keto esters and α-trifluoromethyl ketones reacted with 2-(trimethysilyloxy)furan efficiently to produce γ-substituted butenolides containing adjacent quaternary and tertiary carbon centers in high yields with good diastereoselectivities.

  13. 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

  14. Effects of Medicinal Cake-Separated Moxibustion on Plasma 6-Keto-PGF1α and TXB2 Contents in the Rabbit of Hyperlipemia

    Institute of Scientific and Technical Information of China (English)

    Chang Xiaorong; Yan Jie; Yue Zenghui; Shen Jing; Lin Yaping; Yi Shouxiang; Cao Xiangping

    2005-01-01

    Hyperlipemia rabbit models established with high cholesterol and fat diet were treated with direct moxibustion and medicinal cake-separated moxibustion. The post-treatment plasma 6-keto-prostaglandin F1α (6-keto-PGF1α) and thromboxane B2 (TXB2) contents were determined by radioimmunoassay. Results indicated that the plasma 6-keto-PGF1α content significantly increased, the TXB2 level decreased (P<0.05)and the TXB2 /6-keto-PGF1α ratio also decreased (P<0.01) in the medicinal cake-separated moxibustion group as compared with those in the model group respectively, but there was no significant difference between the medicinal cake-separated moxibustion group and the direct moxibustion group (P>0.05),suggesting that both the medicinal cake-separated moxibustion and direct moxibustion can regulate the plasma 6-keto-PGF1α and TXB2 contents, and the TXB2/6-keto-PGF1α ratio with similar actions, and have a certain protective action on endothelial cells of the aorta in the rabbit of hyperlipemia.

  15. [Optimization of the fermentation conditions for 5-keto-D-gluconic acid production].

    Science.gov (United States)

    Li, Boyi; Pan, Haifeng; Sun, Weirong; Cheng, Yongqing; Xie, Zhipeng; Zhang, Jianguo

    2014-09-01

    Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 5-keto-D-gluconic acid (5-KGA), a precursor of industrially important L(+)-tartaric acid. To increase the yield of 5-KGA, fermentation conditions of 5-KGA production was optimized. Under the optimum medium and culture conditions in the shake flask, the highest 5-KGA production reached 19.7 g/L, increased by 43.8% after optimization. In a 5-L bioreactor, the pH was controlled at 5.5 and dissolved oxygen (DO) at 15%, 5-KGA production reached 46.0 g/L, raised at least 1.3 times than in the shake flask. Glucose feeding fermentation process was further developed, and the highest 5-KGA production of 75.5 g/L with 70% of yield was obtained, 32.0% higher than the highest reported value. Therefore, this newly developed fermentation process provided a practical and effective alternative for the commercial production of 5-KGA, and further of L(+)-tartaric acid.

  16. Aroylhydrazone Cu(II Complexes in keto Form: Structural Characterization and Catalytic Activity towards Cyclohexane Oxidation

    Directory of Open Access Journals (Sweden)

    Manas Sutradhar

    2016-03-01

    Full Text Available The reaction of the Schiff base (3,5-di-tert-butyl-2-hydroxybenzylidene-2-hydroxybenzohydrazide (H3L with a copper(II salt of a base of a strong acid, i.e., nitrate, chloride or sulphate, yielded the mononuclear complexes [Cu(H2L(NO3(H2O] (1, [Cu(H2LCl]·2MeOH (2 and the binuclear complex [{Cu(H2L}2(µ-SO4]·2MeOH (3, respectively, with H2L− in the keto form. Compounds 1–3 were characterized by elemental analysis, Infrared (IR spectroscopy, Electrospray Ionisation Mass Spectrometry (ESI-MS and single crystal X-ray crystallography. All compounds act as efficient catalysts towards the peroxidative oxidation of cyclohexane to cyclohexyl hydroperoxide, cyclohexanol and cyclohexanone, under mild conditions. In the presence of an acid promoter, overall yields (based on the alkane up to 25% and a turnover number (TON of 250 (TOF of 42 h−1 after 6 h, were achieved.

  17. Acetyl-11-keto-β-boswellic acid (AKBA; targeting oral cavity pathogens

    Directory of Open Access Journals (Sweden)

    Shawl Abdul S

    2011-10-01

    Full Text Available Abstract Background Boswellic acids mixture of triterpenic acids obtained from the oleo gum resin of Boswellia serrata and known for its effectiveness in the treatment of chronic inflammatory disease including peritumor edema. Boswellic acids have been extensively studied for a number of activities including anti inflammatory, antitumor, immunomodulatory, and inflammatory bowel diseases. The present study describes the antimicrobial activities of boswellic acid molecules against oral cavity pathogens. Acetyl-11-keto-β-boswellic acid (AKBA, which exhibited the most potent antibacterial activity, was further evaluated in time kill studies, mutation prevention frequency, postantibiotic effect (PAE and biofilm susceptibility assay against oral cavity pathogens. Findings AKBA exhibited an inhibitory effect on all the oral cavity pathogens tested (MIC of 2-4 μg/ml. It exhibited concentration dependent killing of Streptococcus mutans ATCC 25175 up to 8 × MIC and also prevented the emergence of mutants of S.mutans ATCC 25175 at 8× MIC. AKBA demonstrated postantibiotic effect (PAE of 5.7 ± 0.1 h at 2 × MIC. Furthermore, AKBA inhibited the formation of biofilms generated by S.mutans and Actinomyces viscosus and also reduced the preformed biofilms by these bacteria. Conclusions AKBA can be useful compound for the development of antibacterial agent against oral pathogens and it has great potential for use in mouthwash for preventing and treating oral infections.

  18. Cardiovascular and antihypertensive actions of 1-methyl-3-keto-4-phenylquinuclidinium bromide.

    Science.gov (United States)

    Vidrio, H; Hong, E

    1976-01-01

    The sympatholytic and norepinephrine depleting drug 1-methyl-3-keto-4-phenylquinuclidinium bromide (MA540) possessed significant chronic antihypertensive activity in mecamylamine- and renal-hypertensive dogs. The compound was approximately four times more potent than guanethidine in the former model and three times as potent in the latter. MA540 reduced orthostatic blood pressure responses in unanesthetized rabbits, but was approximately ten times less potent than guanethidine. The quinuclidine derivative did not affect cardiac output, heart rate or stroke volume in anesthetized open chest dogs and moderately increased mean blood pressure and total peripheral resistance. It produced diuresis and saluresis in anesthetized dogs, but did not influence water or electrolyte urinary excretion in conscious rats. In the latter test, guanethidine produced antidiuresis and antisaluresis. It was concluded that MA540 is a potent, orally effective antihypertensive agent acting through adrenergic neuron blockade, that it lacks undesirable effects on cardiac and renal functions, and that compared with guanethidine, it is more potent in lowering blood pressure but less so in interfering with orthostatic cardiovascular reflexes.

  19. The Aldo Leopold' Ecological Holism Thought%略论利奥波德的生态整体主义思想

    Institute of Scientific and Technical Information of China (English)

    程李李; 王军; 杨绍陇

    2011-01-01

    As one of the theorists in Ecological Ethics, Aldo Leopold is also a philosopher who initiated the study of ethical holism systematically. In his posthumous work “A Sand County Almanac”, Leopold put forward many theories of ecological ethics, such as “the Value of Wildness”, “the Land Ethic” etc., expatiating his theory system of ecological holism. This article elaborated the establishment and contents of Leopold's ecological holism thoughts.%奥尔多.利奥波德是美国著名的生态伦理学家,也是最早系统深入地研究整体主义伦理观的哲学家.他在其遗著中,提出了"荒野的价值"与"大地伦理"等生态伦理理论,集中阐述了其生态整体主义的理论体系.本文正是对利奥波德生态整体主义思想的形成过程及其内容进行了全面考察.

  20. 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.

  1. Risk factors for mortality in children with diabetic keto acidosis from developing countries.

    Science.gov (United States)

    Poovazhagi, Varadarajan

    2014-12-15

    Diabetic keto acidosis (DKA) is the major cause for mortality in children with Diabetes mellitus (DM). With increasing incidence of type 1 DM worldwide, there is an absolute increase of DM among children between 0-14 year age group and overall incidence among less than 30 years remain the same. This shift towards younger age group is more of concern especially in developing countries where mortality in DKA is alarmingly high. Prior to the era of insulin, DKA was associated with 100% mortality and subsequently mortality rates have come down and is now, 0.15%-0.31% in developed countries. However the scenario in developing countries like India, Pakistan, and Bangladesh are very different and mortality is still high in children with DKA. Prospective studies on DKA in children are lacking in developing countries. Literature on DKA related mortality are based on retrospective studies and are very recent from countries like India, Pakistan and Bangladesh. There exists an urgent need to understand the differences between developed and developing countries with respect to mortality rates and factors associated with increased mortality in children with DKA. Higher mortality rates, increased incidence of cerebral edema, sepsis, shock and renal failure have been identified among DKA in children from developing countries. Root cause for all these complications and increased mortality in DKA could be delayed diagnosis in children from developing countries. This necessitates creating awareness among parents, public and physicians by health education to identify symptoms of DM/DKA in children, in order to decrease mortality in DKA. Based on past experience in Parma, Italy it is possible to prevent occurrence of DKA both in new onset DM and in children with established DM, by simple interventions to increase awareness among public and physicians.

  2. Risk factors for mortality in children with diabetic keto acidosis from developing countries

    Institute of Scientific and Technical Information of China (English)

    Varadarajan; Poovazhagi

    2014-01-01

    Diabetic keto acidosis(DKA) is the major cause for mortality in children with Diabetes mellitus(DM). With increasing incidence of type 1 DM worldwide, there is an absolute increase of DM among children between 0-14 year age group and overall incidence among less than 30 years remain the same. This shift towards younger age group is more of concern especially in developing countries where mortality in DKA is alarmingly high. Prior to the era of insulin, DKA was associated with 100% mortality and subsequently mortality rates have come down and is now, 0.15%-0.31% in developed countries. However the scenario in developing countries like India, Pakistan, and Bangladesh are very different and mortality is still high in children with DKA. Prospective studies on DKA in children are lacking in developing countries. Literature on DKA related mortality are based on retrospective studies and are very recent from countries like India, Pakistan and Bangladesh. There exists an urgent need to understand the differences between developed and developing countries with respect to mortality rates and factors associated with increased mortality in children with DKA. Higher mortality rates, increased incidence of cerebral edema, sepsis, shock and renal failure have been identified among DKA in children from developing countries.Root cause for all these complications and increased mortality in DKA could be delayed diagnosis in children from developing countries. This necessitates creating awareness among parents, public and physicians by health education to identify symptoms of DM/DKA in children, in order to decrease mortality in DKA. Based on past experience in Parma, Italy it is possible to prevent occurrence of DKA both in new onset DM and in children with established DM, by simple interventions to increase awareness among public and physicians.

  3. Short-chain dehydrogenase/reductase catalyzing the final step of noscapine biosynthesis is localized to laticifers in opium poppy.

    Science.gov (United States)

    Chen, Xue; Facchini, Peter J

    2014-01-01

    The final step in the biosynthesis of the phthalideisoquinoline alkaloid noscapine involves a purported dehydrogenation of the narcotinehemiacetal keto moiety. A short-chain dehydrogenase/reductase (SDR), designated noscapine synthase (NOS), that catalyzes dehydrogenation of narcotinehemiacetal to noscapine was identified in opium poppy and functionally characterized. The NOS gene was isolated using an integrated transcript and metabolite profiling strategy and subsequently expressed in Escherichia coli. Noscapine synthase is highly divergent from other characterized members of the NADPH-dependent SDR superfamily involved in benzylisoquinoline alkaloid metabolism, and it exhibits exclusive substrate specificity for narcotinehemiacetal. Kinetic analyses showed that NOS exhibits higher catalytic efficiency with NAD+ as the cofactor compared with NADP+. Suppression of NOS transcript levels in opium poppy plants subjected to virus-induced gene silencing resulted in a corresponding reduction in the accumulation of noscapine and an increase in narcotinehemiacetal levels in the latex. Noscapine and NOS transcripts were detected in all opium poppy organs, but both were most abundant in stems. Unlike other putative biosynthetic genes clustered in the opium poppy genome, and their corresponding proteins, NOS transcripts and the cognate enzyme were abundant in latex, indicating that noscapine metabolism is completed in a distinct cell type compared with the rest of the pathway.

  4. Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Pennacchio, Angela; Giordano, Assunta; Pucci, Biagio; Rossi, Mosè; Raia, Carlo A

    2010-03-01

    The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

  5. 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.

  6. 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

  7. Expression of Mitochondrial Branched-Chain Aminotransferase and α-Keto-Acid Dehydrogenase in Rat Brain: Implications for Neurotransmitter Metabolism

    Directory of Open Access Journals (Sweden)

    Jeffrey Thomas Cole

    2012-05-01

    Full Text Available In the brain, metabolism of the essential branched chain amino acids (BCAAs leucine, isoleucine and valine, is regulated in part by protein synthesis requirements. Excess BCAAs are catabolized or excreted. The first step in BCAA catabolism is catalyzed by the branched chain aminotransferase (BCAT isozymes, mitochondrial BCATm and cytosolic BCATc. A product of this reaction, glutamate, is the major excitatory neurotransmitter and precursor of the major inhibitory neurotransmitter -aminobutyric acid (GABA. The BCATs are thought to participate in an α-keto-acid nitrogen shuttle that provides nitrogen for synthesis of glutamate from -ketoglutarate. The branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC catalyzes the second and first irreversible step in BCAA metabolism, which is oxidative decarboxylation of the branched-chain α-keto acid (BCKA products of the BCAT reaction. Maple Syrup Urine Disease (MSUD results from genetic defects in BCKDC, which leads to accumulation of toxic levels of BCAAs and BCKAs that result in brain swelling. Immunolocalization of BCATm and BCKDC in rats revealed that BCATm is present in astrocytes in white matter and in neuropil, while BCKDC is expressed only in neurons. BCATm appears uniformly distributed in astrocyte cell bodies throughout the brain. The segregation of BCATm to astrocytes and BCKDC to neurons provides further support for the existence of a BCAA-dependent glial-neuronal nitrogen shuttle since the data show that BCKAs produced by glial BCATm must be exported to neurons. Additionally, the neuronal localization of BCKDC suggests that MSUD is a neuronal defect involving insufficient oxidation of BCKAs, with secondary effects extending beyond the neuron.

  8. Effect of ropivacaine-fentanyl combined with spinal-epidural labor analgesia on maternal serum markers andTXB2/6-Keto-PGF1α proportion

    Institute of Scientific and Technical Information of China (English)

    Yan Huang

    2015-01-01

    Objective:To study the effect of ropivacaine-fentanyl combined with spinal–epidural labor analgesia on maternal serum markers and TXB2/6-Keto-PGF1α proportion.Methods:40 cases of puerperas intending to receive vaginal delivery in our hospital were randomly divided into two groups. Combined group received fentanyl subarachnoid injection combined with ropivacaine epidural injection and control group received fentanyl combined with ropivacaine epidural injection. Then levels of serum pain mediators and inflammation related factors as well as TXB2/6-Keto-PGF1α proportion of both groups were detected. Results:In delivery process, contents of serum pain mediatorsβ-EP, NO, SP, PGE2, TNF-α, IL-6, CCL2 and CCR2 of combined group were significantly lower than those of control group, and IL-10 and TGb-βcontents were significantly higher than those of control group; In and after labor, serum TXB2 content and TXB2/6-keto-PGF1α proportion of combined group were lower than those of control group, while 6-keto-PGF1α content was higher than that of control group.Conclusion:Ropivacaine-fentanyl combined with spinal–epidural labor analgesia can achieve more precise analgesia effect, reduce contents of pain mediators, pro-inflammatory cytokines and chemokines, and regulate TXB2/6-Keto-PGF1ααbalance.

  9. Intersystem crossing rates of S1 state keto-amino cytosine at low excess energy

    Science.gov (United States)

    Lobsiger, Simon; Etinski, Mihajlo; Blaser, Susan; Frey, Hans-Martin; Marian, Christel; Leutwyler, Samuel

    2015-12-01

    The amino-keto tautomer of supersonic jet-cooled cytosine undergoes intersystem crossing (ISC) from the v = 0 and low-lying vibronic levels of its S1(1ππ∗) state. We investigate these ISC rates experimentally and theoretically as a function of S1 state vibrational excess energy Eexc. The S1 vibronic levels are pumped with a ˜5 ns UV laser, the S1 and triplet state ion signals are separated by prompt or delayed ionization with a second UV laser pulse. After correcting the raw ISC yields for the relative S1 and T1 ionization cross sections, we obtain energy dependent ISC quantum yields QISC corr = 1 % -5%. These are combined with previously measured vibronic state-specific decay rates, giving ISC rates kISC = 0.4-1.5 ṡ 109 s-1, the corresponding S1⇝S0 internal conversion (IC) rates are 30-100 times larger. Theoretical ISC rates are computed using SCS-CC2 methods, which predict rapid ISC from the S1; v = 0 state with kISC = 3 ṡ 109 s-1 to the T1(3ππ∗) triplet state. The surprisingly high rate of this El Sayed-forbidden transition is caused by a substantial admixture of 1nOπ∗ character into the S1(1ππ∗) wave function at its non-planar minimum geometry. The combination of experiment and theory implies that (1) below Eexc = 550 cm-1 in the S1 state, S1⇝S0 internal conversion dominates the nonradiative decay with kIC ≥ 2 ṡ 1010 s-1, (2) the calculated S1⇝T1 (1ππ∗⇝3ππ∗) ISC rate is in good agreement with experiment, (3) being El-Sayed forbidden, the S1⇝T1 ISC is moderately fast (kISC = 3 ṡ 109 s-1), and not ultrafast, as claimed by other calculations, and (4) at Eexc ˜ 550 cm-1 the IC rate increases by ˜50 times, probably by accessing the lowest conical intersection (the C5-twist CI) and thereby effectively switching off the ISC decay channels.

  10. Single step synthesis of strigolactone analogues from cyclic keto enols, germination stimulants for seeds of parasitic weeds.

    Science.gov (United States)

    Mwakaboko, Alinanuswe S; Zwanenburg, Binne

    2011-08-15

    The single step synthesis of a newly designed series of strigolactones (SLs) from cyclic keto enols is described. The germinating activity of these SL analogues towards seeds of the parasitic weeds Striga and Orobanche spp. is reported. The first of these SL analogues are derived from the hydroxyl γ-pyrones kojic acid and maltol, the second type from hydroxyl α-pyrones, namely, 4-hydroxy-6-methyl-2H-pyran-2-one and 4-hydroxy-coumarin and the third type from 1,3-diketones, namely, 1,3-cyclohexane-dione (dimedone) and tricyclic 1,3-dione. All keto enols are coupled in a single step with the appropriate D-ring precursor in the presence of a base to give the desired SL analogues. All SL analogues are acceptably biologically active in inducing the germination of seeds of Striga hermonthica and Orobanchecernua. Most interesting are the analogues derived from 4-hydroxy coumarin and dimedone, as they have a remarkably high biological activity towards the seeds of parasitic weeds at relatively low concentrations, comparable with that of the general standard stimulant GR24.

  11. Solvent dependency of the UV-Vis spectrum of indenoisoquinolines: role of keto-oxygens as polarity interaction probes.

    Directory of Open Access Journals (Sweden)

    Andrea Coletta

    Full Text Available Indenoisoquinolines are the most promising non-campthotecins topoisomerase IB inhibitors. We present an integrated experimental/computational investigation of the UV-Vis spectra of the IQNs parental compound (NSC314622 and two of its derivatives (NSC724998 and NSC725776 currently undergoing Phase I clinical trials. In all the three compounds a similar dependence of the relative absorption intensities at 270 nm and 290 nm on solvent polarity is found. The keto-oxygens in positions 5 and 11 of the molecular scaffold of the molecule are the principal chromophores involved in this dependence. Protic interactions on these sites are also found to give rise to absorptions at wavelength <250 nm observed in water solution, due to the stabilization of highly polarized tautomers of the molecule. These results suggest that the keto-oxygens are important polarizable groups that can act as useful interactors with the molecular receptor, providing at the same time an useful fingerprint for the monitoring of the drug binding to topoisomerase IB.

  12. 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.

  13. 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.

  14. 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...

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Determination of Solvent Effects on Keto-Enol Equilibria of 1,3-Dicarbonyl Compounds Using NMR: Revisiting a Classic Physical Chemistry Experiment

    Science.gov (United States)

    Cook, A. Gilbert; Feltman, Paul M.

    2007-01-01

    The use of proton NMR to determine the equilibrium position of tautomeric 1,3-dicarbonyl compounds in various solvents has been a classic physical chemistry experiment. We are presenting an expansion of the excellent description of this experiment by Garland, Shoemaker, and Nibler. Often the assumption is made that the keto tautomer is always the…

  1. Determination of pKa and Hydration Constants for a Series of α-Keto-Carboxylic Acids Using Nuclear Magnetic Resonance Spectrometry.

    Science.gov (United States)

    Lopalco, Antonio; Douglas, Justin; Denora, Nunzio; Stella, Valentino J

    2016-02-01

    The determination of the acid-base dissociation constants, and thus the pKa values, of α-keto acids such as pyruvic acid is complex because of the existence of these acids in their hydrated and nonhydrated or oxo state. Equilibria involved in the hydration and dehydration of the α-keto group of pyruvic acid and three other α-keto acids, 3-methyl-2-oxobutanoic acid, 4-methyl-2-oxopentanoic acid, and 2-oxo-2-phenylacetic acid, were investigated by proton and carbon nuclear magnetic resonance spectrometry, at constant ionic strength, 0.15, and 25 °C. Dissociation constants for the oxo (pKa(oxo)) and hydrated (pKa(hyd)) acids of each compound were estimated from the change in the degree of hydration with changes in pH and directly from the changes in chemical shifts of various hydrogen and carbons nuclei with pH. α-Keto acids showed greater hydration in their acidic forms than their carboxylate forms. The degree of hydration was sensitive to steric and electronic/resonance factors. As expected, the oxo forms of the acids were stronger acids compared with their hydrated analogs, and their dissociation constants were also sensitive to steric and electronic factors.

  2. Structural insight into substrate binding and catalysis of a novel 2-keto-3-deoxy-D-arabinonate dehydratase illustrates common mechanistic features of the FAH superfamily

    NARCIS (Netherlands)

    Brouns, S.J.J.; Barends, T.R.M.; Worm, P.; Akerboom, J.; Turnbull, A.P.; Salmon, L.; Oost, van der J.

    2008-01-01

    The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3-deoxy-D-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzym

  3. Deposition Form and Bioaccessibility of Keto-carotenoids from Mamey Sapote (Pouteria sapota), Red Bell Pepper (Capsicum annuum), and Sockeye Salmon (Oncorhynchus nerka) Filet.

    Science.gov (United States)

    Chacón-Ordóñez, Tania; Esquivel, Patricia; Jiménez, Víctor M; Carle, Reinhold; Schweiggert, Ralf M

    2016-03-09

    The ultrastructure and carotenoid-bearing structures of mamey sapote (Pouteria sapota) chromoplasts were elucidated using light and transmission electron microscopy and compared to carotenoid deposition forms in red bell pepper (Capsicum annuum) and sockeye salmon (Oncorhynchus nerka). Globular-tubular chromoplasts of sapote contained numerous lipid globules and tubules embodying unique provitamin A keto-carotenoids in a lipid-dissolved and presumably liquid-crystalline form, respectively. Bioaccessibility of sapotexanthin and cryptocapsin was compared to that of structurally related keto-carotenoids from red bell pepper and salmon. Capsanthin from bell pepper was the most bioaccessible pigment, followed by sapotexanthin and cryptocapsin esters from mamey sapote. In contrast, astaxanthin from salmon was the least bioaccessible keto-carotenoid. Thermal treatment and fat addition consistently enhanced bioaccessibility, except for astaxanthin from naturally lipid-rich salmon, which remained unaffected. Although the provitamin A keto-carotenoids from sapote were highly bioaccessible, their qualitative and quantitative in vivo bioavailability and their conversion to vitamin A remains to be confirmed.

  4. Highly functionalized 1,2-diamino compounds through reductive amination of amino acid-derived β-keto esters.

    Directory of Open Access Journals (Sweden)

    Paula Pérez-Faginas

    Full Text Available 1,2-Diamine derivatives are valuable building blocks to heterocyclic compounds and important precursors of biologically relevant compounds. In this respect, amino acid-derived β-keto esters are a suitable starting point for the synthesis of β,γ-diamino ester derivatives through a two-step reductive amination procedure with either simple amines or α-amino esters. AcOH and NaBH(3CN are the additive and reducing agents of choice. The stereoselectivity of the reaction is still an issue, due to the slow imine-enamine equilibria through which the reaction occurs, affording mixtures of diastereoisomers that can be chromatographically separated. Transformation of the β,γ-diamino esters into pyrrolidinone derivatives allows the configuration assignment of the linear compounds, and constitutes an example of their potential application in the generation of molecular diversity.

  5. Semi-continuous production of 2-keto-gluconic acid by Pseudomonas fluorescens AR4 from rice starch hydrolysate.

    Science.gov (United States)

    Sun, Wen-Jing; Zhou, Yan-Zheng; Zhou, Qiang; Cui, Feng-Jie; Yu, Shi-Lian; Sun, Lei

    2012-04-01

    2-Keto-gluconic acid (2KGA) was produced in a semi-continuous process using Pseudomonas fluorescens AR4 and rice starch hydrolysate (RSH). The bacterium was cultured in medium with an initial glucose concentration of 170g/L supplied as RSH. Once the glucose level had dropped to 20g/L, 60% of the culture volume was replaced with fresh medium containing 190g/L of glucose in the form of RSH. After an additional two cycles of growth and media replacement, a total of 476.88g/L of glucose was consumed and 444.96g/L of 2KGA was produced. A total productivity of 6.74g/L and a yield of 0.93g/g were obtained. These findings suggest that P. fluorescens AR4 is suitable for the production of commercially acceptable levels of 2KGA in semi-continuous culture.

  6. Synthesis, spectroscopic characterization and solution behavior of new tin tetrachloride adducts with γ-keto allyl phosphonates

    Science.gov (United States)

    Elleuch, Haitham; Sanhoury, M. A. K.; Rezgui, F.

    2017-01-01

    Four new octahedral complexes of the type [SnCl4L2] (L = γ-keto allyl phosphonate) (1-4) were prepared and characterized by multinuclear (1H, 13C, 31P and 119Sn) NMR, IR spectroscopy and elemental analysis. The NMR data show, as expected, that these complexes exist in solution as mixtures of cis and trans isomers. More importantly, the solution structure was confirmed by 119Sn NMR spectra which show two triplets corresponding to the two isomers. In addition, the solution behavior of these complexes in the presence of excess ligand was studied by variable temperature NMR using the coalescence temperature method. The metal-ligand exchange activation energies were therefore determined and found to be in the range 57-60 kJ/mol. The effect of remote substituents on the metal-ligand interaction was studied and compared with closely related tin-phosphoryl complexes.

  7. Theoretical study on the reaction mechanism of ozone addition to the double bonds of keto-limonene

    Institute of Scientific and Technical Information of China (English)

    Lei Jiang; Yisheng Xu; Baohui Yin; Zhipeng Bai

    2012-01-01

    The reaction mechanism of ozone (O3) addition to the double bonds of gas phase keto-limonene was investigated using ab initio methods.Two different possibilities for O3 addition to the double bond were considered and two corresponding van der Waais complexes (Complex 1 and Complex 2) were found for 1-endo and 2-endo.The rate constants were calculated using the transition state theory at the CCSD(T)/6-31G(d) + CF//B3LYP/6-31G(d,p) level.The high-pressure limit of the total rate constant at 298 K was 3.51 × 10-16cm3/(molecule.sec),which was in a good agreement with the experimental data.

  8. α-Hydroxy-β-keto acid rearrangement-decarboxylation: impact on thiamine diphosphate-dependent enzymatic transformations.

    Science.gov (United States)

    Beigi, Maryam; Loschonsky, Sabrina; Lehwald, Patrizia; Brecht, Volker; Andrade, Susana L A; Leeper, Finian J; Hummel, Werner; Müller, Michael

    2013-01-14

    The thiamine diphosphate (ThDP) dependent MenD catalyzes the reaction of α-ketoglutarate with pyruvate to selectively form 4-hydroxy-5-oxohexanoic acid 2, which seems to be inconsistent with the assumed acyl donor role of the physiological substrate α-KG. In contrast the reaction of α-ketoglutarate with acetaldehyde gives exclusively the expected 5-hydroxy-4-oxo regioisomer 1. These reactions were studied by NMR and CD spectroscopy, which revealed that with pyruvate the observed regioselectivity is due to the rearrangement-decarboxylation of the initially formed α-hydroxy-β-keto acid rather than a donor-acceptor substrate role variation. Further experiments with other ThDP-dependent enzymes, YerE, SucA, and CDH, verified that this degenerate decarboxylation can be linked to the reduced enantioselectivity of acyloins often observed in ThDP-dependent enzymatic transformations.

  9. Mechanisms of activation of muscle branched-chain alpha-keto acid dehydrogenase during exercise in man

    DEFF Research Database (Denmark)

    Van Hall, Gerrit; MacLean, D A; Saltin, B

    1996-01-01

    1. Exercise leads to activation (dephosphorylation) of the branched-chain alpha-keto acid dehydrogenase (BCKADH). Here we investigate the effect of low pre-exercise muscle glycogen content and of branched-chain amino acid (BCAA) ingestion on the activity of BCKADH at rest and after 90 min of one......-leg knee-extensor exercise at 65% maximal one-leg power output in five subjects. 2. Pre-exercise BCAA ingestion (308 mg BCAAs (kg body wt)-1) caused an increased muscle BCAA uptake, a higher intramuscular BCAA concentration and activation of BCKADH both at rest (9 +/- 1 versus 25 +/- 5% for the control...... and BCAA test, respectively) and after exercise (27 +/- 4 versus 54 +/- 7%). 3. At rest the percentage active BCKADH was not different, 6 +/- 2% versus 5 +/- 1%, in the normal and low glycogen content leg (392 +/- 21 and 147 +/- 34 mumol glycosyl units (g dry muscle)-1, respectively). The post...

  10. Synthesis, spectral studies, antimicrobial, antioxidant and insect antifeedant activities of some 9 H-fluorene-2-yl keto-oxiranes

    Science.gov (United States)

    Thirunarayanan, G.; Vanangamudi, G.

    2011-10-01

    Thirteen ee (α S, β R) 9 H-fluorene-2-yl keto-oxiranes (2-(9 H)-fluorene-4-yl[3-(substituted phenyl)oxiran-2-yl]methanones) have been synthesized by phase transfer catalysed epoxidation of 9 H-fluorene-2-yl chalcones. The yields of oxiranes are more than 95%. The synthesized oxiranes have been characterized by IR, 1H, 13C and GC-MS spectral data. The spectral data are correlated with Hammett substituent constants and Swain-Lupton parameters. From the regression analysis, the effect of substituents on the group frequencies has been predicted. The antimicrobial, antioxidant and insect antifeedant activities of all the synthesized oxiranes have been studied.

  11. 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.

  12. 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.

  13. Evaluation of plasma and urinary levels of 6-keto-prostaglandin F1a as a marker for asymptomatic myxomatous mitral valve disease in dogs

    DEFF Research Database (Denmark)

    Rasmussen, Caroline Elisabeth; Sundqvist, Anna Vilhelmina; Kjempff, Christina Tirsdal;

    2010-01-01

    ) to compare plasma and urinary 6-keto-PGF(1alpha) in dogs with asymptomatic MMVD. The study included two breeds predisposed to MMVD and two control groups (Cairn terriers and dogs of different breeds). Echocardiography was used to estimate the severity of MMVD. The intra- and inter-assay coefficients...... of variation were between 3.1% and 24.5% in the assay range. No echocardiographic parameter was correlated with plasma or urinary 6-keto-PGF(1alpha) (P>0.05), but all control dogs had lower urinary 6-keto-PGF(1alpha) (P

  14. 酮缬氨酸钙与酮异亮氨酸钙对人肾系膜细胞增殖影响及机制研究%Effects of keto-valine-calcium and keto-isoleucine-calcium on proliferation of human mesangial cells and its mechanism study

    Institute of Scientific and Technical Information of China (English)

    李洁; 周平; 胡秀梅; 刘伏友; 彭佑铭

    2008-01-01

    Objective To study the effects of keto-valine-calcium and keto-isoleucine-calcium on(human mesangial cells)HMCs. Method HMCs were stimulated by keto-valine-calcium and keto-isoleucine-calcium. The AT1R and TGF-β1 were detected. MTr method was used to measure the proliferation of HMCs, and cell cycle was studied by flow cytometry. Results The expression of AT1R and TGF-β1was increased in the experiment groups compared with negative and DMSO control groups. Cell cycle G1 attest and cell apoptosis were observed in the experiment groups. Conclusions 10mM keto-valine-calcium and keto-isoleueine-calcium have multiple effects on HMCs in vitro, which not only increased the expression of AT1R,but also the expression of TGF-β1.Furthermore,keto-valine-calcium and keto-isoleucine-calcium can induce cell cycle G1 arrest and cell apoptosis.to%目的 体外观察酮缬氨酸钙和酮异亮氨酸钙对人肾系膜细胞(HMC)的影响.方法 10mM酮缬氨酸钙和酮异亮氨酸钙对HMC体外干预,检测干预后转化生长因子-β1(TGF-β1)和血管紧张素Ⅱ-1型受体(AT1R)的变化,并采用MTT和流式细胞仪检测细胞增殖、凋亡和细胞周期.结果 10mM酮缬氨酸钙和酮异亮氨酸钙干预HMC,实验组与对照组相比,TGF-β1含量增加;细胞AT1R表达亦有增加;同时细胞出现增殖抑制,细胞流式分析发现,实验组干预后出现了细胞周期G1期阻滞并观测到了细胞凋亡.结论 10mM的酮缬氨酸钙和酮异亮氨酸钙可以增加ATlR在HMC的表达,诱导TGF-β1的表达增加;同时诱导细胞周期G1阻滞并伴随细胞凋亡.

  15. 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.

  16. 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.

  17. Sub anno domini 1465... In casa de’ figliuoli di Aldo. Origin and advancements of the printing press in Italy through the exemplars of the Bagnacavallo ‘Giuseppe Taroni’ library

    Directory of Open Access Journals (Sweden)

    Federica Fabbri

    2016-11-01

    Full Text Available The paper contains the descriptions of the ancient books owned by the Public Library ‘Giuseppe Taroni’ in Bagnacavallo, which were selected for the exhibition Sub anno domini 1465... In casa de’ figliuoli di Aldo to celebrate the printing of the first book in Italy by Konrad Sweynheym and Arnold Pannartz with datation included in the colophon (Subiaco, 1465 and the fifth centenary of Aldus Manutius’ death, the major Italian printer and publisher of the Renaissance age (1449-52?-1515. Among the exhibited copies the one belonging to the Roman edition of 1470 of the work of the Christian scholar Lactantius, the only one copy printed by Sweynheym and Pannartz owned by the Library in Bagnacavallo, and a selection of books printed by Aldus Manutius and his heirs, among which the only copy found in this library of a book printed by Aldus The Elder, belonging to Institutiones Grammaticae of 1514.

  18. 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.

  19. 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.

  20. 6-Iso-chlortetracycline or keto form of chlortetracycline? Need for clarification for relevant monitoring of chlortetracycline residues in food.

    Science.gov (United States)

    Gaugain, Murielle; Gautier, Sophie; Bourcier, Sophie; Jacques, Anne-Marie; Laurentie, Michel; Abjean, Jean-Pierre; Hurtaud-Pessel, Dominique; Verdon, Eric

    2015-01-01

    Chlortetracycline (CTC) is a broad-spectrum antibiotic used in veterinary medicine for pulmonary or digestive infections and having a regulatory maximum residue limit (MRL) necessitating an official analytical control method. The purpose of this study was to clarify the identification of different forms of CTC observed in standard solution, in spiked muscle samples and in naturally incurred muscle samples of pigs analysed by LC-MS/MS and to demonstrate the in vivo formation of 6-iso-chlortetracycline and 4-epi-6-iso-CTC as a metabolite of CTC and 4-epi-CTC in muscle. The six following forms were identified, all being isobaric with a protonated molecule at m/z 479 (precursor ion): the keto-enol forms of CTC and the keto-enol forms of 4-epi-chlortetracycline (4-epi-CTC), 6-iso-chlortetracycline (6-iso-CTC) and 4-epi-6-iso-chlortetracycline (4-epi-6-iso-CTC). The 6-iso-CTC and 4-epi-6-iso-CTC were observed only in incurred pig samples so were identified for the first time as metabolites of CTC and 4-epi-CTC. Identification of the different forms was obtained by comparing incurred muscle samples with standard solutions and with spiked samples. Then the differences between the features of the chromatograms obtained by LC-TQ-MS and the fragmentation study of the different forms of CTC obtained by LC-Q-TOF-MS helped us to support this identification. The extraction steps and the LC-MS/MS conditions developed to analyse muscle tissue samples are described. This clarification concerning the rigorous identification of chromatographic peaks allowed us to evaluate the relevance of our monitoring method with regard to the regulations in place in the European Union and could be of help to laboratories involved in official control of antibiotic residues in food of animal origin. Additional results are also presented highlighting the transformation of the CTC when prepared in a mixture with other antibiotics.

  1. Synthesis of Novel β-Keto-Enol Derivatives Tethered Pyrazole, Pyridine and Furan as New Potential Antifungal and Anti-Breast Cancer Agents

    Directory of Open Access Journals (Sweden)

    Smaail Radi

    2015-11-01

    Full Text Available Recently, a new generation of highly promising inhibitors bearing β-keto-enol functionality has emerged. Reported herein is the first synthesis and use of novel designed drugs based on the β-keto-enol group embedded with heterocyclic moieties such as pyrazole, pyridine, and furan, prepared in a one-step procedure by mixed Claisen condensation. All the newly synthesized compounds were characterized by FT-IR, 1H-NMR, 13C-NMR, ESI/LC-MS, elemental analysis, and evaluated for their in vitro antiproliferative activity against breast cancer (MDA-MB241 human cell lines and fungal strains (Fusarium oxysporum f.sp albedinis FAO. Three of the synthesized compounds showed potent activity against fungal strains with IC50 values in the range of 0.055–0.092 µM. The results revealed that these compounds showed better IC50 values while compared with positive controls.

  2. 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.

  3. A novel carbazole-based fluorescent probe:3,6-Bis-[(N-ethylcarbazole-3-y1)-propene-1-keto]-N-ethylcarbazole

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A novel carbazole-based compound 5,3,6-bis[(N-ethylcarbazole-3-yl)-propene-1-keto]-N-ethylcarbazole has been designed, synthesized and characterized.The absorption and fluorescence spectra in solvents of different polarities prove that the compound has a distinct intramolecular charge transfer character.Compound 5 can be used as a new class of fluorescent probe or biosensor due to its sensitivity to the local microenvironment such as solvent polarity.

  4. 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.

  5. 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...

  6. 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

  7. 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...

  8. 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

  9. 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...

  10. 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.

  11. Theoretical and vibrational spectroscopic approach to keto-enol tautomerism in methyl-2-(4-methoxybenzoyl)-3-(4-methoxyphenyl)-3-oxopropanoylcarbamate

    Science.gov (United States)

    Arı, Hatice; Özpozan, Talat; Büyükmumcu, Zeki; Kabacalı, Yiğit; Saçmaci, Mustafa

    2016-10-01

    A carbamate compound having tricarbonyl groups, methyl-2-(4-methoxybenzoyl)-3-(4-methoxyphenyl)-3-oxopropanoylcarbamate (BPOC) was investigated from theoretical and vibrational spectroscopic point of view employing quantum chemical methods. Hybrid Density Functionals (B3LYP, X3LYP and B3PW91) with 6-311 G(d,p) basis set were used for the calculations. Rotational barrier and conformational analyses were performed to find the most stable conformers of keto and enol forms of the molecule. Three transition states for keto-enol tautomerism in gas phase were determined. The results of the calculations show that enol-1 form of BPOC is more stable than keto and enol-2 forms. Hydrogen bonding investigation including Natural bond orbital analysis (NBO) for all the tautomeric structures was employed to compare intra-molecular interactions. The energies of HOMO and LUMO molecular orbitals for all tautomeric forms of BPOC were predicted. Normal Coordinate Analysis (NCA) was carried out for the enol-1 to assign vibrational bands of IR and Raman spectra. The scaling factors were calculated as 0.9721, 0.9697 and 0.9685 for B3LYP, X3LYP and B3PW91 methods, respectively. The correlation graphs of experimental versus calculated vibrational wavenumbers were plotted and X3LYP method gave better frequency agreement than the others.

  12. HPLC Determination of α-keto Acids from Human Serum Using 2,3-Diamino-2,3-dimethylbutane as Derivatizing Reagent

    Directory of Open Access Journals (Sweden)

    Khalida Parveen Mahar

    2013-12-01

    Full Text Available Seven α-keto acids, pyruvic acid (PYR, 2-oxobutyric acid (KB, 3-methyl-2-oxobutyric acid (MKBA, 3-methyl-2-oxovaleric acid (K3MVA, 4-methyl-2-oxovaleric acid (K4MVA, 2-oxoglutaric acid (KG and Phenyl pyruvic acid (PPY as derivatives of 2,3-diamino-2,3-dimethybutane (DDB were separated by HPLC column Zorbax C–18 (4.6x150 mm-id. The compounds were eluted with methanol-water-acetonitrile (40:58:2 V/V/V with flow rate 1 ml/min. UV detection was carried out by photodiode array at 255 nm. Linear calibration plots were obtained with 0.1 to 60 µg/ml with limits of detection (LoD within 0.04-0.4 µg/ml. The method was applied for the analysis of α-keto acids from serum of diabetic patients with blood glucose level 430-458 mg/dl and healthy volunteers. The amounts of α-keto acids observed 3.24-9.71 µg/ml with RSD 1.1-1.9% in diabetic patients were higher than healthy volunteer’s 0.11-1.3 µg/ml with RSD 0.9-2.6%.

  13. Effect of compoundα-keto acid tablets on nutritional status, calcium-phosphorus metabolism and inflammatory factors in patients with maintenance hemodialysis

    Institute of Scientific and Technical Information of China (English)

    Min-Hui Xi; Pei-Ju Mao; Dan-Huan Zhang; Ling-Yun Chen

    2015-01-01

    Objective:To investigate the effect of compoundα-keto acid tablets on nutritional status, calcium-phosphorus metabolism and inflammatory factors in patients with maintenance hemodialysis.Methods: 84 cases with maintenance hemodialysis were collected, according to the order of treatment were randomly divided into the observation group and control group, each of 42 cases. The control group was treated with maintenance hemodialysis, on the basis of this; the observation group was treated with compound-1α-keto acid tablets. The serum total protein(TP), serum albumin(ALB), hemoglobin (HB), transferrin (TRF), calcium (Ca), phosphorus (P), calcium phosphorus product (Ca×P), serum intact parathyroid hormone (iPTH), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and high sensitive C reactive protein (hs-CRP) of two groups were tested before and after treatment.Results:After treatment, the TP, ALB, HB, TRF of observation group were significantly higher than those in the control group (P<0.05), and blood P, blood Ca×P, serum iPTH were significantly lower than the control group (P<0.05), serum IL-6, TNF-alpha and hs-CRP levels were significantly lower than those of the control group (P<0.05).Conclusion:Compoundα-keto acid tablets can effectively improve the nutritional status of hemodialysis patients, maintain the metabolism of calcium and phosphorus balance, and can reduce the inflammatory reaction, is worth the clinical promotion and application.

  14. Effect of adjuvantα-keto acid therapy on serum renal function indexes and nutritional status in patients with early diabetic nephropathy

    Institute of Scientific and Technical Information of China (English)

    Min Zhang; Lu Wang

    2016-01-01

    Objective:To analyze the effect of adjuvantα-keto acid therapy on serum renal function indexes and nutritional status in patients with early diabetic nephropathy.Methods:A total of 86 patients with early diabetic nephropathy were divided into observation group and control group, control group received conventional therapy, observation group received conventional therapy + adjuvantα-keto acid therapy, and then differences in serum renal function indexes, nutrition index, vascular endothelial function indexes, micro-inflammation indexes and so on were compared between two groups of patients after 2 months of treatment.Results: Serum renal function indexes UAER level as well as BUN, SCr, CysC andβ2-MG content of observation group was lower than those of control group while GFR level was higher than that of control group; nutrition indexes ALB, PA and Hb content were higher than those of control group while nPCR level was lower than that of control group; vascular endothelial function indexes NO and CGRP content were higher than those of control group while ET-1, MCP-1 and resistin content were lower than those of control group; micro-inflammation indexes hs-CRP, IL-1β, IL-17, IL-22, TGF-β1 and SAA content were lower than those of control group. Conclusions:Adjuvantα-keto acid therapy helps to improve the renal function and nutritional status of patients with early diabetic nephropathy, and has positive significance in optimizing the overall treatment effect.

  15. The cyclic keto-enol insecticide spirotetramat inhibits insect and spider mite acetyl-CoA carboxylases by interfering with the carboxyltransferase partial reaction.

    Science.gov (United States)

    Lümmen, Peter; Khajehali, Jahangir; Luther, Kai; Van Leeuwen, Thomas

    2014-12-01

    Acetyl-CoA carboxylase (ACC) catalyzes the committed and rate-limiting step in fatty acid biosynthesis. The two partial reactions, carboxylation of biotin followed by carboxyl transfer to the acceptor acetyl-CoA, are performed by two separate domains in animal ACCs. The cyclic keto-enol insecticides and acaricides have been proposed to inhibit insect ACCs. In this communication, we show that the enol derivative of the cylic keto-enol insecticide spirotetramat inhibited ACCs partially purified from the insect species Myzus persicae and Spodoptera frugiperda, as well as the spider mite (Tetranychus urticae) ACC which was expressed in insect cells using a recombinant baculovirus. Steady-state kinetic analysis revealed competitive inhibition with respect to the carboxyl acceptor, acetyl-CoA, indicating that spirotetramat-enol bound to the carboxyltransferase domain of ACC. Interestingly, inhibition with respect to the biotin carboxylase substrate ATP was uncompetitive. Amino acid residues in the carboxyltransferase domains of plant ACCs are important for binding of established herbicidal inhibitors. Mutating the spider mite ACC at the homologous positions, for example L1736 to either isoleucine or alanine, and A1739 to either valine or serine, did not affect the inhibition of the spider mite ACC by spirotetramat-enol. These results indicated different binding modes of the keto-enols and the herbicidal chemical families.

  16. EFFECTS OF HERBAL CAKE-SEPARATED MOXIBUSTION ON BLOOD LIPIDS,PLASMA THROMOXANE B2 AND 6-KETO-PROSTAGLANDIN F1αCONTENTS IN THE RABBIT WITH HYPERLIPEMIA

    Institute of Scientific and Technical Information of China (English)

    沈菁; 常小荣; 严洁; 曹湘萍; 岳增辉

    2004-01-01

    Objective: To observe effects of herbal cake-separated moxibustion on blood lipids, including total cholesterol (TCh), triglyceride (TG), high density lipoprotein-Ch (HDL-Ch), low density lipoprotein-Ch (LDL-Ch), apolipoprotein A (Apo A), apolipoprotein B (Apo B), and plasma thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-keto-PGF1α) contents and analyse its mechanism. Methods: The hyperlipemia rabbit model was established by breeding of high fat forage and injection of bovine serum albumin. They were treated respectively by continuously for 40 days. Blood TCh and TG contents were detected with enzymatic method, LDL-Ch and HDL-Ch with colorimetric method, Apo A and Apo B with electrophoretic method, and TXB2 and 6-keto-PGF1α with radioimmunoassay. Results: Both the herbal cake-separated moxibustion and direct moxibustion could effectively decrease TCh, TG, LDL-Ch, Apo B and TXB2 contents and TXB2/6-keto-PGF1α, and increase HDL-CH and 6-keto-PGF1α contents in the rabbit of hyperlipemia. Conclusion: 6-keto-PGF1α and TXB2 are possibly involved in the mechanism of herbal cake-separated moxibustion decreasing blood lipids.

  17. Engineered Expression Vectors Significantly Enhanced the Production of 2-Keto-D-gluconic Acid by Gluconobacter oxidans.

    Science.gov (United States)

    Shi, Yuan-yuan; Li, Ke-fei; Lin, Jin-ping; Yang, Sheng-li; Wei, Dong-zhi

    2015-06-10

    2-Keto-D-gluconic acid (2KGA), a precursor of the important food antioxidant erythorbic acid, can be produced by Gluconobacter oxidans. To genetically engineer G. oxidans for improved 2KGA production, six new expression vectors with increased copy numbers based on pBBR1MCS-5 were constructed via rational mutagenesis. The utility of the mutant vectors was demonstrated by the increased ga2dh mRNA abundance, enzyme activity, and 2KGA production when the ga2dh gene was overexpressed using these vectors. Among the obtained constructs, G. oxidans/pBBR-3510-ga2dh displayed the highest oxidative activity toward gluconic acid (GA). In a batch biotransformation process, the G. oxidans/pBBR-3510-ga2dh strain exhibited 2KGA productivity (0.63 g/g CWW/h) higher than that obtained using strain G. oxidans/pBBR-ga2dh (0.40 g/g CWW/h). When sufficient oxygen was supplied during the biotransformation, up to 480 g/L GA was exhausted in 45 h by the G. oxidans/pBBR-3510-ga2dh strain and approximately 486 g/L 2KGA was produced, generating the productivity of 0.54 g/g CWW/h.

  18. 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.

  19. 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.

  20. (+)-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.

  1. 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.

  2. 酮基类胡萝卜素的生理功能及应用研究%Physiological function and application research of Keto-carotenoid

    Institute of Scientific and Technical Information of China (English)

    王冉冉; 张慧; 陶正国; 李涛; 张祥敏; 张万青; 王延霞

    2011-01-01

    作为具有较高应用价值和经济价值的添加剂,酮基类胡萝卜素在食品、医药、保健品、饲料和日用化妆品等行业中应用广泛,并引起人们的日益关注.酮基类胡萝卜素是一类具有特殊官能团的类胡萝卜素,它不仅具有共轭双键,而且在共轭双键链的末端还有不饱和酮基,这些结构都具有比较活泼的电子效应,使它与自由基反应,进而更加容易清除自由基,达到抗氧化作用和保护人体健康的目的.本文综述了酮基类胡萝卜素的概念、结构、性质、生理功能、在自然界的分布以及分析检测手段,也介绍了它的生产技术、主要用途及研究进展,并对其应用前景做出了展望,为酮类类胡萝卜素的进一步开发研究提供参考.%As an additive with high application value and economic value, Keto- carotenoid has a widely application in food, medicine, health -care products, animal feed and daily cosmetics industry etc, and it raises more and more attention. Keto - carotenoid is a class of special functional group of carotenoid. It not only has conjugated double bonds and unsaturated ketonic group at the end of the conjugated double bonds. These structures have lively electronic effects which make Keto - carotenoid has a reaction with the free radical, and It is easier to eliminate free radicals, enhance the capacity of antioxidant defense systems and protect human health. The study of the Keto - carotenoid was introduced entirely in this article including basic concept, structure, physical chemistry properties, biological function, and distribution in nature, analysis and measure methods. This paper also introduces the production technology, main application and research progress of Keto - carotenoid, and foretells its application prospect, as reference for further research and development.

  3. 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.

  4. 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.

  5. Production of non-proteinogenic amino acids from α-keto acid precursors with recombinant Corynebacterium glutamicum.

    Science.gov (United States)

    Kim, Ju-Yeon; Lee, Young-A; Wittmann, Christoph; Park, Jin-Byung

    2013-11-01

    In the present work, Corynebacterium glutamicum was metabolically engineered for the enantioselective synthesis of non-proteinogenic amino acids as valuable building blocks for pharmaceuticals and agrochemicals. The novel bio-catalytic activity of C. glutamicum was obtained by heterologous expression of the branched chain aminotransferase IlvE from Escherichia coli. Upon this modification, the recombinant cells converted the α-keto acid precursor 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (HOAE) into the corresponding amino acid 2-(3-hydroxy-1-adamantyl)-(2S)-amino ethanoic acid (HAAE). Similarly, also L-tert-leucine could be obtained from trimethyl pyruvate indicating a broader applicability of the novel strategy. In both cases, the amino group donor glutamate was supplied from the endogenous metabolism of the recombinant producer. Hereby, the uptake of the precursor and secretion of the product was supported by an enhanced cell permeability through treatment of ethambutol, which inhibits arabinosyl transferases involved in cell wall biosynthesis. The excretion of HAAE into the reaction medium was linked to the secretion of glutamate, indicating a similar mechanism for the export of both compounds. On the other hand, the efflux of L-tert-leucine appeared to be driven by active transport. Subsequent bioprocess engineering enabled HAAE and L-tert-leucine to be produced at a rate of 0.21 and 0.42 mmol (g dry cells)⁻¹  h⁻¹, respectively up to a final product titer of 40 mM. Beyond the given examples, integrated metabolic and cell envelop engineering might extend the production of a variety of other non-proteinogenic amino acids as well as chiral amines by C. glutamicum.

  6. Mitochondrial complex II participates in normoxic and hypoxic regulation of α-keto acids in the murine heart.

    Science.gov (United States)

    Mühling, Jörg; Tiefenbach, Martina; López-Barneo, José; Piruat, José I; García-Flores, Paula; Pfeil, Uwe; Gries, Barbara; Mühlfeld, Christian; Weigand, Markus A; Kummer, Wolfgang; Weissmann, Norbert; Paddenberg, Renate

    2010-12-01

    α-Keto acids (α-KAs) are not just metabolic intermediates but are also powerful modulators of different cellular pathways. Here, we tested the hypothesis that α-KA concentrations are regulated by complex II (succinate dehydrogenase=SDH), which represents an intersection between the mitochondrial respiratory chain for which an important function in cardiopulmonary oxygen sensing has been demonstrated, and the Krebs cycle, a central element of α-KA metabolism. SDH subunit D heterozygous (SDHD(+/-)) and wild-type (WT) mice were housed at normoxia or hypoxia (10% O(2)) for 4 days or 3 weeks, and right ventricular pressure, right ventricle/(left ventricle+septum) ratio, cardiomyocyte ultrastructure, pulmonary vascular remodelling, ventricular complex II subunit expression, SDH activity and α-KA concentrations were analysed. In both strains, hypoxia induced increases in right ventricular pressure and enhanced muscularization of distal pulmonary arteries. Right ventricular hypertrophy was less severe in SDHD(+/-) mice although the cardiomyocyte ultrastructure and mitochondrial morphometric parameters were unchanged. Protein amounts of SDHA, SDHB and SDHC, and SDH activity were distinctly reduced in SDHD(+/-) mice. In normoxic SDHD(+/-) mice, α-ketoisocaproate concentration was lowered to 50% as compared to WT animals. Right/left ventricular concentration differences and the hypoxia-induced decline in individual α-KAs were less pronounced in SDHD(+/-) animals indicating that mitochondrial complex II participates in the adjustment of cardiac α-KA concentrations both under normoxic and hypoxic conditions. These characteristics are not related to the hemodynamic consequences of hypoxia-induced pulmonary vascular remodelling, since its extent and right ventricular pressure were not affected in SDHD(+/-) mice albeit right ventricular hypertrophy was attenuated.

  7. Enhancement of 5-keto-d-gluconate production by a recombinant Gluconobacter oxydans using a dissolved oxygen control strategy.

    Science.gov (United States)

    Yuan, Jianfeng; Wu, Mianbin; Lin, Jianping; Yang, Lirong

    2016-07-01

    The rapid and incomplete oxidation of sugars, alcohols, and polyols by the gram-negative bacterium Gluconobacter oxydans facilitates a wide variety of biological applications. For the conversion of glucose to 5-keto-d-gluconate (5-KGA), a promising precursor of the industrial substance L-(+)-tartaric acid, G. oxydans DSM2343 was genetically engineered to strain ZJU2, in which the GOX1231 and GOX1081 genes were knocked out in a markerless fashion. Then, a secondary alcohol dehydrogenase (GCD) from Xanthomonas campestris DSM3586 was heterologously expressed in G. oxydans ZJU2. The 5-KGA production and cell yield were increased by 10% and 24.5%, respectively. The specific activity of GCD towards gluconate was 1.75±0.02 U/mg protein, which was 7-fold higher than that of the sldAB in G. oxydans. Based on the analysis of kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (qs) and specific 5-KGA production rate (qp), a dissolved oxygen (DO) control strategy was proposed. Finally, batch fermentation was carried out in a 15-L bioreactor using an initial agitation speed of 600 rpm to obtain a high μ for cell growth. Subsequently, DO was continuously maintained above 20% to achieve a high qp to ensure a high accumulation of 5-KGA. Under these conditions, the maximum concentration of 5-KGA reached 117.75 g/L with a productivity of 2.10 g/(L·h).

  8. Acetyl-11-keto-beta-boswellic acid, a constituent of a herbal medicine from Boswellia serrata resin, attenuates experimental ileitis.

    Science.gov (United States)

    Krieglstein, C F; Anthoni, C; Rijcken, E J; Laukötter, M; Spiegel, H U; Boden, S E; Schweizer, S; Safayhi, H; Senninger, N; Schürmann, G

    2001-04-01

    The gum resin extract from Boswellia serrata (H15), an herbal product, was recently shown to have positive therapeutic effects in inflammatory bowel disease (IBD). However, the mechanisms and constituents responsible for these effects are poorly understood. This study examined the effect of the Boswellia extract and its single constituent acetyl-11-keto-beta-boswellic acid (AKBA) on leukocyte-endothelial cell interactions in an experimental model of IBD. Ileitis was induced by two subcutaneous injections of indomethacin (7.5 mg/kg) in Sprague-Dawley rats 24 h apart. Rats also received oral treatment with the Boswellia extract (H15) or AKBA at two different doses (low and high) equivalent to recommendations in human disease over 2 days. Controls received only the carriers NaHCO3 (subcutaneously) and tylose (orally). Effects of treatment were assessed by intravital microscopy in ileal submucosal venules for changes in the number of rolling and adherent leukocytes and by macroscopic and histological scoring. Increased leukocyte-endothelial cell adhesive interactions and severe tissue injury accompanied indomethacin-induced ileitis. Treatment with the Boswellia extract or AKBA resulted in a dose-dependent decrease in rolling (up to 90%) and adherent (up to 98%) leukocytes. High-dose Boswellia extract as well as both low- and high-dose AKBA significantly attenuated tissue injury scores. Oral therapy with the Boswellia extract or AKBA significantly reduces macroscopic and microcirculatory inflammatory features normally associated with indomethacin administration, indicating that the anti-inflammatory actions of the Boswellia extract in IBD may be due in part to boswellic acids such as AKBA.

  9. 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.

  10. 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.

  11. 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.

  12. Improved training tolerance by supplementation with α-Keto acids in untrained young adults: a randomized, double blind, placebo-controlled trial

    Directory of Open Access Journals (Sweden)

    Liu Yuefei

    2012-08-01

    Full Text Available Abstract Background Exercise causes a variety of physiological and metabolic changes that can in turn reduce exercise tolerance. One of the potential mechanisms responsible for fatigue is “exercise-induced hyperammonemia”. Previous studies have shown that supplementation with amino acids can increase training tolerance. The α-keto acids are biochemical analogs of amino acids and can be converted to amino acids through transamination, thus reducing the cellular ammonia level. This double blind, placebo-controlled study was designed to investigate the effects of α-keto acid supplementation (KAS on training tolerance, training effect, and stress-recovery state. Methods Thirty-three untrained young male adults underwent four weeks of training (5 sessions/week; 30 minutes running at the individual anaerobic threshold followed by 3 x 3 minute sprints/each session. Throughout the 4 weeks of training and one week of recovery, subjects took α-ketoglutarate (AKG group, 0.2 g/kg/d, n = 9, branched-chain keto acids (BCKA group, 0.2 g/kg/d, n = 12 or isocaloric placebo (control group, n = 12 daily. Results The 4th week training volume, maximum power output and muscle torque were higher in the AKG group (175 ± 42 min, 412 ± 49 Watts and 293 ± 58 Newton meters, respectively, Prd week of training increased significantly in the control group (P Conclusions Under KAS, subjects could bear a higher training volume and reach a higher power output and peak muscle torque, accompanied by a better stress-recovery-state. Thus, KAS improves exercise tolerance and training effects along with a better stress-recovery state. Whether the improved training tolerance by KAS is associated with effects on ammonia homeostasis requires further observation.

  13. Chiral-at-Metal Rh(III) Complex-Catalyzed Decarboxylative Michael Addition of β-Keto Acids with α,β-Unsaturated 2-Acyl Imidazoles or Pyridine.

    Science.gov (United States)

    Li, Shi-Wu; Gong, Jun; Kang, Qiang

    2017-03-17

    A newly prepared chiral-at-metal Rh(III) complex-catalyzed, highly efficient enantioselective decarboxylative Michael addition of β-keto acids with α,β-unsaturated 2-acyl imidazoles or pyridine has been developed, affording the corresponding adducts in 94-98% yield with up to 96% enantioselectivity. This protocol exhibits remarkable reactivity, as the complex with a Rh(III) loading as low as 0.05 mol % can catalyze the decarboxylative Michael addition on a gram scale without loss of enantioselectivity.

  14. Uso de cetoanálogo na terapia da insuficiência renal canina Use of keto analogues in therapy of the renal failure in dogs

    Directory of Open Access Journals (Sweden)

    J.C.C. Veado

    2002-10-01

    Full Text Available The efficacy of keto acids and essential amino acids on a two-year-old female dog, Labrador, suffering from Leishmaniasis and acute renal failure was evaluated by clinical and laboratorial analyses based on urea and creatinine dosages. An improving in the animal general condition and an increasing in the appetite, activity and weight gain, were observed and favorably contributed to the treatment of the primary disease. Ketoanalogueswere oraly administered during 76 days and the levels of urea and creatinine remained within acceptable limits, even after this period of time. Ketoanaloguescontributed positively for the treatment of acute renal failure and appears to be an important alternative for the leishmaniasis treatment.

  15. 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.

  16. Fast regeneration of carotenoids from radical cations by isoflavonoid dianions: importance of the carotenoid keto group for electron transfer.

    Science.gov (United States)

    Han, Rui-Min; Chen, Chang-Hui; Tian, Yu-Xi; Zhang, Jian-Ping; Skibsted, Leif H

    2010-01-14

    Electron transfer to radical cations of beta-carotene, zeaxanthin, canthaxanthin, and astaxanthin from each of the three acid/base forms of the diphenolic isoflavonoid daidzein and its C-glycoside puerarin, as studied by laser flash photolysis in homogeneous methanol/chloroform (1/9) solution, was found to depend on carotenoid structures and more significantly on the deprotonation degree of the isoflavonoids. None of the carotenoid radical cations reacted with the neutral forms of the isoflavonoids while the monoanionic and dianionic forms of the isoflavonoids regenerated the oxidized carotenoid. Electron transfer to the beta-carotene radical cation from the puerarin dianion followed second order kinetics with the rate constant at 25 degrees C k(2) = 5.5 x 10(9) M(-1) s(-1), zeaxanthin 8.5 x 10(9) M(-1) s(-1), canthaxanthin 6.5 x 10(10) M(-1) s(-1), and astaxanthin 11.1 x 10(10) M(-1) s(-1) approaching the diffusion limit and establishing a linear free energy relationship between rate constants and driving force. Comparable results found for the daidzein dianion indicate that the steric hindrance from the glucoside is not important suggesting the more reducing but less acidic 4'-OH/4'-O(-) as electron donors. On the basis of the rate constants obtained from kinetic analyses, the keto group of carotenoids is concluded to facilitate electron transfer. The driving force was estimated from oxidation potentials, as determined by cyclic-voltametry for puerarin and daidzein in aqueous solutions at varying pH conditions, which led to the standard reduction potentials E degrees = 1.13 and 1.10 V versus NHE corresponding to the uncharged puerarin and daidzein. For pH > pK(a2), the apparent potentials of both puerarin and daidzein became constants and were E degrees = 0.69 and 0.65 V, respectively. Electron transfer from isoflavonoids to the carotenoid radical cation, as formed during oxidative stress, is faster for astaxanthin than for the other carotenoids, which may relate

  17. Crystal structures of four δ-keto esters and a Cambridge Structural Database analysis of cyano-halogen interactions.

    Science.gov (United States)

    Kamal, Kulsoom; Maurya, Hardesh K; Gupta, Atul; Vasudev, Prema G

    2015-10-01

    The revived interest in halogen bonding as a tool in pharmaceutical cocrystals and drug design has indicated that cyano-halogen interactions could play an important role. The crystal structures of four closely related δ-keto esters, which differ only in the substitution at a single C atom (by H, OMe, Cl and Br), are compared, namely ethyl 2-cyano-5-oxo-5-phenyl-3-(piperidin-1-yl)pent-2-enoate, C19H22N2O3, (1), ethyl 2-cyano-5-(4-methoxyphenyl)-5-oxo-3-(piperidin-1-yl)pent-2-enoate, C20H24N2O4, (2), ethyl 5-(4-chlorophenyl)-2-cyano-5-oxo-3-(piperidin-1-yl)pent-2-enoate, C19H21ClN2O3, (3), and the previously published ethyl 5-(4-bromophenyl)-2-cyano-5-oxo-3-(piperidin-1-yl)pent-2-enoate, C19H21BrN2O3, (4) [Maurya, Vasudev & Gupta (2013). RSC Adv. 3, 12955-12962]. The molecular conformations are very similar, while there are differences in the molecular assemblies. Intermolecular C-H...O hydrogen bonds are found to be the primary interactions in the crystal packing and are present in all four structures. The halogenated derivatives have additional aromatic-aromatic interactions and cyano-halogen interactions, further stabilizing the molecular packing. A database analysis of cyano-halogen interactions using the Cambridge Structural Database [CSD; Groom & Allen (2014). Angew. Chem. Int. Ed. 53, 662-671] revealed that about 13% of the organic molecular crystals containing both cyano and halogen groups have cyano-halogen interactions in their packing. Three geometric parameters for the C-X...N[triple-bond]C interaction (X = F, Cl, Br or I), viz. the N...X distance and the C-X...N and C-N...X angles, were analysed. The results indicate that all the short cyano-halogen contacts in the CSD can be classified as halogen bonds, which are directional noncovalent interactions.

  18. 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.

  19. 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.

  20. Verification of a novel NADH-binding motif: combinatorial mutagenesis of three amino acids in the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase.

    Science.gov (United States)

    Banta, Scott; Anderson, Stephen

    2002-12-01

    A screening method has been developed to support randomized mutagenesis of amino acids in the cofactor-binding pocket of the NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase. Such an approach could enable the isolation of an enzyme that can better catalyze the reduction of 2,5-DKG to 2-keto-L-gulonic acid (2-KLG) using NADH as a cofactor. 2-KLG is a valuable precursor to ascorbic acid, or vitamin C, and an enzyme with increased activity with NADH may be able to improve two potential vitamin C production processes. Previously we have identified three amino acid residues that can be mutated to improve activity with NADH as a cofactor. As a pilot study to show feasibility, a library was made with these three amino acids randomized, and 300 random colonies were screened for increased NADH activity. The activities of seven mutants with apparent improvements were verified using activity-stained native gels, and sequencing showed that the amino acids obtained were similar to some of those already discovered using rational design. The four most active mutants were purified and kinetically characterized. All of the new mutations resulted in apparent kcat values that were equal to or higher than that of the best mutant obtained through rational design. At saturating levels of cofactor, the best mutant obtained was almost twice as active with NADH as a cofactor as the wild-type enzyme is with NADPH. This screen is a valuable tool for improving 2,5-DKG reductase, and it could easily be modified for improving other aspects of this protein or similar enzymes.

  1. Design, optimization, and biological evaluation of novel keto-benzimidazoles as potent and selective inhibitors of phosphodiesterase 10A (PDE10A).

    Science.gov (United States)

    Hu, Essa; Kunz, Roxanne K; Chen, Ning; Rumfelt, Shannon; Siegmund, Aaron; Andrews, Kristin; Chmait, Samer; Zhao, Sharon; Davis, Carl; Chen, Hang; Lester-Zeiner, Dianna; Ma, Ji; Biorn, Christopher; Shi, Jianxia; Porter, Amy; Treanor, James; Allen, Jennifer R

    2013-11-14

    Our development of PDE10A inhibitors began with an HTS screening hit (1) that exhibited both high p-glycoprotein (P-gp) efflux ratios in rat and human and poor metabolic stability. On the basis of cocrystal structure of 1 in human PDE10A enzyme, we designed a novel keto-benzimidazole 26 with comparable PDE10A potency devoid of efflux liabilities. On target in vivo coverage of PDE10A in rat brain was assessed using our previously reported LC-MS/MS receptor occupancy (RO) technology. Compound 26 achieved 55% RO of PDE10A at 30 mg/kg po and covered PDE10A receptors in rat brain in a dose-dependent manner. Cocrystal structure of 26 in PDE10A confirmed the binding mode of the novel scaffold. Further optimization resulted in the identification of keto-benzimidazole 34, which showed an increased in vivo efficacy of 57% RO in rats at 10 mg/kg po and an improved in vivo rat clearance and oral bioavailability.

  2. Nuclear Magnetic Resonance (NMR), Infrared (IR) and Mass Spectrometry (MS) study of keto-enol tautomerism of isobenzofuran-1(3H)-one derivatives

    Science.gov (United States)

    Pires, Diego Arantes Teixeira; Pereira, Wagner Luiz; Teixeira, Róbson Ricardo; Figueroa-Villar, José Daniel; Nascimento, Claudia Jorge do

    2016-06-01

    The keto-enol tautomerism of 3-(2-hydroxy-4,4-dimethyl-6-oxo-cyclohexen-1-yl)isobenzofuran-1(3H-one (1), 3-(2-hydroxy-6-oxocyclohex-1-enyl)isobenzofuran-1(3H)-one (2), 3-(2-hydroxy-4-methyl-6-oxocyclohex-1-enyl)isobenzofuran-1(3H)-one (3), 3-(2-hydroxy-5-oxocyclopent-1-enyl)isobenzofuran-1(3H)-one (4) and 2-(3-oxo-1,3-dihydroisobenzofuran-1-yl)-1H-indene-1,3(2H)-dione (5) were investigated. We noticed that for compounds 1 to 4 only the enol form is observed in solid, in solution or in the gas phase. Their tautomeric equilibria are not affected by the solvent, temperature or physical state. Compound 5 was observed in its keto form in solution (NMR) and solid state (IR). The enol species of 5 was also observed upon Mass Spectrometry analysis. These findings were supported by NMR, IR, MS/MS and molecular modeling analyses.

  3. Structure-activity relationships of the nonredox-type non-competitive leukotriene biosynthesis inhibitor acetyl-11-keto-β-boswellic acid.

    Science.gov (United States)

    Sailer, E R; Hoernlein, R F; Ammon, H P; Safayhi, H

    1996-05-01

    Acetyl-11-keto-β-boswellic acid (AKBA) from Boswellia serrata Roxb. and italics Boswellia carterii Birdw. is the first selective, direct, non-competitive and non-redox-type inhibitor of 5-lipoxygenase, the key enzyme for leukotriene biosynthesis (Safayhi et al., 1992). Previously, we showed that AKBA interacts with the 5-lipoxygenase via a pentacyclic triterpene selective effector site (Safayhi et al., 1995). In order to study the impact of AKBA's functional groups on enzyme inhibition, natural and synthetic analogues of this boswellic acid were tested for 5-lipoxygenase inhibition in intact rat neutrophils (Sailer et al., 1996 a). The results reveal that the carboxylic group of AKBA combined with the 11-keto-group is essential for enzyme inhibition, whereas the acetoxy-group on position C-3 α increases the affinity of AKBA to its effector site. Furthermore, other experiments demonstrated that minor structural modifications could cause a total loss of binding affinity and/or inhibitory activity of these compounds.

  4. 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.

  5. Applications of Carboxylic Acid Reductases in Oleaginous Microbes

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-04-24

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

  6. 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.

  7. 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 ...

  8. 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...

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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...

  16. 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.

  17. 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.

  18. 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.

  19. The agro issue on Aldo Ferrer's perspective. A re-evaluation of "La Economía Argentina" political discurse and the history practice during the nationalist openess (1970-1971 La problemática del agro en la perspectiva de Aldo Ferrer: Una reevaluación del discurso político de 'La Economía Argentina' y la práctica histórica durante la apertura nacionalista [1970-1971

    Directory of Open Access Journals (Sweden)

    Tomás Elías Zeitler

    2010-01-01

    Full Text Available The current importance that the studies have Historiographies on the questions of the Argentina agro in the XXth century and the growth of works of investigation and of balance sheets on the above mentioned production they generate a propitious context for the critical reappraisal of the contributions that have been done to the history of the Argentine agro from the perspective cepalina, mas specifically that one expressed in the wide production of the economist Aldo Ferrer: his explanation of the agrarian problematics from the analysis of the internal weaknesses of the productive Argentina system and the basic failings of the functioning of the capitalist economy constitutes a speech in historical perspective that demonstrates the representations identitarias that the capitalist national sectors had of the Argentine agro at the end of the decade of sixty. The analysis of his classic work The Economy Argentina (1963 allows to argue that in his theoretical and methodological boarding of the economic Argentine development the agrarian question occupies the center of the problems that they have afflicted to the productive Argentine system during all the 20th century and specially during the stage called of "nationalistic opening" (1970-1971 in which the author had an active political participation as Secretary of the Treasury of the Nation Argentina.La actualidad que tienen los estudios historiográficos sobre las cuestiones del agro argentino en el siglo XX y el crecimiento de trabajos de investigación y de balances sobre dicha producción generan un contexto propicio para la reevaluación crítica de los aportes que se han hecho a la historia del agro argentino desde la perspectiva cepalina, mas específicamente aquella expresada en la amplia producción del economista Aldo Ferrer: su explicación de la problemática agraria desde el análisis de las debilidades internas del sistema productivo argentino y las falencias básicas del

  20. Domain analysis of 3 Keto Acyl-CoA synthase for structural variations in Vitis vinifera and Oryza brachyantha using comparative modelling.

    Science.gov (United States)

    Sagar, Mamta; Pandey, Neetesh; Qamar, Naseha; Singh, Brijendra; Shukla, Akanksha

    2015-03-01

    The long chain fatty acids incorporated into plant lipids are derived from the iterative addition of C2 units which is provided by malonyl-CoA to an acyl-CoA after interactions with 3-ketoacyl-CoA synthase (KCS), found in several plants. This study provides functional characterization of three 3 ketoacyl CoA synthase like proteins in Vitis vinifera (one) and Oryza brachyantha (two proteins). Sequence analysis reveals that protein of Oryza brachyantha shows 96% similarity to a hypothetical protein in Sorghum bicolor; total 11 homologs were predicted in Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type III polyketide synthase-like protein, Thiolase-like, subgroup; Thiolase-like and 3-Oxoacyl-ACP synthase III, C-terminal and chalcone synthase like domain but very long chain 3-keto acyl CoA domain is absent. All three proteins were found to have Chalcone and stilbene synthases C terminal domain which is similar to domain of thiolase and β keto acyl synthase. Its N terminal domain is absent in J3M9Z7 protein of Oryza brachyantha and F6HH63 protein of Vitis vinifera. Differences in N-terminal domain is responsible for distinguish activity. The J3MF16 protein of Oryza brachyantha contains N terminal domain and C terminal domain and characterized using annotation of these domains. Domains Gcs (streptomyces coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase (Gerbera hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in three proteins. This similarity points toward anthocyanin biosynthetic process. Similarity to chalcone synthase 2 reveals its possible role in Naringenine and Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza brachyantha. Active site residues C-240, H-407, N-447 are present in J3MF16 protein that are common in these three protein at different positions. Structural variations among dimer interface, product binding site, malonyl-CoA binding sites, were predicted in

  1. ANTI-INFLAMMATORY EFFECTS OF LOW PROTEIN DIET SUPPLEMENTED WITH KETO-AMINO ACID IN THE TREATMENT OF TYPE 2 DIABETIC NEPHROPATHY

    Directory of Open Access Journals (Sweden)

    Nan Chen

    2012-06-01

    Full Text Available Recent clinical research strongly approves that low-protein diet supplemented with keto-amino acid can effectively delay progression of type 2 diabetic nephropathy (DN. Anti-inflammation is one of these effects, but the mechanism is still controversial. This study is designed to further explore roles of ketogenic diets in regulation of inflammation status of type 2 DN. Twenty-one patients with type 2 DN (mean age at 65.14±7.34 years, were followed-up for 52 weeks in this study. All patients were in CKD stages 3–4 with glomerular filtration rates 26–55 ml/min/1.73 m2 and were all on a low-protein diet containing 0.8 g protein/kg BW per day and 30–35 Kcal /kg BW per day. The diet was randomly supplemented with keto-amino acids at a dosage of 100 mg/kg BW per day in 10 patients, who were assigned into Group II. Other 11 patients were assigned into Group I. At the end of this study, related clinical data showed there was a significant increase in the serum level of TNF-α which could mediate inflammation systemically in Group I (from 230.25±54.34 to 332.11 pg/ml, P 0.05. The level of CRP, which is produced in response to inflammation, rose greatly in Group I (from 7.5±1.07 to 20.4±3.72 ug/ml, P 0.05. Nutritional markers including serum albumin, hemoglobin and basal metabolic index showed no malnutrition happened during the follow-up period. In conclusion, low-protein diet supplemented with keto-amino acids contribute to ameliorate inflammation in the progression of type 2 diabetic nephropathy through regulating inflammatory factors production, including TNF-α, CRP and adiponectin.

  2. 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.

  3. 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.

  4. 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.

  5. Characterization of 3alpha-acetyl-11-keto-alpha-boswellic acid, a pentacyclic triterpenoid inducing apoptosis in vitro and in vivo.

    Science.gov (United States)

    Büchele, Berthold; Zugmaier, Waltraud; Estrada, Aidee; Genze, Felicitas; Syrovets, Tatiana; Paetz, Christian; Schneider, Bernd; Simmet, Thomas

    2006-11-01

    3Alpha-acetyl-11-keto-alpha-boswellic acid (3alpha-acetoxy-11-oxo-olean-12-en-24-oic acid, 1) was synthesized by a radical-type reaction using bromine and 3alpha-acetyl-alpha-boswellic acid isolated from the oleo-gum-resin of Boswellia carterii. 1D and 2D NMR (COSY, HMBC, ROESY) at 500 MHz were used for shift assignments and structure verification. The compound investigated is present in a herbal preparation extracted from Boswellia serrata oleo-gum-resin, it inhibits the growth of chemotherapy-resistant human PC-3 prostate cancer cells in vitro and induces apoptosis as shown by activation of caspase 3 and the induction of DNA fragmentation. In addition, compound 1 is active IN VIVO as shown by inhibition of proliferation and induction of apoptosis in PC-3 prostate cancer cells xenotransplanted onto the chick chorioallantoic membrane.

  6. Optimisation of a system for the co-translational incorporation of a keto amino acid and its application to a tumour-specific Anticalin.

    Science.gov (United States)

    Reichert, Andreas J; Poxleitner, Gabriele; Dauner, Martin; Skerra, Arne

    2015-12-01

    The bioorthogonal keto group has attracted interest for the site-specific chemical conjugation of recombinant proteins under mild conditions, e.g. with aminooxy-functionalised fluorescent probes, radiometal chelates, toxins or polymers. However, the cotranslational incorporation of the corresponding non-canonical amino acid p-acetyl-L-phenylalanine (Apa) into proteins expressed in Escherichia coli by means of amber suppression using a previously described system with a mutated tRNA and an engineered tyrosyl-tRNA synthetase from Methanococcus jannaschii shows limited efficiency and considerable promiscuity towards endogenous amino acids. Employing a one-plasmid system that encodes all three components required for selection, i.e. the modified aminoacyl-tRNA synthetase (aaRS), the cognate amber suppressor tRNA and the enhanced green fluorescent protein equipped with an amber stop codon and serving as reporter, we have generated an Apa-specific aaRS&tRNA pair with considerably improved efficiency (17-fold increased expression) and also fidelity (6-fold). To this end, both the aaRS and the tRNA were subjected to doped random mutagenesis and selection in altogether four evolutionary cycles using fluorescence-activated bacterial cell sorting as well as automated screening of microcultures. The resulting aaRS&tRNA pair was applied to the functionalisation of an Anticalin with specificity towards oncofetal fibronectin by introducing a keto group at a permissible site for subsequent conjugation with a fluorescent dye, thus allowing visualisation of this tumour target under the microscope.

  7. Crystal Structures of Apo and Liganded 4-Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous β-Keto Acid.

    Science.gov (United States)

    Guimarães, Samuel L; Coitinho, Juliana B; Costa, Débora M A; Araújo, Simara S; Whitman, Christian P; Nagem, Ronaldo A P

    2016-05-10

    The enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria Pseudomonas putida G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg(2+) as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent β-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous β-keto acid decarboxylase and the first crystal structure of a 4-OD.

  8. 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.

  9. 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.

  10. 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...

  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

    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...

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Generation of human endometrial knockout cell lines with the CRISPR/Cas9 system confirms the prostaglandin F2α synthase activity of aldo-ketoreductase 1B1.

    Science.gov (United States)

    Lacroix Pépin, Nicolas; Chapdelaine, Pierre; Rodriguez, Yoima; Tremblay, Jacques-P; Fortier, Michel A

    2014-07-01

    Prostaglandins (PGs) are important regulators of female reproductive function. The primary PGs produced in the endometrium are PGE2 and PGF2α. Relatively little is known about the biosynthetic pathways leading to the formation of PGF2α. We have described the role of aldo-ketoreductase (AKR)1B1 in increased PGF2α production by human endometrial cells following stimulation with interleukin-1β (IL-1β). However, alternate PGF synthases are expressed concurrently in endometrial cells. A definite proof of the role of AKR1B1 would require gene knockout; unfortunately, this gene has no direct equivalent in the mouse. Recently, an efficient genome-editing technology using RNA-guided DNase Cas9 and the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed. We have adapted this approach to knockout AKR1B1 gene expression in human endometrial cell lines. One clone (16-2) of stromal origin generated by the CRISPR/Cas9 system exhibited a complete loss of AKR1B1 protein and mRNA expression, whereas other clones presented with partial edition. The present report focuses on the characterization of clone 16-2 exhibiting deletion of 68 and 2 nucleotides, respectively, on each of the alleles. Cells from this clone lost their ability to produce PGF2α but maintained their original stromal cell (human endometrial stromal cells-2) phenotype including the capacity to decidualize in the presence of progesterone (medroxyprogesterone acetate) and 8-bromo-cAMP. Knockout cells also maintained their ability to increase PGE2 production in response to IL-1β. In summary, we demonstrate that the new genome editing CRISPR/Cas9 system can be used in human cells to generate stable knockout cell line models. Our results suggest that genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells. Our results also confirm that AKR1B1 is involved in the synthesis of PGF2α.

  19. 激光椎间盘减压术对盘源性腰痛患者血清中6-keto-PGE1α水平的影响%The Influence of Percutaneous Laser Disc Decompression on The Level of Serum 6-keto-PGE1α of Discogenic Back Pain Patients

    Institute of Scientific and Technical Information of China (English)

    都芳涛; 尚博; 张劼; 张骞; 李帮国; 李庆富; 任鹏; 丁艳玲

    2011-01-01

    目的 观察经皮激光椎间盘减压术(percutaneous laser disc decompression,PLDD)对椎间盘源性腰痛(discogenic low back pain,DLBP)患者血清中6-酮-前列腺素Ela(6-keto-PGEla)水平的影响,为临床应用、推广这一治疗方法提供理论依据.方法 30例DLBP患者分为治疗前(A)及治疗后(B)两组,进行术前及术后1周血清中6-ke-to-PGEla水平的检测,对30例健康者(C组)也进行其检测.采用疼痛视觉模拟评分(visual analog scale,VAS)作为腰痛缓解疗效评价指标,并对手术前后评分进行统计学分析.采用中华医学会脊柱学组腰背痛手术评定标准作为术后功能评定标准.结果 A组(54.745 0±12.663)pg/mL明显高于B组(47.307 5±5.316)Pg/mL及C组(45.338 0±7.909)pg/mL,差异具有统计学意义(P<0.05).对A、B两组资料进行配对t检验及双尾检验,差异具有统计学意义.VAS评分B组(1.0±1.8)分比A组(7.7±1.0)分明显下降,有非常显著的差异(P<0.01),功能评定优21例,良7例,可1例,差1例,优良率93.3%.结论 前列腺素E2与PLDD治疗DLBP的机制密切相关.%Objective To study the influence of percutaneous laser disc decompression on the level of serum 6-keto-PGElα of discogenic back pain Patients ,to offer a theoretical basis of clinical application and promotion. Methods 30 cases of DLBP were divided into 2 groups: pretreatment and post-treament, serum 6-keto-PGElαconcentration were measured before therapy and after therapy 1 week. 30 volunteer peoples who were health blood donor as the control group and measured serum 6-keto-PGElα. The visual analogue scale (VAS) and the operation evaluation criteria of lumbago and backache of Chinese Medical Association Spine Group was taken to value the therapeutic effect. Results the concentration of serum 6-keto-PGElαin Group A (54. 745 0± 12. 663) pg/mL is much higher than in group B(47. 307 5±5. 316) pg/mL and C(45. 338 0±7. 909) pg/mL,the difference is significant (P<0. 0S). By paired

  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

    Directory of Open Access Journals (Sweden)

    José Neptuno Rodríguez-López

    2009-12-01

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

  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

    Directory of Open Access Journals (Sweden)

    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. Aldo Leopold: A Bio-Bibliography

    Science.gov (United States)

    1990-10-01

    consciousness, evident in the widespread acceptance of the American Environmental Protection Act or the growth of the European Green Parties. Leopold did not...34* "An Outline Plan for Game Management in Wisconsin" " Feathered vs Human Predators" "Coon Valley: An Adventure in Cooperative Conservation" "Leopold on...34 "Clandeboye" "Wilderness as a Land Laboratory" "Prairie Birthday" 29 "Lakes in Relation to Terrestrial Life Patterns" "A Raptor Tally in the Northwest

  18. Assessment of the degree of oxidative stress injury, renin-angiotensin system activity and podocyte loss after combined treatment of keto acid with low protein diet for patients with diabetic nephropathy

    Institute of Scientific and Technical Information of China (English)

    Yuan-Hua Xu; Mei Wang

    2016-01-01

    Objective:To analyze the degree of oxidative stress injury, RAS activity and podocyte loss after patients with diabetic nephropathy received keto acid combined with low protein diet. Methods:A total of 106 cases of patients with diabetic nephropathy who received hospital treatment in our hospital from September 2012 to July 2015 were selected as research subjects and randomly divided into observation group and control group according to different treatment, each group with 53 cases. Control group received low protein diet treatment alone, observation group received keto acid combined with low protein diet treatment, and then the degree of oxidative stress injury, RAS activity and podocyte loss of two groups were compared. Results:Serum MDA and AOPP levels of observation group after treatment were lower than those of control group, and levels of SOD and T-AOC were higher than those of control group;PRA, AngⅡand Aldosterone levels of observation group after treatment were lower than those of control group;mRNA expression levels of podocin and synaptopodin in urine sediment of observation group after treatment were lower than those of control group. Conclusion:Keto acid combined with low protein diet treatment for patients with diabetic nephropathy can reduce the degree of oxidative stress injury and RAS activity, decrease podocyte loss and optimize patients’ condition.

  19. Quantification of the Keto-Hydroperoxide (HOOCH2OCHO) and Other Elusive Intermediates during Low-Temperature Oxidation of Dimethyl Ether

    KAUST Repository

    Moshammer, Kai

    2016-09-17

    This work provides new temperature-dependent mole fractions of elusive intermediates relevant to the low-temperature oxidation of dimethyl ether (DME). It extends the previous study of Moshammer et al. [ J. Phys. Chem. A 2015, 119, 7361–7374] in which a combination of a jet-stirred reactor and molecular beam mass spectrometry with single-photon ionization via tunable synchrotron-generated vacuum-ultraviolet radiation was used to identify (but not quantify) several highly oxygenated species. Here, temperature-dependent concentration profiles of 17 components were determined in the range of 450–1000 K and compared to up-to-date kinetic modeling results. Special emphasis is paid toward the validation and application of a theoretical method for predicting photoionization cross sections that are hard to obtain experimentally but essential to turn mass spectral data into mole fraction profiles. The presented approach enabled the quantification of the hydroperoxymethyl formate (HOOCH2OCH2O), which is a key intermediate in the low-temperature oxidation of DME. The quantification of this keto-hydroperoxide together with the temperature-dependent concentration profiles of other intermediates including H2O2, HCOOH, CH3OCHO, and CH3OOH reveals new opportunities for the development of a next-generation DME combustion chemistry mechanism.

  20. Bromopyruvate, an active site-directed inactivator of E. coli 2-keto-4-hydroxyglutarate(KHG) aldolase, modifies glutamic acid residue-45

    Energy Technology Data Exchange (ETDEWEB)

    Vlahos, C.J.; Dekker, E.E.

    1987-05-01

    E. coli KHG-aldolase (2-keto-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate), a novel trimeric Class I aldolase, requires one active-site lysine residue (Lys 133)/subunit for Schiff-base formation as well as one arginine residue (Arg 49)/subunit for catalytic activity. The substrate analog, 3-bromopyruvate (BRPY), causes a time- and concentration-dependent loss of KHG-aldolase activity. This inactivation is regarded as active site-directed since: (a) BRPY modification results in complete loss of enzymatic activity; (b) saturation kinetics are exhibited, suggesting that a reversible complex is formed between the aldolase and BRPY prior to the rate-limiting inactivation step; (c) over 90% of the initial aldolase activity is protected by either substrate, pyruvate or KHG; (d) 1.1 mol of /sup 14/C-BRPY is bound/enzyme subunit. Peptide isolation and sequencing show that the incorporated radioactivity is associated with residue Glu-45. Denaturation of the enzyme with guanidine x HCl following treatment with excess /sup 14/C-BRPY allows for the incorporation of carbon-14 at Cys-159 and Cys-180 as well. The presence of pyruvate protects Glu-45 from being esterified but does not prevent the alkylation of the two cysteine residues. These results suggest that Glu-45 is essential for the catalytic activity of E. coli KHG-aldolase, most likely functioning as the active-site amphoteric proton donor/acceptor moiety that is involved in the overall mechanism of the reaction catalyzed by this enzyme.

  1. Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-L-gulonic acid from D-sorbitol.

    Science.gov (United States)

    Gao, Lili; Hu, Yudong; Liu, Jie; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2014-07-01

    2-Keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C, is currently produced by a two-step fermentation route from D-sorbitol. However, this route involves three bacteria, making the mix-culture system complicated and redundant. Thus, replacement of the conventional two-step fermentation process with a one-step process could be revolutionary in vitamin C industry. In this study, different combinations of five L-sorbose dehydrogenases (SDH) and two L-sorbosone dehydrogenases (SNDH) from Ketogulonicigenium vulgare WSH-001 were introduced into Gluconobacter oxydans WSH-003, an industrial strain used for the conversion of d-sorbitol to L-sorbose. The optimum combination produced 4.9g/L of 2-KLG. In addition, 10 different linker peptides were used for the fusion expression of SDH and SNDH in G. oxydans. The best recombinant strain (G. oxydans/pGUC-k0203-GS-k0095) produced 32.4g/L of 2-KLG after 168h. Furthermore, biosynthesis of pyrroloquinoline quinine (PQQ), a cofactor of those dehydrogenases, was enhanced to improve 2-KLG production. With the stepwise metabolic engineering of G. oxydans, the final 2-KLG production was improved to 39.2g/L, which was 8.0-fold higher than that obtained using independent expression of the dehydrogenases. These results bring us closer to the final one-step industrial-scale production of vitamin C.

  2. Inhibitory effect of acetyl-11-keto-beta-boswellic acid on androgen receptor by interference of Sp1 binding activity in prostate cancer cells.

    Science.gov (United States)

    Yuan, Hui-Qing; Kong, Feng; Wang, Xiao-Ling; Young, Charles Y F; Hu, Xiao-Yan; Lou, Hong-Xiang

    2008-06-01

    Androgen receptor (AR)-mediated signaling is crucial for the development and progression of prostate cancer (PCa). Naturally occurring phytochemicals that target the AR signaling offer significant protection against this disease. Acetyl-11-keto-beta-boswellic acid (AKBA), a compound isolated from the gum-resin of Boswellia carterii, caused G1-phase cell cycle arrest with an induction of p21(WAF1/CIP1), and a reduction of cyclin D1 as well in prostate cancer cells. AKBA-mediated cellular proliferation inhibition was associated with a decrease of AR expression at mRNA and protein levels. Furthermore, the functional biomarkers used in evaluation of AR transactivity showed suppressions of prostate-specific antigen promoter-dependent and androgen responsive element-dependent luciferase activities. Additionally, down-regulation of an AR short promoter mainly containing a Sp1 binding site suggested the essential role of Sp1 for the reduction of AR expression in cells exposed to AKBA. Interruption effect of AKBA on Sp1 binding activity but not Sp1 protein levels was further confirmed by EMSA and transient transfection with a luciferase reporter driven by three copies of the Sp1 binding site of the AR promoter. Therefore, anti-AR properties ascribed to AKBA suggested that AKBA-containing drugs could be used for the development of novel therapeutic chemicals.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    Hu Peng

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

  10. Una forma de intervención del Estado a comienzos de los años 1930s.: Poder político, represión e indiferencia. Alrededor de la publicación de El Fusilamiento de Joaquín Penina de Aldo Oliva

    Directory of Open Access Journals (Sweden)

    Diego P. Roldán

    2007-12-01

    Full Text Available A partir de la publicación del libro El fusilamiento de Joaquín Penina de Aldo Oliva, el presente artículo da a conocer la documentación oficial producida por el Municipio de Rosario en torno a este acontecimiento. El trabajo muestra una de las modalidades olvidadas de intervención que el Estado tuvo a comienzo de la década de 1930. Igualmente, intenta reflexionar sobre las condiciones de posibilidad para la producción de prácticas y sentidos en torno al asesinato político y la privación de la identidad que sufrió Penina en el plano simbólico durante los "debates" en el seno del Concejo Deliberante de Rosario, una vez repuestos parcialmente los mecanismos formales de un sistema político restringido

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Una forma de intervención del Estado a comienzos de los años 1930s.: Poder político, represión e indiferencia. Alrededor de la publicación de El Fusilamiento de Joaquín Penina de Aldo Oliva

    Directory of Open Access Journals (Sweden)

    Diego P. Roldán

    2007-12-01

    Full Text Available A partir de la publicación del libro El fusilamiento de Joaquín Penina de Aldo Oliva, el presente artículo da a conocer la documentación oficial producida por el Municipio de Rosario en torno a este acontecimiento. El trabajo muestra una de las modalidades olvidadas de intervención que el Estado tuvo a comienzo de la década de 1930. Igualmente, intenta reflexionar sobre las condiciones de posibilidad para la producción de prácticas y sentidos en torno al asesinato político y la privación de la identidad que sufrió Penina en el plano simbólico durante los "debates" en el seno del Concejo Deliberante de Rosario, una vez repuestos parcialmente los mecanismos formales de un sistema político restringidoFrom the publication of the book El fusilamiento de Joaquín Penina de Aldo Oliva, the present article brings to light the official documentation produced by the Municipality of Rosario around this event. The work shows one of the intervention modalities forgot that the State had at the beginning of the years 1930s. Likewise it tries to reflect on the conditions of possibility for the production of practices and senses around the political murder and the deprivation of the identity that suffered Penina in the symbolic plan during the "debates" in the Deliberative Council of Rosario rehabilitated partly of the formal mechanisms of a restricted political system

  16. 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

  17. Cuminaldehyde: Aldose Reductase and alpha-Glucosidase Inhibitor Derived from Cuminum cyminum L. Seeds.

    Science.gov (United States)

    Lee, Hoi-Seon

    2005-04-06

    The inhibitory activity of Cuminum cyminum seed-isolated component was evaluated against lens aldose reductase and alpha-glucosidase isolated from Sprague-Dawley male rats and compared to that of 11 commercially available components derived from C. cyminum seed oil, as well as quercitrin as an aldose reductase inhibitor and acarbose as an alpha-glucosidase inhibitor. The biologically active constituent of C. cyminum seed oil was characterized as cuminaldehyde by various spectral analyses. The IC(50) value of cuminaldehyde is 0.00085 mg/mL against aldose reductase and 0.5 mg/mL against alpha-glucosidase, respectively. Cuminaldehyde was about 1.8 and 1.6 times less in inhibitory activity than acarbose and quercitin, respectively. Nonetheless, cuminaldehyde may be useful as a lead compound and a new agent for antidiabetic therapeutics.

  18. [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.

  19. 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.

  20. 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...

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. Concentrations of oxytocin in the intercavernous sinus of mares during luteolysis: temporal relationship with concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha.

    Science.gov (United States)

    Vanderwall, D K; Silvia, W J; Fitzgerald, B P

    1998-03-01

    The reproductive tracts of nine thoroughbred mares were examined by ultrasound to determine the day of ovulation (day 0). Mares were fitted with intercavernous sinus cannulae on the day before the start of sample collection of pituitary venous effluent rich in oxytocin. Intercavernous sinus blood samples were collected for at least 36 h at 5 min intervals beginning at noon on day 13 (n = 2), day 15 (n = 5) or day 16 (n = 2) after ovulation. Concentrations of oxytocin and 13,14-dihydro-15-keto prostaglandin F2 alpha (PGFM) in plasma were determined by radioimmunoassay. Three high-magnitude surges of PGFM (> 1 ng ml-1) were found in these samples. Three high magnitude pulses of oxytocin (> 200 pg ml-1) were also observed, one associated with each of the PGFM surges. In each of these cases, the oxytocin pulse appeared to follow or coincide with the onset of the PGFM surge. Lower magnitude pulses of both hormones were detected throughout the bleeding period in every mare. The average interval between these pulses was 122.3 min for oxytocin and 121.0 min for PGFM. The interval between pulses for individual mares varied from 90 to 199 min for oxytocin, and from 87 to 213 min for PGFM. However, there was no correlation between PGFM and oxytocin pulse intervals among mares. Within each mare, there was no discernable association between low magnitude pulses of oxytocin and PGFM. From these data, it was concluded that high-magnitude surges of PGF2 alpha are associated with similar surges of oxytocin from the posterior pituitary gland, and that PGF2 alpha may induce their secretion. The posterior pituitary gland also appears to secrete oxytocin in a pulsatile manner at a frequency of approximately 1 pulse every 2 h but these pulses do not appear to be associated with the low magnitude pulses of PGF2 alpha secreted from the uterus.

  9. Detection and Identification of the Keto-Hydroperoxide (HOOCH 2 OCHO) and Other Intermediates during Low-Temperature Oxidation of Dimethyl Ether

    KAUST Repository

    Moshammer, Kai

    2015-07-16

    In this paper we report the detection and identification of the keto-hydroperoxide (hydroperoxymethyl formate, HPMF, HOOCH2OCHO) and other partially oxidized intermediate species arising from the low-temperature (540 K) oxidation of dimethyl ether (DME). These observations were made possible by coupling a jet-stirred reactor with molecular-beam sampling capabilities, operated near atmospheric pressure, to a reflectron time-of-flight mass spectrometer that employs single-photon ionization via tunable synchrotron-generated vacuum-ultraviolet radiation. On the basis of experimentally observed ionization thresholds and fragmentation appearance energies, interpreted with the aid of ab initio calculations, we have identified HPMF and its conceivable decomposition products HC(O)O(O)CH (formic acid anhydride), HC(O)OOH (performic acid), and HOC(O)OH (carbonic acid). Other intermediates that were detected and identified include HC(O)OCH3 (methyl formate), cycl-CH2-O-CH2-O- (1,3-dioxetane), CH3OOH (methyl hydroperoxide), HC(O)OH (formic acid), and H2O2 (hydrogen peroxide). We show that the theoretical characterization of multiple conformeric structures of some intermediates is required when interpreting the experimentally observed ionization thresholds, and a simple method is presented for estimating the importance of multiple conformers at the estimated temperature (∼100 K) of the present molecular beam. We also discuss possible formation pathways of the detected species: for example, supported by potential energy surface calculations, we show that performic acid may be a minor channel of the O2 + CH2OCH2OOH reaction, resulting from the decomposition of the HOOCH2OCHOOH intermediate, which predominantly leads to the HPMF. © 2015 American Chemical Society.

  10. Functional characteristics of spirilloxanthin and keto-bearing Analogues in light-harvesting LH2 complexes from Rhodobacter sphaeroides with a genetically modified carotenoid synthesis pathway.

    Science.gov (United States)

    Niedzwiedzki, Dariusz M; Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Bocian, David F; Holten, Dewey; Hunter, C Neil

    2015-01-01

    Light-harvesting 2 (LH2) complexes from a genetically modified strain of the purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. Carotenoid synthesis in the Rba. sphaeroides strain was engineered to redirect carotenoid production away from spheroidene into the spirilloxanthin synthesis pathway. The strain assembles LH2 antennas with substantial amounts of spirilloxanthin (total double-bond conjugation length N=13) if grown anaerobically and of keto-bearing long-chain analogs [2-ketoanhydrorhodovibrin (N=13), 2-ketospirilloxanthin (N=14) and 2,2'-diketospirilloxanthin (N=15)] if grown semi-aerobically (with ratios that depend on growth conditions). We present the photophysical, electronic, and vibrational properties of these carotenoids, both isolated in organic media and assembled within LH2 complexes. Measurements of excited-state energy transfer to the array of excitonically coupled bacteriochlorophyll a molecules (B850) show that the mean lifetime of the first singlet excited state (S1) of the long-chain (N≥13) carotenoids does not change appreciably between organic media and the protein environment. In each case, the S1 state appears to lie lower in energy than that of B850. The energy-transfer yield is ~0.4 in LH2 (from the strain grown aerobically or semi-aerobically), which is less than half that achieved for LH2 that contains short-chain (N≤11) analogues. Collectively, the results suggest that the S1 excited state of the long-chain (N≥13) carotenoids participates little if at all in carotenoid-to-BChl a energy transfer, which occurs predominantly via the carotenoid S2 excited state in these antennas.

  11. 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...

  12. 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.

  13. 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

  14. 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...

  15. 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....

  16. 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).

  17. 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.

  18. 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

  19. 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

  20. 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

  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. Concentrations of prostaglandins E2, F2 alpha and 6-keto-prostaglandin F1 alpha in the utero-ovarian venous plasma of nonpregnant and early pregnant ewes.

    Science.gov (United States)

    Silvia, W J; Ottobre, J S; Inskeep, E K

    1984-05-01

    The effect of pregnancy on concentrations of prostaglandins E2, F2 alpha and 6-keto-prostaglandin F1 alpha (PGE2, PGF2 alpha and 6-keto-PGF1 alpha) in utero-ovarian venous plasma was examined in ewes on Days 10 through 14 after estrus, an interval which includes the critical period for maternal recognition of pregnancy. The utero-ovarian vein ipsilateral to a corpus luteum was catheterized on Day 9 or 10 in 6 pregnant and 8 nonpregnant ewes. Five blood samples were collected at 30-min intervals for 2 h beginning at 0500 and 1700 h daily. Sampling began at 0500 h on the day after catheterization. The mean and variance within each 2-h collection period were calculated for each ewe. The natural logarithm of the variance in each collection period (ln variance) was used as an estimate of the fluctuations in secretory activity by the endometrial-conceptus complex. Patterns of the mean concentrations of PGE2 were different between pregnant and nonpregnant ewes (P less than 0.01); PGE2 being higher in the pregnant ewes beginning on Day 13. There was a trend for the patterns of ln variance in PGE2 to differ (P less than 0.1) with pregnancy status over the entire period; ln variance was greater in pregnant ewes beginning on Day 13. The patterns of the mean concentrations and ln variances for PGF2 alpha and 6-keto-PGF1 alpha did not differ between pregnant and nonpregnant ewes. There were significant increases in both of these prostaglandins over time, independent of pregnancy status (P less than 0.01). The association of higher concentrations of PGE2 in utero-ovarian venous plasma with early pregnancy is consistent with the hypothesis that PGE2, originating from the uterus and/or conceptus, is one factor involved in maintenance of the corpus luteum of pregnancy.

  3. 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.

  4. 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

  5. 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.

  6. 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.

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

    DEFF Research Database (Denmark)

    Panagiotou, Gianni; Christakopoulos, P.

    2004-01-01

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

  8. 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.

  9. Cloning, expression and antigenicity of the L. donovani reductase

    DEFF Research Database (Denmark)

    Jensen, A T; Kemp, K; Theander, T G

    2001-01-01

    (K). Only 2 of 22 plasma samples from patients with visceral leishmaniasis were found to have detectable anti-reductase antibodies and peripheral blood mononuclear cells (PBMC) from one of three individuals previously infected with visceral leishmaniasis proliferated in the presence of recombinant...

  10. 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...

  11. 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.

  12. 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.

  13. 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.

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

    DEFF Research Database (Denmark)

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

    1998-01-01

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

  15. 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

  16. 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...

  17. One-Pot Synthesis of Pyrrolo[1,2-a]indoles by Chiral N-Triflyl Phosphoramide Catalyzed Friedel-Crafts Alkylation of 4,7-Dihydroindole with ②, y-Unsaturated a-Keto Esters

    Institute of Scientific and Technical Information of China (English)

    曾蜜; 张玮; 游书力

    2012-01-01

    Chiral N-triflyl phosphoramide was found an efficient catalyst for the enantioselective Friedel-Crafts alkylation reaction of 4,7-dihydroindole with β, γ-unsaturated a-keto esters. In the presence of 5 tool% of the optimized catalyst, various pyrrolo[1,2-a]indoles were obtained in excellent enantioselectivity, moderate yields and up to 3 : 1 di- astereoselectivity based on the one-pot synthesis including the Friedel-Crafts alkylation reaction and the subsequent p-benzoquinone oxidation.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. A novel bacteriophage KSL-1 of 2-Keto-gluconic acid producer Pseudomonas fluorescens K1005: isolation, characterization and its remedial action

    Directory of Open Access Journals (Sweden)

    Sun Wen-Jing

    2012-06-01

    Full Text Available Abstract Background Bacteriophages have the destructive damage on the industrial bioprocess. 2-Keto-gluconic acid (2KGA producing bacteria had also been attacked and lysed by bacteriophages which lowered the glucose consumption and 2KGA yield and even stopped the fermentation process. In this study, we presented the characteristics of a novel virulent bacteriophage specifically infecting Pseudomonas fluorescens K1005 and proposed an efficient remedial action for this phage infection to reduce the production loss. Results The phage KSL-1 of Pseudomonas fluorescens K1005 was isolated from abnormal 2KGA fermentation broth. It belonged to the Siphoviridae family with a hexagonal head diameter of about 99 nm and a non-contractile tail of about 103 nm × 39 nm. The genome size of phage KSL-1 was estimated to be approximately 53 kbp. Its optimal MOI to infect P. fluorescens K1005 was about 0.001. One-step growth curve gave its latent and burst periods of 90 min and 75 min with a burst size of 52 phage particles per infected cell. This phage was stable with a pH range of 7.0–10.0, and sensitive to thermal treatment. Finally, a simple remedial action was proposed by feeding fresh seed culture. Compared with the infected 2KGA fermentation, the remedial experiments restored 2KGA fermentation performance by increasing the produced 2KGA concentration to 159.89 g/L and shortening the total fermentation time of 80 h with the productivity and yield of 2.0 g/L.h and 0.89 g/g. The obtained data proved that this method was effective to combat the phage infections problems during the 2KGA fermentation. Conclusion The phage KSL-1 was a novel bacteriophage specifically infecting Pseudomonas fluorescens K1005. The remedial action of feeding fresh seed culture to the infected broth was an easily-operating and effective method to maintain a high 2KGA yield and avoid the draft of infected broth.

  3. 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.

  4. 3 Beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase from bovine adrenals: mechanism of inhibition by 3-oxo-4-aza steroids and kinetic mechanism of the dehydrogenase.

    Science.gov (United States)

    Brandt, M; Levy, M A

    1989-01-10

    Several 3-oxo-4-aza steroids (1) have been identified as inhibitors of the 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase catalyzed conversion of pregnenolone to progesterone. By kinetically decoupling the two enzyme activities isolated from bovine adrenal cortex, it has been demonstrated that inhibition by 1 occurs through interference of both activities. A preferred ordered association of substrates to the 3 beta-hydroxy-delta 5-steroid dehydrogenase in which the cofactor binds prior to steroid was determined by isotope exchange at equilibrium. With this result, the dead-end inhibition patterns of 1 with the dehydrogenase were interpreted to originate from a preferred association of inhibitor within an enzyme ternate containing NADH; this proposal is supported by data from multiple inhibition analysis indicating synergistic binding of NADH and 1. Similarly, inhibition of the 3-keto-delta 5-steroid isomerase by the 3-oxo-4-aza steroids was enhanced in the presence of the positive effector NADH. On the basis of pH profiles upon Vm, Vm/Km, and 1/Ki for both enzyme activities, inhibition is proposed to result from the structural similarity of 1 to intermediate states formed upon enzyme catalysis.

  5. Beta-keto amphetamines: studies on the metabolism of the designer drug mephedrone and toxicological detection of mephedrone, butylone, and methylone in urine using gas chromatography-mass spectrometry.

    Science.gov (United States)

    Meyer, Markus R; Wilhelm, Jens; Peters, Frank T; Maurer, Hans H

    2010-06-01

    In recent years, a new class of designer drugs has appeared on the drugs of abuse market in many countries, namely, the so-called beta-keto (bk) designer drugs such as mephedrone (bk-4-methylmethamphetamine), butylone (bk-MBDB), and methylone (bk-MDMA). The aim of the present study was to identify the metabolites of mephedrone in rat and human urine using GC-MS techniques and to include mephedrone, butylone, and methylone within the authors' systematic toxicological analysis (STA) procedure. Six phase I metabolites of mephedrone were detected in rat urine and seven in human urine suggesting the following metabolic steps: N-demethylation to the primary amine, reduction of the keto moiety to the respective alcohol, and oxidation of the tolyl moiety to the corresponding alcohols and carboxylic acid. The STA procedure allowed the detection of mephedrone, butylone, methylone, and their metabolites in urine of rats treated with doses corresponding to those reported for abuse of amphetamines. Besides macro-based data evaluation, an automated evaluation using the automated mass spectral deconvolution and identification system was performed. Mephedrone and butylone could be detected also in human urine samples submitted for drug testing. Assuming similar kinetics in humans, the described STA procedure should be suitable for proof of an intake of the bk-designer drugs in human urine.

  6. 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.

  7. Induction of quinone reductase (QR) by withanolides isolated from Physalis pubescens L. (Solanaceae).

    Science.gov (United States)

    Ji, Long; Yuan, Yonglei; Ma, Zhongjun; Chen, Zhe; Gan, Lishe; Ma, Xiaoqiong; Huang, Dongsheng

    2013-09-01

    In the present study, it was demonstrated that the dichloromethane extract of Physalis pubescens L. (DEPP) had weak potential quinone reductase (QR) inducing activity, but an UPLC-ESI-MS method with glutathione (GSH) as the substrate revealed that the DEPP had electrophiles (with an α,β-unsaturated ketone moiety). These electrophiles could induce quinone reductase (QR) activity, which might be attributed to the modification of the highly reactive cysteine residues in Keap1. Herein, four withanolides, including three new compounds physapubescin B (2), physapubescin C (3), physapubescin D (4), together with one known steroidal compound physapubescin (1) were isolated. Structures of these compounds were determined by spectroscopic analysis and that of physapubescin C (3) was confirmed by a combination of molecular modeling and quantum chemical DFT-GIAO calculations. Evaluation of the QR inducing activities of all withanolides indicated potent activities of compounds 1 and 2, which had a common α,β-unsaturated ketone moiety.

  8. 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...

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  10. 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...

  11. 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.

  12. A conservative region of the mercuric reductase gene (mera) as a molecular marker of bacterial mercury resistance

    Science.gov (United States)

    Sotero-Martins, Adriana; de Jesus, Michele Silva; Lacerda, Michele; Moreira, Josino Costa; Filgueiras, Ana Luzia Lauria; Barrocas, Paulo Rubens Guimarães

    2008-01-01

    The most common bacterial mercury resistance mechanism is based on the reduction of Hg(II) to Hg0, which is dependent of the mercuric reductase enzyme (MerA) activity. The use of a 431 bp fragment of a conservative region of the mercuric reductase (merA) gene was applied as a molecular marker of this mechanism, allowing the identification of mercury resistant bacterial strains. PMID:24031221

  13. 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...

  14. 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...

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

    Science.gov (United States)

    Balotf, Sadegh; Kavoosi, Gholamreza; Kholdebarin, Bahman

    2016-01-01

    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.

  16. 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.

  17. Transgenic Tobacco Overexpressing Tea cDNA Encoding Dihydroflavonol 4-Reductase and Anthocyanidin Reductase Induces Early Flowering and Provides Biotic Stress Tolerance.

    Directory of Open Access Journals (Sweden)

    Vinay Kumar

    Full Text Available Flavan-3-ols contribute significantly to flavonoid content of tea (Camellia sinensis L.. Dihydroflavonol 4-reductase (DFR and anthocyanidin reductase (ANR are known to be key regulatory enzymes of flavan-3-ols biosynthesis. In this study, we have generated the transgenic tobacco overexpressing individually tea cDNA CsDFR and CsANR encoding for DFR and ANR to evaluate their influence on developmental and protective abilities of plant against biotic stress. The transgenic lines of CsDFR and CsANR produced early flowering and better seed yield. Both types of transgenic tobacco showed higher content of flavonoids than control. Flavan-3-ols such as catechin, epicatechin and epicatechingallate were found to be increased in transgenic lines. The free radical scavenging activity of CsDFR and CsANR transgenic lines was improved. Oxidative stress was observed to induce lesser cell death in transgenic lines compared to control tobacco plants. Transgenic tobacco overexpressing CsDFR and CsANR also showed resistance against infestation by a tobacco leaf cutworm Spodoptera litura. Results suggested that the overexpression of CsDFR and CsANR cDNA in tobacco has improved flavonoids content and antioxidant potential. These attributes in transgenic tobacco have ultimately improved their growth and development, and biotic stress tolerance.

  18. 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.

  19. 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.

  20. 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 ...

  1. 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.

  2. 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...

  3. 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...

  4. 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.  

  5. 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.  

  6. 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...

  7. 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.

  8. 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....

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Study on Film Properties of Keto-Aldehyde Resin Based Polyurethane Dispersion%基于醛酮树脂的聚氨酯分散体的涂膜性能研究

    Institute of Scientific and Technical Information of China (English)

    徐鹏; 孙东成

    2013-01-01

    以二羟甲基丁酸(DMBA)和新型聚氧乙烯醚作为亲水单体,以聚己二酸新戊二醇酯(PNA)、醛酮树脂、异佛尔酮二异氰酸酯(IPDI)为主要原料,采用预聚体法合成了阴/非离子型聚氨酯分散体(PUD),并以此为主要组分制备了水性涂料.研究表明,醛酮改性的聚氨酯分散体具有较小的粒径和较低的黏度,耐热性良好,适用于涂料产品;当亲水基团含量为25 mmol/100 g,醛酮树脂含量为40%,n(-NCO)∶n(-OH)=1.3时涂膜的综合性能最好,涂膜硬度可达4H,且涂膜附着力、柔韧性、耐冲击性和耐水性优异.%An anionic/non - ionic type polyurethane dispersion(PUD) was synthesized by using 2,2 -dimethylolbutyric acid(DMBA) and a new polyoxyethylene ether as hydrophilic monomer, polyneopentane-diol adipate( PNA) , keto - aldehyde resin and isophorone diisocyanate (IPDI) as raw materials by a prepoly-merization process. Which was then used as a main material to prepare the waterborne coatings. Results showed that keto - aldehyde resin modified polyurethane dispersions had advantages of small particle size, low viscosity and good heat resistance, so it were suitable for use in coating products. When the hydrophilic group content was 25 mmol/100 g, keto - aldehyde resin content 40% , and -NCO/-OH molar ratio 1. 3 , the film showed the best comprehensive performance, such as pencil hardness(4H) , adhesion, flexibility, impact resistance and water resistance.

  12. Síntese de β-cetoésteres cíclicos: novo procedimento para ciclizações de Dieckmann empregando ALCL3 e trietilamina Synthesis of β-keto esters: new easy procedure for dieckmann cyclization employing aluminum chloride and triethylamine

    Directory of Open Access Journals (Sweden)

    Emerson P. Peçanha

    1997-08-01

    Full Text Available In this communication we describe a new methodology to Dieckmann cyclization of diethyl adipate (1 and diethyl pimelate (3 applying "push-pull" strategy using anhydrous aluminium trichloride and triethylamine in dichloromethane at room temperature. This method is very efficient, simple, safe and reproducible, giving the corresponding cyclic β-keto ester derivatives in 84% and 71% yield, respectively.

  13. CESIUM DITHIOLATE BASED SYNTHESES OF KETO-FUNCTIONALIZED THIO-CROWN ETHERS EMPLOYING THE NOVEL BUILDING-BLOCK 1,3-DIMERCAPTOACETONE - MOLECULAR-STRUCTURES OF 2,5,9,12-TETRATHIA-7-OXO-(13)-M-BENZENOPHANE AND 1,4,7,10,13-PENTATHIACYCLOHEXADECAN-15-ONE

    NARCIS (Netherlands)

    EDEMA, JJH; SCHOONBEEK, FS; MEETSMA, A; VANBOLHUIS, F; KELLOGG, RM

    1993-01-01

    Cyclization of the bis cesium thiolate (generated in situ) of 1,3-dimercaptoacetone (1) with alpha,omega-dihalides in DMF affords the corresponding cyclic thioethers in good yields, providing an efficient alternative route to keto-functionalized thio-crown ethers. A previously suggested dimeric stru

  14. 乙酰基-11-酮-β-乳香酸抗肿瘤活性的研究进展%Research progress of acetyl-11-keto-β-boswellic acid on its anti-cancer activity

    Institute of Scientific and Technical Information of China (English)

    陆益彬; 何明芳

    2012-01-01

    Acetyl-11-keto-β-boswellic acid (AKBA) is one of the triterpenes in the gum resin of the Boswellia serrata and Boswellia carterii,also known as Salai guggal or Indian frankincense.There has been growing interst in anti-tumor activity of AKBA.This review will summarize the latest advances of AKBA on anti-tumor activity for the better understanding of this compound and its further applications.%乳香中三萜类成分乙酰基-11-酮-β-乳香酸(AKBA)具有非常强的抗肿瘤作用,是乳香中抗肿瘤活性成分研究的焦点.对AKBA的各类抗肿瘤活性的机制研究的最新进展进行综述,以期为乳香和AKBA的临床研究开发提供参考.

  15. 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...

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  16. 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.

  17. 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...

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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...

  4. 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...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-03-08

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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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...

  11. 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...

  12. 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.

  13. 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.

  14. 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).

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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...

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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).

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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) (-).

  2. 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.

  3. 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.

  4. 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.

  5. The effect of α-keto acid on diabetes nephropathy%复方α-酮酸联合氯沙坦钾对糖尿病肾病的疗效分析

    Institute of Scientific and Technical Information of China (English)

    杨方才

    2011-01-01

    Objective: To evaluate the effect of α-keto acid on diabetes nephroqathy. Methods: Under the use of low-protein diet and Losartan Potassium, sixty-two patients with Ⅲ, Ⅳ plase diabetie nephropathy were divided into two groups, experimental group(n =32) (α-keto acid for 24 weeks) and control group( n = 30). Results: After treatment, the 24 h urine albumin excretion decreased significantly in both of the two groups, and the effect in experimental group was better than in control groups. There were no obvions change in the index of nutrition in experimental group, but ALB, PA, Hb and MAMC in the control groups were decreessed significantly ( P < 0.05 ). Couclusion: The α-keto acid can reduce urine albumin more effectively, which is beneficial to the control of blood pressure, renal damage and malnutrition.%目的:评价在低蛋白饮食和氯沙坦钾治疗基础上用复方α-酮酸对糖尿病肾病的影响.方法:选择62例Ⅲ、Ⅳ期2型糖尿病肾病病人,在低蛋白饮食加氯沙坦钾基础上控制血压、血糖和血脂平稳后,随机分为观察组(32例)和对照组(30例),观察组病人给予复方α-酮酸,两组均随访观察24周.结果:两组病人治疗后24h尿清蛋白量均较治疗前明显减少,观察组优于对照组,差异有显著性意义(P<0.01);两组病人治疗前后血尿素氮(BUN)、肌酐(Cr)、肾小球滤过率等均无明显改变;观察组病人治疗前后各项营养指标均无明显改变,而对照组病人清蛋白、前清蛋白、血红蛋白和上臂肌围均有明显下降(P<0.05);观察组病人治疗后收缩压较治疗前明显下降(P<0.05).结论:复方α-酮酸联合氯沙坦钾能更有效地减少尿蛋白,可有利于控制血压,延缓肾功能进一步损害,可有效地避免低蛋白饮食造成的营养不良.

  6. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  8. 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...

  9. 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

  10. 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...

  11. 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.

  12. 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

  13. 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...

  14. 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%.

  15. 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.

  16. 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

  17. 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.

  18. 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...

  19. [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.

  20. 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