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Sample records for acid dehydrogenase activity

  1. Efficient screening for new amino acid dehydrogenase activity: directed evolution of Bacillus sphaericus phenylalanine dehydrogenase towards activity with an unsaturated non-natural amino acid.

    Science.gov (United States)

    Chen, Sihong; Engel, Paul C

    2009-06-15

    Mutants of phenylalanine dehydrogenase (PheDH) from Bacillus sphaericus with improved activity and specificity towards an unsaturated non-natural amino acid of commercial interest, propargylglycine, have been obtained by directed evolution after screening 10,000 colonies. An effective high-throughput screening assay for the detection of amino acid dehydrogenase activity using spectrophotometric measurement of formazan colour has been developed and explored. Parallel over-expression of the target protein library in 96-well plates was optimized by using auto-induction medium. Using these methodologies and medium/high mutation frequency error-prone polymerase chain reaction, combinations of mutations and multi-site saturation mutagenesis targeting two "hot-spots" close to the active site of the enzyme, one mutant was obtained with 7.4-fold improved catalytic efficiency with the target substrate together with 612-fold improvement of selectivity between the target substrate and the natural one. In the high mutation frequency epPCR and saturation mutagenesis, a robotic colony picker and automation workstation were employed for the high-throughput screening.

  2. Potentiation of insulin release in response to amino acid methyl esters correlates to activation of islet glutamate dehydrogenase activity

    DEFF Research Database (Denmark)

    Kofod, Hans; Lernmark, A; Hedeskov, C J

    1986-01-01

    Column perifusion of mouse pancreatic islets was used to study the ability of amino acids and their methyl esters to influence insulin release and activate islet glutamate dehydrogenase activity. In the absence of L-glutamine, L-serine and the methyl ester of L-phenylalanine, but neither L...... glutamate dehydrogenase activity showed that only the two methyl esters of L-phenylalanine and L-serine activated the enzyme. It is concluded that the mechanism by which methyl esters of amino acids potentiate insulin release is most likely to be mediated by the activation of pancreatic beta-cell glutamate...

  3. 15-hydroxyprostaglandin dehydrogenase activity in vitro in lung and kidney of essential fatty acid-deficient rats

    DEFF Research Database (Denmark)

    Hansen, Harald S.; Toft, B.S.

    1978-01-01

    Weanling rats were fed for 6 months on a diet deficient in essential fatty acids: either fat-free, or with 28% (w/w) partially hydrogenated fish oil. Control rats were fed a diet with 28% (w/w) arachis oil for 6 months. 15-Hydroxyprostaglandin dehydrogenase activity was determined as initial rates...... of the two groups on diets deficient in essential fatty acids as compared to the control group. No difference was observed in dehydrogenase activity in the kidneys. The dehydrogenase may be of importance for the regulation of the level of endogenous prostaglandins and, thus, a decrease in activity could...

  4. 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...

  5. Regulation of lactate dehydrogenase activity by ascorbic acid in organs of rats with hypoxia of closed space

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    Олександр Костянтинович Будняк

    2015-07-01

    Full Text Available The research of the effects of different doses of ascorbic acid (AA on activity of lactate dehydrogenase (LDG in the organs of white nonlinear rats with hypoxia of closed space (HCS has been conducted. Injections of АА to rats for 30 minutes of HCS reduced the LDG activity in all organs of hypoxic animals

  6. Mycophenolic acid exposure and complement fraction C3 influence inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus.

    Science.gov (United States)

    Mino, Yasuaki; Naito, Takafumi; Shimoyama, Kumiko; Ogawa, Noriyoshi; Kawakami, Junichi

    2017-07-01

    Background Mycophenolate mofetil has recently been reported to be effective against systemic lupus erythematosus. The influence of the pharmacokinetics of mycophenolic acid, the active form of mycophenolate mofetil and the major inactive mycophenolic acid phenolic glucuronide on the activity of the target enzyme inosine 5'-monophosphate dehydrogenase, is expected to be revealed. The aim of this study was to identify the factors associated with inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus patients. Methods Fifty systemic lupus erythematosus patients in remission maintenance phase (29 received mycophenolate mofetil [MMF+] and 21 did not [MMF-]) were enrolled. Median and interquartile range of dose of mycophenolate mofetil were 1500 and 1000-1500 mg/day, respectively. Stepwise multiple linear regression analysis was performed to assess the dependence between inosine 5'-monophosphate dehydrogenase activity and 25 predictor values including predose plasma concentrations of free mycophenolic acid and mycophenolic acid phenolic glucuronide. Results Median and interquartile range of predose total plasma concentrations of mycophenolic acid and mycophenolic acid phenolic glucuronide were 2.73 and 1.43-5.73 and 25.5 and 13.1-54.7  µg/mL, respectively. Predose inosine 5'-monophosphate dehydrogenase activity was significantly higher in MMF+ than MMF- patients (median 38.3 and 20.6 nmoL xanthosine 5'-monophosphate/g haemoglobin/h, Psystemic lupus erythematosus patients receiving mycophenolate mofetil therapy. Inosine 5'-monophosphate dehydrogenase activity may be determined by mycophenolic acid exposure and complement fraction C3 in systemic lupus erythematosus patients.

  7. Regulation of the activity of lactate dehydrogenases from four lactic acid bacteria

    NARCIS (Netherlands)

    Feldman-Salit, A.; Hering, S.; Messiha, H.L.; Veith, N.; Cojocaru, V.; Sieg, A.; Westerhoff, H.V.; Kreikemeyer, B.; Wade, R.C.; Fiedler, T.

    2013-01-01

    Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the

  8. Lactic acid production by Rhizopus oryzae transformants with modified lactate dehydrogenase activity.

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    Skory, C D

    2004-04-01

    Rhizopus oryzae is capable of producing high levels of lactic acid by the fermentation of glucose. Yields typically vary over 60-80%, with the remaining glucose diverted primarily into ethanol fermentation. The goal of this work was to increase lactate dehydrogenase (LDH) activity, so lactic acid fermentation could more effectively compete for available pyruvate. Three different constructs, pLdhA71X, pLdhA48XI, and pLdhA89VII, containing various lengths of the ldhA gene fragment, were transformed into R. oryzae. This fungus rarely integrates DNA used for transformation, but instead relies on extra-chromosomal replication in a high-copy number. Plasmid pLdhA48XI was linearized prior to transformation in order to facilitate integration into the pyrG gene used for selection. Isolates transformed with ldhA containing plasmid were compared with both the wild-type parent strain and the auxotrophic recipient strain containing vector only. All isolates transformed with pLdhA71X or pLdhA48XI had multiple copies of the ldhA gene that resulted in ldhA transcript accumulation, LDH specific activity, and lactic acid production higher than the controls. Integration of plasmid pLdhA48XI increased the stability of the strain, but did not seem to offer any benefit for increasing lactic acid production. Since lactic acid fermentation competes with ethanol and fumaric acid production, it was not unexpected that increased lactic acid production was always concomitant with decreased ethanol and fumaric acid. Plasmid pLdhA71X, containing a large ldhA fragment (6.1 kb), routinely yielded higher levels of lactic acid than the smaller region (3.3 kb) used to construct plasmid pLdhA48XI. The greatest levels of ldhA transcript and enzyme production occurred with isolates transformed with plasmid pLdhA89VII. However, these transformants always produced less lactic acid and higher amounts of ethanol, fumaric, and glycerol compared with the control.

  9. Regulation of the activity of lactate dehydrogenases from four lactic acid bacteria.

    Science.gov (United States)

    Feldman-Salit, Anna; Hering, Silvio; Messiha, Hanan L; Veith, Nadine; Cojocaru, Vlad; Sieg, Antje; Westerhoff, Hans V; Kreikemeyer, Bernd; Wade, Rebecca C; Fiedler, Tomas

    2013-07-19

    Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (Pi), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (Kact) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of Pi are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of Pi inhibits E. faecalis LDH2, whereas in the absence of FBP, Pi is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDHs of different LABs.

  10. Palladium alpha-lipoic acid complex formulation enhances activities of Krebs cycle dehydrogenases and respiratory complexes I-IV in the heart of aged rats.

    Science.gov (United States)

    Sudheesh, N P; Ajith, T A; Janardhanan, K K; Krishnan, C V

    2009-08-01

    Age-related decline in the capacity to withstand stress, such as ischemia and reperfusion, results in congestive heart failure. Though the mechanisms underlying cardiac decay are not clear, age dependent somatic damages to mitochondrial DNA (mtDNA), loss of mitochondrial function, and a resultant increase in oxidative stress in heart muscle cells may be responsible for the increased risk for cardiovascular diseases. The effect of a safe nutritional supplement, POLY-MVA, containing the active ingredient palladium alpha-lipoic acid complex, was evaluated on the activities of the Krebs cycle enzymes such as isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, II, III, and IV in heart mitochondria of aged male albino rats of Wistar strain. Administration of 0.05 ml/kg of POLY-MVA (which is equivalent to 0.38 mg complexed alpha-lipoic acid/kg, p.o), once daily for 30 days, was significantly (pKrebs cycle dehydrogenases, and mitochondrial electron transport chain complexes. The unique electronic and redox properties of palladium alpha-lipoic acid complex appear to be a key to this physiological effectiveness. The results strongly suggest that this formulation might be effective to protect the aging associated risk of cardiovascular and neurodegenerative diseases.

  11. Thiamine Induces Long-Term Changes in Amino Acid Profiles and Activities of 2-Oxoglutarate and 2-Oxoadipate Dehydrogenases in Rat Brain.

    Science.gov (United States)

    Tsepkova, P M; Artiukhov, A V; Boyko, A I; Aleshin, V A; Mkrtchyan, G V; Zvyagintseva, M A; Ryabov, S I; Ksenofontov, A L; Baratova, L A; Graf, A V; Bunik, V I

    2017-06-01

    Molecular mechanisms of long-term changes in brain metabolism after thiamine administration (single i.p. injection, 400 mg/kg) were investigated. Protocols for discrimination of the activities of the thiamine diphosphate (ThDP)-dependent 2-oxoglutarate and 2-oxoadipate dehydrogenases were developed to characterize specific regulation of the multienzyme complexes of the 2-oxoglutarate (OGDHC) and 2-oxoadipate (OADHC) dehydrogenases by thiamine. The thiamine-induced changes depended on the brain-region-specific expression of the ThDP-dependent dehydrogenases. In the cerebral cortex, the original levels of OGDHC and OADHC were relatively high and not increased by thiamine, whereas in the cerebellum thiamine upregulated the OGDHC and OADHC activities, whose original levels were relatively low. The effects of thiamine on each of the complexes were different and associated with metabolic rearrangements, which included (i) the brain-region-specific alterations of glutamine synthase and/or glutamate dehydrogenase and NADP+-dependent malic enzyme, (ii) the brain-region-specific changes of the amino acid profiles, and (iii) decreased levels of a number of amino acids in blood plasma. Along with the assays of enzymatic activities and average levels of amino acids in the blood and brain, the thiamine-induced metabolic rearrangements were assessed by analysis of correlations between the levels of amino acids. The set and parameters of the correlations were tissue-specific, and their responses to the thiamine treatment provided additional information on metabolic changes, compared to that gained from the average levels of amino acids. Taken together, the data suggest that thiamine decreases catabolism of amino acids by means of a complex and long-term regulation of metabolic flux through the tricarboxylic acid cycle, which includes coupled changes in activities of the ThDP-dependent dehydrogenases of 2-oxoglutarate and 2-oxoadipate and adjacent enzymes.

  12. Genetic analysis of central carbon metabolism unveils an amino acid substitution that alters maize NAD-dependent isocitrate dehydrogenase activity.

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    Nengyi Zhang

    Full Text Available BACKGROUND: Central carbon metabolism (CCM is a fundamental component of life. The participating genes and enzymes are thought to be structurally and functionally conserved across and within species. Association mapping utilizes a rich history of mutation and recombination to achieve high resolution mapping. Therefore, applying association mapping in maize (Zea mays ssp. mays, the most diverse model crop species, to study the genetics of CCM is a particularly attractive system. METHODOLOGY/PRINCIPAL FINDINGS: We used a maize diversity panel to test the CCM functional conservation. We found heritable variation in enzyme activity for every enzyme tested. One of these enzymes was the NAD-dependent isocitrate dehydrogenase (IDH, E.C. 1.1.1.41, in which we identified a novel amino-acid substitution in a phylogenetically conserved site. Using candidate gene association mapping, we identified that this non-synonymous polymorphism was associated with IDH activity variation. The proposed mechanism for the IDH activity variation includes additional components regulating protein level. With the comparison of sequences from maize and teosinte (Zea mays ssp. Parviglumis, the maize wild ancestor, we found that some CCM genes had also been targeted for selection during maize domestication. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate the efficacy of association mapping for dissecting natural variation in primary metabolic pathways. The considerable genetic diversity observed in maize CCM genes underlies heritable phenotypic variation in enzyme activities and can be useful to identify putative functional sites.

  13. Inhibition of snowshoe hare succinate dehydrogenase activity as a mechanism of deterrence for papyriferic acid in birch.

    Science.gov (United States)

    Forbey, Jennifer Sorensen; Pu, Xinzhu; Xu, Dong; Kielland, Knut; Bryant, John

    2011-12-01

    The plant secondary metabolite papyriferic acid (PA) deters browsing by snowshoe hares (Lepus americanus) on the juvenile developmental stage of the Alaska paper birch (Betula neoalaskana). However, the physiological mechanism that reduces browsing remains unknown. We used pharmacological assays and molecular modeling to test the hypothesis that inhibition of succinate dehydrogenase (SDH) is a mode of action (MOA) of toxicity of PA in snowshoe hares. We tested this hypothesis by measuring the effect of PA on the activity of SDH in liver mitochondria isolated from wild hares. In addition, we used molecular modeling to determine the specific binding site of PA on SDH. We found that PA inhibits SDH from hares by an uncompetitive mechanism in a dose-dependent manner. Molecular modeling suggests that inhibition of SDH is a result of binding of PA at the ubiquinone binding sites in complex II. Our results provide a MOA for toxicity that may be responsible for the concentration-dependent anti-feedant effects of PA. We propose that snowshoe hares reduce the dose-dependent toxic consequences of PA by relying on efflux transporters and metabolizing enzymes that lower systemic exposure to dietary PA.

  14. Reduction of lipoic acid by lipoamide dehydrogenase.

    NARCIS (Netherlands)

    Biewenga, G.Ph.; Dorstijn, M.A.; Verhagen, J.V.; Haenen, G.R.M.M.; Bast, A.

    1996-01-01

    Racemic lipoic acid is therapeutically applied in pathologies in which free radicals are involved. The in vivo reduction of lipoic acid may play an essential role in its antioxidant effect. It was found that mitochondrial lipoamide dehydrogenase (LipDH, EC 1.8.1.4.) reduces the R-enantiomer 28 times

  15. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci

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    Guillermo Hugo Peralta

    Full Text Available ABSTRACT Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor.

  16. STUDIES CONCERNING THE INFLUENCE OF SOME AMINO ACIDS ON THE DYNAMICS OF KREBS CYCLE DEHYDROGENASES ACTIVITY AT MONILINIA LAXA (ADERH.& RUHL. HONEY PARASITE ON PLUM TREES

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    Elena Tutu

    2011-11-01

    Full Text Available As ubiquitous organisms, fungi grow on a large number of organic substrate, alive or dead, confronting therefore with a wide variety of carbohydrates and various physical factors, and their versatility to adapt and be able to use a large number of these compounds could provide them the chance to survive. Given that, these fungi have a rich enzyme equipment that allows them to operate on different metabolic pathways, this study aims to monitor the dynamics activity of some Krebs cycle dehydrogenases in Monilinia laxa (Aderh & Ruhl. Honey species parasitic on various species of plum trees. To this end, the fungus was cultivated in vitro on media enriched with different carbohydrates and the isocitrate dehydrogenase, �-cetoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase activity in the fungus mycelium was followed, at 7, respectively, 14 days after the inoculation of the culture medium and determined using the spectrophotometric Sîsoev and Krasna method (Cojocaru, D.C., 2009. Data revealed obvious differences depending on the type of carbohydrate introduced into the medium and the age of the culture mycelia.

  17. INFLUENCE OF SELECTED PHARMACEUTICALS ON ACTIVATED SLUDGE DEHYDROGENASE ACTIVITY

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    Agnieszka Tomska

    2016-06-01

    The aim of this work was to evaluate the effect of selected antibiotics - sulfanilamide and erythromycin on activated sludge dehydrogenase activity with use of trifenyltetrazolinum chloride (TTC test. Dehydrogenases activity is an indicator of biochemical activity of microorganisms present in activated sludge or the ability to degrade organic compounds in waste water. TTC test is particularly useful for the regularity of the course of treatment, in which the presence of inhibitors of biochemical reactions and toxic compounds are present. It was observed that the dehydrogenase activity decreases with the increase of a antibiotics concentration. The lowest value of the dehydrogenase activity equal to 32.4 μmol TF / gMLSS obtained at sulfanilamide concentration 150mg / l. For this sample, an inhibition of dehydrogenase activity was 31%.

  18. [Effects of infrasound on activities of 3beta hydroxysteroid dehydrogenase and acid phosphatase of polygonal cells in adrenal cortex zona fasciculate in mice].

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    Dang, Wei-min; Wang, Sheng; Tian, Shi-xiu; Chen, Bing; Sun, Fei; Li, Wei; Jiao, Yan; He, Li-hua

    2007-02-01

    To explore the biological effects of infrasound on the polygonal cells in adrenal cortex zona fasciculation in mice. The biological effects of infrasound on the activities of 3beta hydroxysteroid dehydrogenase (3-betaHSDH) and acid phosphatase(ACP) of the polygonal cells in adrenal cortex zona fasciculate were observed when exposure to 8 and 16 Hz infrasound at 80, 90, 100, 110, 120 and 130 dB for 1 day, 7 days and 14 days or 14 days after the exposure. When exposure to 8 Hz infrasound, the enzyme activities of 3-betaHSDH increase as the sound pressure levels increase. Only when the sound pressure levels reach 130 dB, the enzyme activities began to decrease exceptionally. When exposure to 16 Hz, 80 dB infrasound, no significant difference between the treatment and control group in the activities of 3-betaHSDH could be observed, but the injury of the polygonal cells had appeared. When exposure to 16 Hz, 100 dB infrasound, the activities of 3-betaHSDH started to increase. The cell injury still existed. When exposed to 16 Hz, 120 dB infrasound, the local tissue damage represented. Fourteen days after the mice exposure to 8 Hz, 90 dB and 130 dB infrasound for 14 days continuously, the local tissue injury of the adrenal cortex zona fasciculation began to recover at certain extent, but the higher the exposure sound pressure level, the poorer the tissue recovery. The biological effects of infrasound on the polygonal cells in adrenal cortex zona fasciculation response to the frequency of the infrasound are found at certain action strength range, but this characteristic usually is covered by the severe tissue injury. When exposure to infrasound is stopped for a period of time, the local tissue injury of the adrenal cortex zona fasciculation could recovers at certain extent, but the higher the exposure sound pressure level, the more poorer the tissue recovery.

  19. Inhibition of snowshoe hare succinate dehydrogenase activity as a mechanism of deterrence for papyriferic acid in birch

    Science.gov (United States)

    Jennifer Sorensen Forbey; Xinzhu Pu; Dong Xu; Knut Kielland; John. Bryant

    2011-01-01

    The plant secondary metabolite papyriferic acid (PA) deters browsing by snowshoe hares (Lepus americanus) on the juvenile developmental stage of the Alaska paper birch (Betula neoalaskana). However, the physiological mechanism that reduces browsing remains unknown. We used pharmacological assays and molecular modeling to test the...

  20. Metabolism of branched-chain amino acids in fibroblasts from patients with maple syrup urine disease and other abnormalities of branched-chain ketoacid dehydrogenase activity

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, I.; Sweetman, L.; Nyhan, W.L.

    1986-02-01

    The metabolism of branched-chain amino acids was studied in cultured fibroblasts from patients with branched-chain ketoacid dehydrogenase deficiency using 1-/sup 14/C- and UL-/sup 14/C-leucine and valine. The formation of /sup 14/CO/sub 2/ from 1-/sup 14/C-valine or 1-/sup 14/C-leucine was 1-3% of normal. In fibroblasts of patients with associated lactic acidemia the values were 4-29% of control. Analysis of organic acid products revealed that in both patients and controls the amount of labeled alpha-ketoisovalerate recovered after incubation with 1-/sup 14/C-valine was one-third of the amount of alpha-ketoisocaproate recovered after incubation with 1-/sup 14/C-leucine. Very little alpha-hydroxyisocaproate was produced, while the amount of alpha-hydroxyisovalerate was about 10% of the alpha-ketoisovalerate. Unexpectedly beta-hydroxyisobutyrate was found to be the major metabolic product of UL-/sup 14/C-valine in normal fibroblasts. Large accumulations of beta-hydroxyisovalerate were found in normal fibroblasts using UL-/sup 14/C-leucine. There were little or no conversions to these compounds in fibroblasts of patients with branched-chain ketoacid dehydrogenase deficiency. There were substantial conversions in the patients in whom dehydrogenase deficiency was associated with lactic acidemia.

  1. In vivo regulation of alcohol dehydrogenase and lactate dehydrogenase in Rhizopus oryzae to improve L-lactic acid fermentation.

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    Thitiprasert, Sitanan; Sooksai, Sarintip; Thongchul, Nuttha

    2011-08-01

    Rhizopus oryzae is becoming more important due to its ability to produce an optically pure L: -lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4'-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways.

  2. Comparison of benzyl alcohol dehydrogenases and benzaldehyde dehydrogenases from the benzyl alcohol and mandelate pathways in Acinetobacter calcoaceticus and from the TOL-plasmid-encoded toluene pathway in Pseudomonas putida. N-terminal amino acid sequences, amino acid compositions and immunological cross-reactions.

    Science.gov (United States)

    Chalmers, R M; Keen, J N; Fewson, C A

    1991-01-01

    1. N-Terminal sequences were determined for benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase I and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus N.C.I.B. 8250, benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by the TOL plasmid pWW53 in Pseudomonas putida MT53 and yeast K(+)-activated aldehyde dehydrogenase. Comprehensive details of the sequence determinations have been deposited as Supplementary Publication SUP 50161 (5 pages) at the British Library Document Supply Centre, Boston Spa. Wetherby. West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273. 5. The extent of sequence similarity suggests that the benzyl alcohol dehydrogenases are related to each other and also to established members of the family of long-chain Zn2(+)-dependent alcohol dehydrogenases. Benzaldehyde dehydrogenase II from Acinetobacter appears to be related to the Pseudomonas TOL-plasmid-encoded benzaldehyde dehydrogenase. The yeast K(+)-activated aldehyde dehydrogenase has similarity of sequence with the mammalian liver cytoplasmic class of aldehyde dehydrogenases but not with any of the Acinetobacter or Pseudomonas enzymes. 2. Antisera were raised in rabbits against the three Acinetobacter enzymes and both of the Pseudomonas enzymes, and the extents of the cross-reactions were determined by immunoprecipitation assays with native antigens and by immunoblotting with SDS-denatured antigens. Cross-reactions were detected between the alcohol dehydrogenases and also among the aldehyde dehydrogenases. This confirms the interpretation of the N-terminal sequence comparisons and also indicates that benzaldehyde dehydrogenase I from Acinetobacter may be related to the other two benzaldehyde dehydrogenases. 3. The amino acid compositions of the Acinetobacter and the Pseudomonas enzymes were determined and the numbers of amino acid residues per subunit were calculated to be: benzyl alcohol dehydrogenase

  3. Effects of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity.

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    Li, Sha; Gan, Li-Qin; Li, Shu-Ke; Zheng, Jie-Cong; Xu, Dong-Ping; Li, Hua-Bin

    2014-01-01

    Various alcoholic beverages containing different concentrations of ethanol are widely consumed, and excessive alcohol consumption may result in serious health problems. The consumption of alcoholic beverages is often accompanied by non-alcoholic beverages, such as herbal infusions, tea and carbonated beverages to relieve drunk symptoms. The aim of this study was to supply new information on the effects of these beverages on alcohol metabolism for nutritionists and the general public, in order to reduce problems associated with excessive alcohol consumption. The effects of 57 kinds of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity were evaluated. Generally, the effects of these beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity are very different. The results suggested that some beverages should not be drank after excessive alcohol consumption, and several beverages may be potential dietary supplements for the prevention and treatment of problems related to excessive alcohol consumption.

  4. Discrimination of aliphatic substrates by a single amino acid substitution in Bacillus badius and Bacillus sphaericus phenylalanine dehydrogenases.

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    Tachibana, Shinjiro; Kuwamori, Yuko; Asano, Yasuhisa

    2009-03-23

    Replacement of glycine by serine at positions 123 and 124 of phenylalanine dehydrogenases from Bacillus badius and Bacillus sphaericus respectively strikingly decreased enzyme activity toward aromatic amino acids and resulted in an elevation of relative activity toward aliphatic amino acids. The mutant from B. badius preferentially dehydrogenated branched-chain amino acids, while that from B. sphaericus acted on amino acids with straight-chain amino acids.

  5. Variants of glycerol dehydrogenase having D-lactate dehydrogenase activity and uses thereof

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    Wang, Qingzhao; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    2017-08-29

    The present invention provides methods of designing and generating glycerol dehydrogenase (GlyDH) variants that have altered function as compared to a parent polypeptide. The present invention further provides nucleic acids encoding GlyDH polypeptide variants having altered function as compared to the parent polypeptide. Host cells comprising polynucleotides encoding GlyDH variants and methods of producing lactic acids are also provided in various aspects of the invention.

  6. A bioluminescence assay for aldehyde dehydrogenase activity.

    Science.gov (United States)

    Duellman, Sarah J; Valley, Michael P; Kotraiah, Vinayaka; Vidugiriene, Jolanta; Zhou, Wenhui; Bernad, Laurent; Osterman, Jean; Kimball, Joshua J; Meisenheimer, Poncho; Cali, James J

    2013-03-15

    The aldehyde dehydrogenase (ALDH) family of enzymes is critical for cell survival and adaptation to cellular and environmental stress. These enzymes are of interest as therapeutic targets and as biomarkers of stem cells. This article describes a novel, homogeneous bioluminescence assay to study the activity of the ALDH enzymes. The assay is based on a proluciferin-aldehyde substrate that is recognized and utilized by multiple ALDH enzyme isoforms to generate luciferin. A detection reagent is added to inactivate ALDH and generate light from the luciferin product. The luminescent signal is dependent on the ALDH enzyme concentration and the incubation time in the ALDH reaction; moreover, the luminescent signal generated with the detection reagent is stable for greater than 2 h. This assay provides many advantages over standard NADH fluorescence assays. It is more sensitive and the signal stability provided allows convenient assay setup in batch mode-based high-throughput screens. The assay also shows an accurate pharmacological response for a common ALDH inhibitor and is robust, with a large assay window (S/B=64) and Z'=0.75. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.

    Science.gov (United States)

    Wang, Qingzhao; Ingram, Lonnie O; Shanmugam, K T

    2011-11-22

    Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates.

  8. Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum.

    Science.gov (United States)

    Tesfaye, M; Temple, S J; Allan, D L; Vance, C P; Samac, D A

    2001-12-01

    Al toxicity is a severe impediment to production of many crops in acid soil. Toxicity can be reduced through lime application to raise soil pH, however this amendment does not remedy subsoil acidity, and liming may not always be practical or cost-effective. Addition of organic acids to plant nutrient solutions alleviates phytotoxic Al effects, presumably by chelating Al and rendering it less toxic. In an effort to increase organic acid secretion and thereby enhance Al tolerance in alfalfa (Medicago sativa), we produced transgenic plants using nodule-enhanced forms of malate dehydrogenase and phosphoenolpyruvate carboxylase cDNAs under the control of the constitutive cauliflower mosaic virus 35S promoter. We report that a 1.6-fold increase in malate dehydrogenase enzyme specific activity in root tips of selected transgenic alfalfa led to a 4.2-fold increase in root concentration as well as a 7.1-fold increase in root exudation of citrate, oxalate, malate, succinate, and acetate compared with untransformed control alfalfa plants. Overexpression of phosphoenolpyruvate carboxylase enzyme specific activity in transgenic alfalfa did not result in increased root exudation of organic acids. The degree of Al tolerance by transformed plants in hydroponic solutions and in naturally acid soil corresponded with their patterns of organic acid exudation and supports the concept that enhancing organic acid synthesis in plants may be an effective strategy to cope with soil acidity and Al toxicity.

  9. Structural basis for the dysfunctioning of human 2-oxo acid dehydrogenase complexes

    NARCIS (Netherlands)

    Hengeveld, A.F.; Kok, de A.

    2002-01-01

    2-oxo acid dehydrogenase complexes are a ubiquitous family of multienzyme systems that catalyse the oxidative decarboxylation of various 2-oxo acid substrates. They play a key role in the primary energy metabolism: in glycolysis (pyruvate dehydrogenase complex), the citric acid cycle (2-oxoglutarate

  10. 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...

  11. Lactic acid production by Saccharomyces cerevisiae expressing a Rhizopus oryzae lactate dehydrogenase gene.

    Science.gov (United States)

    Skory, Christopher D

    2003-01-01

    This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2 micro -containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25-0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased.

  12. RECIPIENT PRETRANSPLANT INOSINE MONOPHOSPHATE DEHYDROGENASE ACTIVITY IN NONMYELOABLATIVE HCT

    Science.gov (United States)

    Bemer, Meagan J.; Risler, Linda J.; Phillips, Brian R.; Wang, Joanne; Storer, Barry E.; Sandmaier, Brenda M.; Duan, Haichuan; Raccor, Brianne S.; Boeckh, Michael J.; McCune, Jeannine S.

    2014-01-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5’- monophosphate (IMP) to xanthosine 5’-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNC) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T-cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation, but not with chronic GVHD, relapse, non-relapse mortality, or overall mortality. We conclude that quantitation of the recipient’s pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient’s sensitivity to MMF, but confirmatory studies are needed. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

  13. Glucose -6- phosphate dehydrogenase (g6pd) activity and ...

    African Journals Online (AJOL)

    The activity of red blood cell Glucose 6-phosphate dehydrogenase (G6PD) in one hundred and twenty six healthy male individuals who are Nigerians residing in Jos was evaluated. The enzyme activity was determined quantitatively by spectrophotometer assay method. The activity of red cell G6PD enzyme was subnormal ...

  14. Study on the triphenyl tetrazolium chloride– dehydrogenase activity ...

    African Journals Online (AJOL)

    The results indicate that dehydrogenase activity (DHA) can effectively facilitate the biochemical reaction of tomato paste wastewater treatment upon analysis of the influences of various DHA and kinetic factors. The biological activity of the activated sludge by TTC-DHA was changed to become applicable to aeration and ...

  15. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    Directory of Open Access Journals (Sweden)

    Koji Sode

    2012-11-01

    Full Text Available Mutagenesis studies on glucose oxidases (GOxs were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe and Aspergillus niger GOx (PDB ID; 1cf3. We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.

  16. Improved production of propionic acid in Propionibacterium jensenii via combinational overexpression of glycerol dehydrogenase and malate dehydrogenase from Klebsiella pneumoniae.

    Science.gov (United States)

    Liu, Long; Zhuge, Xin; Shin, Hyun-Dong; Chen, Rachel R; Li, Jianghua; Du, Guocheng; Chen, Jian

    2015-04-01

    Microbial production of propionic acid (PA), an important chemical building block used as a preservative and chemical intermediate, has gained increasing attention for its environmental friendliness over traditional petrochemical processes. In previous studies, we constructed a shuttle vector as a useful tool for engineering Propionibacterium jensenii, a potential candidate for efficient PA synthesis. In this study, we identified the key metabolites for PA synthesis in P. jensenii by examining the influence of metabolic intermediate addition on PA synthesis with glycerol as a carbon source under anaerobic conditions. We also further improved PA production via the overexpression of the identified corresponding enzymes, namely, glycerol dehydrogenase (GDH), malate dehydrogenase (MDH), and fumarate hydratase (FUM). Compared to those in wild-type P. jensenii, the activities of these enzymes in the engineered strains were 2.91- ± 0.17- to 8.12- ± 0.37-fold higher. The transcription levels of the corresponding enzymes in the engineered strains were 2.85- ± 0.19- to 8.07- ± 0.63-fold higher than those in the wild type. The coexpression of GDH and MDH increased the PA titer from 26.95 ± 1.21 g/liter in wild-type P. jensenii to 39.43 ± 1.90 g/liter in the engineered strains. This study identified the key metabolic nodes limiting PA overproduction in P. jensenii and further improved PA titers via the coexpression of GDH and MDH, making the engineered P. jensenii strain a potential industrial producer of PA. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. A double mutant of highly purified Geobacillus stearothermophilus lactate dehydrogenase recognises l-mandelic acid as a substrate.

    Science.gov (United States)

    Binay, Barış; Sessions, Richard B; Karagüler, Nevin Gül

    2013-05-10

    Lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) (bsLDH) has a crucial role in producing chirally pure hydroxyl compounds. α-Hydroxy acids are used in many industrial situations, ranging from pharmaceutical to cosmetic dermatology products. One drawback of this enzyme is its limited substrate specificity. For instance, l-lactate dehydrogenase exhibits no detectable activity towards the large side chain of 2-hydroxy acid l-mandelic acid, an α-hydroxy acid with anti-bacterial activity. Despite many attempts to engineer bsLDH to accept α-hydroxy acid substrates, there have been no attempts to introduce the industrially important l-mandelic acid to bsLDH. Herein, we describe attempts to change the reactivity of bsLDH towards l-mandelic acid. Using the Insight II molecular modelling programme (except 'program' in computers) and protein engineering techniques, we have successfully introduced substantial mandelate dehydrogenase activity to the enzyme. Energy minimisation modelling studies suggested that two mutations, T246G and I240A, would allow the enzyme to utilise l-mandelic acid as a substrate. Genes encoding for the wild-type and mutant enzymes were constructed, and the resulting bsLDH proteins were overexpressed in Escherichia coli and purified using the TAGZyme system. Enzyme assays showed that insertion of this double mutation into highly purified bsLDH switched the substrate specificity from lactate to l-mandelic acid. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Monovalent Cation Activation of Plant Pyruvate Dehydrogenase Kinase.

    Science.gov (United States)

    Schuller, K. A.; Gemel, J.; Randall, D. D.

    1993-05-01

    The pyruvate dehydrogenase kinase-catalyzed inactivation of the pyruvate dehydrogenase complex was studied using dialyzed, soluble proteins from mitochondria purified from green leaf tissue of Pisum sativum L. seedlings. At subsaturating ATP concentrations, K+ or NH4+, but not Na+, stimulated the pyruvate dehydrogenase kinase by lowering the Km(ATP). Micromolar concentrations of NH4+ were required to produce the same effect as millimolar concentrations of K+. This is apparent from the observations that the activation constant (Kact) for NH4+ was 0.1 mM, whereas the Kact(K+) was 0.7 mM. Maximal pyruvate dehydrogenase kinase velocities attained with NH4+ were higher than those with K+, and, therefore, NH4+ was able to stimulate PDH kinase further in the presence of saturating K+. This result supports our conclusion that photorespiratory NH4+ production in plant mitochondria may be involved in regulating the entry of carbon into the Krebs cycle by way of the pyruvate dehydrogenase complex.

  19. Generation of oxamic acid libraries: antimalarials and inhibitors of Plasmodium falciparum lactate dehydrogenase.

    Science.gov (United States)

    Choi, Seoung-ryoung; Beeler, Aaron B; Pradhan, Anupam; Watkins, E Blake; Rimoldi, John M; Tekwani, Babu; Avery, Mitchell A

    2007-01-01

    Lactate dehydrogenase (LDH) is a key enzyme in the glycolytic pathway of Plasmodium falciparum (pf) and has several unique amino acids, related to other LDHs, at the active site, making it an attractive target for antimalarial agents. Oxamate, a competitive inhibitor, shows high substrate affinity for pfLDH. This class of compounds has been viewed as potential antimalarial agents. Thus, we have developed an effective automated synthetic strategy for the rapid synthesis of oxamic acid and ester libraries to screen for potential lead inhibitors. One hundred sixty-seven oxamic acids were synthesized using a "catch and release" method with overall yields of 20-70%. Most of the compounds synthesized had some inhibitory effects, but compounds 5 and 6 were the most active against both chloroquine- and mefloquine-resistant strains with IC50 values of 15.4 and 9.41 microM and 20.4 and 8.40 microM, respectively. Some oxamic acids showed activities against pfLDH and mammalian LDH (mLDH) at the micromolar range. These oxamic acids selectively inhibited pfLDH 2-5 fold over mLDH. Oxamic acid 21 was the most active against pfLDH at IC50 = 14 and mLDH at IC50 = 25 microM, suggesting that oxamic acid derivatives are potential inhibitors of pfLDH and that further study is required to develop selective inhibitors of pfLDH over mLDH.

  20. Production of optically pure L-phenyllactic acid by using engineered Escherichia coli coexpressing L-lactate dehydrogenase and formate dehydrogenase.

    Science.gov (United States)

    Zheng, Zhaojuan; Zhao, Mingyue; Zang, Ying; Zhou, Ying; Ouyang, Jia

    2015-08-10

    L-Phenyllactic acid (L-PLA) is a novel antiseptic agent with broad and effective antimicrobial activity. In addition, L-PLA has been used for synthesis of poly(phenyllactic acid)s, which exhibits better mechanical properties than poly(lactic acid)s. However, the concentration and optical purity of L-PLA produced by native microbes was rather low. An NAD-dependent L-lactate dehydrogenase (L-nLDH) from Bacillus coagulans NL01 was confirmed to have a good ability to produce L-PLA from phenylpyruvic acid (PPA). In the present study, l-nLDH gene and formate dehydrogenase gene were heterologously coexpressed in Escherichia coli. Through two coupled reactions, 79.6mM l-PLA was produced from 82.8mM PPA in 40min and the enantiomeric excess value of L-PLA was high (>99%). Therefore, this process suggested a promising alternative for the production of chiral l-PLA. Copyright © 2015. Published by Elsevier B.V.

  1. Stereoselective biotransformation of racemic mandelic acid using immobilized laccase and (S)-mandelate dehydrogenase

    National Research Council Canada - National Science Library

    Chen, Xing; Yang, Chengli; Wang, Peng; Zhang, Xuan; Bao, Bingxin; Li, Dali; Shi, Ruofu

    2017-01-01

    (S)-Mandelate dehydrogenase (SMDH) and laccase were immobilized on chitosan. The bi-enzymatic system with immobilized SMDH and immobilized laccase was taken to catalyze the stereoselective transformation of racemic mandelic acid and (R...

  2. Dimerization and enzymatic activity of fungal 17β-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily

    Directory of Open Access Journals (Sweden)

    Kristan Katja

    2005-12-01

    Full Text Available Abstract Background 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl is a member of the short-chain dehydrogenase/reductase (SDR superfamily. SDR proteins usually function as dimers or tetramers and 17β-HSDcl is also a homodimer under native conditions. Results We have investigated here which secondary structure elements are involved in the dimerization of 17β-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the αE-helices interact with the Asp121, Glu117 and Asp187 residues from the αE and αF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17β-HSDcl monomeric, while the mutant 17β-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity. Conclusion We have shown by site-directed mutagenesis and structure/function analysis that 17β-HSDcl dimerization involves the αE and αF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor.

  3. Pistacia lentiscus Oleoresin: Virtual Screening and Identification of Masticadienonic and Isomasticadienonic Acids as Inhibitors of 11β-Hydroxysteroid Dehydrogenase 1.

    Science.gov (United States)

    Vuorinen, Anna; Seibert, Julia; Papageorgiou, Vassilios P; Rollinger, Judith M; Odermatt, Alex; Schuster, Daniela; Assimopoulou, Andreana N

    2015-04-01

    In traditional medicine, the oleoresinous gum of Pistacia lentiscus var. chia, so-called mastic gum, has been used to treat multiple conditions such as coughs, sore throats, eczema, dyslipidemia, and diabetes. Mastic gum is rich in triterpenes, which have been postulated to exert antidiabetic effects and improve lipid metabolism. In fact, there is evidence of oleanonic acid, a constituent of mastic gum, acting as a peroxisome proliferator-activated receptor γ agonist, and mastic gum being antidiabetic in mice in vivo. Despite these findings, the exact antidiabetic mechanism of mastic gum remains unknown. Glucocorticoids play a key role in regulating glucose and fatty acid metabolism, and inhibition of 11β-hydroxysteroid dehydrogenase 1 that converts inactive cortisone to active cortisol has been proposed as a promising approach to combat metabolic disturbances including diabetes. In this study, a pharmacophore-based virtual screening was applied to filter a natural product database for possible 11β-hydroxysteroid dehydrogenase 1 inhibitors. The hit list analysis was especially focused on the triterpenoids present in Pistacia species. Multiple triterpenoids, such as masticadienonic acid and isomasticadienonic acid, main constituents of mastic gum, were identified. Indeed, masticadienonic acid and isomasticadienonic acid selectively inhibited 11β-hydroxysteroid dehydrogenase 1 over 11β-hydroxysteroid dehydrogenase 2 at low micromolar concentrations. These findings suggest that inhibition of 11β-hydroxysteroid dehydrogenase 1 contributes to the antidiabetic activity of mastic gum. Georg Thieme Verlag KG Stuttgart · New York.

  4. Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production

    Science.gov (United States)

    Dave, Khyati K.; Punekar, Narayan S.

    2015-01-01

    Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production. PMID:26683313

  5. Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production.

    Science.gov (United States)

    Dave, Khyati K; Punekar, Narayan S

    2015-01-01

    Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production.

  6. Orthodontic Force Application in Correlation with Salivary Lactate Dehydrogenase Activity

    Directory of Open Access Journals (Sweden)

    Erik Husin

    2013-07-01

    Full Text Available Orthodontic tooth movement generate mechanical forces to periodontal ligament and alveolar bone. The forces correlate with initial responses of periodontal tissues and involving many metabolic changes. One of the metabolic changes detected in saliva is lactate dehydrogenase (LDH activity. Objectives: To evaluate the correlation between orthodontic interrupted force application, lactate dehydrogenase activity and the distance of tooth movement. Methods: upper premolar, pre-retraction of upper canine and 1, 7, 14, 21 and 28 days post-retraction of upper canine with 100g interrupted orthodontic force. Results: duration of force (F=11.926 p 14 and 28 days post-retraction of canine. The region of retraction correlated with the distance of tooth movement (F=7.377 p=0.007. The duration of force correlated with the distance of tooth movement (F=66.554 p=0.000. retraction of canine. Conclusion: This study concluded that orthodontic interrupted force application on canine could increase the distance of tooth movement and LDH activity in saliva.

  7. Efficient production of (R-2-hydroxy-4-phenylbutyric acid by using a coupled reconstructed D-lactate dehydrogenase and formate dehydrogenase system.

    Directory of Open Access Journals (Sweden)

    Binbin Sheng

    Full Text Available (R-2-hydroxy-4-phenylbutyric acid [(R-HPBA] is a key precursor for the production of angiotensin-converting enzyme inhibitors. However, the product yield and concentration of reported (R-HPBA synthetic processes remain unsatisfactory.The Y52L/F299Y mutant of NAD-dependent D-lactate dehydrogenase (D-nLDH in Lactobacillus bulgaricus ATCC 11842 was found to have high bio-reduction activity toward 2-oxo-4-phenylbutyric acid (OPBA. The mutant D-nLDHY52L/F299Y was then coexpressed with formate dehydrogenase in Escherichia coli BL21 (DE3 to construct a novel biocatalyst E. coli DF. Thus, a novel bio-reduction process utilizing whole cells of E. coli DF as the biocatalyst and formate as the co-substrate for cofactor regeneration was developed for the production of (R-HPBA from OPBA. The biocatalysis conditions were then optimized.Under the optimum conditions, 73.4 mM OPBA was reduced to 71.8 mM (R-HPBA in 90 min. Given its high product enantiomeric excess (>99% and productivity (47.9 mM h(-1, the constructed coupling biocatalysis system is a promising alternative for (R-HPBA production.

  8. Solution structures of lipoyl domains of the 2-oxo acid dehydrogenase complexes from Azotobacter vinelandii : implications for molecular recognition

    NARCIS (Netherlands)

    Berg, A.

    1997-01-01

    The 2-oxo acid dehydrogenase complexes are large multienzyme complexes that catalyse the irreversible oxidative decarboxylation of a specific 2-oxo acid to the corresponding acyl-CoA derivative. The pyruvate dehydrogenase complex (PDHC) converts the product of the glycolysis, pyruvate, to

  9. Inhibition of dehydrogenase activity in petroleum refinery wastewater bacteria by phenolic compounds

    National Research Council Canada - National Science Library

    Gideon C. Okpokwasili; Christian Okechukwu Nweke

    2010-01-01

    .... At low concentrations, 2-nitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol stimulated dehydrogenase activity and at sufficient concentrations, phenolic compounds...

  10. Circadian rhythm in succinate dehydrogenase activity in Neurospora crassa

    Directory of Open Access Journals (Sweden)

    Claudia Patricia Álvarez Barón

    2004-07-01

    Full Text Available Neurospora crassa is a widely studied model of circadian rhythmicity. In this fungus, metabolism is controlled by multiple factors which include development, medium characteristics and the circadian clock. The study of the circadian control of metabolism in this fungus could be masked by the use of restrictive media that inhibit growth and development. In this report, the presence of a circadian rhythm in the activity of the enzyme Succinate Dehydrogenase in Neurospora crassa is demonstrated. Rhythmic and arrhythmic Neurospora strains were grown in complete medium without conidiation restriction. A circadian change in the enzymatic activity was found with high values in hours corresponding to the night and a low level during the day. This finding highlights the importance of deeper studies in the circadian control of metabolism in this fungus, given the existence of multiple pathways of regulation of metabolic enzymes and a circadian clock control at the transcriptional and post-transcriptional levels.

  11. Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

    Science.gov (United States)

    Lignin is a significant recalcitrant in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired...

  12. New insights in dihydropyrimidine dehydrogenase deficiency: a pivotal role for beta-aminoisobutyric acid?

    NARCIS (Netherlands)

    van Kuilenburg, André B. P.; Stroomer, Alida E. M.; van Lenthe, Henk; Abeling, Nico G. G. M.; van Gennip, Albert H.

    2004-01-01

    DPD (dihydropyrimidine dehydrogenase) constitutes the first step of the pyrimidine degradation pathway, in which the pyrimidine bases uracil and thymine are catabolized to beta-alanine and file R-enantiomer of beta-AIB (beta-aminoisobutyric acid) respectively. The S-enantiomer of beta-AIB is

  13. Stable shRNA Silencing of Lactate Dehydrogenase A (LDHA) in Human MDA-MB-231 Breast Cancer Cells Fails to Alter Lactic Acid Production, Glycolytic Activity, ATP or Survival.

    Science.gov (United States)

    Mack, Nzinga; Mazzio, Elizabeth A; Bauer, David; Flores-Rozas, Hernan; Soliman, Karam F A

    2017-03-01

    In the US, African Americans have a high death rate from triple-negative breast cancer (TNBC), characterized by lack of hormone receptors (ER, PR, HER2/ERRB2) which are otherwise valuable targets of chemotherapy. There is a need to identify novel targets that negatively impact TNBC tumorigenesis. TNBCs release an abundance of lactic acid, under normoxic, hypoxic and hyperoxic conditions; this referred to as the Warburg effect. Accumulated lactic acid sustains peri-cellular acidity which propels metastatic invasion and malignant aggressive transformation. The source of lactic acid is believed to be via conversion of pyruvate by lactate dehydrogenase (LDH) in the last step of glycolysis, with most studies focusing on the LDHA isoform. In this study, LDHA was silenced using long-term MISSION® shRNA lentivirus in human breast cancer MDA-MB-231 cells. Down-regulation of LDHA transcription and protein expression was confirmed by western blot, immunocytochemistry and qPCR. A number of parameters were measured in fully viable vector controls versus knock-down (KD) clones, including levels of lactic acid produced, glucose consumed, ATP and basic metabolic rates. The data show that lentivirus V-165 generated a knock-down clone most effective in reducing both gene and protein levels to less than 1% of vector controls. Stable KD showed absolutely no changes in cell viability, lactic acid production, ATP, glucose consumption or basic metabolic rate. Given the complete absence of impact on any observed parameter by LDH-A KD and this being somewhat contrary to findings in the literature, further analysis was required to determine why. Whole-transcriptome analytic profile on MDA-MB-231 for LDH subtypes using Agilent Human Genome 4×44k microarrays, where the data show the following component breakdown. Transcripts: 30.47 % LDHA, 69.36% LDHB, 0.12% LDHC and 0.05% LDHD. These findings underscore the importance of alternative isoforms of LDH in cancer cells to produce lactic acid

  14. Colorimetric Enzymatic Assay of L-Malic Acid Using Dehydrogenase from Baker’s Yeast

    Directory of Open Access Journals (Sweden)

    Cecilia Laluce

    2008-01-01

    Full Text Available A colorimetric method has been developed and optimized to measure L-malic acid in samples of fruit juices and wine. This method is based on oxidation of the analyte, catalyzed by malate dehydrogenase (MDH from dry baker’s yeast, and in combination with the reduction of a tetrazolium salt (MTT: 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide. In the present study, the method exhibited sensitivity in the range of 500–4000 mM of L-malic acid in the reaction cuvette, with the lower detection limit of 6.7·10^-2 g/L, the upper limit of 53.6·10^-2 g/L and a maximum standard deviation of only 2.5 % for the analyzed samples. The MDH activity from baker’s yeast was also optimized, the enzyme showed a high stability at pH=8.0–9.0 and the activity was maintained completely at temperatures up to 40 °C for 1 hour. The results show that the colorimetric method using enzymatic preparations from dry baker’s yeast is a simple and low-cost method with possibility of wide application

  15. 17 beta-hydroxysteroid dehydrogenase activity in canine pancreas

    Energy Technology Data Exchange (ETDEWEB)

    Mendoza-Hernandez, G.; Lopez-Solache, I.; Rendon, J.L.; Diaz-Sanchez, V.; Diaz-Zagoya, J.C.

    1988-04-15

    The mitochondrial fraction of the dog pancreas showed NAD(H)-dependent enzyme activity of 17 beta-hydroxysteroid dehydrogenase. The enzyme catalyzes oxidoreduction between androstenedione and testosterone. The apparent Km value of the enzyme for androstenedione was 9.5 +/- 0.9 microM, the apparent Vmax was determined as 0.4 nmol mg-1 min-1, and the optimal pH was 6.5. In phosphate buffer, pH 7.0, maximal rate of androstenedione reduction was observed at 37 degrees C. The oxidation of testosterone by the enzyme proceeded at the same rate as the reduction of the androstenedione at a pH of 6.8-7.0. The apparent Km value and the optimal pH of the enzyme for testosterone were 3.5 +/- 0.5 microM and 7.5, respectively.

  16. A Case of Hyperammonemia Associated with High Dihydropyrimidine Dehydrogenase Activity

    Directory of Open Access Journals (Sweden)

    Keiki Nagaharu

    2016-01-01

    Full Text Available Over the past decades, 5-Fluorouracil (5-FU has been widely used to treat several types of carcinoma, including esophageal squamous cell carcinoma. In addition to its common side effects, including diarrhea, mucositis, neutropenia, and anemia, 5-FU treatment has also been reported to cause hyperammonemia. However, the exact mechanism responsible for 5-FU-induced hyperammonemia remains unknown. We encountered an esophageal carcinoma patient who developed hyperammonemia when receiving 5-FU-containing chemotherapy but did not exhibit any of the other common adverse effects of 5-FU treatment. At the onset of hyperammonemia, laboratory tests revealed high dihydropyrimidine dehydrogenase (DPD activity and rapid 5-FU clearance. Our findings suggested that 5-FU hypermetabolism may be one of the key mechanisms responsible for hyperammonemia during 5-FU treatment.

  17. Evaluation of Serum Lactate Dehydrogenase Activity in a Virtual Environment

    Directory of Open Access Journals (Sweden)

    V.M.T. Trindade

    2013-05-01

    Full Text Available Introduction: Lactate dehydrogenase is a citosolic enzyme involved in reversible transformation of pyruvate to lactate. It participates in anaerobic glycolysis of skeletal muscle and red blood cells, in liver gluconeogenesis and in aerobic metabolism of heart muscle. The determination of its activity helps in the diagnosis of various diseases, because it is increased in serum of patients suffering from myocardial infarction, acute hepatitis, muscular dystrophy and cancer. This paper presents a learning object, mediated by computer, which contains the simulation of the laboratory determination serum lactate dehydrogenase activity measured by the spectrophotometric method, based in the decrease of absorbance at 340 nm. Materials and Methods: Initially, pictures and videos were obtained recording the procedure of the methodology. The most representative images were selected, edited and inserted into an animation developed with the aid of the tool Adobe ® Flash ® CS3. The validation of the object was performed by the students of Biochemistry I (Pharmacy-UFRGS from the second semester of 2009 and both of 2010. Results and Discussion: The analysis of students' answers revealed that 80% attributed the excellence of the navigation program, the display format and to aid in learning. Conclusion: Therefore, this software can be considered an adequate teaching resource as well as an innovative support in the construction of theoretical and practical knowledge of Biochemistry. Available at: http://www6.ufrgs.br/gcoeb/LDH

  18. A quantitative histochemical study of lactate dehydrogenase and succinate dehydrogenase activities in the membrana granulosa of the ovulatory follicle of the rat.

    Science.gov (United States)

    Zoller, L C; Enelow, R

    1983-11-01

    Using a microdensitometer, lactate dehydrogenase and succinate dehydrogenase activities were measured in the membrana granulosa of the rat ovulatory follicle. Ovaries were removed on each day of the oestrous cycle; oestrus, dioestrus-1, dioestrus-2, and proestrus; and enzyme activities measured in the membrana granulosa as a whole and in four regions within it: peripheral (PR), antral (AR), cumulus oophorus (CO) and corona radiata (CR). Throughout the cycle, lactate dehydrogenase activity was greatest in PR. On oestrus, lactate dehydrogenase activity was progressively less in AR, CO and CR. On dioestrus-1, activity was identical in AR and CO and less in CR. On dioestrus-2, activity was greater in AR than in CO or CR. By proestrus, activity was equal in AR, CO and CR. In the membrana granulosa as a whole, and in each region, lactate dehydrogenase activity declined as ovulation approached. In contrast, succinate dehydrogenase activity in the membrana granulosa as a whole and in PR was constant throughout the cycle. Activity fluctuated in the other regions. Succinate dehydrogenase activity on oestrus was greatest in PR, less in AR and CO and least in CR. On the remaining days, succinate dehydrogenase activity was greatest in PR and less but equal in the remainder of the membrana granulosa.

  19. The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites.

    Science.gov (United States)

    Klimacek, Mario; Nidetzky, Bernd

    2009-12-23

    The side chains of Asn191 and Asn300 constitute a characteristic structural motif of the active site of Pseudomonas fluorescens mannitol 2-dehydrogenase that lacks precedent in known alcohol dehydrogenases and resembles the canonical oxyanion binding pocket of serine proteases. We have used steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in substrates and solvent on the enzymatic rates to delineate catalytic consequences resulting from individual and combined replacements of the two asparagine residues by alanine. The rate constants for the overall hydride transfer to and from C-2 of mannitol, which were estimated as approximately 5 x 102 s-1 and approximately 1.5 x 103 s-1 in the wild-type enzyme respectively, were selectively slowed, between 540- and 2700-fold, in single-site mannitol 2-dehydrogenase mutants. These effects were additive in the corresponding doubly mutated enzyme, suggesting independent functioning of the two asparagine residues in catalysis. Partial disruption of the oxyanion hole in single-site mutants caused an upshift, by >or=1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme-NAD+-mannitol complex. The oxyanion hole of mannitol 2-dehydrogenase is suggested to drive a precatalytic conformational equilibrium at the ternary complex level in which the reactive group of the substrate is 'activated' for chemical conversion through its precise alignment with the unprotonated side chain of Lys295 (mannitol oxidation) and C=O bond polarization by the carboxamide moieties of Asn191 and Asn300 (fructose reduction). In the subsequent hydride transfer step, the two asparagine residues provide approximately 40 kJ/mol of electrostatic stabilization.

  20. Changes in native alcohol dehydrogenase activity of Drosophila ...

    Indian Academy of Sciences (India)

    tribpo

    and within the Mexican populations of the insect after treatment with the denaturants. Keywords. Drosophila melanogaster; alcohol dehydrogenase; guanidine hydrochloride; urea; heat. Introduction. The existence of clines in the frequency of alcohol dehydrogenase (Adh) alleles in. Drosophila under various environmental ...

  1. Alcohol dehydrogenase of acetic acid bacteria: structure, mode of action, and applications in biotechnology.

    Science.gov (United States)

    Yakushi, Toshiharu; Matsushita, Kazunobu

    2010-05-01

    Pyrroquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH) of acetic acid bacteria is a membrane-bound enzyme involved in the acetic acid fermentation by oxidizing ethanol to acetaldehyde coupling with reduction of membranous ubiquinone (Q), which is, in turn, re-oxidized by ubiquinol oxidase, reducing oxygen to water. PQQ-ADHs seem to have co-evolved with the organisms fitting to their own habitats. The enzyme consists of three subunits and has a pyrroloquinoline quinone, 4 heme c moieties, and a tightly bound Q as the electron transfer mediators. Biochemical, genetic, and electrochemical studies have revealed the unique properties of PQQ-ADH since it was purified in 1978. The enzyme is unique to have ubiquinol oxidation activity in addition to Q reduction. This mini-review focuses on the molecular properties of PQQ-ADH, such as the roles of the subunits and the cofactors, particularly in intramolecular electron transport of the enzyme from ethanol to Q. Also, we summarize biotechnological applications of PQQ-ADH as to enantiospecific oxidations for production of the valuable chemicals and bioelectrocatalysis for sensors and fuel cells using indirect and direct electron transfer technologies and discuss unsolved issues and future prospects related to this elaborate enzyme.

  2. Calcium regulates glutamate dehydrogenase and poly-γ-glutamic acid synthesis in Bacillus natto.

    Science.gov (United States)

    Meng, Yonghong; Dong, Guiru; Zhang, Chen; Ren, Yuanyuan; Qu, Yuling; Chen, Weifeng

    2016-04-01

    To study the effect of Ca(2+) on glutamate dehydrogenase (GDH) and its role in poly-γ-glutamic acid (γ-PGA) synthesis in Bacillus natto HSF 1410. When the concentration of Ca(2+) varied from 0 to 0.1 g/l in the growth medium of B. natto HSF 1410, γ-PGA production increased from 6.8 to 9.7 g/l, while GDH specific activity and NH4Cl consumption improved from 183 to 295 U/mg and from 0.65 to 0.77 g/l, respectively. GDH with α-ketoglutarate as substrate primarily used NADPH as coenzyme with a K m of 0.08 mM. GDH was responsible for the synthesis of endogenous glutamate. The specific activity of GDH remained essentially unchanged in the presence of CaCl2 (0.05-0.2 g/l) in vitro. However, the specific activity of GDH and its expression was significantly increased by CaCl2 in vivo. Therefore, the regulation of GDH and PGA synthesis by Ca(2+) is an intracellular process. Calcium regulation may be an effective approach for producing γ-PGA on an industrial scale.

  3. The activity of class I, II, III, and IV of alcohol dehydrogenase isoenzymes and aldehyde dehydrogenase in pancreatic cancer.

    Science.gov (United States)

    Jelski, Wojciech; Chrostek, Lech; Szmitkowski, Maciej

    2007-08-01

    The pancreas can metabolize ethanol via oxidative pathway involving the enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) as well as the nonoxidative pathway. In this study, we compared the activity of ADH isoenzymes and ALDH in the pancreatic cancer with the activity in normal tissue. In addition, the differences between enzyme activities of drinkers and nondrinkers were compared. For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, we used the fluorometric methods. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. The samples were taken from 56 pancreatic cancer patients (22 drinkers and 34 nondrinkers) and 56 healthy subjects. The activity of class III ADH was significantly higher in cancer than in healthy tissues. Total activities of ADH and ALDH were not significantly different in cancer and normal cells. The differences between enzymes of drinkers and nondrinkers in both cancer and healthy tissue were not significant. Pancreatic cancer tissue exhibits higher activity of class III ADH isoenzyme than healthy tissue, and we consider that oxidative pathway of ethanol metabolism via ADH and ALDH does not play a role in pancreatic carcinogenesis.

  4. A ketogenic diet increases succinic dehydrogenase activity in aging cardiomyocytes.

    Science.gov (United States)

    Balietti, Marta; Fattoretti, Patrizia; Giorgetti, Belinda; Casoli, Tiziana; Di Stefano, Giuseppina; Solazzi, Moreno; Platano, Daniela; Aicardi, Giorgio; Bertoni-Freddari, Carlo

    2009-08-01

    Impairment of energy metabolism and an increase of reactive oxygen species (ROS) production seem to play a major role in age-related apoptotic loss of cardiomyocytes. Succinic dehydrogenase (SDH) is an important marker of the mitochondrial capability to provide an adequate amount of ATP. Moreover, because of its unique redox properties, SDH activity contributes to maintain the reduced state of the ubiquinone pool. Recent reports have shown that ketone body intake improves cardiac metabolic efficiency and exerts a cardioprotective antioxidant action, we therefore performed a cytochemical investigation of SDH activity in cardiomyocytes of late-adult (19-month-old) rats fed for 8 weeks with a medium-chain triglycerides ketogenic diet (MCT-KD). Young, age-matched and old animals fed with a standard chow were used as controls. The overall area of the precipitates (PA) from SDH activity and the area of the SDH-positive mitochondria (MA) were measured. The percent ratios PA/MA and MA/total myocardial tissue area (MA/TA) were the parameters taken into account. We found that PA/MA was significantly higher in young control rats and in MCT-KD-fed rats versus late-adult and old control rats and in young control versus MCT-KD-fed rats. MA/TA of MCT-KD-fed rats was significantly higher versus age-matched and old control rats and tended to be higher versus young control rats; this parameter was significantly higher in young versus old control rats. Thus, MCT-KD intake partially recovers age-related decrease of SDH activity and increases the myocardial area occupied by metabolically active mitochondria. These effects might counteract metabolic alterations leading to apoptosis-induced myocardial atrophy and failure during aging.

  5. Loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolism

    Science.gov (United States)

    Lussey-Lepoutre, Charlotte; Hollinshead, Kate E. R.; Ludwig, Christian; Menara, Mélanie; Morin, Aurélie; Castro-Vega, Luis-Jaime; Parker, Seth J.; Janin, Maxime; Martinelli, Cosimo; Ottolenghi, Chris; Metallo, Christian; Gimenez-Roqueplo, Anne-Paule; Favier, Judith; Tennant, Daniel A.

    2015-01-01

    The tricarboxylic acid (TCA) cycle is a central metabolic pathway responsible for supplying reducing potential for oxidative phosphorylation and anabolic substrates for cell growth, repair and proliferation. As such it thought to be essential for cell proliferation and tissue homeostasis. However, since the initial report of an inactivating mutation in the TCA cycle enzyme complex, succinate dehydrogenase (SDH) in paraganglioma (PGL), it has become clear that some cells and tissues are not only able to survive with a truncated TCA cycle, but that they are also able of supporting proliferative phenotype observed in tumours. Here, we show that loss of SDH activity leads to changes in the metabolism of non-essential amino acids. In particular, we demonstrate that pyruvate carboxylase is essential to re-supply the depleted pool of aspartate in SDH-deficient cells. Our results demonstrate that the loss of SDH reduces the metabolic plasticity of cells, suggesting vulnerabilities that can be targeted therapeutically. PMID:26522426

  6. Loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolism.

    Science.gov (United States)

    Lussey-Lepoutre, Charlotte; Hollinshead, Kate E R; Ludwig, Christian; Menara, Mélanie; Morin, Aurélie; Castro-Vega, Luis-Jaime; Parker, Seth J; Janin, Maxime; Martinelli, Cosimo; Ottolenghi, Chris; Metallo, Christian; Gimenez-Roqueplo, Anne-Paule; Favier, Judith; Tennant, Daniel A

    2015-11-02

    The tricarboxylic acid (TCA) cycle is a central metabolic pathway responsible for supplying reducing potential for oxidative phosphorylation and anabolic substrates for cell growth, repair and proliferation. As such it thought to be essential for cell proliferation and tissue homeostasis. However, since the initial report of an inactivating mutation in the TCA cycle enzyme complex, succinate dehydrogenase (SDH) in paraganglioma (PGL), it has become clear that some cells and tissues are not only able to survive with a truncated TCA cycle, but that they are also able of supporting proliferative phenotype observed in tumours. Here, we show that loss of SDH activity leads to changes in the metabolism of non-essential amino acids. In particular, we demonstrate that pyruvate carboxylase is essential to re-supply the depleted pool of aspartate in SDH-deficient cells. Our results demonstrate that the loss of SDH reduces the metabolic plasticity of cells, suggesting vulnerabilities that can be targeted therapeutically.

  7. Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver.

    Science.gov (United States)

    Tran, Steven; Nowicki, Magda; Chatterjee, Diptendu; Gerlai, Robert

    2015-01-02

    Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Design of novel dihydroxynaphthoic acid inhibitors of Plasmodium falciparum lactate dehydrogenase.

    Science.gov (United States)

    Megnassan, Eugene; Keita, Melalie; Bieri, Cecile; Esmel, Akori; Frecer, Vladimir; Miertus, Stanislav

    2012-09-01

    We have studied inhibition of Plasmodium falciparum lactate dehydrogenase (pfLDH) by dihydroxynaphthoic acid (DHNA) analogues derivatives of hemigossypol-sesquiterpene found in cottonseed known to exhibit antimalarial activity. Molecular models of pfLDH-DHNA complexes were prepared from high-resolution crystal structures containing DHNA and azole inhibitors and binding affinities of the inhibitors were computed by molecular mechanics - polarizable continuum model of solvation (MM-PCM) approach. The 3D structures of the pfLDH-DHNA complexes were validated by a QSAR model, which confirmed consistency between the computed binding affinities and experimental inhibition constants for a training set and validation set of twelve DHNA inhibitors obtained from literature. Novel more potent DHNA analogs were identified by structure-based molecular design and predicted to inhibit pfLDH in the low nanomolar concentration range. In addition, the designed DHNA analogs displayed favorable predicted ADME-related profiles and an elevated selectivity for the pfLDH over the human isoform.

  9. The Diagnostic Significance of Serum Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase Activity in Urinary Bladder Cancer Patients.

    Science.gov (United States)

    Orywal, Karolina; Jelski, Wojciech; Werel, Tadeusz; Szmitkowski, Maciej

    2017-07-01

    The aim of this study was to investigate a potential role of alcohol dehydrogenase and aldehyde dehydrogenase as tumor markers for urinary bladder cancer. Serum samples were obtained from 41 patients with bladder cancer and 52 healthy individuals. Class III and IV of ADH and total ADH activity were measured by the photometric method. For measurement of class I and II ADH and ALDH activity, the fluorometric method was employed. Significantly higher total activity of ADH was found in sera of both, low-grade and high-grade bladder cancer patients. The diagnostic sensitivity for total ADH activity was 81.5%, specificity 98.1%, positive (PPV) and negative (NPV) predictive values were 97.4% and 92.3% respectively. Area under ROC curve for total ADH activity was 0.848. A potential role of total ADH activity as a marker for bladder cancer, is herein proposed. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  10. Effect of different mulch materials on the soil dehydrogenase activity (DHA) in an organic pepper crop

    Science.gov (United States)

    Moreno, Marta M.; Peco, Jesús; Campos, Juan; Villena, Jaime; González, Sara; Moreno, Carmen

    2016-04-01

    The use biodegradable materials (biopolymers of different composition and papers) as an alternative to conventional mulches has increased considerably during the last years mainly for environmental reason. In order to assess the effect of these materials on the soil microbial activity during the season of a pepper crop organically grown in Central Spain, the soil dehydrogenase activity (DHA) was measured in laboratory. The mulch materials tested were: 1) black polyethylene (PE, 15 μm); black biopolymers (15 μm): 2) Mater-Bi® (corn starch based), 3) Sphere 4® (potato starch based), 4) Sphere 6® (potato starch based), 5) Bioflex® (polylactic acid based), 6) Ecovio® (polylactic acid based), 7) Mimgreen® (black paper, 85 g/m2). A randomized complete block design with four replications was adopted. The crop was drip irrigated following the water demand of each treatment. Soil samples (5-10 cm depth) under the different mulches were taken at different dates (at the beginning of the crop cycle and at different dates throughout the crop season). Additionally, samples of bare soil in a manual weeding and in an untreated control were taken. The results obtained show the negative effect of black PE on the DHA activity, mainly as result of the higher temperature reached under the mulch and the reduction in the gas interchange between the soil and the atmosphere. The values corresponding to the biodegradable materials were variable, although highlighting the low DHA activity observed under Bioflex®. In general, the uncovered treatments showed higher values than those reached under mulches, especially in the untreated control. Keywords: mulch, biodegradable, biopolymer, paper, dehydrogenase activity (DHA). Acknowledgements: the research was funded by Project RTA2011-00104-C04-03 from the INIA (Spanish Ministry of Economy and Competitiveness).

  11. High performance liquid chromatography method for the determination of cinnamyl alcohol dehydrogenase activity in soybean roots.

    Science.gov (United States)

    dos Santos, W D; Ferrarese, Maria de Lourdes Lucio; Ferrarese-Filho, O

    2006-01-01

    This study proposes a simple, quick and reliable method for determining the cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity in soybean (Glycine max L. Merr.) roots using reversed-phase high performance liquid chromatography (RP-HPLC). The method includes a single extraction of the tissue and conduction of the enzymatic reaction at 30 degrees C with cinnamaldehydes (coniferyl or sinapyl), substrates of CAD. Disappearance of the substrates in the reaction mixture is monitored at 340 nm (for coniferaldehyde) or 345 nm (for sinapaldehyde) by isocratic elution with methanol/acetic acid through a GLC-ODS (M) column. This HPLC technique furnishes a rapid and reliable measure of cinnamaldehyde substrates, and may be used as an alternative tool to analyze CAD activity in enzyme preparation without previous purification.

  12. INFLUENCE OF LIMING AND WASTE ORGANIC MATERIALS ON THE ACTIVITY OF UREASE AND DEHYDROGENASE IN SOIL CONTAMINATED WITH NICKEL

    Directory of Open Access Journals (Sweden)

    Dorota Kalembasa

    2014-10-01

    Full Text Available In soil sampled after a two-year pot experiment, the activity of urease and dehydrogenase and content of nitrogen have been determined. The experiment included three factors: 1. the amount of nickel added to the soil (0, 75, 150 and 225 mg · kg-1 of soil, 2. liming (0 and Ca according to 1 Hh hydrolytic acidity, 3. organic materials (straw of rye and brown coal. Test plant was cocksfoot, which four cuts were collected in each growing season. It was found that nickel added to the soli in dose of 75 mg · kg-1 activates enzymes studied, whereas higher doses cause them explicit deactivation. Both liming and waste organic materials limited the negative effect of higher doses of nickel on the activity of dehydrogenase and urease. Simultaneously, both straw and brown coal caused a slight increase in the amount of nitrogen in the soil.

  13. Assessment of the activity of glucose-6-phosphate dehydrogenase ...

    African Journals Online (AJOL)

    Glucose-6-phosphate dehydrogenase (G-6-PD) is an enzyme in the pentose phosphate pathway (PPP) which reduces NADP to NADPH while oxidizing glucose-6-phosphate. In turn, NADPH then provides reducing equivalents needed for the conversion of oxidized glutathione to reduced glutathione, which protects against ...

  14. Assessment of creatine kinase and lactate dehydrogenase activities ...

    African Journals Online (AJOL)

    Ina bid to investigate the influence of menopausal on coronary heart disease, plasma creatine kinase (CK) and lactate dehydrogenase (LDH) enzymes were analysed on a prospective cohort of 100 women attending Irrua Specialist Teaching Hospital (ISTH), Irrua, Edo state-Nigeria. They were divided into two groups; ...

  15. Not only osmoprotectant: betaine increased lactate dehydrogenase activity and L-lactate production in lactobacilli.

    Science.gov (United States)

    Zou, Huibin; Wu, Zaiqiang; Xian, Mo; Liu, Hui; Cheng, Tao; Cao, Yujin

    2013-11-01

    Lactobacilli are commonly used for industrial production of polymer-grade L-lactic acid. The present study tested the Tween 80 alternative betaine in L-lactate production by several industrial lactobacilli. In flask fermentation of Lactobacillus casei, Lactobacillus buchneri, Lactobacillus lactis and Lactobacillus rhamnosus, the betaine addition (2g/l) had similar osmoprotectant effect with Tween 80 but had increased the lactate dehydrogenase activities and L-lactate production than Tween 80 control. In fed-batch fermentation of L. casei, betaine supplementation improved the L-lactic acid titer to 190 g/l, the yield to 95.5% (g L-lactic acid/g glucose), the productivity to 2.6g/lh, and the optical purity to 97.0%. The results demonstrated that supplementation of Tween 80 alternative - betaine in the fermentation medium is feasible for industrial l-lactic acid fermentation by lactobacilli, which will improve the lactate production but will not increase the process costs and modify any process conditions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon

    Science.gov (United States)

    2013-01-01

    Background Lignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production. Results We developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and increased stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1 downregulated and control plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4 downregulated plants exhibited a reduction in total lignin content and decreased Maule staining of syringyl units in stem. Ethanol yield by microbial fermentation was enhanced in amiR-cad1-8 plants. Conclusion These results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater stem biomass yield and bioconversion efficiency. PMID:23902793

  17. SDHAF4 promotes mitochondrial succinate dehydrogenase activity and prevents neurodegeneration

    OpenAIRE

    Van Vranken, Jonathan G.; Bricker, Daniel K.; Dephoure, Noah; Gygi, Steven P.; Cox, James E.; Thummel, Carl S.; Rutter, Jared

    2014-01-01

    Succinate dehydrogenase (SDH) occupies a central place in cellular energy production, linking the tricarboxylic cycle with the electron transport chain. As a result, a subset of cancers and neuromuscular disorders result from mutations affecting any of the four SDH structural subunits or either of two known SDH assembly factors. Herein we characterize a novel evolutionarily conserved SDH assembly factor designated Sdh8/SDHAF4, using yeast, Drosophila, and mammalian cells. Sdh8 interacts speci...

  18. Native and modified lactate dehydrogenase expression in a fumaric acid producing isolate Rhizopus oryzae 99-880.

    Science.gov (United States)

    Skory, Christopher D; Ibrahim, Ashraf S

    2007-07-01

    Rhizopus oryzae is subdivided into two groups based on genetic and phenotypic differences. Type-I isolates accumulate primarily lactic acid when grown in the presence of a fermentable carbon source and contain two lactate dehydrogenase genes, ldhA and ldhB. Type-II isolates synthesize predominantly fumaric acid and only have an ldhB gene. In this study, we determined that ldhB transcript is only minimally expressed in the Type-II isolate R. oryzae 99-880. LdhB enzyme purified from gene clones isolated from the Type-I isolate R. oryzae NRRL 395 and the Type-II isolate R. oryzae 99-880 each showed reductive LDH activity (pyruvate to lactate), while no oxidative LDH activity (lactate to pyruvate) was detected in either preparation. A transformation system was then developed for the first time with R. oryzae 99-880, using a uracil auxotrophic isolate that could be complemented with an orotate phosphoribosyltransferase gene, pyrF, isolated in this study. Transformation of this Type-II isolate with the ldhA gene from R. oryzae NRRL 395 resulted in reductive LDH activity between 1.0 and 1.8 U/mg total protein. Additionally, transformed isolates grown with glucose accumulated up to 27 g lactic acid/l with a concurrent decrease in fumaric acid, ethanol, and glycerol compared with the untransformed and vector-transformed control strains.

  19. Enzymatic activity toward poly(L-lactic acid) implants

    NARCIS (Netherlands)

    Schakenraad, J.M.; Hardonk, M.J.; Feijen, Jan; Molenaar, I.; Nieuwenhuis, P.

    1990-01-01

    Tissue reactions toward biodegradable poly(L-lactic acid) implants were monitored by studying the activity pattern of seven enzymes as a function of time: alkaline phosphatase, acid phosphatase, -naphthyl acetyl esterase, -glucuronidase, ATP-ase, NADH-reductase, and lactate dehydrogenase. Cell types

  20. Structural insights into the enzymatic activity and potential substrate promiscuity of human 3-phosphoglycerate dehydrogenase (PHGDH).

    Science.gov (United States)

    Unterlass, Judith E; Wood, Robert J; Baslé, Arnaud; Tucker, Julie; Cano, Céline; Noble, Martin M E; Curtin, Nicola J

    2017-11-28

    Cancer cells reprogram their metabolism and energy production to sustain increased growth, enable metastasis and overcome resistance to cancer treatments. Although primary roles for many metabolic proteins have been identified, some are promiscuous in regards to the reaction they catalyze. To efficiently target these enzymes, a good understanding of their enzymatic function and structure, as well as knowledge regarding any substrate or catalytic promiscuity is required. Here we focus on the characterization of human 3-phosphoglycerate dehydrogenase (PHGDH). PHGDH catalyzes the NAD + -dependent conversion of 3-phosphoglycerate to phosphohydroxypyruvate, which is the first step in the de novo synthesis pathway of serine, a critical amino acid for protein and nucleic acid biosynthesis. We have investigated substrate analogues to assess whether PHGDH might possess other enzymatic roles that could explain its occasional over-expression in cancer, as well as to help with the design of specific inhibitors. We also report the crystal structure of the catalytic subunit of human PHGDH, a dimer, solved with bound cofactor in one monomer and both cofactor and L -tartrate in the second monomer. In vitro enzyme activity measurements show that the catalytic subunit of PHGDH is still active and that PHGDH activity could be significantly inhibited with adenosine 5'-diphosphoribose.

  1. Lowering effect of firefly squid powder on triacylglycerol content and glucose-6-phosphate dehydrogenase activity in rat liver.

    Science.gov (United States)

    Takeuchi, Hiroyuki; Morita, Ritsuko; Shirai, Yoko; Nakagawa, Yoshihisa; Terashima, Teruya; Ushikubo, Shun; Matsuo, Tatsuhiro

    2014-01-01

    Effects of dietary firefly squid on serum and liver lipid levels were investigated. Male Wistar rats were fed a diet containing 5% freeze-dried firefly squid or Japanese flying squid for 2 weeks. There was no significant difference in the liver triacylglycerol level between the control and Japanese flying squid groups, but the rats fed the firefly squid diet had a significantly lower liver triacylglycerol content than those fed the control diet. No significant difference was observed in serum triacylglycerol levels between the control and firefly squid groups. The rats fed the firefly squid had a significantly lower activity of liver glucose-6-phosphate dehydrogenase compared to the rats fed the control diet. There was no significant difference in liver fatty acid synthetase activity among the three groups. Hepatic gene expression and lipogenic enzyme activity were investigated; a DNA microarray showed that the significantly enriched gene ontology category of down-regulated genes in the firefly squid group was "lipid metabolic process". The firefly squid group had lower mRNA level of glucose-6-phosphate dehydrogenase compared to the controls. These results suggest that an intake of firefly squid decreases hepatic triacylglycerol in rats, and the reduction of mRNA level and enzyme activity of glucose-6-phosphate dehydrogenase might be related to the mechanisms.

  2. Recombinant expression, characterization and application of a dihydrolipoamide dehydrogenase with diaphorase activity from Bacillus sphaericus.

    Science.gov (United States)

    Kianmehr, Anvarsadat; Mahdizadeh, Rahman; Oladnabi, Morteza; Ansari, Javad

    2017-06-01

    Diaphorases are flavin-containing enzymes with potential applications in biotransfomation reactions, biosensor design and in vitro diagnostic tests. In this communication, we describe recombinant expression, characterization and application of a lipoamide dehydrogenase (DLD) with diaphorase activity from a strain of Bacillus sphaericus. The DLD gene consisting of 1413 bp encoding a protein of 470 amino acids was expressed in Escherichia coli BL21 (DE3) and the recombinant enzyme was characterized. B. sphaericus DLD catalyzed the reduction of NAD+ by dihydrolipoamide and exhibited NADH-dependent diaphorase activity. The molecular weight of purified enzyme was about 50 kDa, and determined to be a monomeric protein. Diaphorase was active and stable from pH 7.0 to 9.0 with an optimal activity at pH 8.5. It showed its maximal activity at temperature of 30 °C and was almost stable at temperatures between 25 and 30 °C. Different metal ions and inhibitors showed no influence on the activity of target enzyme. The K m and V max values for NADH were estimated to be 0.33 mM and 200.0 U/ml, respectively. Moreover, recombinant B. sphaericus diaphorase exhibited considerable potential to be used as a component of diagnostic tests for the quantification of metabolites. In conclusion, considering the properties of diaphorase from B. sphaericus PAD-91, it can have potential application as a diagnostic enzyme.

  3. Semi-Rational Design of Geobacillus stearothermophilus L-Lactate Dehydrogenase to Access Various Chiral α-Hydroxy Acids.

    Science.gov (United States)

    Aslan, Aşkın Sevinç; Birmingham, William R; Karagüler, Nevin Gül; Turner, Nicholas J; Binay, Barış

    2016-06-01

    Chiral α-hydroxy acids (AHAs) are rapidly becoming important synthetic building blocks, in particular for the production of pharmaceuticals and other fine chemicals. Chiral compounds of a variety of functionalities are now often derived using enzymes, and L-lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (bsLDH) has the potential to be employed for the industrial synthesis of chiral α-hydroxy acids. Despite the thorough characterization of this enzyme, generation of variants with high activity on non-natural substrates has remained difficult and therefore limits the use of bsLDH in industry. Here, we present the engineering of bsLDH using semi-rational design as a method of focusing screening in a small and smart library for novel biocatalysts. In this study, six mutant libraries were designed in an effort to expand the substrate range of bsLDH. The eight variants identified as having enhanced activity toward the selected α-keto acids belonged to the same library, which targeted two positions simultaneously. These new variants now may be useful biocatalysts for chiral synthesis of α-hydroxy acids.

  4. PHARMACOKINETIC AND PHARMACODYNAMIC ANALYSIS OF INOSINE MONOPHOSPHATE DEHYDROGENASE (IMPDH) ACTIVITY IN MMF-TREATED HCT RECIPIENTS

    Science.gov (United States)

    Li, Hong; Mager, Donald E.; Sandmaier, Brenda M.; Storer, Barry E.; Boeckh, Michael J.; Bemer, Meagan J.; Phillips, Brian R.; Risler, Linda J.; McCune, Jeannine S.

    2014-01-01

    A novel approach to personalizing postgrafting immunosuppression in hematopoietic cell transplant (HCT) recipients is evaluating inosine monophosphate dehydrogenase (IMPDH) activity as a drug-specific biomarker of mycophenolic acid (MPA)-induced immunosuppression. This prospective study evaluated total MPA, unbound MPA, and total MPA glucuronide plasma concentrations and IMPDH activity in peripheral blood mononuclear cells (PMNC) at five time points after the morning dose of oral mycophenolate mofetil (MMF) on day +21 in 56 nonmyeloablative HCT recipients. Substantial interpatient variability in the pharmacokinetics and pharmacodynamics was observed and accurately characterized by the population pharmacokinetic/dynamic model. IMPDH activity decreased with increasing MPA plasma concentration, with maximum inhibition coinciding with maximum MPA concentration in most patients. The overall relationship between MPA concentration and IMPDH activity was described by a direct inhibitory Emax model with an IC50 = 3.23 mg/L total MPA and 57.3 ng/mL unbound MPA. The day +21 IMPDH area under the effect curve (AUEC) was associated with cytomegalovirus reactivation, non-relapse mortality, and overall mortality. In conclusion, a pharmacokinetic/dynamic model was developed that relates plasma MPA concentrations with PMNC IMPDH activity after an MMF dose in HCT recipients. Future studies should validate this model and confirm that day +21 IMPDH AUEC is a predictive biomarker. PMID:24727337

  5. Recipient pretransplant inosine monophosphate dehydrogenase activity in nonmyeloablative hematopoietic cell transplantation.

    Science.gov (United States)

    Bemer, Meagan J; Risler, Linda J; Phillips, Brian R; Wang, Joanne; Storer, Barry E; Sandmaier, Brenda M; Duan, Haichuan; Raccor, Brianne S; Boeckh, Michael J; McCune, Jeannine S

    2014-10-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5'-monophosphate to xanthosine 5'-monophosphate (XMP). We developed a highly sensitive liquid chromatography-mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNCs) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation but not with chronic GVHD, relapse, nonrelapse mortality, or overall mortality. We conclude that quantitation of the recipient's pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient's sensitivity to MMF. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  6. Cytosolic malate dehydrogenase activity helps support glycolysis in actively proliferating cells and cancer.

    Science.gov (United States)

    Hanse, E A; Ruan, C; Kachman, M; Wang, D; Lowman, X H; Kelekar, A

    2017-07-06

    Increased glucose consumption is a hallmark of cancer cells. The increased consumption and subsequent metabolism of glucose during proliferation creates the need for a constant supply of NAD, a co-factor in glycolysis. Regeneration of the NAD required to support enhanced glycolysis has been attributed to the terminal glycolytic enzyme, lactate dehydrogenase (LDH). However, loss of glucose carbons to biosynthetic pathways early in glycolysis reduces the carbon supply to LDH. Thus, alternative routes for NAD regeneration must exist to support the increased glycolytic rate while allowing for the diversion of glucose to generate biomass and support proliferation. Here we demonstrate, using a variety of cancer cell lines as well as activated primary T cells, that cytosolic malate dehydrogenase 1 (MDH1) is an alternative to LDH as a supplier of NAD. Moreover, our results indicate that MDH1 generates malate with carbons derived from glutamine, thus enabling utilization of glucose carbons for glycolysis and for biomass. Amplification of MDH1 occurs at an impressive frequency in human tumors and correlates with poor prognosis. Together, our findings suggest that proliferating cells rely on both MDH1 and LDH to replenish cytosolic NAD, and that therapies designed at targeting glycolysis must consider both dehydrogenases.

  7. Soil dehydrogenase activity of natural macro aggregates in a toposequence of forest soil

    Directory of Open Access Journals (Sweden)

    Maira Kussainova

    2013-01-01

    Full Text Available The main objective of this study was to determine changes in soil dehydrogenase activity in natural macro aggregates development along a slope in forest soils. This study was carried out in Kocadag, Samsun, Turkey. Four landscape positions i.e., summit, shoulder backslope and footslope, were selected. For each landseape position, soil macro aggregates were separated into six aggregate size classes using a dry sieving method and then dehydrogenase activity was analyzed. In this research, topography influenced the macroaggregate size and dehydrogenase activity within the aggregates. At all landscape positions, the contents of macro aggregates (especially > 6.3 mm and 2.00–4.75 mm in all soil samples were higher than other macro aggregate contents. In footslope position, the soils had generally the higher dehydrogenase activity than the other positions at all landscape positions. In all positions, except for shoulder, dehydrogenase activity was greater macro aggregates of <1 mm than in the other macro aggregate size.

  8. Brain aldehyde dehydrogenase activity in rat strains with high and low ethanol preferences.

    Science.gov (United States)

    Inoue, K; Rusi, M; Lindros, K O

    1981-01-01

    The activity of aldehyde dehydrogenase in subcellular fractions of whole brain homogenates from the AA and ANA rat strains developed respectively for high and low ethanol preferences has been studied. No significant strain or sex differences between naive AA and ANA rats were found. In ethanol-experienced rats some strain and sex differences were found, the most consistent being higher enzyme activity in AA females than in males both with aliphatic and aromatic aldehyde substrates. However, contrary to previous findings no relation between brain aldehyde dehydrogenase activity and drinking behavior was found in the AA and ANA rat strains.

  9. Dehydrogenase activity in association with poised potential during biohydrogen production in single chamber microbial electrolysis cell.

    Science.gov (United States)

    Venkata Mohan, S; Lenin Babu, M

    2011-09-01

    Variation in the dehydrogenase (DH) activity and its simultaneous influence on hydrogen (H2) production, substrate degradation rate (SDR) and volatile fatty acid (VFA) generation was investigated with respect to varying poised potential in single chambered membrane-less microbial electrolysis cell (MEC) using anaerobic consortia as biocatalyst. Poised potential showed significant influence on H2 production and DH activity. Maximum H2 production was observed at 1.0V whereas the control system showed least H2 production among the experimental variations studied. DH activity was observed maximum at 0.6V followed by 0.8, 0.9 and 1.0V, suggests the influence of poised potential on the microbial metabolism. Almost complete degradation of substrate was observed in all the experimental conditions studied irrespective of the applied potential. Experimental data was also analysed employing multiple regression analysis and 3D-surface plots to find out the best theoretical poised potential for maximum H2 production and DH activity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. SDHAF4 promotes mitochondrial succinate dehydrogenase activity and prevents neurodegeneration

    Science.gov (United States)

    Van Vranken, Jonathan G.; Bricker, Daniel K.; Dephoure, Noah; Gygi, Steven P.; Cox, James E.; Thummel, Carl S.; Rutter, Jared

    2014-01-01

    SUMMARY Succinate dehydrogenase (SDH) occupies a central place in cellular energy production, linking the tricarboxylic cycle with the electron transport chain. As a result, a subset of cancers and neuromuscular disorders result from mutations affecting any of the four SDH structural subunits or either of two known SDH assembly factors. Herein we characterize a novel evolutionarily conserved SDH assembly factor designated Sdh8/SDHAF4, using yeast, Drosophila, and mammalian cells. Sdh8 interacts specifically with the catalytic Sdh1 subunit in the mitochondrial matrix, facilitating its association with Sdh2 and the subsequent assembly of the SDH holocomplex. These roles for Sdh8 are critical for preventing motility defects and neurodegeneration in Drosophila as well as the excess ROS generated by free Sdh1. These studies provide insights into the mechanisms by which SDH is assembled and raise the possibility that some forms of neuromuscular disease may be associated with mutations that affect this SDH assembly factor. PMID:24954416

  11. Retinoic acids and trichostatin A (TSA), a histone deacetylase inhibitor, induce human pyruvate dehydrogenase kinase 4 (PDK4) gene expression.

    Science.gov (United States)

    Kwon, Hye-Sook; Huang, Boli; Ho Jeoung, Nam; Wu, Pengfei; Steussy, Calvin N; Harris, Robert A

    2006-01-01

    Induction of pyruvate dehydrogenase kinase 4 (PDK4) conserves glucose and substrates for gluconeogenesis and thereby helps regulate blood glucose levels during starvation. We report here that retinoic acids (RA) as well as Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), regulate PDK4 gene expression. Two retinoic acid response elements (RAREs) to which retinoid X receptor alpha (RXRalpha) and retinoic acid receptor alpha (RARalpha) bind and activate transcription are present in the human PDK4 (hPDK4) proximal promoter. Sp1 and CCAAT box binding factor (CBF) bind to the region between two RAREs. Mutation of either the Sp1 or the CBF site significantly decreases basal expression, transactivation by RXRalpha/RARalpha/RA, and the ability of TSA to stimulate hPDK4 gene transcription. By the chromatin immunoprecipitation assay, RA and TSA increase acetylation of histones bound to the proximal promoter as well as occupancy of CBP and Sp1. Interaction of p300/CBP with E1A completely prevented hPDK4 gene activation by RXRalpha/RARalpha/RA and TSA. The p300/CBP may enhance acetylation of histones bound to the hPDK4 promoter and cooperate with Sp1 and CBF to stimulate transcription of the hPDK4 gene in response to RA and TSA.

  12. Magnetic-Resonance Studies of the Geometry of Bound Substrate, Coenzyme and Activator on Bovine-Liver Glutamate Dehydrogenase

    NARCIS (Netherlands)

    Zantema, Alt; de Smet, Marie-José; Robillard, George T.

    ADP and ATP with a spin-label linked to the terminal phosphate are activators of glutamate dehydrogenase and bind to the same site as the activator ADP. There is hardly any interaction with the coenzyme site. Glutamate dehydrogenase can be modified with a ketone spin-label at a site in the active

  13. A new metabolic link. The acyl carrier protein of lipid synthesis donates lipoic acid to the pyruvate dehydrogenase complex in Escherichia coli and mitochondria.

    Science.gov (United States)

    Jordan, S W; Cronan, J E

    1997-07-18

    Lipoic acid is an essential enzyme cofactor that requires covalent attachment to its cognate proteins to confer biological activity. The major lipoylated proteins are highly conserved enzymes of central metabolism, the pyruvate and alpha-ketoglutarate dehydrogenase complexes. The classical lipoate ligase uses ATP to activate the lipoate carboxyl group followed by attachment of the cofactor to a specific subunit of each dehydrogenase complex, and it was assumed that all lipoate attachment proceeded by this mechanism. However, our previous work indicated that Escherichia coli could form lipoylated proteins in the absence of detectable ATP-dependent ligase activity raising the possibility of a class of enzyme that attaches lipoate to the dehydrogenase complexes by a different mechanism. We now report that E. coli and mitochondria contain lipoate transferases that use lipoyl-acyl carrier protein as the lipoate donor. This finding demonstrates a direct link between fatty acid synthesis and lipoate attachment and also provides the first direct demonstration of a role for the enigmatic acyl carrier proteins of mitochondria.

  14. Expression and characterization of a class III alcohol dehydrogenase gene from Gluconobacter frateurii in the presence of methanol during glyceric acid production from glycerol.

    Science.gov (United States)

    Sato, Shun; Morita, Naoki; Kitamoto, Dai; Habe, Hiroshi

    2013-01-01

    Some acetic acid bacteria have been shown to produce large amounts of glyceric acid (GA) from glycerol, which is a by-product of biodiesel fuel (BDF) production. Previously, a Gluconobacter strain was found that produced decreased amounts of GA from glycerol in the presence of methanol, a major ingredient of raw glycerol derived from the BDF industry. Thus, a comparative transcriptome analysis of Gluconobacter frateurii NBRC103465 was performed to investigate changes in gene expression during GA production from glycerol in the presence of methanol. Cells grown with methanol showed upregulated expression of a class III alcohol dehydrogenase homolog (adhC(Gf)) and decreased GA production. adhC(Gf) was cloned and expressed heterologously in Escherichia coli, and the presence of an additional protein with an approximate molecular mass of 39 kDa in the cytosol of the recombinant E. coli cells was identified by SDS-PAGE. Activity measurements of the cytosol revealed that the translational product of adhC(Gf) exhibited formaldehyde dehydrogenase activity in the presence of nicotinamide adenine dinucleotide and glutathione. Gluconobacter frateurii cells grown in 1% methanol-containing glycerol were found to have fivefold higher formaldehyde dehydrogenase activity than cells grown without methanol, suggesting that adhC(Gf) in G. frateurii cells functions in the dissimilation of methanol-derived formaldehyde.

  15. Retinol dehydrogenase-10 regulates pancreas organogenesis and endocrine cell differentiation via paracrine retinoic acid signalling

    DEFF Research Database (Denmark)

    Arregi, Igor; Climent, Maria; Iliev, Dobromir

    2016-01-01

    Vitamin A-derived retinoic acid (RA) signals are critical for the development of several organs, including the pancreas. However, the tissue-specific control of RA synthesis in organ and cell lineage development has only poorly been addressed in vivo. Here we show that Retinol dehydrogenase-10 (Rdh......10), a key enzyme in embryonic RA production, has important functions in pancreas organogenesis and endocrine cell differentiation. Rdh10 was expressed in the developing pancreas epithelium and surrounding mesenchyme. Rdh10 null mutant mouse embryos exhibited dorsal pancreas agenesis...... glucagon(+) and insulin(+) cells. During the secondary transition, the reduction of Neurogenin3(+) endocrine progenitors in the mutant dorsal pancreas accounted for fewer α- and β-cells. Changes in the expression of α- and β-cell-specific transcription factors indicated that Rdh10 might also participate...

  16. Spectrophotometric assay for sensitive detection of glycerol dehydratase activity using aldehyde dehydrogenase.

    Science.gov (United States)

    Park, Eul-Soo; Park, Sunghoon; Shin, Jong-Shik

    2017-04-01

    Glycerol dehydratase (GDHt) is a pivotal enzyme for fermentative utilization of glycerol by catalyzing radical-mediated conversion of glycerol into 3-hydroxypropionaldehyde (3-HPA). Precise and sensitive monitoring of cellular GDHt activity during the fermentation process is a prerequisite for reliable metabolic analysis to afford efficient cellular engineering and process optimization. Here we report a new spectrophotometric assay for the sensitive measurement of the GDHt activity with a sub-nanomolar limit of detection (LOD). The assay method employs aldehyde dehydrogenase (ALDH) as a reporter enzyme, so the readout of the GDHt activity is recorded at 340 nm as an increase in UV absorbance which results from NADH generation accompanied by oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP). The GDHt assay was performed under the reaction conditions where the ALDH activity overwhelms the GDHt activity (i.e., 50-fold higher activity of ALDH relative to GDHt activity), affording sensitive detection of GDHt with 360 pM LOD. The ALDH-coupled assay was used to determine kinetic parameters of GDHt for glycerol, leading to KM = 0.73 ± 0.09 mM and kcat = 400 ± 20 s-1 which are in reasonable agreements with the previous reports. Our assay method allowed measurement of even a 104-fold decrease in the cellular GDHt activity during fermentative production of 3-HP, which demonstrates the detection sensitivity much higher than the previous methods. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  17. Biosynthesis of Germacrene A Carboxylic Acid in Chicory Roots. Demonstration of a Cytochrome P450 (+)-Germacrene A Hydroxylase and NADP+-Dependent Sesquiterpenoid Dehydrogenase(s) Involved in Sesquiterpene Lactone Biosynthesis

    Science.gov (United States)

    de Kraker, Jan-Willem; Franssen, Maurice C. R.; Dalm, Marcella C. F.; de Groot, Aede; Bouwmeester, Harro J.

    2001-01-01

    Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-Germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates β-elemene with a modest degree of enantioselectivity. PMID:11299372

  18. Determination of malic Acid using a malate dehydrogenase reactor after purification and immobilization in non-denaturing conditions and staining with ponceau S.

    Science.gov (United States)

    Shimazaki, Youji; Sakikawa, Takahiro

    2010-08-01

    Mouse liver cytosolic malate dehydrogenase was separated by non-denaturing two-dimensional electrophoresis and identified. Furthermore, the activity of the enzyme was preserved even after separation, electroblotting onto a membrane and staining with Ponceau S in acidic buffer solution (pH 5.1). Using the membrane-immobilized enzyme, the malic acid content was estimated by measuring absorbance changes due to the conversion of nicotinamide adenine dinucleotide (NAD) to NADH. These results indicate that enzyme reactors can be systematically produced after purification, immobilization and staining with Ponceau S.

  19. Structure and function of Plasmodium falciparum malate dehydrogenase: role of critical amino acids in co-substrate binding pocket.

    Science.gov (United States)

    Pradhan, Anupam; Tripathi, Abhai K; Desai, Prashant V; Mukherjee, Prasenjit K; Avery, Mitchell A; Walker, Larry A; Tekwani, Babu L

    2009-01-01

    The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining the co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining the co-substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD, a synthetic analog of NAD), since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in catalytic activity, and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co

  20. Succinic semialdehyde dehydrogenase deficiency: Decrease in 4-OH-butyric acid levels with low doses of vigabatrin

    NARCIS (Netherlands)

    Escalera, G.I.; Ferrer, I.; Marina, L.C.; Sala, P.R.; Salomons, G.S.; Jakobs, C.; Perez-Cerda, C.

    2010-01-01

    Succinic semialdehyde dehydrogenase deficiency (gamma-hydroxybutyric aciduria) is a rare neurometabolic disease caused by a deficiency in gamma-aminobutyric degradation, resulting in an increase in gamma-hydroxybutyric acid in biological fluids. The clinical spectrum is heterogeneous, including a

  1. Positive correlation between rat brain glutamate concentrations and mitochondrial 2-oxoglutarate dehydrogenase activity.

    Science.gov (United States)

    Mkrtchyan, Garik V; Graf, Anastasia; Trofimova, Lidia; Ksenofontov, Alexander; Baratova, Ludmila; Bunik, Victoria

    2018-01-08

    Glutamate is a key metabolite and major excitatory neurotransmitter, degraded through transformation to 2-oxoglutarate which is further catabolized by 2-oxoglutarate dehydrogenase complex (OGDHC). Both the glutamate excitotoxicity and impaired OGDHC activity are hallmarks of neurodegeneration. This work quantifies a relationship between the brain OGDHC activity and glutamate levels, assessing its diagnostic value to characterize (patho)physiology. A moderate to strong positive correlation of the two parameters determined under varied physiological settings (brain regions, seasons, gender, pregnancy, rat line), is revealed. Mitochondrial impairment (OGDHC inhibition or acute hypobaric hypoxia) decreases the interdependence, even when the parameter means do not change significantly. Compared to the cortex, the cerebellum exhibits a lower inter-individual glutamate variation and a weaker glutamate-OGDHC interdependence. Specific metabolism of the brain regions is also characterized by a positive correlation between glutamate and γ-aminobutyric acid (GABA) concentrations in the cortex but not in the cerebellum. In contrast, a strong positive correlation between glutamate and glutamine is present in both the cortex and cerebellum. The differences in metabolic correlations are in line with transcriptomics data which suggest that glutamate distribution between competitive pathways contributes to the brain-region-specific features of the interdependences of glutamate and OGDHC or GABA. Copyright © 2018. Published by Elsevier Inc.

  2. Characterization of the glutamate dehydrogenase activity of Gigantocotyle explanatum and Gastrothylax crumenifer (Trematoda: Digenea).

    Science.gov (United States)

    Abidi, S M A; Khan, P; Saifullah, M K

    2009-12-01

    Glutamate dehydrogenase (GLDH) (EC 1.4.1.3) is a ubiquitous enzyme, which is present at the protein and carbohydrate metabolism crossroads. The enzyme activity was investigated in biliary and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer, respectively, infecting the Indian water buffalo Bubalus bubalis. The enzyme activity was consistently higher in G. explanatum as compared to G. crumenifer, where NAD(H) was utilized as coenzyme and the pH optima was recorded at 8. The K(m) and V(max) values for α-ketoglutarate were 2.1 mM and 9.09 units in G. explanatum, whereas 3.03 mM and 1.90 units in G. crumenifer, respectively. Among the allosteric modulator nucleotides, AMP, ADP, ATP, GMP, CMP and UMP, only AMP enhanced GLDH activity in G. crumenifer while ADP was stimulatory in G. explanatum. The amino acid leucine stimulated the GLDH activity in both the amphistomes while alanine was stimulatory only in G. crumenifer. Pronounced interspecific differences in response to different metabolic inhibitors like diethyldithiocarbamate, semicarbazide hydrochloride and mercurial ions were also observed. The osmotic stress alters the enzyme activity, particularly in hypertonic saline the GLDH activity increased significantly (p < 0.01) in G. explanatum, while insignificant effects were observed in rumen dwelling G. crumenifer. Histoenzymology revealed region/tissue specific distribution of GLDH with prominent staining in tissues like vitellaria, lymph system and tegument/subtegument, thus showing specific distribution of GLDH indicating differential metabolic state. Such intergeneric differences in GLDH activity could also be a consequence of occupying different microenvironments within the same host.

  3. Enhancement of cell growth and glycolic acid production by overexpression of membrane-bound alcohol dehydrogenase in Gluconobacter oxydans DSM 2003.

    Science.gov (United States)

    Zhang, Huan; Shi, Lulu; Mao, Xinlei; Lin, Jinping; Wei, Dongzhi

    2016-11-10

    Membrane-bound alcohol dehydrogenase (mADH) was overexpressed in Gluconobacter oxydans DSM 2003, and the effects on cell growth and glycolic acid production were investigated. The transcription levels of two terminal ubiquinol oxidases (bo3 and bd) in the respiratory chain of the engineered strain G. oxydans-adhABS were up-regulated by 13.4- and 3.8-fold, respectively, which effectively enhanced the oxygen uptake rate, resulting in higher resistance to acid. The cell biomass of G. oxydans-adhABS could increase by 26%-33% when cultivated in a 7L bioreactor. The activities of other major membrane-bound dehydrogenases were also increased to some extent, particularly membrane-bound aldehyde dehydrogenase (mALDH), which is involved in the catalytic oxidation of aldehydes to the corresponding acids and was 1.26-fold higher. Relying on the advantages of the above, G. oxydans-adhABS could produce 73.3gl(-1) glycolic acid after 45h of bioconversion with resting cells, with a molar yield 93.5% and a space-time yield of 1.63gl(-1)h(-1). Glycolic acid production could be further improved by fed-batch fermentation. After 45h of culture, 113.8gl(-1) glycolic acid was accumulated, with a molar yield of 92.9% and a space-time yield of 2.53gl(-1)h(-1), which is the highest reported glycolic acid yield to date. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. L-lactic acid production from D-xylose with Candida sonorensis expressing a heterologous lactate dehydrogenase encoding gene.

    Science.gov (United States)

    Koivuranta, Kari T; Ilmén, Marja; Wiebe, Marilyn G; Ruohonen, Laura; Suominen, Pirkko; Penttilä, Merja

    2014-08-08

    Bioplastics, like polylactic acid (PLA), are renewable alternatives for petroleum-based plastics. Lactic acid, the monomer of PLA, has traditionally been produced biotechnologically with bacteria. With genetic engineering, yeast have the potential to replace bacteria in biotechnological lactic acid production, with the benefits of being acid tolerant and having simple nutritional requirements. Lactate dehydrogenase genes have been introduced to various yeast to demonstrate this potential. Importantly, an industrial lactic acid producing process utilising yeast has already been implemented. Utilisation of D-xylose in addition to D-glucose in production of biochemicals such as lactic acid by microbial fermentation would be beneficial, as it would allow lignocellulosic raw materials to be utilised in the production processes. The yeast Candida sonorensis, which naturally metabolises D-xylose, was genetically modified to produce L-lactic acid from D-xylose by integrating the gene encoding L-lactic acid dehydrogenase (ldhL) from Lactobacillus helveticus into its genome. In microaerobic, CaCO3-buffered conditions a C. sonorensis ldhL transformant having two copies of the ldhL gene produced 31 g l-1 lactic acid from 50 g l-1 D-xylose free of ethanol.Anaerobic production of lactic acid from D-xylose was assessed after introducing an alternative pathway of D-xylose metabolism, i.e. by adding a xylose isomerase encoded by XYLA from Piromyces sp. alone or together with the xylulokinase encoding gene XKS1 from Saccharomyces cerevisiae. Strains were further modified by deletion of the endogenous xylose reductase encoding gene, alone or together with the xylitol dehydrogenase encoding gene. Strains of C. sonorensis expressing xylose isomerase produced L-lactic acid from D-xylose in anaerobic conditions. The highest anaerobic L-lactic acid production (8.5 g l-1) was observed in strains in which both the xylose reductase and xylitol dehydrogenase encoding genes had been

  5. Species-related inhibition of human and rat dihydroorotate dehydrogenase by immunosuppressive isoxazol and cinchoninic acid derivatives.

    Science.gov (United States)

    Knecht, W; Löffler, M

    1998-11-01

    The isoxazol leflunomide (N-(4-trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide) and its active metabolite A77-1726 (N-(4-trifluoromethyl)-phenyl-2-cyano-3-hydroxy-crotonic acidamide) are promising disease-modifying antirheumatic drugs now in clinical trials. The malononitrilamides MNA279 (2-cyano-3-cyclopropyl-3-oxo-(4-cyanophenyl)propionamide) MNA715(N-(4-trifluoromethyl)-phenyl-2-cyano-3-hydroxy-hept-2-en-6- in-carboxylic acidamide) and HR325 (1(3-methyl-4-trifluoro methylphenyl-carbamoyl)-2-cyclopropyl-2oxo-propionitrile) were shown to block rejection after allograft and xenograft transplantation in animals. Brequinar and other cinchoninic acid derivatives have also been evaluated as immuno-suppressive agents. A77-1726, HR325 and brequinar have been shown to have strong inhibitory effects on mitochondrial dihydroorotate dehydrogenase [EC 1.3.99.11], the fourth enzyme of pyrimidine de novo synthesis, with concomitant reduction of pyrimidine nucleotide pools. Pyrimidine nucleotides are essential for normal immune cell functions. Because most investigations had been carried out with cells, cell homogenates or mitochondrial fractions, it was the rationale of the present study to differentiate, under standardized conditions, the effect of leflunomide, A77-1726, MNA279, MNA715, HR 325 and brequinar on the recombinant rat and human enzymes, which were purified in our laboratory. Whereas leflunomide was a relatively weak inhibitor of the rat (IC50 = 6.3 microM) and human (IC50 = 98 microM) dihydroorotate dehydrogenase, the influence of A77-1726, MNA 279, MNA715 and HR325 was of comparable efficacy for either the rat (range of IC50, 19-53 nM) or the human enzyme (range of IC50, 0.5-2.3 microM). From the IC50 values, it was deduced that brequinar was a more potent inhibitor of the human dihydroorotate dehydrogenase activity (IC50 = 10 nM) than of the rat enzyme (IC50 = 367 nM). The rat enzyme was influenced by all isoxazol derivatives to a greater extent (IC50 = 19

  6. Inhibition of dehydrogenase activity in petroleum refinery wastewater bacteria by phenolic compounds

    Directory of Open Access Journals (Sweden)

    Gideon C. Okpokwasili

    2010-04-01

    Full Text Available The toxicity of phenol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol on Pseudomonas, Bacillus and Escherichia species isolated from petroleum refinery wastewater was assessed via inhibition of dehydrogenase enzyme activity. At low concentrations, 2-nitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol stimulated dehydrogenase activity and at sufficient concentrations, phenolic compounds inhibited dehydrogenase activities. Generally, phenol is less toxic than substituted phenols. Estimations of the degree of inhibition/stimulation of dehydrogenase activities showed significant dose-dependent responses that are describable by logistic functions. The toxicity thresholds varied significantly (P < 0.05 among the bacterial strains and phenolic compounds. The median inhibitory concentrations (IC50s ranged from 4.118 ± 0.097 mg.L-1 for 4-nitrophenol against Pseudomonas sp. DAF1 to 1407.997 ± 7.091 mg.L-1 for phenol against Bacillus sp. DISK1. This study suggested that the organisms have moderate sensitivity to phenols and have the potential to be used as indicators for assessment of chemical toxicity. They could also be used as catalysts for degradation of phenols in effluents.

  7. In vitro effects of metals and pesticides on dehydrogenase activity in ...

    African Journals Online (AJOL)

    Effects of heavy metals and pesticides on cowpea (Vigna unquiculata) rhizoplane microbial community were assessed in vitro via dehydrogenase activity. The microbial community was exposed to various concentrations of heavy metals and pesticides in a nutrient broth-glucose-2,3,5-triphenyl chloride (TTC) medium.

  8. Papyriferic acid, an antifeedant triterpene from birch trees, inhibits succinate dehydrogenase from liver mitochondria.

    Science.gov (United States)

    McLean, Stuart; Richards, Stephen M; Cover, Siow-Leng; Brandon, Sue; Davies, Noel W; Bryant, John P; Clausen, Thomas P

    2009-10-01

    Papyriferic acid (PA) is a triterpene that is secreted by glands on twigs of the juvenile ontogenetic phase of resin producing tree birches (e.g., Betula neoalaskana, B. pendula) and that deters browsing by mammals such as the snowshoe hare (Lepus americanus). We investigated the pharmacology of PA as a first step in understanding its antifeedant effect. After oral administration to rats, PA and several metabolites were found in feces but not urine, indicating that little was absorbed systemically. Metabolism involved various combinations of hydrolysis of its acetyl and malonyl ester groups, and hydroxylation of the terpene moiety. The presence of a malonyl group suggested a possible interaction with succinate dehydrogenase (SDH), a mitochondrial enzyme known to be competitively inhibited by malonic acid. The effect of PA on the oxidation of succinate by SDH was examined in mitochondrial preparations from livers of ox, rabbit, and rat. In all three species, PA was a potent inhibitor of SDH. Kinetic analysis indicated that, unlike malonate, PA acted by an uncompetitive mechanism, meaning that it binds to the enzyme-substrate complex. The hydrolysis product of PA, betulafolienetriol oxide, was inactive on SDH. Overall, the evidence suggests that PA acts as the intact molecule and interacts at a site other than the succinate binding site, possibly binding to the ubiquinone sites on complex II. Papyriferic acid was potent (K(iEIS) ranged from 25 to 45 microM in the three species) and selective, as malate dehydrogenase was unaffected. Although rigorous proof will require further experiments, we have a plausible mechanism for the antifeedant effect of PA: inhibition of SDH in gastrointestinal cells decreases mitochondrial energy production resulting in a noxious stimulus, 5-HT release, and sensations of nausea and discomfort. There is evidence that the co-evolution of birches and hares over a large and geographically-diverse area in Northern Europe and America has

  9. A role for AMPK in the inhibition of glucose-6-phosphate dehydrogenase by polyunsaturated fatty acids

    Energy Technology Data Exchange (ETDEWEB)

    Kohan, Alison B.; Talukdar, Indrani; Walsh, Callee M. [Department of Biochemistry, West Virginia University, Morgantown, WV (United States); Salati, Lisa M., E-mail: lsalati@hsc.wvu.edu [Department of Biochemistry, West Virginia University, Morgantown, WV (United States)

    2009-10-09

    Both polyunsaturated fatty acids and AMPK promote energy partitioning away from energy consuming processes, such as fatty acid synthesis, towards energy generating processes, such as {beta}-oxidation. In this report, we demonstrate that arachidonic acid activates AMPK in primary rat hepatocytes, and that this effect is p38 MAPK-dependent. Activation of AMPK mimics the inhibition by arachidonic acid of the insulin-mediated induction of G6PD. Similar to intracellular signaling by arachidonic acid, AMPK decreases insulin signal transduction, increasing Ser{sup 307} phosphorylation of IRS-1 and a subsequent decrease in AKT phosphorylation. Overexpression of dominant-negative AMPK abolishes the effect of arachidonic acid on G6PD expression. These data suggest a role for AMPK in the inhibition of G6PD by polyunsaturated fatty acids.

  10. NAD(P-DEPENDENT DEHYDROGENASE ACTIVITY IN PERIPHERAL BLOOD LYMPHOCYTES OF INFANTS WITH ENLARGEMENT OF PHARYNGEAL TONSILS

    Directory of Open Access Journals (Sweden)

    L. M. Kurtasova

    2014-01-01

    Full Text Available We have observed and examined 57 children 1 to 3 years old diagnosed with enlargement of pharyngeal tonsils. A control group was presented by 35 healthy children. Bioluminescence technique was applied for studying NAD(P-dependent dehydrogenase activity in peripheral blood lymphocytes. Activation of aerobic respiration and increasing activity of pentose phosphate cycle-dependent plastic processes were registered in blood lymphocytes of children with hypertrophic pharyngeal tonsils; along with decreased function of malate-aspartate shunt in energy metabolism of the cells, diminished anaerobic reaction of NADHdependent LDH, lower interaction between Krebs cycle and reactions of amino acid metabolism, and reduced activity of glutathione reductase.

  11. A Unique Short-Chain Dehydrogenase/Reductase in Arabidopsis Glucose Signaling and Abscisic Acid Biosynthesis and Functions

    Science.gov (United States)

    Cheng, Wan-Hsing; Endo, Akira; Zhou, Li; Penney, Jessica; Chen, Huei-Chi; Arroyo, Analilia; Leon, Patricia; Nambara, Eiji; Asami, Tadao; Seo, Mitsunori; Koshiba, Tomokazu; Sheen, Jen

    2002-01-01

    Glc has hormone-like functions and controls many vital processes through mostly unknown mechanisms in plants. We report here on the molecular cloning of GLUCOSE INSENSITIVE1 (GIN1) and ABSCISIC ACID DEFICIENT2 (ABA2) which encodes a unique Arabidopsis short-chain dehydrogenase/reductase (SDR1) that functions as a molecular link between nutrient signaling and plant hormone biosynthesis. SDR1 is related to SDR superfamily members involved in retinoid and steroid hormone biosynthesis in mammals and sex determination in maize. Glc antagonizes ethylene signaling by activating ABA2/GIN1 and other abscisic acid (ABA) biosynthesis and signaling genes, which requires Glc and ABA synergistically. Analyses of aba2/gin1 null mutants define dual functions of endogenous ABA in inhibiting the postgermination developmental switch modulated by distinct Glc and osmotic signals and in promoting organ and body size and fertility in the absence of severe stress. SDR1 is sufficient for the multistep conversion of plastid- and carotenoid-derived xanthoxin to abscisic aldehyde in the cytosol. The surprisingly restricted spatial and temporal expression of SDR1 suggests the dynamic mobilization of ABA precursors and/or ABA. PMID:12417697

  12. Dietary fatty acid composition alters 11β-hydroxysteroid dehydrogenase type 1 gene expression in rat retroperitoneal white adipose tissue

    Directory of Open Access Journals (Sweden)

    Qadri Syed SYH

    2010-10-01

    Full Text Available Abstract The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1 amplifies intracellular glucocorticoid action by converting inactive glucocorticoids to their active forms in vivo. Adipose-specific overexpression of 11β-HSD1 induces metabolic syndrome in mice, whereas 11β-HSD1 null mice are resistant to it. Dietary trans and saturated fatty acids (TFAs and SFAs are involved in the development of metabolic syndrome, whereas polyunsaturated fatty acids (PUFA offer protection against this. Here, we report the effects of chronic feeding of different diets containing vanaspati (TFA rich, palm oil (SFA rich and sunflower oil (PUFA rich at 10%level on 11β-HSD1 gene expression in rat retroperitoneal adipose tissue. 11β-HSD1 gene expression was significantly higher in TFA rich diet-fed rats compared to SFA rich diet-fed rats, which in turn was significantly higher than PUFA rich diet-fed rats. Similar trend was observed in the expression of CCAAT-enhancer binding protein-α (C/EBP-α, the main transcription factor required for the expression of 11β-HSD1. We propose that TFAs and SFAs increase local amplification of glucocorticoid action in adipose tissue by upregulating 11β-HSD1 by altering C/EBP-α-gene expression. The increased levels of glucocorticoids in adipose tissue may lead to development of obesity and insulin resistance, thereby increasing the risk of developing metabolic syndrome.

  13. Dietary fatty acid composition alters 11β-hydroxysteroid dehydrogenase type 1 gene expression in rat retroperitoneal white adipose tissue.

    Science.gov (United States)

    Vara Prasad, Sakamuri S S; Jeya Kumar, Shanmugam S; Kumar, Putcha Uday; Qadri, Syed S Y H; Vajreswari, Ayyalasomayajula

    2010-10-08

    The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) amplifies intracellular glucocorticoid action by converting inactive glucocorticoids to their active forms in vivo. Adipose-specific overexpression of 11β-HSD1 induces metabolic syndrome in mice, whereas 11β-HSD1 null mice are resistant to it. Dietary trans and saturated fatty acids (TFAs and SFAs) are involved in the development of metabolic syndrome, whereas polyunsaturated fatty acids (PUFA) offer protection against this. Here, we report the effects of chronic feeding of different diets containing vanaspati (TFA rich), palm oil (SFA rich) and sunflower oil (PUFA rich) at 10%level on 11β-HSD1 gene expression in rat retroperitoneal adipose tissue. 11β-HSD1 gene expression was significantly higher in TFA rich diet-fed rats compared to SFA rich diet-fed rats, which in turn was significantly higher than PUFA rich diet-fed rats. Similar trend was observed in the expression of CCAAT-enhancer binding protein-α (C/EBP-α), the main transcription factor required for the expression of 11β-HSD1. We propose that TFAs and SFAs increase local amplification of glucocorticoid action in adipose tissue by upregulating 11β-HSD1 by altering C/EBP-α-gene expression. The increased levels of glucocorticoids in adipose tissue may lead to development of obesity and insulin resistance, thereby increasing the risk of developing metabolic syndrome.

  14. The role of hydrophobic amino acids of K-segments in the cryoprotection of lactate dehydrogenase by dehydrins.

    Science.gov (United States)

    Hara, Masakazu; Endo, Takuya; Kamiya, Keita; Kameyama, Ayuko

    2017-03-01

    Dehydrins, which are group 2 late embryogenesis abundant (LEA) proteins, accumulate in plants during the development of the embryo and exposure to abiotic stresses including low temperature. Dehydrins exhibit cryoprotection of freezing-sensitive enzymes, e.g. lactate dehydrogenase (LDH). Although it has been reported that K-segments conserved in dehydrins are related to their cryoprotection activity, it has not been determined which sequence features of the K-segments contribute to the cryoprotection. A cryoprotection assay using LDH indicated that 13 K-segments including 12 K-segments found in Arabidopsis dehydrins and a typical K-segment (TypK, EKKGIMEKIKEKLPG) derived from the K-segments of many plants showed similar cryoprotective activities. Mutation of the TypK sequence demonstrated that hydrophobic amino acids were clearly involved in preventing the cryoinactivation, cryoaggregation, and cryodenaturation of LDH. We propose that the cryoprotective activities of dehydrins may be made possible by the hydrophobic residues of the K-segments. Copyright © 2016 Elsevier GmbH. All rights reserved.

  15. Carbohydrate metabolism during prolonged exercise and recovery: interactions between pyruvate dehydrogenase, fatty acids, and amino acids

    DEFF Research Database (Denmark)

    Mourtzakis, Marina; Saltin, B.; Graham, T.

    2006-01-01

    in pyruvate production could affect tricarboxycylic acid cycle flux as well as gluconeogenesis. To enhance our understanding of these interactions, we studied the time course of changes in substrate utilization in six men who cycled at 44 ± 1% peak oxygen consumption (mean ± SE) until exhaustion (exhaustion...

  16. Characterization of the glutamate dehydrogenase activity of Gigantocotyle explanatum and Gastrothylax crumenifer (Trematoda: Digenea)

    OpenAIRE

    Abidi, S. M. A.; Khan, P.; Saifullah, M. K.

    2009-01-01

    Glutamate dehydrogenase (GLDH) (EC 1.4.1.3) is a ubiquitous enzyme, which is present at the protein and carbohydrate metabolism crossroads. The enzyme activity was investigated in biliary and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer, respectively, infecting the Indian water buffalo Bubalus bubalis. The enzyme activity was consistently higher in G. explanatum as compared to G. crumenifer, where NAD(H) was utilized as coenzyme and the pH optima was recorded at 8. ...

  17. [Cloning, soluble expression and mutant activity analysis of lactate dehydrogenase gene from Plasmodium falciparum].

    Science.gov (United States)

    Xu, Xiao-Ling; Yang, Rui-Yi; Yang, Xue-Qin; Feng, Li-Ling; Zeng, Qing-Ping

    2007-07-01

    To establish a platform for high throughput screening and in vitro evaluating novel metabolic enzyme-targeted inhibitors towards anti-malarial drugs, a lactate dehydrogenase gene of Plasmodium falciparum (PfLDH) was amplified from the Hainan isolate FCC1/HN. The fusion expression vectors, pGEX-2TK and pET-29a( + ), were utilized to introduce the PfLDH gene into strains of Escherichia coli, BL21 and BL21 (DE3), for over-expression. Consequently, the enzymatic activity of PfLDH was successfully detected in the suspension of lytic bacteria. The PfLDH gene cloned in pGEX-2TK was mainly expressed as inclusion bodies, while the same gene cloned in pET-29a( + ) was nearly expressed in a soluble form of PfLDH, demonstrating the latter vehicle might be more suitable for the large-scale preparation of recombinant PfLDH. Furthermore, according to the electrophoregram of SDS-PAGE and the sequencing data, a series of truncated PfLDH sequences generated randomly from gene amplification were screened and cloned, from which four pre-matured genes with a terminator mutation, PfLDH-delta271, -delta236, -delta167 and -delta53 coding for 45, 80, 149 and 263 amino acid residues, were individually recovered. Through the gene expression and enzymatic activity measurement, the effect of pre-matured terminator mutation on the activity of PfLDH was evaluated, which should pave the way for probing the relationship between structure and function of PfLDH.

  18. (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (FadB’) from fatty acid degradation operon of Ralstonia eutropha H16

    Science.gov (United States)

    2014-01-01

    In this study (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (H16_A0461/FadB’, gene ID: 4247876) from one of two active fatty acid degradation operons of Ralstonia eutropha H16 has been heterologously expressed in Escherichia coli, purified as protein possessing a His-Tag and initially characterized. FadB’ is an enzyme with two catalytic domains exhibiting a single monomeric structure and possessing a molecular weight of 86 kDa. The C-terminal part of the enzyme harbors enoyl-CoA hydratase activity and is able to convert trans-crotonyl-CoA to 3-hydroxybutyryl-CoA. The N-terminal part of FadB’ comprises an NAD+ binding site and is responsible for 3-hydroxyacyl-CoA dehydrogenase activity converting (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA. Enoyl-CoA hydratase activity was detected spectrophotometrically with trans-crotonyl-CoA. (S)-3-Hydroxyacyl-CoA dehydrogenase activity was measured in both directions with acetoacetyl-CoA and 3-hydroxybutyryl-CoA. FadB’ was found to be strictly stereospecific to (S)-3-hydroxybutyryl-CoA and to prefer NAD+. The Km value for acetoacetyl-CoA was 48 μM and Vmax 149 μmol mg−1 min−1. NADP(H) was utilized at a rate of less than 10% in comparison to activity with NAD(H). FadB’ exhibited optimal activity at pH 6–7 and the activity decreased at alkaline and acidic pH values. Acetyl-CoA, propionyl-CoA and CoA were found to have an inhibitory effect on FadB’. This study is a first report on biochemical properties of purified (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase with the inverted domain order from R. eutropha H16. In addition to fundamental information about FadB’ and fatty acid metabolism, FadB’ might be also interesting for biotechnological applications. PMID:25401070

  19. (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (FadB') from fatty acid degradation operon of Ralstonia eutropha H16.

    Science.gov (United States)

    Volodina, Elena; Steinbüchel, Alexander

    2014-01-01

    In this study (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (H16_A0461/FadB', gene ID: 4247876) from one of two active fatty acid degradation operons of Ralstonia eutropha H16 has been heterologously expressed in Escherichia coli, purified as protein possessing a His-Tag and initially characterized. FadB' is an enzyme with two catalytic domains exhibiting a single monomeric structure and possessing a molecular weight of 86 kDa. The C-terminal part of the enzyme harbors enoyl-CoA hydratase activity and is able to convert trans-crotonyl-CoA to 3-hydroxybutyryl-CoA. The N-terminal part of FadB' comprises an NAD(+) binding site and is responsible for 3-hydroxyacyl-CoA dehydrogenase activity converting (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA. Enoyl-CoA hydratase activity was detected spectrophotometrically with trans-crotonyl-CoA. (S)-3-Hydroxyacyl-CoA dehydrogenase activity was measured in both directions with acetoacetyl-CoA and 3-hydroxybutyryl-CoA. FadB' was found to be strictly stereospecific to (S)-3-hydroxybutyryl-CoA and to prefer NAD(+). The K m value for acetoacetyl-CoA was 48 μM and V max 149 μmol mg(-1) min(-1). NADP(H) was utilized at a rate of less than 10% in comparison to activity with NAD(H). FadB' exhibited optimal activity at pH 6-7 and the activity decreased at alkaline and acidic pH values. Acetyl-CoA, propionyl-CoA and CoA were found to have an inhibitory effect on FadB'. This study is a first report on biochemical properties of purified (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase with the inverted domain order from R. eutropha H16. In addition to fundamental information about FadB' and fatty acid metabolism, FadB' might be also interesting for biotechnological applications.

  20. Changes in the zonation of lactate dehydrogenase activity in lobules of rat liver after experimentally induced colon carcinoma metastases

    NARCIS (Netherlands)

    Griffini, P.; Freitas, I.; Vigorelli, E.; van Noorden, C. J.

    1994-01-01

    Visualization of lactate dehydrogenase (LDH) activity with Neotetrazolium as final electron acceptor under anaerobic conditions and an incubation medium containing polyvinyl alcohol showed that under normal physiological conditions a zonal distribution of LDH activity is present in the liver lobule

  1. Enhancement of the activity of enzyme immobilized on polydopamine-coated iron oxide nanoparticles by rational orientation of formate dehydrogenase.

    Science.gov (United States)

    Gao, Xin; Ni, Kefeng; Zhao, Chengcheng; Ren, Yuhong; Wei, Dongzhi

    2014-10-20

    Immobilization of enzymes onto nanoparticles and retention of their structure and activity, which may be related to the orientation of enzymes on nanoparticles, remain a challenge. Here, we developed a novel enzyme-orientation strategy to enhance the activity of formate dehydrogenase immobilized on polydopamine-coated iron oxide nanoparticles via site-directed mutation. Seven mutants were constructed based on homology modeling of formate dehydrogenase and immobilized on polydopamine-coated iron oxide nanoparticles to investigate the influence of these mutations on immobilization. The immobilized mutant C242A/C275V/C363V/K389C demonstrated the highest immobilization yield and retained 90% of its initial activity, which was about 3-fold higher than that of wild-type formate dehydrogenase. Moreover, co-immobilization of formate dehydrogenase and leucine dehydrogenase was performed for the synthesis of l-tert-leucine. The catalytic efficiency of the co-immobilized mutant C242A/C275V/C363V/K389C and leucine dehydrogenase increased by more than 4-fold compared to that of co-immobilized wild-type formate dehydrogenase and leucine dehydrogenase. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L.

    Science.gov (United States)

    Kobayashi, Keiichi; Hattori, Takasumi; Hayashi, Rie; Kirimura, Kohtaro

    2014-01-01

    In the tricarboxylic acid (TCA) cycle, NADP(+)-specific isocitrate dehydrogenase (NADP(+)-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP(+) as a cofactor. We constructed an NADP(+)-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP(+)-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP(+)-ICDH activity. Therefore, NADP(+)-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.

  3. Retinol Dehydrogenase-10 Regulates Pancreas Organogenesis and Endocrine Cell Differentiation via Paracrine Retinoic Acid Signaling.

    Science.gov (United States)

    Arregi, Igor; Climent, Maria; Iliev, Dobromir; Strasser, Jürgen; Gouignard, Nadège; Johansson, Jenny K; Singh, Tania; Mazur, Magdalena; Semb, Henrik; Artner, Isabella; Minichiello, Liliana; Pera, Edgar M

    2016-12-01

    Vitamin A-derived retinoic acid (RA) signals are critical for the development of several organs, including the pancreas. However, the tissue-specific control of RA synthesis in organ and cell lineage development has only poorly been addressed in vivo. Here, we show that retinol dehydrogenase-10 (Rdh10), a key enzyme in embryonic RA production, has important functions in pancreas organogenesis and endocrine cell differentiation. Rdh10 was expressed in the developing pancreas epithelium and surrounding mesenchyme. Rdh10 null mutant mouse embryos exhibited dorsal pancreas agenesis and a hypoplastic ventral pancreas with retarded tubulogenesis and branching. Conditional disruption of Rdh10 from the endoderm caused increased mortality, reduced body weight, and lowered blood glucose levels after birth. Endodermal Rdh10 deficiency led to a smaller dorsal pancreas with a reduced density of early glucagon(+) and insulin(+) cells. During the secondary transition, the reduction of Neurogenin3(+) endocrine progenitors in the mutant dorsal pancreas accounted for fewer α- and β-cells. Changes in the expression of α- and β-cell-specific transcription factors indicated that Rdh10 might also participate in the terminal differentiation of endocrine cells. Together, our results highlight the importance of both mesenchymal and epithelial Rdh10 for pancreogenesis and the first wave of endocrine cell differentiation. We further propose a model in which the Rdh10-expressing exocrine tissue acts as an essential source of RA signals in the second wave of endocrine cell differentiation.

  4. Serum creatine kinase and lactate dehydrogenase activities in ...

    African Journals Online (AJOL)

    Background and Objectives: There is the recognition of a pattern of elevations of serum enzymes in hyperthyroid and hypothyroid patients. The aims of this study were to determine the activities of serum creatine kinase (CK) and lactate deydrogenase (LDH) in thyroid disorders, and to evaluate the relationship between CK, ...

  5. Production of L-lactic acid by the yeast Candida sonorensis expressing heterologous bacterial and fungal lactate dehydrogenases.

    Science.gov (United States)

    Ilmén, Marja; Koivuranta, Kari; Ruohonen, Laura; Rajgarhia, Vineet; Suominen, Pirkko; Penttilä, Merja

    2013-05-25

    Polylactic acid is a renewable raw material that is increasingly used in the manufacture of bioplastics, which offers a more sustainable alternative to materials derived from fossil resources. Both lactic acid bacteria and genetically engineered yeast have been implemented in commercial scale in biotechnological production of lactic acid. In the present work, genes encoding L-lactate dehydrogenase (LDH) of Lactobacillus helveticus, Bacillus megaterium and Rhizopus oryzae were expressed in a new host organism, the non-conventional yeast Candida sonorensis, with or without the competing ethanol fermentation pathway. Each LDH strain produced substantial amounts of lactate, but the properties of the heterologous LDH affected the distribution of carbon between lactate and by-products significantly, which was reflected in extra-and intracellular metabolite concentrations. Under neutralizing conditions C. sonorensis expressing L. helveticus LDH accumulated lactate up to 92 g/l at a yield of 0.94 g/g glucose, free of ethanol, in minimal medium containing 5 g/l dry cell weight. In rich medium with a final pH of 3.8, 49 g/l lactate was produced. The fermentation pathway was modified in some of the strains studied by deleting either one or both of the pyruvate decarboxylase encoding genes, PDC1 and PDC2. The deletion of both PDC genes together abolished ethanol production and did not result in significantly reduced growth characteristic to Saccharomyces cerevisiae deleted of PDC1 and PDC5. We developed an organism without previous record of genetic engineering to produce L-lactic acid to a high concentration, introducing a novel host for the production of an industrially important metabolite, and opening the way for exploiting C. sonorensis in additional biotechnological applications. Comparison of metabolite production, growth, and enzyme activities in a representative set of transformed strains expressing different LDH genes in the presence and absence of a functional

  6. Succinate dehydrogenase activity and soma size of motoneurons innervating different portions of the rat tibialis anterior

    Science.gov (United States)

    Ishihara, A.; Roy, R. R.; Edgerton, V. R.

    1995-01-01

    The spatial distribution, soma size and oxidative enzyme activity of gamma and alpha motoneurons innervating muscle fibres in the deep (away from the surface of the muscle) and superficial (close to the surface of the muscle) portions of the tibialis anterior in normal rats were determined. The deep portion had a higher percentage of high oxidative fibres than the superficial portion of the muscle. Motoneurons were labelled by retrograde neuronal transport of fluorescent tracers: Fast Blue and Nuclear Yellow were injected into the deep portion and Nuclear Yellow into the superficial portion of the muscle. Therefore, motoneurons innervating the deep portion were identified by both a blue fluorescent cytoplasm and a golden-yellow fluorescent nucleus, while motoneurons innervating the superficial portion were identified by only a golden-yellow fluorescent nucleus. After staining for succinate dehydrogenase activity on the same section used for the identification of the motoneurons, soma size and succinate dehydrogenase activity of the motoneurons were measured. The gamma and alpha motoneurons innervating both the deep and superficial portions were located primarily at L4 and were intermingled within the same region of the dorsolateral portion of the ventral horn in the spinal cord. Mean soma size was similar for either gamma or alpha motoneurons in the two portions of the muscle. The alpha motoneurons innervating the superficial portion had a lower mean succinate dehydrogenase activity than those innervating the deep portion of the muscle. An inverse relationship between soma size and succinate dehydrogenase activity of alpha, but not gamma, motoneurons innervating both the deep and superficial portions was observed. Based on three-dimensional reconstructions within the spinal cord, there were no apparent differences in the spatial distribution of the motoneurons, either gamma or alpha, associated with the deep and superficial compartments of the muscle. The data

  7. Active succinate dehydrogenase (SDH) and lack of SDHD mutations in sporadic paragangliomas.

    Science.gov (United States)

    Braun, Simone; Riemann, Kathrin; Kupka, Susan; Leistenschneider, Peter; Sotlar, Karl; Schmid, Heide; Blin, Nikolaus

    2005-01-01

    Paragangliomas are benign, slow-growing tumours of the head and neck region. The candidate gene for familial and some sporadic paragangliomas, SDHD (succinate dehydrogenase, subunit D), has been mapped to the PGL1 locus in 11q23.3. Normal and tumour DNA of 17 patients with sporadic paragangliomas were analysed by sequencing (SDHD, SDHB and SDHC genes), fluorescence in situ hybridisation (FISH). In addition, loss of heterozygosity (LOH) and succinate dehydrogenase (SDH) enzyme activity assays were performed. Only two patients from our collective showed SDH gene mutations, one in SDHD and one in SDHB, respectively. Moreover, SDH activity detected in 5/8 patients confirmed the fact that SDH inactivation is not a major event in sporadic paragangliomas. LOH and FISH analysis demonstrated a frequent loss of regions within chromosome 11, indicating that additional genes in 11q may play a role in tumour genesis of sporadic paragangliomas.

  8. Hypoxia and anoxia effects on alcohol dehydrogenase activity and hemoglobin content in Chironomus riparius Meigen, 1804

    OpenAIRE

    Valentina Grazioli; Bruno Rossaro; Paolo Parenti; Roberto Giacchini; Valeria Lencioni

    2016-01-01

    The metabolic effects of low oxygen content on alcohol-dehydrogenase (ADH) activity and hemoglobin (Hb) concentration were investigated in IV-instar larvae of Chironomus riparius (Diptera: Chironomidae) from an Italian stream. Two series of short-term (48 h) experiments were carried out: exposure to (1) progressive hypoxia (95 to 5% of oxygen saturation) and (2) anoxia (at <5% of oxygen saturation). In (1), Hb amount increased with increasing oxygen depletion up to a critical value of oxyg...

  9. In vitro effects of metals and pesticides on dehydrogenase activity in ...

    African Journals Online (AJOL)

    AJB SERVER

    2007-01-04

    Jan 4, 2007 ... medium. At 0.2 mM, iron and cadmium stimulated the dehydrogenase activity of the microbial community. For all the metal ions, there was progressive inhibition with each successive increase in the concentration of metal ion, reaching near 100% at 0.6, 0.8, 1.2, 0.12 and 12 mM for cobalt, cadmium,.

  10. Rationally re-designed mutation of NAD-independent l-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jiang Tianyi

    2012-11-01

    Full Text Available Abstract Background NAD-independent l-lactate dehydrogenase (l-iLDH from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids. Results Val-108 of l-iLDH was changed to Ala by rationally site-directed mutagenesis. The l-iLDH mutant exhibited much higher activity than wide-type l-iLDH towards l-mandelate, an aromatic 2-hydroxycarboxylic acid. Using the engineered Escherichia coli expressing the mutant l-iLDH as a biocatalyst, 40 g·L-1 of dl-mandelic acid was converted to 20.1 g·L-1 of d-mandelic acid (enantiomeric purity higher than 99.5% and 19.3 g·L-1 of benzoylformic acid. Conclusions A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally re-design mutagenesis. Two building block intermediates (optically pure d-mandelic acid and benzoylformic acid were efficiently produced by the one-pot biotransformation system.

  11. A simple spectrophotometric assay for long-chain acyl-CoA dehydrogenase activity measurements in human skin fibroblasts

    NARCIS (Netherlands)

    IJlst, L.; Wanders, R. J.

    1993-01-01

    Long-chain acyl-CoA dehydrogenase (LCAD) deficiency is an autosomal recessive disorder of fatty acid metabolism characterized by hypoglycemia, muscle weakness and hepato- and cardiomegaly to varying extents. Analysis of organic acids in urine usually reveals dicarboxylic aciduria with elevated

  12. SDR-type human hydroxysteroid dehydrogenases involved in steroid hormone activation.

    Science.gov (United States)

    Wu, Xiaoqiu; Lukacik, Petra; Kavanagh, Kathryn L; Oppermann, Udo

    2007-02-01

    Hydroxysteroid dehydrogenases catalyze the NAD(P)(H)-dependent oxidoreduction of hydroxyl and oxo-functions at distinct positions of steroid hormones. This reversible reaction constitutes an important pre-receptor control mechanism for nuclear receptor ligands of the androgen, estrogen and glucocorticoid classes, since the conversion "switches" between receptor ligands and their inactive metabolites. The major reversible activities found in mammals acting on steroid hormones comprise 3alpha-, 11beta- and 17beta-hydroxysteroid dehydrogenases, and for each group several distinct isozymes have been described. The enzymes differ in their expression pattern, nucleotide cofactor preference, steroid substrate specificity and subcellular localization, and thus constitute a complex system ensuring cell-specific adaptation and regulation of steroid hormone levels. Several isoforms constitute promising drug targets, of particular importance in cancer, metabolic diseases, neurodegeneration and immunity.

  13. Cytophotometric analysis of reaction rates of succinate and lactate dehydrogenase activity in rat liver, heart muscle and tracheal epithelium

    NARCIS (Netherlands)

    van Noorden, C. J.; Vogels, I. M.

    1989-01-01

    Reaction rates of succinate and lactate dehydrogenase activity in cryostat sections of rat liver, tracheal epithelium and heart muscle were monitored by continuous measurement of formazan formation by cytophotometry at room temperature. Incubation media contained polyvinyl alcohol as tissue

  14. Anisaldehyde production and aryl-alcohol oxidase and dehydrogenase activities in ligninolytic fungi of the genus Pleurotus.

    Science.gov (United States)

    Gutiérrez, A; Caramelo, L; Prieto, A; Martínez, M J; Martínez, A T

    1994-01-01

    A variety of simple aromatic compounds were identified in liquid cultures of the basidiomycetes Pleurotus cornucopiae, P. eryngii, P. floridanus, P. pulmonarius, P. ostreatus, and P. sajor-caju by using gas chromatography-mass spectrometry. Such compounds were detected in fungal cultures on lignin- and straw-containing media, but it was found that they were also produced in the absence of aromatic precursors. Anisylic and hydroxybenzylic compounds (such as alcohols, aldehydes, and acids) were identified, p-anisaldehyde being the most characteristic extracellular metabolite synthesized by these ligninolytic fungi. Small amounts of 3-chloro-p-anisaldehyde were also detected in several species. It is postulated that the balance between the more-or-less-oxidized aromatic compounds can be explained in terms of the activity of fungal enzymes, including aryl-alcohol oxidase and dehydrogenase. The former enzyme shows high affinity for p-anisyl alcohol, which is oxidized to p-anisaldehyde with production of H2O2. The aryl-alcohol dehydrogenase was detected only in the mycelium, where it reduces aromatic aldehydes in the presence of NADPH. Both enzymes could be involved in the redox cycling of these aromatic compounds, providing H2O2 to ligninolytic peroxidases. PMID:8031078

  15. Towards a systematic analysis of human short-chain dehydrogenases/reductases (SDR): Ligand identification and structure-activity relationships.

    Science.gov (United States)

    Bhatia, Chitra; Oerum, Stephanie; Bray, James; Kavanagh, Kathryn L; Shafqat, Naeem; Yue, Wyatt; Oppermann, Udo

    2015-06-05

    Short-chain dehydrogenases/reductases (SDRs) constitute a large, functionally diverse branch of enzymes within the class of NAD(P)(H) dependent oxidoreductases. In humans, over 80 genes have been identified with distinct metabolic roles in carbohydrate, amino acid, lipid, retinoid and steroid hormone metabolism, frequently associated with inherited genetic defects. Besides metabolic functions, a subset of atypical SDR proteins appears to play critical roles in adapting to redox status or RNA processing, and thereby controlling metabolic pathways. Here we present an update on the human SDR superfamily and a ligand identification strategy using differential scanning fluorimetry (DSF) with a focused library of oxidoreductase and metabolic ligands to identify substrate classes and inhibitor chemotypes. This method is applicable to investigate structure-activity relationships of oxidoreductases and ultimately to better understand their physiological roles. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. Crystal structures of the active and alloxanthine-inhibited forms of xanthine dehydrogenase from Rhodobacter capsulatus.

    Science.gov (United States)

    Truglio, James J; Theis, Karsten; Leimkühler, Silke; Rappa, Roberto; Rajagopalan, K V; Kisker, Caroline

    2002-01-01

    Xanthine dehydrogenase (XDH), a complex molybdo/iron-sulfur/flavoprotein, catalyzes the oxidation of hypoxanthine to xanthine followed by oxidation of xanthine to uric acid with concomitant reduction of NAD+. The 2.7 A resolution structure of Rhodobacter capsulatus XDH reveals that the bacterial and bovine XDH have highly similar folds despite differences in subunit composition. The NAD+ binding pocket of the bacterial XDH resembles that of the dehydrogenase form of the bovine enzyme rather than that of the oxidase form, which reduces O(2) instead of NAD+. The drug allopurinol is used to treat XDH-catalyzed uric acid build-up occurring in gout or during cancer chemotherapy. As a hypoxanthine analog, it is oxidized to alloxanthine, which cannot be further oxidized but acts as a tight binding inhibitor of XDH. The 3.0 A resolution structure of the XDH-alloxanthine complex shows direct coordination of alloxanthine to the molybdenum via a nitrogen atom. These results provide a starting point for the rational design of new XDH inhibitors.

  17. Substrate Specificity and Allosteric Regulation of a D-Lactate Dehydrogenase from a Unicellular Cyanobacterium are Altered by an Amino Acid Substitution.

    Science.gov (United States)

    Ito, Shoki; Takeya, Masahiro; Osanai, Takashi

    2017-11-08

    Lactate/lactic acid is an important chemical compound for the manufacturing of bioplastics. The unicellular cyanobacterium Synechocystis sp. PCC 6803 can produce lactate from carbon dioxide and possesses D-lactate dehydrogenase (Ddh). Here, we performed a biochemical analysis of the Ddh from this cyanobacterium (SyDdh) using recombinant proteins. SyDdh was classified into a cyanobacterial clade similar to those from Gram-negative bacteria, although it was distinct from them. SyDdh can use both pyruvate and oxaloacetate as a substrate and is activated by fructose-1,6-bisphosphate and repressed by divalent cations. An amino acid substitution based on multiple sequence alignment data revealed that the glutamine at position 14 and serine at position 234 are important for the allosteric regulation by Mg 2+ and substrate specificity of SyDdh, respectively. These results reveal the characteristic biochemical properties of Ddh in a unicellular cyanobacterium, which are different from those of other bacterial Ddhs.

  18. Major Role of NAD-Dependent Lactate Dehydrogenases in the Production of l-Lactic Acid with High Optical Purity by the Thermophile Bacillus coagulans.

    Science.gov (United States)

    Wang, Limin; Cai, Yumeng; Zhu, Lingfeng; Guo, Honglian; Yu, Bo

    2014-12-01

    Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production-NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)-were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  19. Scanning mutagenesis of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

    Directory of Open Access Journals (Sweden)

    Nagib eAhsan

    2012-07-01

    Full Text Available The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1α subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated Ser-residue and the sequences of the flanking amino acids are highly conserved. We have used the synthetic peptide-based kinase client assay plus recombinant pyruvate dehydrogenase E1α and E1α-kinase to perform scanning mutagenesis of the residues flanking the site of phosphorylation. Consistent with the results from phylogenetic analysis of the flanking sequences, the direct peptide-based kinase assays tolerated very few changes. Even conservative changes such as Leu, Ile, or Val for Met, or Glu for Asp, gave very marked reductions in phosphorylation. Overall the results indicate that regulation of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation is an extreme example of multiple, interdependent instances of co-evolution.

  20. Novel biohybrids of layered double hydroxide and lactate dehydrogenase enzyme: Synthesis, characterization and catalytic activity studies

    Science.gov (United States)

    Djebbi, Mohamed Amine; Braiek, Mohamed; Hidouri, Slah; Namour, Philippe; Jaffrezic-Renault, Nicole; Ben Haj Amara, Abdesslem

    2016-02-01

    The present work introduces new biohybrid materials involving layered double hydroxides (LDH) and biomolecule such as enzyme to produce bioinorganic system. Lactate dehydrogenase (Lac Deh) has been chosen as a model enzyme, being immobilized onto MgAl and ZnAl LDH materials via direct ion-exchange (adsorption) and co-precipitation methods. The immobilization efficiency was largely dependent upon the immobilization methods. A comparative study shows that the co-precipitation method favors the immobilization of great and tunable amount of enzyme. The structural behavior, chemical bonding composition and morphology of the resulting biohybrids were determined by X-ray diffraction (XRD) study, Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM), respectively. The free and immobilized enzyme activity and kinetic parameters were also reported using UV-Visible spectroscopy. However, the modified LDH materials showed a decrease in crystallinity as compared to the unmodified LDH. The change in activity of the immobilized lactate dehydrogenase was considered to be due, to the reduced accessibility of substrate molecules to the active sites of the enzyme and the partial conformational change of the Lac Deh molecules as a result of the immobilization way. Finally, it was proven that there is a correlation between structure/microstructure and enzyme activity dependent on the immobilization process.

  1. Xanthine Dehydrogenase (XDH) cross-reacting material in mutants of Drosophila melanogaster deficient in XDH activity.

    Science.gov (United States)

    Browder, L W; Tucker, L; Wilkes, J

    1982-02-01

    Rocket immunoelectrophoresis was used to estimate xanthine dehydrogenase cross-reacting material (XDH-CRM) in strains containing the cin and cin mutant genes, which are deficient in XDH enzymatic activity. CRM levels were determined as percentages of CRM in the Oregon-R wild-type strain. The mutant strains contain 72 and 76% of Oregon-R CRM, respectively. CRM levels in strains containing the XDH-deficient mutant genes lxd and mal are 93 and 105%, respectively. The high levels of CRM in these four mutant strains indicate that the primary effects of the mutant genes are on the function of XDH protein rather than its accumulation.

  2. [Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

    Science.gov (United States)

    Voloshchuk, O N; Kopylchuk, G P

    2016-01-01

    Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage.

  3. Co-expression of two heterologous lactate dehydrogenases genes in Kluyveromyces marxianus for l-lactic acid production.

    Science.gov (United States)

    Lee, Jae Won; In, Jung Hoon; Park, Joon-Bum; Shin, Jonghyeok; Park, Jin Hwan; Sung, Bong Hyun; Sohn, Jung-Hoon; Seo, Jin-Ho; Park, Jin-Byoung; Kim, Soo Rin; Kweon, Dae-Hyuk

    2017-01-10

    Lactic acid (LA) is a versatile compound used in the food, pharmaceutical, textile, leather, and chemical industries. Biological production of LA is possible by yeast strains expressing a bacterial gene encoding l-lactate dehydrogenase (LDH). Kluyveromyces marxianus is an emerging non-conventional yeast with various phenotypes of industrial interest. However, it has not been extensively studied for LA production. In this study, K. marxianus was engineered to express and co-express various heterologous LDH enzymes that were reported to have different pH optimums. Specifically, three LDH enzymes originating from Staphylococcus epidermidis (SeLDH; optimal at pH 5.6), Lactobacillus acidophilus (LaLDH; optimal at pH 5.3), and Bos taurus (BtLDH; optimal at pH 9.8) were functionally expressed individually and in combination in K. marxianus, and the resulting strains were compared in terms of LA production. A strain co-expressing SeLDH and LaLDH (KM5 La+SeLDH) produced 16.0g/L LA, whereas the strains expressing those enzymes individually produced only 8.4 and 6.8g/L, respectively. This co-expressing strain produced 24.0g/L LA with a yield of 0.48g/g glucose in the presence of CaCO3. Our results suggest that co-expression of LDH enzymes with different pH optimums provides sufficient LDH activity under dynamic intracellular pH conditions, leading to enhanced production of LA compared to individual expression of the LDH enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Dehydroepiandrosterone supplement increases malate dehydrogenase activity and decreases NADPH-dependent antioxidant enzyme activity in rat hepatocellular carcinogenesis

    Science.gov (United States)

    Kim, Sook-Hee; Choi, Haymie

    2008-01-01

    Beneficial effects of dehydroepiandrosterone (DHEA) supplement on age-associated chronic diseases such as cancer, cardiovascular disease, insulin resistance and diabetes, have been reported. However, its mechanism of action in hepatocellular carcinoma in vivo has not been investigated in detail. We have previously shown that during hepatocellular carcinogenesis, DHEA treatment decreases formation of preneoplastic glutathione S-transferase placental form-positive foci in the liver and has antioxidant effects. Here we aimed to determine the mechanism of actions of DHEA, in comparison to vitamin E, in a chemically-induced hepatocellular carcinoma model in rats. Sprague-Dawley rats were administered with control diet without a carcinogen, diets with 1.5% vitamin E, 0.5% DHEA and both of the compounds with a carcinogen for 6 weeks. The doses were previously reported to have anti-cancer effects in animals without known toxicities. With DHEA treatment, cytosolic malate dehydrogenase activities were significantly increased by ~5 fold and glucose 6-phosphate dehydrogenase activities were decreased by ~25% compared to carcinogen treated group. Activities of Se-glutathione peroxidase in the cytotol was decreased significantly with DHEA treatment, confirming its antioxidative effect. However, liver microsomal cytochrome P-450 content and NADPH-dependent cytochrome P-450 reductase activities were not altered with DHEA treatment. Vitamin E treatment decreased cytosolic Se-glutathione peroxidase activities in accordance with our previous reports. However, vitamin E did not alter glucose 6-phosphate dehydrogenase or malate dehydrogenase activities. Our results suggest that DHEA may have decreased tumor nodule formation and reduced lipid peroxidation as previously reported, possibly by increasing the production of NADPH, a reducing equivalent for NADPH-dependent antioxidant enzymes. DHEA treatment tended to reduce glucose 6-phosphate dehydrogenase activities, which may have

  5. The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: benzyl viologen oxidoreductase activity

    Directory of Open Access Journals (Sweden)

    Trchounian Armen

    2011-08-01

    Full Text Available Abstract Background Escherichia coli synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Two of the formate dehydrogenases (Fdh-N and Fdh-O and two of the hydrogenases (Hyd-1 and Hyd-2 have their respective catalytic subunits located in the periplasm and these enzymes have been shown previously to oxidize formate and hydrogen, respectively, and thus function in energy metabolism. Mutants unable to synthesize the [NiFe]-hydrogenases retain a H2: benzyl viologen oxidoreductase activity. The aim of this study was to identify the enzyme or enzymes responsible for this activity. Results Here we report the identification of a new H2: benzyl viologen oxidoreductase enzyme activity in E. coli that is independent of the [NiFe]-hydrogenases. This enzyme activity was originally identified after non-denaturing polyacrylamide gel electrophoresis and visualization of hydrogen-oxidizing activity by specific staining. Analysis of a crude extract derived from a variety of E. coli mutants unable to synthesize any [NiFe]-hydrogenase-associated enzyme activity revealed that the mutants retained this specific hydrogen-oxidizing activity. Enrichment of this enzyme activity from solubilised membrane fractions of the hydrogenase-negative mutant FTD147 by ion-exchange, hydrophobic interaction and size-exclusion chromatographies followed by mass spectrometric analysis identified the enzymes Fdh-N and Fdh-O. Analysis of defined mutants devoid of selenocysteine biosynthetic capacity or carrying deletions in the genes encoding the catalytic subunits of Fdh-N and Fdh-O demonstrated that both enzymes catalyze hydrogen activation. Fdh-N and Fdh-O can also transfer the electrons derived from oxidation of hydrogen to other redox dyes. Conclusions The related respiratory molybdo-selenoproteins Fdh-N and Fdh-O of Escherichia coli have hydrogen-oxidizing activity. These findings

  6. Structure of D-lactate dehydrogenase from Aquifex aeolicus complexed with NAD(+) and lactic acid (or pyruvate).

    Science.gov (United States)

    Antonyuk, Svetlana V; Strange, Richard W; Ellis, Mark J; Bessho, Yoshitaka; Kuramitsu, Seiki; Inoue, Yumiko; Yokoyama, Shigeyuki; Hasnain, S Samar

    2009-12-01

    The crystal structure of D-lactate dehydrogenase from Aquifex aeolicus (aq_727) was determined to 2.12 A resolution in space group P2(1)2(1)2(1), with unit-cell parameters a = 90.94, b = 94.43, c = 188.85 A. The structure was solved by molecular replacement using the coenzyme-binding domain of Lactobacillus helveticus D-lactate dehydrogenase and contained two homodimers in the asymmetric unit. Each subunit of the homodimer was found to be in a ;closed' conformation with the NADH cofactor bound to the coenzyme-binding domain and with a lactate (or pyruvate) molecule bound at the interdomain active-site cleft.

  7. Peroxisome proliferator-activated receptor ɣ activation induces 11β-hydroxysteroid dehydrogenase type 1 activity in human alternative macrophages

    Science.gov (United States)

    Chinetti-Gbaguidi, Giulia; Bouhlel, Mohamed Amine; Copin, Corinne; Duhem, Christian; Derudas, Bruno; Neve, Bernardette; Noel, Benoit; Eeckhoute, Jerome; Lefebvre, Philippe; Seckl, Jonathan R.; Staels, Bart

    2012-01-01

    Objectives 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyses the intracellular reduction of inactive cortisone to active cortisol, the natural ligand activating the glucocorticoid receptor (GR). Peroxisome Proliferator-Activated Receptor gamma (PPARγ) is a nuclear receptor controlling inflammation, lipid metabolism and the macrophage polarization state. In this study, we investigated the impact of macrophage polarization on the expression and activity of 11β-HSD1 and the role of PPAR therein. Methods and Results 11β-HSD1 gene expression is higher in pro-inflammatory M1 and anti-inflammatory M2 macrophages than in resting macrophages (RM), whereas its activity is highest in M2 macrophages. Interestingly, PPARγ activation induces 11β-HSD1 enzyme activity in M2 macrophages, but not in RM or M1 macrophages. Consequently, human M2 macrophages displayed enhanced responsiveness to the 11β-HSD1 substrate cortisone, an effect amplified by PPAR -induction of 11β-HSD1 activity, as illustrated by an increased expression of GR target genes. Conclusions Our data identify a positive cross-talk between PPARγ and GR in human M2 macrophages via the induction of 11β-HSD1 expression and activity. PMID:22207732

  8. Changes in dehydrogenases activity, number and ultrastructure of mitochondria in Aspergillus niger mycelium growing on molasses media.

    Science.gov (United States)

    Gabara, B; Zakowska, Z

    1993-01-01

    Effects of toxic molasses compounds and of the antifoamer (Spumol BJ) on dehydrogenases activity, on the number and ultrastructure of mitochondria in A. niger mycelium of two strains characterized by different tolerance to toxic agents, were observed. In spite of significantly higher dehydrogenases activity in the intolerant strain (R-16) mycelia developing both on productive molasses in the presence of the defoamer and on non-productive molasses are characterized by marked reduction in the activity of these enzymes. Changes in respiratory enzymes activity are partly correlated with the number of mitochondria but mostly with abnormalities in their ultrastructure.

  9. Pharmacokinetic and pharmacodynamic analysis of inosine monophosphate dehydrogenase activity in hematopoietic cell transplantation recipients treated with mycophenolate mofetil.

    Science.gov (United States)

    Li, Hong; Mager, Donald E; Sandmaier, Brenda M; Storer, Barry E; Boeckh, Michael J; Bemer, Meagan J; Phillips, Brian R; Risler, Linda J; McCune, Jeannine S

    2014-08-01

    A novel approach to personalizing postgrafting immunosuppression in hematopoietic cell transplantation (HCT) recipients is evaluating inosine monophosphate dehydrogenase (IMPDH) activity as a drug-specific biomarker of mycophenolic acid (MPA)-induced immunosuppression. This prospective study evaluated total MPA, unbound MPA, and total MPA glucuronide plasma concentrations and IMPDH activity in peripheral blood mononuclear cells (PMNCs) at 5 time points after the morning dose of oral mycophenolate mofetil (MMF) on day +21 in 56 nonmyeloablative HCT recipients. Substantial interpatient variability in pharmacokinetics and pharmacodynamics was observed and accurately characterized by the population pharmacokinetic-dynamic model. IMPDH activity decreased with increasing MPA plasma concentration, with maximum inhibition coinciding with maximum MPA concentration in most patients. The overall relationship between MPA concentration and IMPDH activity was described by a direct inhibitory maximum effect model with an IC50 of 3.23 mg/L total MPA and 57.3 ng/mL unbound MPA. The day +21 IMPDH area under the effect curve (AUEC) was associated with cytomegalovirus reactivation, nonrelapse mortality, and overall mortality. In conclusion, a pharmacokinetic-dynamic model was developed that relates plasma MPA concentrations with PMNC IMPDH activity after an MMF dose in HCT recipients. Future studies should validate this model and confirm that day +21 IMPDH AUEC is a predictive biomarker. Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  10. A nitrogen-doped graphene/gold nanoparticle/formate dehydrogenase bioanode for high power output membrane-less formic acid/O2 biofuel cells.

    Science.gov (United States)

    Gai, Panpan; Ji, Yusheng; Chen, Yun; Zhu, Cheng; Zhang, Jianrong; Zhu, Jun-Jie

    2015-03-21

    A high power output, membrane-less formic acid/O2 enzymatic biofuel cell was fabricated, in which nitrogen-doped graphene was proven to be effective for recycling the NAD(+)/NADH cofactor at a nitrogen-doped graphene/gold nanoparticle/formate dehydrogenase bioanode in the catalytic oxidation of formic acid.

  11. [Cytosol malate dehydrogenase in Calicophoron ijimai trematodes and the effect of antiparasitic preparations on its activity].

    Science.gov (United States)

    Vykhrestiuk, N P; Khamatova, A Iu

    1983-01-01

    The activity and properties of malate dehydrogenase (MDH; EC 1.1.1.37) and of "malic" enzyme (EC 1.1.1.40) in cytosole of the trematode C. ijimai were determined. The activity of MDH directed to oxaloacetate formation was shown to be 14 times and maximum velocity 13 times lower than that of the reverse reaction. The apparent KM was one order higher in the direct reaction. This confirms the possibility of glycolytic pathway in C. ijimai via CO2 fixation into phosphoenolpyruvate to form oxaloacatate which is readily eliminated by active MDH. The presence of "malic" enzyme in C. ijimai testifies to the occurrence of different pathways of succinate formation in this species.

  12. Designing a highly active soluble PQQ-glucose dehydrogenase for efficient glucose biosensors and biofuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Durand, Fabien [Universite de Bordeaux, Centre de Recherche Paul Pascal (CRPP), UPR 8641, Avenue Albert Schweitzer, 33600 Pessac (France); Stines-Chaumeil, Claire [Universite de Bordeaux, CNRS, Institut de Biochimie et de Genetique Cellulaires, 1 rue Camille Saint Saens, 33077 Bordeaux Cedex (France); Flexer, Victoria [Universite de Bordeaux, Centre de Recherche Paul Pascal (CRPP), UPR 8641, Avenue Albert Schweitzer, 33600 Pessac (France); Andre, Isabelle [Universite de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse (France); CNRS, UMR5504, F-31400 Toulouse (France); INRA, UMR 792 Ingenierie des Systemes Biologiques et des Procedes, F-31400 Toulouse (France); Mano, Nicolas, E-mail: mano@crpp-bordeaux.cnrs.fr [Universite de Bordeaux, Centre de Recherche Paul Pascal (CRPP), UPR 8641, Avenue Albert Schweitzer, 33600 Pessac (France)

    2010-11-26

    Research highlights: {yields} A new mutant of PQQ-GDH designed for glucose biosensors application. {yields} First mutant of PQQ-GDH with higher activity for D-glucose than the Wild type. {yields} Position N428 is a key point to increase the enzyme activity. {yields} Molecular modeling shows that the N428 C mutant displays a better interaction for PQQ than the WT. -- Abstract: We report for the first time a soluble PQQ-glucose dehydrogenase that is twice more active than the wild type for glucose oxidation and was obtained by combining site directed mutagenesis, modelling and steady-state kinetics. The observed enhancement is attributed to a better interaction between the cofactor and the enzyme leading to a better electron transfer. Electrochemical experiments also demonstrate the superiority of the new mutant for glucose oxidation and make it a promising enzyme for the development of high-performance glucose biosensors and biofuel cells.

  13. The esg locus of Myxococcus xanthus encodes the E1 alpha and E1 beta subunits of a branched-chain keto acid dehydrogenase.

    Science.gov (United States)

    Toal, D R; Clifton, S W; Roe, B A; Downard, J

    1995-04-01

    The esg locus of Myxococcus xanthus appears to control the production of a signal that must be transmitted between cells for the completion of multicellular development. DNA sequence analysis suggested that the esg locus encodes the E1 decarboxylase (composed of E1 alpha and E1 beta subunits) of a branched-chain keto acid dehydrogenase (BCKAD) that is involved in branched-chain amino acid (BCAA) metabolism. The properties of an esg::Tn5 insertion mutant supported this conclusion. These properties include: (i) the growth yield of the mutant was reduced with increasing concentrations of the BCAAs in the medium while the growth yield of wild-type cells increased, (ii) mutant extracts were deficient in BCKAD activity, and (iii) growth of the mutant in media with short branched-chain fatty acids related to the expected products of the BCKAD helped to correct the mutant defects in growth, pigmentation and development. The esg BCKAD appears to be involved in the synthesis of long branched-chain fatty acids since the mutant contained reduced levels of this class of compounds. Our results are consistent with a model in which the esg-encoded enzyme is involved in the synthesis of branched-chain fatty acids during vegetative growth, and these compounds are used later in cell-cell signalling during development.

  14. EFFECT OF DIVALENT CATIONS ON THE ACTIVITY AND CONFORMATION OF YEAST ALCOHOL DEHYDROGENASE

    Directory of Open Access Journals (Sweden)

    Rizwanul Haque

    2012-12-01

    Full Text Available The divalent metal chlorides affect the activity of yeast alcohol dehydrogenase (YADH. The activity of the enzyme in the presence of 20 µM of these metal chlorides decreased in the following order: CaCl2 > MgCl2 > no salt > MnCl2 > CoCl2 > CuCl2 > CdCl2 . The EC50 values for the incubation of YADH in the presence of CaCl 2 , MgCl2 , MnCl2 , CoCl2 , CuCl2 and CdCl2 were 14.5, 37.8, 0.25, 0.21, 0.18 and 0.005 mM, respectively. The most potent inhibitor was CdCl2 . Fluorescence studies revealed that the conformation of YADH undergoes huge changes in the presence of these metal chlorides. By far UV-CD spectra, some secondary structural changes were observed in YADH in the presence of CaCl2 , MgCl2 , MnCl2 and CoCl2 , however the changes were very significant in case of CdCl2 and CuCl2 . The removal of zinc from YADH resulted in complete loss of enzymatic activity which is regained back by addition of Zn. Mg, Ca and Mn can substitute Zn in YADH, and the enzyme exhibits comparable activity as in presence of Zn. However, Co, Cu, and Cd cannot substitute Zn in YADH. The results obtained here might be useful in the biotechnological applications of alcohol dehydrogenase.

  15. Loss of NADH Oxidase Activity in Streptococcus mutans Leads to Rex-Mediated Overcompensation in NAD+ Regeneration by Lactate Dehydrogenase.

    Science.gov (United States)

    Baker, J L; Derr, A M; Faustoferri, R C; Quivey, R G

    2015-12-01

    Previous studies of the oral pathogen Streptococcus mutans have determined that this Gram-positive facultative anaerobe mounts robust responses to both acid and oxidative stresses. The water-forming NADH oxidase (Nox; encoded by nox) is thought to be critical for the regeneration of NAD(+), for use in glycolysis, and for the reduction of oxygen, thereby preventing the formation of damaging reactive oxygen species. In this study, the free NAD(+)/NADH ratio in a nox deletion strain (Δnox) was discovered to be remarkably higher than that in the parent strain, UA159, when the strains were grown in continuous culture. This unanticipated result was explained by significantly elevated lactate dehydrogenase (Ldh; encoded by ldh) activity and ldh transcription in the Δnox strain, which was mediated in part by the redox-sensing regulator Rex. cDNA microarray analysis of S. mutans cultures exposed to simultaneous acid stress (growth at a low pH) and oxidative stress (generated through the deletion of nox or the addition of exogenous oxygen) revealed a stress response synergistically heightened over that with either stress alone. In the Δnox strain, this elevated stress response included increased glucose phosphoenolpyruvate phosphotransferase system (PTS) activity, which appeared to be due to elevated manL transcription, mediated in part, like elevated ldh transcription, by Rex. While the Δnox strain does possess a membrane composition different from that of the parent strain, it did not appear to have defects in either membrane permeability or ATPase activity. However, the altered transcriptome and metabolome of the Δnox strain were sufficient to impair its ability to compete with commensal peroxigenic oral streptococci during growth under aerobic conditions. Streptococcus mutans is an oral pathogen whose ability to outcompete commensal oral streptococci is strongly linked to the formation of dental caries. Previous work has demonstrated that the S. mutans water

  16. Dramatic reduction of erythrocyte glucose-6-phosphate dehydrogenase activity in athletes participating in the ultradistance foot race "Spartathlon".

    Science.gov (United States)

    Schulpis, K H; Tsironi, M; Skenderi, K; Lazaropoulou, C; Parthimos, N; Reclos, G; Goussetis, E; Tsakiris, S; Papassotiriou, I

    2008-01-01

    To investigate the effect of a long-distance endurance exercise "Spartathlon" on erythrocyte glucose-6-phosphate dehydrogenase (G(6)PD) activity. The study comprised 15 male runners, median age 36.5 years. Blood samples were obtained in the 15 min before the race and again within 15 min after the end of the race. Erythrocyte glutathione (GSH and GSSG) and plasma malonyldialdehyde were measured with HPLC methods, and total antioxidant capacity (TAC), total hyperoxides and G(6)PD activity with commercial kits. Lipids, uric acid and total bilirubin were determined with a clinical chemistry analyser. Total hyperoxides were found statistically reduced, whereas total bilirubin was measured elevated post-race. Interestingly, GSSG levels were found increased (167.3+/-12.0 versus 219.5+/-20.3 micromol/L; prace. In contrast, G(6)PD activity was found remarkably decreased (8.72+/-3.10 versus 3.8+/-2.5 U/g Hb; prace as a consequence of the modulation of NADP/NADPH levels and elevation of the erythrocyte GSSG, and especially GSSG/GSH ratio, resulting in an impairment of the hexose monophosphate shunt.

  17. 5´AMP activated protein kinase α2 controls substrate metabolism during post-exercise recovery via regulation of pyruvate dehydrogenase kinase 4

    DEFF Research Database (Denmark)

    Fritzen, Andreas Mæchel; Lundsgaard, Annemarie; Jeppesen, Jacob

    2015-01-01

    in muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in WT and AMPKα2 KO was observed following exercise, which is consistent with AMPKα2 -deficiency not affecting the exercise-induced activation of the PDK4 transcriptional regulators, HDAC4 and SIRT1. Interestingly, PDK4 protein content...... regulates muscle metabolism post-exercise through inhibition of the PDH complex and hence glucose oxidation, subsequently creating conditions for increased fatty acid oxidation. This article is protected by copyright. All rights reserved....

  18. Estrogen modulates xanthine dehydrogenase/xanthine oxidase activity by a receptor-independent mechanism.

    Science.gov (United States)

    Budhiraja, Rohit; Kayyali, Usamah S; Karamsetty, Mallik; Fogel, Michael; Hill, Nicholas S; Chalkley, Roger; Finlay, Geraldine A; Hassoun, Paul M

    2003-12-01

    Hypoxia causes up-regulation and activation of xanthine dehydrogenase/xanthine oxidase (XDH/XO) in vitro and in the lungs in vivo. This up-regulation, and the likely corresponding production of reactive oxygen species, may underlie the pathogenesis of an array of disorders. Thus, compounds that prevent hypoxia-induced increase in XDH/XO activity may provide a therapeutic strategy in such disorders. The antioxidant properties of estrogens have been demonstrated in several studies. However, the effect of these compounds on XDH/XO has not been explored previously. The aim of this study was to investigate the effects of estrogen on hypoxia-induced increase in XDH/XO activity. Rat pulmonary artery microvascular endothelial cells were exposed to normoxia or hypoxia in the presence or absence of 17beta- or 17alpha-estradiol. The XDH/XO enzyme and gene promoter activities were measured in different groups of cells. Hypoxia caused a twofold increase in XDH/XO enzymatic and promoter activity. Either of the estradiol stereoisomers prevented the hypoxia-induced increase in XDH/XO enzymatic activity, but not the promoter activity. ICI 182,780, an antagonist of the estrogen receptor, failed to block the inhibitory effect of estradiol on XDH/XO. In conclusion, 17alpha- and 17beta-estradiol modulate the hypoxia-induced regulation of XDH/XO activity at a posttranscriptional level by a receptor-independent mechanism.

  19. Evidence of activation of the renal glutamate dehydrogenase pathway in intact acidotic dogs.

    Science.gov (United States)

    Lombardo, J V; Risquez, A; McCarthy, M; Preuss, H G

    1981-04-01

    To determine if activity of the renal glutamate dehydrogenase (GD) pathway changes during chronic acidosis in intact dogs, we assessed the deamination of glutamate formed within renal cells during glutamine and alanine infusions. Infusing glutamine into chronically acidotic, normal and acutely alkalotic dogs enhanced renal ammonia production; more was formed as glutamine loading increased. In 4 acidotic dogs, the ratio of ammonia produced to glutamine extracted by the kidneys during exogenous glutamine loading was 1.93 compared with 0.99 for 5 alkalotic dogs and 1.23 for 2 control dogs. Little glutamate and alanine were released into the renal vein in acidotic dogs, whereas over 50% of the exogenous glutamine extracted in acutely alkalotic dogs could be accounted for as glutamate and alanine released into the renal vein. Renal glutamate concentrations were not elevated in acidosis compared with alkalosis despite greater deamidation. When glutamine infusions increased renal ammoniagenesis in acutely alkalotic and control dogs to levels seen in chronically acidotic dogs receiving no exogenous glutamine, approximately 4 to 6 times more glutamate was released from the kidneys. Infusing alanine into 7 chronically acidotic dogs enhanced ammoniagenesis significantly (p less than 0.01), but lesser augmentation was seen in 3 control dogs and no augmentation was seen in 6 acutely alkalotic dogs. The increases were secondary to enhanced glutamate deamination, not secondary to any changes in glutamine extraction and/or transaminase activity. We conclude that the glutamate dehydrogenase pathway is more active in intact acidotic dogs than it is in control and alkalotic dogs.

  20. Cloning of D-lactate dehydrogenase genes of Lactobacillus delbrueckii subsp. bulgaricus and their roles in D-lactic acid production.

    Science.gov (United States)

    Huang, Yanna; You, Chunping; Liu, Zhenmin

    2017-07-01

    Lactobacillus delbrueckii subsp. bulgaricus is a heterogenous lactic acid bacterium that converts pyruvate mainly to D-lactic acid using D-lactate dehydrogenases (D-LDHs), whose functional properties remain poorly characterized. Here, the D-LDHs genes (ldb0101, ldb0813, ldb1010, ldb1147 and ldb2021) were cloned and overexpressed in Escherichia coli JM109 from an inducible pUC18 vector, respectively, and the resulting strains were compared in terms of D-lactic acid production. The strain expressing ldb0101 and ldb1010 gene individually produced more D-lactate than other three strains. Further study revealed that Ldb0101 activity was down-regulated by the oxygen and, therefore, achieved a highest titer of D-lactate (1.94 g/L) under anaerobic condition, and introduction of ldb1010 gene enhanced D-lactate formation (0.94 and 0.85 g/L, respectively) both in aerobic and anaerobic conditions due to a relatively stable q d-lactate. Our results suggested that the enzyme Ldb0101 and Ldb1010 played a role of more importance in D-lactate formation. To the best of our knowledge, we demonstrate for the first time the roles of different D-LDH homologs from L. bulgaricus in D-lactic acid production.

  1. Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

    Science.gov (United States)

    Llanos, R M; Harris, C J; Hillier, A J; Davidson, B E

    1993-01-01

    The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon. Images PMID:8478320

  2. Identification of a metabolically stable triazolopyrimidine-based dihydroorotate dehydrogenase inhibitor with antimalarial activity in mice.

    Science.gov (United States)

    Gujjar, Ramesh; Marwaha, Alka; El Mazouni, Farah; White, John; White, Karen L; Creason, Sharon; Shackleford, David M; Baldwin, Jeffrey; Charman, William N; Buckner, Frederick S; Charman, Susan; Rathod, Pradipsinh K; Phillips, Margaret A

    2009-04-09

    Plasmodium falciparum causes 1-2 million deaths annually. Yet current drug therapies are compromised by resistance. We previously described potent and selective triazolopyrimidine-based inhibitors of P. falciparum dihydroorotate dehydrogenase (PfDHODH) that inhibited parasite growth in vitro; however, they showed no activity in vivo. Here we show that lack of efficacy against P. berghei in mice resulted from a combination of poor plasma exposure and reduced potency against P. berghei DHODH. For compounds containing naphthyl (DSM1) or anthracenyl (DSM2), plasma exposure was reduced upon repeated dosing. Phenyl-substituted triazolopyrimidines were synthesized leading to identification of analogs with low predicted metabolism in human liver microsomes and which showed prolonged exposure in mice. Compound 21 (DSM74), containing p-trifluoromethylphenyl, suppressed growth of P. berghei in mice after oral administration. This study provides the first proof of concept that DHODH inhibitors can suppress Plasmodium growth in vivo, validating DHODH as a new target for antimalarial chemotherapy.

  3. Enhancement of xylitol production by attenuation of intracellular xylitol dehydrogenase activity in Candida tropicalis.

    Science.gov (United States)

    Ko, Byoung Sam; Kim, Dong-Min; Yoon, Byoung Hoon; Bai, Suk; Lee, Hyeon Yong; Kim, Jung Hoe; Kim, Il-Chul

    2011-06-01

    To construct Candida tropicalis strains that produce a high yield of xylitol with no requirement for co-substrates, we engineered the yeast with an attenuated xylitol dehydrogenase (XDH) and then assessed the efficiency of xylitol production The mutants, strains XDH-5 (with only one copy of the XDH gene), and ARSdR-16 (with a mutated XDH gene) showed 70 and 40% of wild type (WT) XDH activity, respectively. Conversions of xylose to xylitol by WT, XDH-5, and ARSdR-16 were 62, 64, and 75%, respectively, with productivities of 0.52, 0.54, and 0.62 g l(-1) h(-1), respectively. The ARSdR-16 mutant strain produced xylitol with high yield and high productivity in a simple process that required no co-substrates, such as glycerol. This strain represents a promising alternative for efficient and cost-effective xylitol production.

  4. The hydrogenase activity of the molybdenum/copper-containing carbon monoxide dehydrogenase of Oligotropha carboxidovorans.

    Science.gov (United States)

    Wilcoxen, Jarett; Hille, Russ

    2013-12-13

    The reaction of the air-tolerant CO dehydrogenase from Oligotropha carboxidovorans with H2 has been examined. Like the Ni-Fe CO dehydrogenase, the enzyme can be reduced by H2 with a limiting rate constant of 5.3 s(-1) and a dissociation constant Kd of 525 μM; both kred and kred/Kd, reflecting the breakdown of the Michaelis complex and the reaction of free enzyme with free substrate in the low [S] regime, respectively, are largely pH-independent. During the reaction with H2, a new EPR signal arising from the Mo/Cu-containing active site of the enzyme is observed which is distinct from the signal seen when the enzyme is reduced by CO, with greater g anisotropy and larger hyperfine coupling to the active site (63,65)Cu. The signal also exhibits hyperfine coupling to at least two solvent-exchangeable protons of bound substrate that are rapidly exchanged with solvent. Proton coupling is also evident in the EPR signal seen with the dithionite-reduced native enzyme, and this coupling is lost in the presence of bicarbonate. We attribute the coupled protons in the dithionite-reduced enzyme to coordinated water at the copper site in the native enzyme and conclude that bicarbonate is able to displace this water from the copper coordination sphere. On the basis of our results, a mechanism for H2 oxidation is proposed which involves initial binding of H2 to the copper of the binuclear center, displacing the bound water, followed by sequential deprotonation through a copper-hydride intermediate to reduce the binuclear center.

  5. Caffeine alters mitochondrial dehydrogenase and alkaline phosphatase activity of human gingival fibroblasts in vitro.

    Science.gov (United States)

    Bozchaloei, Shabnam Soltani; Gong, Siew-Ging; Dehpour, Ahmad R; Farrokh, Parisa; Khoshayand, Mohammad R; Oskoui, Mahvash

    2013-11-01

    Caffeine is one of the most widely consumed behaviorally active substances in the world. Although its effects on the central nervous system and bone metabolism have been documented, as yet there is no report on its effect on tissues in the oral cavity. In this study we analyzed the viability of human gingival fibroblasts (HGF) and alkaline phosphatase (ALP) enzyme activity after exposure to different concentrations of caffeine for different exposure time periods. The HGF were cultured with different concentrations of caffeine. Viability of cells exposed to caffeine was analyzed by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay to assess mitochondrial dehydrogenase activity. The activity of ALP was analyzed at specific time intervals after caffeine addition. Our results showed that caffeine of concentrations caffeine at 5 and 10 mm dramatically decreased the viability and ALP activity of the cells after 4 days such that, by day 9, the viability of cells declined to near zero in the 10 mm group. These results provided evidence that caffeine in high concentrations can decrease cellular viability and ALP activity in HGF. © 2012 Wiley Publishing Asia Pty Ltd.

  6. Glyceraldehyde-3-phosphate dehydrogenase from Chironomidae showed differential activity towards metals.

    Science.gov (United States)

    Chong, Isaac K W; Ho, Wing S

    2013-09-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to interact with different biomolecules and was implicated in many novel cellular activities including programmed cell death, nuclear RNA transport unrelated to the commonly known carbohydrate metabolism. We reported here the purification of GAPDH from Chironomidae larvae (Insecta, Diptera) that showed different biologic activity towards heavy metals. It was inhibited by copper, cobalt nickel, iron and lead but was activated by zinc. The GAPDH was purified by ammonium sulphate fractionation and Chelating Sepharose CL-6B chromatography followed by Blue Sepharose CL-6B chromatography. The 150-kDa tetrameric GAPDH showed optimal activity at pH 8.5 and 37°C. The multiple alignment of sequence of the Chironomidae GAPDH with other known species showed 78 - 88% identity to the conserved regions of the GADPH. Bioinformatic analysis unveils substantial N-terminal sequence similarity of GAPDH of Chironomidae larvae to mammalian GADPHs. However, the GADPH of Chironomidae larvae showed different biologic activities and cytotoxicity towards heavy metals. The GAPDH enzyme would undergo adaptive molecular changes through binding at the active site leading to higher tolerance to heavy metals.

  7. EFFECTS OF AMARANTHS’ SEEDS ON DEHYDROGENASE ACTIVITY AND GASES EMISSION IN METHANOGENIC BIOREACTORS

    Directory of Open Access Journals (Sweden)

    Victor COVALIOV

    2015-12-01

    Full Text Available The influence of amaranths‘ seeds as the source of squalene on the dehydrogenase activity and efficiency of methane production were investigated in methanogenic bench-scale (5000 ml bioreactors used to treat the mixture of distillery wastes and farmyard manure. The adding of amaranth seeds to the methanogenic bioreactor has an inhibitory effect on the dehydrogenase activity and stimulates the process of methanogenesis. Dehydrogenase activity decreased with the increase of doses of squalene and its trend had a close connection with doses (R2=0.77-0.78. The methane content in the total amount of gases is 65.3-71.3% in a bioreactor with the additive of amaranth seeds in a dose of 50 mg l-1, which is 22.1% higher than in the the control bioreactor without additives. The increase in squalene concentration higher than 0.0005% is not rational because its stimulating effect on the methanogenic process decreases. Anaerobic digestion of alcohol distillery industry wastes with manure is a complex nonlinear time-varying microbiological process. Dehydrogenase activity trends in the experiment are described by the power function for 5 hours observations and by the logarithmic function for 120 hours of observations. Trends of CH4 are described by the polynomial function in all periods of testing. Correlation coefficients are 0.37 and 0.70 for CH4 after 5 and 120 hours of the anaerobic digestion. Dehydrogenase activity is in the close negative connection with the amount of gases, including methane. Correlation analysis between dehydrogenase activity and the release of gases has revealed the moderate and strongly negative link during 24 hours after the start of the experiment.EFECTUL SEMINŢELOR DE AMARANT ASUPRA ACTIVITĂŢII DEHIDROGENAZEI ŞI EMISIEI GAZELOR ÎN BIOREACTOARELE METANOGENEÎn bioreactoare metanogene unite consecutiv, cu volum de 5000 ml, utilizate pentru tratarea amestecului de borhot de la distilarea alcoolului cu gunoi de grajd, a fost

  8. Teneligliptin Decreases Uric Acid Levels by Reducing Xanthine Dehydrogenase Expression in White Adipose Tissue of Male Wistar Rats

    Directory of Open Access Journals (Sweden)

    Chihiro Moriya

    2016-01-01

    Full Text Available We investigated the effects of teneligliptin on uric acid metabolism in male Wistar rats and 3T3-L1 adipocytes. The rats were fed with a normal chow diet (NCD or a 60% high-fat diet (HFD with or without teneligliptin for 4 weeks. The plasma uric acid level was not significantly different between the control and teneligliptin groups under the NCD condition. However, the plasma uric acid level was significantly decreased in the HFD-fed teneligliptin treated rats compared to the HFD-fed control rats. The expression levels of xanthine dehydrogenase (Xdh mRNA in liver and epididymal adipose tissue of NCD-fed rats were not altered by teneligliptin treatment. On the other hand, Xdh expression was reduced significantly in the epididymal adipose tissue of the HFD-fed teneligliptin treated rats compared with that of HFD-fed control rats, whereas Xdh expression in liver did not change significantly in either group. Furthermore, teneligliptin significantly decreased Xdh expression in 3T3-L1 adipocytes. DPP-4 treatment significantly increased Xdh expression in 3T3-L1 adipocytes. With DPP-4 pretreatment, teneligliptin significantly decreased Xdh mRNA expression compared to the DPP-4-treated 3T3-L1 adipocytes. In conclusion, our studies suggest that teneligliptin reduces uric acid levels by suppressing Xdh expression in epididymal adipose tissue of obese subjects.

  9. A new high phenyl lactic acid-yielding Lactobacillus plantarum IMAU10124 and a comparative analysis of lactate dehydrogenase gene.

    Science.gov (United States)

    Zhang, Xiqing; Zhang, Shuli; Shi, Yan; Shen, Fadi; Wang, Haikuan

    2014-07-01

    Phenyl lactic acid (PLA) has been widely reported as a new natural antimicrobial compound. In this study, 120 Lactobacillus plantarum strains were demonstrated to produce PLA using high-performance liquid chromatography. Lactobacillus plantarum IMAU10124 was screened with a PLA yield of 0.229 g L(-1) . Compared with all previous reports, this is the highest PLA-producing lactic acid bacteria (LAB) when grown in MRS broth without any optimizing conditions. When 3.0 g L(-1) phenyl pyruvic acid (PPA) was added to the medium as substrate, PLA production reached 2.90 g L(-1) , with the highest 96.05% conversion rate. A lowest PLA-yielding L. plantarum IMAU40105 (0.043 g L(-1) ) was also screened. It was shown that the conversion from PPA to PLA by lactic dehydrogenase (LDH) is the key factor in the improvement of PLA production by LAB. Comparing the LDH gene of two strains, four amino acid mutation sites were found in this study in the LDH of L. plantarum IMAU10124. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  10. Teneligliptin Decreases Uric Acid Levels by Reducing Xanthine Dehydrogenase Expression in White Adipose Tissue of Male Wistar Rats.

    Science.gov (United States)

    Moriya, Chihiro; Satoh, Hiroaki

    2016-01-01

    We investigated the effects of teneligliptin on uric acid metabolism in male Wistar rats and 3T3-L1 adipocytes. The rats were fed with a normal chow diet (NCD) or a 60% high-fat diet (HFD) with or without teneligliptin for 4 weeks. The plasma uric acid level was not significantly different between the control and teneligliptin groups under the NCD condition. However, the plasma uric acid level was significantly decreased in the HFD-fed teneligliptin treated rats compared to the HFD-fed control rats. The expression levels of xanthine dehydrogenase (Xdh) mRNA in liver and epididymal adipose tissue of NCD-fed rats were not altered by teneligliptin treatment. On the other hand, Xdh expression was reduced significantly in the epididymal adipose tissue of the HFD-fed teneligliptin treated rats compared with that of HFD-fed control rats, whereas Xdh expression in liver did not change significantly in either group. Furthermore, teneligliptin significantly decreased Xdh expression in 3T3-L1 adipocytes. DPP-4 treatment significantly increased Xdh expression in 3T3-L1 adipocytes. With DPP-4 pretreatment, teneligliptin significantly decreased Xdh mRNA expression compared to the DPP-4-treated 3T3-L1 adipocytes. In conclusion, our studies suggest that teneligliptin reduces uric acid levels by suppressing Xdh expression in epididymal adipose tissue of obese subjects.

  11. 3D-QSAR Studies on a Series of Dihydroorotate Dehydrogenase Inhibitors: Analogues of the Active Metabolite of Leflunomide

    OpenAIRE

    Li, Shun-Lai; He, Mao-Yu; Du, Hong-Guang

    2011-01-01

    The active metabolite of the novel immunosuppressive agent leflunomide has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. Self-organizing molecular field analysis (SOMFA), a simple three-dimensional quantitative structure-activity relationship (3D-QSAR) method is used to study the correlation between the molecular properties and the biological activities of a series of analogues of the active met...

  12. Catalytic electrochemistry of xanthine dehydrogenase.

    Science.gov (United States)

    Kalimuthu, Palraj; Leimkühler, Silke; Bernhardt, Paul V

    2012-09-27

    We report the mediated electrocatalytic voltammetry of the molybdoenzyme xanthine dehydrogenase (XDH) from Rhodobacter capsulatus at a thiol-modified Au electrode. The 2-electron acceptor N-methylphenazinium methanesulfonate (phenazine methosulfate, PMS) is an effective artificial electron transfer partner for XDH instead of its native electron acceptor NAD(+). XDH catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and xanthine to uric acid. Cyclic voltammetry was used to generate the active (oxidized) form of the mediator. Simulation of the catalytic voltammetry across a broad range of substrate and PMS concentrations at different sweep rates was achieved with the program DigiSim to yield a set of consistent rate and equilibrium constants that describe the catalytic system. This provides the first example of the mediated electrochemistry of a xanthine dehydrogenase (or oxidase) that is uncomplicated by interference from product oxidation. A remarkable two-step, sequential oxidation of hypoxanthine to uric acid via xanthine by XDH is observed.

  13. A newborn lethal defect due to inactivation of retinaldehyde dehydrogenase type 3 is prevented by maternal retinoic acid treatment

    Science.gov (United States)

    Dupé, Valérie; Matt, Nicolas; Garnier, Jean-Marie; Chambon, Pierre; Mark, Manuel; Ghyselinck, Norbert B.

    2003-01-01

    The retinoic acid (RA) signal, produced locally from vitamin A by retinaldehyde dehydrogenase (Raldh) and transduced by the nuclear receptors for retinoids (RA receptor and 9-cis-RA receptor), is indispensable for ontogenesis and homeostasis of numerous tissues. We demonstrate that Raldh3 knockout in mouse suppresses RA synthesis and causes malformations restricted to ocular and nasal regions, which are similar to those observed in vitamin A-deficient fetuses and/or in retinoid receptor mutants. Raldh3 knockout notably causes choanal atresia (CA), which is responsible for respiratory distress and death of Raldh3-null mutants at birth. CA is due to persistence of nasal fins, whose rupture normally allows the communication between nasal and oral cavities. This malformation, which is similar to isolated congenital CA in humans and may result from impaired RA-controlled down-regulation of Fgf8 expression in nasal fins, can be prevented by a simple maternal treatment with RA. PMID:14623956

  14. A sulfurtransferase is essential for activity of formate dehydrogenases in Escherichia coli.

    Science.gov (United States)

    Thomé, Rémi; Gust, Alexander; Toci, René; Mendel, Ralf; Bittner, Florian; Magalon, Axel; Walburger, Anne

    2012-02-10

    l-Cysteine desulfurases provide sulfur to several metabolic pathways in the form of persulfides on specific cysteine residues of an acceptor protein for the eventual incorporation of sulfur into an end product. IscS is one of the three Escherichia coli l-cysteine desulfurases. It interacts with FdhD, a protein essential for the activity of formate dehydrogenases (FDHs), which are iron/molybdenum/selenium-containing enzymes. Here, we address the role played by this interaction in the activity of FDH-H (FdhF) in E. coli. The interaction of IscS with FdhD results in a sulfur transfer between IscS and FdhD in the form of persulfides. Substitution of the strictly conserved residue Cys-121 of FdhD impairs both sulfur transfer from IscS to FdhD and FdhF activity. Furthermore, inactive FdhF produced in the absence of FdhD contains both metal centers, albeit the molybdenum cofactor is at a reduced level. Finally, FdhF activity is sulfur-dependent, as it shows reversible sensitivity to cyanide treatment. Conclusively, FdhD is a sulfurtransferase between IscS and FdhF and is thereby essential to yield FDH activity.

  15. A Sulfurtransferase Is Essential for Activity of Formate Dehydrogenases in Escherichia coli*

    Science.gov (United States)

    Thomé, Rémi; Gust, Alexander; Toci, René; Mendel, Ralf; Bittner, Florian; Magalon, Axel; Walburger, Anne

    2012-01-01

    l-Cysteine desulfurases provide sulfur to several metabolic pathways in the form of persulfides on specific cysteine residues of an acceptor protein for the eventual incorporation of sulfur into an end product. IscS is one of the three Escherichia coli l-cysteine desulfurases. It interacts with FdhD, a protein essential for the activity of formate dehydrogenases (FDHs), which are iron/molybdenum/selenium-containing enzymes. Here, we address the role played by this interaction in the activity of FDH-H (FdhF) in E. coli. The interaction of IscS with FdhD results in a sulfur transfer between IscS and FdhD in the form of persulfides. Substitution of the strictly conserved residue Cys-121 of FdhD impairs both sulfur transfer from IscS to FdhD and FdhF activity. Furthermore, inactive FdhF produced in the absence of FdhD contains both metal centers, albeit the molybdenum cofactor is at a reduced level. Finally, FdhF activity is sulfur-dependent, as it shows reversible sensitivity to cyanide treatment. Conclusively, FdhD is a sulfurtransferase between IscS and FdhF and is thereby essential to yield FDH activity. PMID:22194618

  16. Alcohol dehydrogenase activity in Lactococcus chungangensis: application in cream cheese to moderate alcohol uptake.

    Science.gov (United States)

    Konkit, Maytiya; Choi, Woo Jin; Kim, Wonyong

    2015-09-01

    Many human gastrointestinal facultative anaerobic and aerobic bacteria possess alcohol dehydrogenase (ADH) activity and are therefore capable of oxidizing ethanol to acetaldehyde. However, the ADH activity of Lactococcus spp., except Lactococcus lactis ssp. lactis, has not been widely determined, though they play an important role as the starter for most cheesemaking technologies. Cheese is a functional food recognized as an aid to digestion. In the current study, the ADH activity of Lactococcus chungangensis CAU 28(T) and 11 reference strains from the genus Lactococcus was determined. Only 5 strains, 3 of dairy origin, L. lactis ssp. lactis KCTC 3769(T), L. lactis ssp. cremoris KCCM 40699(T), and Lactococcus raffinolactis DSM 20443(T), and 2 of nondairy origin, Lactococcus fujiensis NJ317(T) and Lactococcus chungangensis CAU 28(T) KCTC 13185(T), showed ADH activity and possessed the ADH gene. All these strains were capable of making cheese, but the highest level of ADH activity was found in L. chungangensis, with 45.9nmol/min per gram in tryptic soy broth and 65.8nmol/min per gram in cream cheese. The extent that consumption of cheese, following imbibing alcohol, reduced alcohol uptake was observed by following the level of alcohol in the serum of mice. The results show a potential novel benefit of cheese as a dairy functional food. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  17. Influence of spaceflight on succinate dehydrogenase activity and soma size of rat ventral horn neurons

    Science.gov (United States)

    Ishihara, A.; Ohira, Y.; Roy, R. R.; Nagaoka, S.; Sekiguchi, C.; Hinds, W. E.; Edgerton, V. R.

    1996-01-01

    Succinate dehydrogenase (SDH) activities and soma cross-sectional areas (CSA) of neurons in the dorsolateral region of the ventral horn at the L5 segmental level of the spinal cord in the rat were determined after 14 days of spaceflight and after 9 days of recovery on earth. The results were compared to those in age-matched ground-based control rats. Spinal cords were quick-frozen, and the SDH activity and CSA of a sample of neurons with a visible nucleus were determined using a digitizer and a computer-assisted image analysis system. An inverse relationship between CSA and SDH activity of neurons was observed in all groups of rats. No change in mean CSA or mean SDH activity or in the size distribution of neurons was observed following spaceflight or recovery. However, there was a selective decrease in the SDH activity of neurons with soma CSA between 500 and 800 microns2 in the flight rats, and this effect persisted for at least 9 days following return to 1 g. It remains to be determined whether the selected population of motoneurons or the specific motor pools affected by spaceflight may be restricted to specific muscles.

  18. Metabolic engineering of Escherichia coli cell factory for highly active xanthine dehydrogenase production.

    Science.gov (United States)

    Wang, Cheng-Hua; Zhang, Chong; Xing, Xin-Hui

    2017-12-01

    The aim of this work was to demonstrate the first proof-of-concept for the use of ab initio-aided assembly strategy intensifying in vivo biosynthesis process to construct Escherichia coli cell factory overproducing highly active xanthine dehydrogenase (XDH). Three global regulator (IscS, TusA and NarJ) and four chaperone proteins (DsbA, DsbB, NifS and XdhC) were overexpressed to aid the formation and ordered assembly of three redox center cofactors of Rhodobacter capsulatus XDH in E. coli. The NifS, IscS and DsbB enhanced the specific activity of RcXDH by 30%, 94% and 49%, respectively. The combinatorial expression of NarJ and IscS synergistically increased the specific activity by 129% and enhanced the total enzyme activity by a remarkable 3.9-fold. The crude enzyme showed nearly the same coupling efficiency of electron transfer and product formation as previously purified XDHs, indicating an integrity and efficient assembly of highly active XDH. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Functional assignment of Glu386 and Arg388 in the active site of l-galactono-¿-lactone dehydrogenase

    NARCIS (Netherlands)

    Leferink, N.G.H.; Jose, M.D.F.; Berg, van den W.A.M.; Berkel, van W.J.H.

    2009-01-01

    The flavoenzyme l-galactono-¿-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plants. Little is known about the catalytic mechanism of GALDH and related aldonolactone oxidoreductases. Here we identified an essential Glu–Arg pair in the active site of GALDH from

  20. The effect of fullerenol C60(OH)~30 on the alcohol dehydrogenase activity irradiated with X-rays

    Science.gov (United States)

    Krokosz, Anita; Grebowski, Jacek; Rodacka, Aleksandra; Pasternak, Beata; Puchala, Mieczyslaw

    2014-04-01

    In the present study the effect of X-irradiation on the alcohol dehydrogenase (ADH) activity in the presence of nanoparticles of fullerenol C60(OH)~30 under aerobic conditions was investigated in order to assess the potential radioprotective properties of fullerenol.

  1. Xanthine dehydrogenase-1 silencing inAedes aegyptimosquitoes promotes a blood feeding-induced adulticidal activity.

    Science.gov (United States)

    Isoe, Jun; Petchampai, Natthida; Isoe, Yurika E; Co, Katrina; Mazzalupo, Stacy; Scaraffia, Patricia Y

    2017-06-01

    Aedes aegypti has 2 genes encoding xanthine dehydrogenase (XDH). We analyzed XDH1 and XDH2 gene expression by real-time quantitative PCR in tissues from sugar- and blood-fed females. Differential XDH1 and XDH2 gene expression was observed in tissues dissected throughout a time course. We next exposed females to blood meals supplemented with allopurinol, a well-characterized XDH inhibitor. We also tested the effects of injecting double-stranded RNA (dsRNA) against XDH1, XDH2, or both. Disruption of XDH by allopurinol or XDH1 by RNA interference significantly affected mosquito survival, causing a disruption in blood digestion, excretion, oviposition, and reproduction. XDH1-deficient mosquitoes showed a persistence of serine proteases in the midgut at 48 h after blood feeding and a reduction in the uptake of vitellogenin by the ovaries. Surprisingly, analysis of the fat body from dsRNA-XDH1-injected mosquitoes fell into 2 groups: one group was characterized by a reduction of the XDH1 transcript, whereas the other group was characterized by an up-regulation of several transcripts, including XDH1, glutamine synthetase, alanine aminotransferase, catalase, superoxide dismutase, ornithine decarboxylase, glutamate receptor, and ammonia transporter. Our data demonstrate that XDH1 plays an essential role and that XDH1 has the potential to be used as a metabolic target for Ae. aegypti vector control.-Isoe, J., Petchampai, N., Isoe, Y. E., Co, K., Mazzalupo, S., Scaraffia, P. Y. Xanthine dehydrogenase-1 silencing in Aedes aegypti mosquitoes promotes a blood feeding-induced adulticidal activity. © FASEB.

  2. Higher thermostability of l-lactate dehydrogenases is a key factor in decreasing the optical purity of d-lactic acid produced from Lactobacillus coryniformis.

    Science.gov (United States)

    Gu, Sol-A; Jun, Chanha; Joo, Jeong Chan; Kim, Seil; Lee, Seung Hwan; Kim, Yong Hwan

    2014-05-10

    Lactobacillus coryniformis is known to produce d-lactic acid as a dominant fermentation product at a cultivation temperature of approximately 30°C. However, the considerable production of l-lactic acid is observed when the fermentation temperature is greater than 40°C. Because optically pure lactates are synthesized from pyruvate by the catalysis of chiral-specific d- or l-lactate dehydrogenase, the higher thermostability of l-LDHs is assumed to be one of the key factors decreasing the optical purity of d-lactic acid produced from L. coryniformis at high temperature. To verify this hypothesis, two types of d-ldh genes and six types of l-ldh genes based on the genomic information of L. coryniformis were synthesized and expressed in Escherichia coli. Among the LDHs tested, five LDHs showed activity and were used to construct polyclonal antibodies. d-LDH1, l-LDH2, and l-LDH3 were found to be expressed in L. coryniformis by Western blotting analysis. The half-life values (t1/2) of the LDHs at 40°C were estimated to be 10.50, 41.76, and 2311min, and the T50(10) values were 39.50, 39.90, and 58.60°C, respectively. In addition, the Tm values were 36.0, 41.0, and 62.4°C, respectively, which indicates that l-LDH has greater thermostability than d-LDH. The higher thermostability of l-LDHs compared with that of d-LDH1 may be a major reason why the enantiopurity of d-lactic acid is decreased at high fermentation temperatures. The key enzymes characterized will suggest a direction for the design of genetically modified lactic acid bacteria to produce optically pure d-lactic acid. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast.

    Science.gov (United States)

    Schifferdecker, Anna Judith; Siurkus, Juozas; Andersen, Mikael Rørdam; Joerck-Ramberg, Dorte; Ling, Zhihao; Zhou, Nerve; Blevins, James E; Sibirny, Andriy A; Piškur, Jure; Ishchuk, Olena P

    2016-04-01

    Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we developed an auxotrophic transformation system and an expression vector, which enabled the manipulation of D. bruxellensis, thereby improving its fermentative performance. Its gene ADH3, coding for alcohol dehydrogenase, was cloned and overexpressed under the control of the strong and constitutive promoter TEF1. Our recombinant D. bruxellensis strain displayed 1.4 and 1.7 times faster specific glucose consumption rate during aerobic and anaerobic glucose fermentations, respectively; it yielded 1.2 times and 1.5 times more ethanol than did the parental strain under aerobic and anaerobic conditions, respectively. The overexpression of ADH3 in D. bruxellensis also reduced the inhibition of fermentation by anaerobiosis, the "Custer effect". Thus, the fermentative capacity of D. bruxellensis could be further improved by metabolic engineering.

  4. Novel inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase with anti-malarial activity in the mouse model.

    Science.gov (United States)

    Booker, Michael L; Bastos, Cecilia M; Kramer, Martin L; Barker, Robert H; Skerlj, Renato; Sidhu, Amar Bir; Deng, Xiaoyi; Celatka, Cassandra; Cortese, Joseph F; Guerrero Bravo, Jose E; Crespo Llado, Keila N; Serrano, Adelfa E; Angulo-Barturen, Iñigo; Jiménez-Díaz, María Belén; Viera, Sara; Garuti, Helen; Wittlin, Sergio; Papastogiannidis, Petros; Lin, Jing-Wen; Janse, Chris J; Khan, Shahid M; Duraisingh, Manoj; Coleman, Bradley; Goldsmith, Elizabeth J; Phillips, Margaret A; Munoz, Benito; Wirth, Dyann F; Klinger, Jeffrey D; Wiegand, Roger; Sybertz, Edmund

    2010-10-22

    Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED(50) values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

  5. Novel Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Anti-malarial Activity in the Mouse Model

    Energy Technology Data Exchange (ETDEWEB)

    Booker, Michael L.; Bastos, Cecilia M.; Kramer, Martin L.; Barker, Jr., Robert H.; Skerlj, Renato; Sidhu, Amar Bir; Deng, Xiaoyi; Celatka, Cassandra; Cortese, Joseph F.; Guerrero Bravo, Jose E.; Crespo Llado, Keila N.; Serrano, Adelfa E.; Angulo-Barturen, Iñigo; Jiménez-Díaz, María Belén; Viera, Sara; Garuti, Helen; Wittlin, Sergio; Papastogiannidis, Petros; Lin, Jing-wen; Janse, Chris J.; Khan, Shahid M.; Duraisingh, Manoj; Coleman, Bradley; Goldsmith, Elizabeth J.; Phillips, Margaret A.; Munoz, Benito; Wirth, Dyann F.; Klinger, Jeffrey D.; Wiegand, Roger; Sybertz, Edmund (Leiden-MC); (Puerto Rico); (STPHI); (Harvard); (GSK); (Genzyme); (UTSMC)

    2010-11-22

    Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED{sub 50} values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

  6. Green Tea Polyphenols Control Dysregulated Glutamate Dehydrogenase in Transgenic Mice by Hijacking the ADP Activation Site

    Energy Technology Data Exchange (ETDEWEB)

    Li, Changhong; Li, Ming; Chen, Pan; Narayan, Srinivas; Matschinsky, Franz M.; Bennett, Michael J.; Stanley, Charles A.; Smith, Thomas J. (CH-PA); (UPENN); (Danforth)

    2012-05-09

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic {beta}-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

  7. Enzyme-dependent fluorescence recovery of NADH after photobleaching to assess dehydrogenase activity of isolated perfused hearts

    Science.gov (United States)

    Moreno, Angel; Kuzmiak-Glancy, Sarah; Jaimes, Rafael; Kay, Matthew W.

    2017-03-01

    Reduction of NAD+ by dehydrogenase enzymes to form NADH is a key component of cellular metabolism. In cellular preparations and isolated mitochondria suspensions, enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH has been shown to be an effective approach for measuring the rate of NADH production to assess dehydrogenase enzyme activity. Our objective was to demonstrate how dehydrogenase activity could be assessed within the myocardium of perfused hearts using NADH ED-FRAP. This was accomplished using a combination of high intensity UV pulses to photobleach epicardial NADH. Replenishment of epicardial NADH fluorescence was then imaged using low intensity UV illumination. NADH ED-FRAP parameters were optimized to deliver 23.8 mJ of photobleaching light energy at a pulse width of 6 msec and a duty cycle of 50%. These parameters provided repeatable measurements of NADH production rate during multiple metabolic perturbations, including changes in perfusate temperature, electromechanical uncoupling, and acute ischemia/reperfusion injury. NADH production rate was significantly higher in every perturbation where the energy demand was either higher or uncompromised. We also found that NADH production rate remained significantly impaired after 10 min of reperfusion after global ischemia. Overall, our results indicate that myocardial NADH ED-FRAP is a useful optical non-destructive approach for assessing dehydrogenase activity.

  8. Glucose-6-Phosphate Dehydrogenase Activity and Protein Oxidative Modification in Patients with Type 2 Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Aida A. Mahmoud

    2013-01-01

    Full Text Available Objectives. The aim of the present investigation was to study the activity of glucose-6-phosphate dehydrogenase (G6PD and correlate its activity to protein oxidation markers in type 2 diabetic patients under poor glycemic control. Methods. G6PD activity, protein carbonyl group concentration, and total thiol group content were measured in blood samples of 40 patients with type 2 diabetes mellitus under poor glycemic control and 20 healthy control subjects. Results. G6PD activity and total thiol group content decreased significantly while glycated hemoglobin (HbA1C and protein carbonyl group concentration increased significantly in diabetic patients than in the controls (P<0.001. In addition, Obtained results revealed that, in diabetics, G6PD activity negatively correlated to protein carbonyl and HbA1C (r=−0.77 and −0.65, resp., while positively correlated to total thiol (r=0.66 and protein carbonyl negatively correlated to total thiol (r=−0.85, while positively correlated to HbA1C (r=0.43. Also in controls, G6PD activity negatively correlated to protein carbonyl and HbA1C (r=−0.57 and −0.56, resp., while positively correlated to total thiol (r=0.5 and protein carbonyl negatively correlated to total thiol (r=−0.48, while positively correlated to HbA1C (r=0.68. Conclusions. We concluded that G6PD activity decreased in diabetics than in controls and was negatively correlated to oxidative stress markers and HbA1C. G6PD activity can be taken as a biomarker of oxidative stress and poor glycemic control in type 2 diabetic patients.

  9. Acrylonitrile irreversibly inactivates glyceraldehyde-3-phosphate dehydrogenase by alkylating the catalytically active cysteine 149.

    Science.gov (United States)

    Campian, E Cristian; Cai, Jian; Benz, Frederick W

    2002-08-15

    Acrylonitrile (AN) is a vinyl monomer used in the production of synthetic fibers, rubber and plastics. AN is acutely toxic but its mechanism of toxicity remains to be established. AN is metabolized to cyanide in vivo but cyanide production alone cannot explain acute AN toxicity. Previous work in our laboratory has shown that AN can alkylate highly reactive cysteine residues in proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a critical enzyme involved in glycolysis, has a catalytically active cysteine 149 in its active site. We report that AN irreversibly inhibits GAPDH with second-order rate constants, at pH 7.4, of 3.7 and 9.2 M(-1) s(-1) at 25 and 37 degrees C, respectively. A combination of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and electrospray ionization-mass spectrometry-mass spectrometry (ESI-MS-MS) was used to show that AN inactivates GAPDH by covalently binding to cysteine 149 in the active site of the enzyme. Inactivation of GAPDH by AN would be expected to impair glycolytic ATP production and when coupled with the inhibition of mitochondrial ATP synthesis by the AN metabolite cyanide would result in metabolic arrest. The brain can withstand metabolic arrest for only a few minutes thus these combined actions may account for the acute toxicity of AN in vivo.

  10. 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.

  11. Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies.

    Science.gov (United States)

    Penna-Coutinho, Julia; Cortopassi, Wilian Augusto; Oliveira, Aline Alves; França, Tanos Celmar Costa; Krettli, Antoniana Ursine

    2011-01-01

    The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH) has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH) all exhibit ∼90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH) and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole) were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2). The IC(50) values for each drug in both tests were similar, were lowest for posaconazole (<5 µM) and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use.

  12. Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies.

    Directory of Open Access Journals (Sweden)

    Julia Penna-Coutinho

    Full Text Available The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH all exhibit ∼90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2. The IC(50 values for each drug in both tests were similar, were lowest for posaconazole (<5 µM and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use.

  13. Interaction between chronic arsenic exposure via drinking water and plasma lactate dehydrogenase activity.

    Science.gov (United States)

    Karim, Md Rezaul; Salam, Kazi Abdus; Hossain, Ekhtear; Islam, Khairul; Ali, Nurshad; Haque, Abedul; Saud, Zahangir Alam; Yeasmin, Tanzima; Hossain, Mostaque; Miyataka, Hideki; Himeno, Seiichiro; Hossain, Khaled

    2010-12-15

    Arsenic is a potent environmental pollutant that has caused one of the largest public health poisonings in the history of human civilization, affecting tens of millions of people worldwide especially in Bangladesh. Lactate dehydrogenase (LDH) in blood plays an important role in predicting cell or organ damage and as an important clue to the diagnosis of a variety of cancers. However, effect of chronic arsenic exposure on the LDH level in blood has not yet been documented. Since the chronic arsenic exposure is associated with organ damages and multi-site cancers, this research aimed at assaying the plasma level of LDH activity in the population who were exposed to arsenic chronically in Bangladesh. A total of 185 individuals living in arsenic-exposed areas and 121 individuals living in non-exposed area in Bangladesh were recruited as study subjects. Arsenic content in drinking water, hair and nails were estimated by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) and LDH activity was assayed by a spectrophotometer. Significant increase in LDH activity was observed with increasing concentrations of arsenic in water, hair and nails. Further, the study subjects were split into four groups based on the three ways of each exposure metrics (water, hair and nail arsenic concentrations) where the study subjects in the non-exposed area were used as a reference (lowest exposure) group. LDH activity was found to be increased in the higher exposure groups of water and hair arsenic concentrations. LDH activity was also increased at low to medium exposure groups of nail arsenic concentrations.Thus, the elevated plasma LDH activity might be helpful for the early prognosis of organ or tissue damage in the individuals who were exposed to arsenic chronically. Copyright © 2010 Elsevier B.V. All rights reserved.

  14. Antitrypanosomal compounds from the essential oil and extracts of Keetia leucantha leaves with inhibitor activity on Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase.

    Science.gov (United States)

    Bero, J; Beaufay, C; Hannaert, V; Hérent, M-F; Michels, P A; Quetin-Leclercq, J

    2013-02-15

    Keetia leucantha is a West African tree used in traditional medicine to treat several diseases among which parasitic infections. The dichloromethane extract of leaves was previously shown to possess growth-inhibitory activities on Plasmodium falciparum, Trypanosoma brucei brucei and Leishmania mexicana mexicana with low or no cytotoxicity (>100 μg/ml on human normal fibroblasts) (Bero et al. 2009, 2011). In continuation of our investigations on the antitrypanosomal compounds from this dichloromethane extract, we analyzed by GC-FID and GC-MS the essential oil of its leaves obtained by hydrodistillation and the major triterpenic acids in this extract by LC-MS. Twenty-seven compounds were identified in the oil whose percentages were calculated using the normalization method. The essential oil, seven of its constituents and the three triterpenic acids were evaluated for their antitrypanosomal activity on Trypanosoma brucei brucei bloodstream forms (Tbb BSF) and procyclic forms (Tbb PF) to identify an activity on the glycolytic process of trypanosomes. The oil showed an IC(50) of 20.9 μg/ml on Tbb BSF and no activity was observed on Tbb PF. The best antitrypanosomal activity was observed for ursolic acid with IC(50) of 2.5 and 6.5 μg/ml respectively on Tbb BSF and Tbb PF. The inhibitory activity on a glycolytic enzyme of T. brucei, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was also evaluated for betulinic acid, olenaolic acid, ursolic acid, phytol, α-ionone and β-ionone. The three triterpenic acids and β-ionone showed inhibitory activities on GAPDH with oleanolic acid being the most active with an inhibition of 72.63% at 20 μg/ml. This paper reports for the first time the composition and antitrypanosomal activity of the essential oil of Keetia leucantha. Several of its constituents and three triterpenic acids present in the dichloromethane leaves extract showed a higher antitrypanosomal activity on bloodstream forms of Tbb as compared to procyclic forms

  15. Evaluation on the inhibition of pyrrol-2-yl ethanone derivatives to lactate dehydrogenase and anticancer activities

    Science.gov (United States)

    Lu, Na-Na; Weng, Zhao-Yue; Chen, Qiu-Yun; Boison, Daniel; Xiao, Xin-Xin; Gao, Jing

    2016-08-01

    Lactate dehydrogenase A (LDH-A) is a potentially important metabolic target for the inhibition of the highly activated glycolysis pathway in cancer cells. In order to develop bifunctional compounds as inhibitor of LDH-A and anticancer agents, two pyrrol-2-yl methanone (or ethanone) derivatives (PM1 and PM2) were synthesized and evaluated as inhibitors of LDH-A based on the enzyme assay and cell assay by spectroscopy analysis. Fluorescence and CD spectra results demonstrated that both the change of second structure of LDH-A and the affinity interaction for compounds to LDH-A gave great effect on the activity of LDH-A. In particular, low concentration of compounds (1 μμ-25 μμ) could change the level of pyruvate in cancer cells. Moreover, the in vitro assay results demonstrated that pyrrol-2-yl ethanone derivatives can inhibit the proliferation of cancer cells. Therefore, pyrrol-2-yl ethanone derivatives (PM2) can be both LDH-A inhibitor and anticancer agents.

  16. Acyl carrier protein-specific 4'-phosphopantetheinyl transferase activates 10-formyltetrahydrofolate dehydrogenase.

    Science.gov (United States)

    Strickland, Kyle C; Hoeferlin, L Alexis; Oleinik, Natalia V; Krupenko, Natalia I; Krupenko, Sergey A

    2010-01-15

    4'-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4'-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G(1) arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions.

  17. Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis.

    Science.gov (United States)

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V

    2015-02-05

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)(+) dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP(+) yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis.

  18. Kinetic and structural investigation of the cytokinin oxidase/dehydrogenase active site.

    Science.gov (United States)

    Kopečný, David; Končitíková, Radka; Popelka, Hana; Briozzo, Pierre; Vigouroux, Armelle; Kopečná, Martina; Zalabák, David; Šebela, Marek; Skopalová, Jana; Frébort, Ivo; Moréra, Solange

    2016-01-01

    Cytokinins are hormones that regulate plant development and their environmental responses. Their levels are mainly controlled by the cytokinin oxidase/dehydrogenase (CKO), which oxidatively cleaves cytokinins using redox-active electron acceptors. CKO belongs to the group of flavoproteins with an 8α-N1-histidyl FAD covalent linkage. Here, we investigated the role of seven active site residues, H105, D169, E288, V378, E381, P427 and L492, in substrate binding and catalysis of the CKO1 from maize (Zea mays, ZmCKO1) combining site-directed mutagenesis with kinetics and X-ray crystallography. We identify E381 as a key residue for enzyme specificity that restricts substrate binding as well as quinone electron acceptor binding. We show that D169 is important for catalysis and that H105 covalently linked to FAD maintains the enzyme's structural integrity, stability and high rates with electron acceptors. The L492A mutation significantly modulates the cleavage of aromatic cytokinins and zeatin isomers. The high resolution X-ray structures of ZmCKO1 and the E381S variant in complex with N6-(2-isopentenyl)adenosine reveal the binding mode of cytokinin ribosides. Those of ZmCKO2 and ZmCKO4a contain a mobile domain, which might contribute to binding of the N9 substituted cytokinins. © 2015 FEBS.

  19. Stability and activity of lactate dehydrogenase on biofunctional layers deposited by activated vapor silanization (AVS) and immersion silanization (IS)

    Science.gov (United States)

    Calvo, Jorge Nieto-Márquez; Elices, Manuel; Guinea, Gustavo V.; Pérez-Rigueiro, José; Arroyo-Hernández, María

    2017-09-01

    The interaction between surfaces and biological elements, in particular, proteins is critical for the performance of biomaterials and biosensors. This interaction can be controlled by modifying the surface in a process known as biofunctionalization. In this work, the enzyme lactate dehydrogenase (LDH) is used to study the stability of the interaction between a functional protein and amine-functionalized surfaces. Two different functionalization procedures were compared: Activated Vapor Silanization (AVS) and Immersion Silanization (IS). Adsorption kinetics is shown to follow the Langmuir model for AVS-functionalized samples, while IS-functionalized samples show a certain instability if immersed in an aqueous medium for several hours. In turn, the enzymatic activity of LDH is preserved for longer times by using glutaraldehyde as crosslinker between the AVS biofunctional surface and the enzyme.

  20. Regulation of xanthine dehydrogenase and xanthine oxidase activity and gene expression in cultured rat pulmonary endothelial cells.

    OpenAIRE

    Dupont, G P; Huecksteadt, T P; Marshall, B C; Ryan, U S; Michael, J R; Hoidal, J R

    1992-01-01

    The central importance of xanthine dehydrogenase (XDH) and xanthine oxidase (XO) in the pathobiochemistry of a number of clinical disorders underscores the need for a comprehensive understanding of the regulation of their expression. This study was undertaken to examine the effects of cytokines on XDH/XO activity and gene expression in pulmonary endothelial cells. The results indicate that IFN-gamma is a potent inducer of XDH/XO activity in rat lung endothelial cells derived from both the mic...

  1. Nicotine promotes Streptococcus mutans extracellular polysaccharide synthesis, cell aggregation and overall lactate dehydrogenase activity.

    Science.gov (United States)

    Huang, R; Li, M; Gregory, R L

    2015-08-01

    Several epidemiology studies have reported a positive relationship between smoking and dental caries. Nicotine, an alkaloid component of tobacco, has been demonstrated to stimulate biofilm formation and metabolic activity of Streptococcus mutans, one of the most important pathogens of dental caries. The first aim of the present study was to explore the possible mechanisms leading to increased biofilm by nicotine treatment from three aspects, extracellular polysaccharides (EPS) synthesis, glucosyltransferase (Gtf) synthesis and glucan-binding protein (Gbp) synthesis at the mRNA and protein levels. The second aim was to investigate how nicotine affects S. mutans virulence, particular in lactate dehydrogenase (LDH) activity. Confocal laser scanning microscopy results demonstrated that both biofilm bacterial cell numbers and EPS were increased by nicotine. Gtf and GbpA protein expression of S. mutans planktonic cells were upregulated while GbpB protein expression of biofilm cells were downregulated by nicotine. The mRNA expression trends of those genes were mostly consistent with results on protein level but not statistically significant, and gtfD and gbpD of biofilm cells were inhibited. Nicotine was not directly involved in S. mutans LDH activity. However, since it increases the total number of bacterial cells in biofilm, the overall LDH activity of S. mutans biofilm is increased. In conclusion, nicotine stimulates S. mutans planktonic cell Gtf and Gbp expression. This leads to more planktonic cells attaching to the dental biofilm. Increased cell numbers within biofilm results in higher overall LDH activity. This contributes to caries development in smokers. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

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    Bo Fu

    2015-12-01

    Full Text Available The in vitro maturation (IVM efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+ oocytes with low glucose-6-phosphate dehydrogenase (G6PDH activity have shown superior quality than BCB negative (− oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9 and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

  3. Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development.

    Science.gov (United States)

    Niederreither, K; McCaffery, P; Dräger, U C; Chambon, P; Dollé, P

    1997-02-01

    Retinaldehyde dehydrogenase type 2 (RALDH-2) was identified as a major retinoic acid generating enzyme in the early embryo. Here we report the expression domains of the RALDH-2 gene during mouse embryogenesis, which are likely to indicate regions of endogenous retinoic acid (RA) synthesis. During early gastrulation, RALDH-2 is expressed in the mesoderm adjacent to the node and primitive streak. At the headfold stage, mesodermal expression is restricted to posterior regions up to the base of the headfolds. Later, RALDH-2 is transiently expressed in the undifferentiated somites and the optic vesicles, and more persistently along the lateral walls of the intraembryonic coelom and around the hindgut diverticulum. The RALDH-2 expression domains in differentiating limbs, which include presumptive interdigital regions, coincide with, but slightly precede, those of the RA-inducible RAR beta gene. The RALDH-2 gene is also expressed in specific regions of the developing head, including the tooth buds, inner ear, meninges and pituitary gland, and in several viscera. Administration of a teratogenic dose of RA at embryonic day 8.5 results in downregulation of RALDH-2 transcript levels in caudal regions of the embryo, and may reflect a mechanism of negative feedback regulation of RA synthesis.

  4. The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions.

    Science.gov (United States)

    Hartmann, Tobias; Schrapers, Peer; Utesch, Tillmann; Nimtz, Manfred; Rippers, Yvonne; Dau, Holger; Mroginski, Maria Andrea; Haumann, Michael; Leimkühler, Silke

    2016-04-26

    Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase.

  5. Hypoxia and anoxia effects on alcohol dehydrogenase activity and hemoglobin content in Chironomus riparius Meigen, 1804

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    Valentina Grazioli

    2016-02-01

    Full Text Available The metabolic effects of low oxygen content on alcohol-dehydrogenase (ADH activity and hemoglobin (Hb concentration were investigated in IV-instar larvae of Chironomus riparius (Diptera: Chironomidae from an Italian stream. Two series of short-term (48 h experiments were carried out: exposure to (1 progressive hypoxia (95 to 5% of oxygen saturation and (2 anoxia (at <5% of oxygen saturation. In (1, Hb amount increased with increasing oxygen depletion up to a critical value of oxygenation (about 70% of oxygen saturation. Below this percentage, the Hb amount declined to values comparable with those present in the control. The respiration rate (R remained almost constant at oxygen saturation >50% and decreased significantly only after 48 h of treatment (= <5% of oxygen saturation reaching values <100 mmolO2 gAFDW-1 h-1. ADH activity showed two phases of growth, within the first 14 h and over 18 h of exposure. Overall, we inferred that i Hb might function as short-term oxygen storage, enabling animals to delay the on-set of anaerobiosis; and ii alcoholic fermentation co-occurs for a short time with aerobic respiration, becoming the prevalent metabolic pathway below 5% of oxygen saturation (<1 mg L-1. These considerations were supported also by results from anoxia exposure (2. In such condition, larvae were visibly stressed, becoming immobile after few minutes of incubation, and ADH reached higher values than in the hypoxia treatment (2.03±0.15 UADH mg prot-1. Overall, this study showed a shift from aerobic to anaerobic activity in C. riparius larvae exposed to poorly oxygenated water with an associated alteration of ADH activity and the Hb amount. Such metabolites might be valid candidate biomarkers for the environmental monitoring of running waters.

  6. Rutin and meloxicam attenuate paw inflammation in mice: Affecting sorbitol dehydrogenase activity.

    Science.gov (United States)

    Mohammad F, Ebtehal; Hasan, Wedad A; Mohamed, Eman G

    2018-01-05

    Rutin, naturally occurring flavonoid, has reported to cover interesting multiple pharmacological properties. This study evaluated rutin or/and meloxicam effects in paw inflammation induced by formalin in mice. Mice were divided into four groups: I-Formalin group, II-Rutin 60 mg/kg (p.o.), III-Meloxicam 10 mg/kg (p.o.), plus IV-Combined rutin and meloxicam. Therapies were administered once a day for 7 days. The curative effects were assessed on inflammatory, oxidative stress, and apoptosis. Both rutin and/or meloxicam induced marked improvement in paw licking time on the 1st day and by combined treatment only on the 3rd day as well reduction in paw edema% on the 3rd day. Moreover, noticeable progress in liver malondialdehyde content, superoxide dismutase, and sorbitol dehydrogenase activities as well decline in paw interleukin-1β level and extent of apoptosis. The results spot light on the good influence of combined rutin and meloxicam in formalin-induced mice paw inflammation to a better extent than either rutin or meloxicam lonely. © 2018 Wiley Periodicals, Inc.

  7. Estrogen-related receptor alpha modulates lactate dehydrogenase activity in thyroid tumors.

    Science.gov (United States)

    Mirebeau-Prunier, Delphine; Le Pennec, Soazig; Jacques, Caroline; Fontaine, Jean-Fred; Gueguen, Naig; Boutet-Bouzamondo, Nathalie; Donnart, Audrey; Malthièry, Yves; Savagner, Frédérique

    2013-01-01

    Metabolic modifications of tumor cells are hallmarks of cancer. They exhibit an altered metabolism that allows them to sustain higher proliferation rates in hostile environment outside the cell. In thyroid tumors, the expression of the estrogen-related receptor α (ERRα), a major factor of metabolic adaptation, is closely related to the oxidative metabolism and the proliferative status of the cells. To elucidate the role played by ERRα in the glycolytic adaptation of tumor cells, we focused on the regulation of lactate dehydrogenases A and B (LDHA, LDHB) and the LDHA/LDHB ratio. Our study included tissue samples from 10 classical and 10 oncocytic variants of follicular thyroid tumors and 10 normal thyroid tissues, as well as samples from three human thyroid tumor cell lines: FTC-133, XTC.UC1 and RO82W-1. We identified multiple cis-acting promoter elements for ERRα, in both the LDHA and LDHB genes. The interaction between ERRα and LDH promoters was confirmed by chromatin immunoprecipitation assays and in vitro analysis for LDHB. Using knock-in and knock-out cellular models, we found an inverse correlation between ERRα expression and LDH activity. This suggests that thyroid tumor cells may reprogram their metabolic pathways through the up-regulation of ERRα by a process distinct from that proposed by the recently revisited Warburg hypothesis.

  8. A new bianthron glycoside as inhibitor of Trypanosoma cruzi glyceraldehyde 3-phosphate dehydrogenase activity

    Energy Technology Data Exchange (ETDEWEB)

    Macedo, Edangelo M.S. de; Silva, Maria G.V. [Universidade Federal do Ceara (UFC), Fortaleza, CE (Brazil). Dept. de Quimica Analitica e Fisico-Quimica; Wiggers, Helton J.; Montanari, Carlos A. [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Quimica; Braz-Filho, Raimundo [Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, (Brazil). Setor de Quimica de Produtos Naturais; Andricopulo, Adriano D. [Universidade de Sao Paulo (USP), Sao Carlos SP (Brazil). Inst. de Fisica

    2009-07-01

    A phytochemical investigation of the ethanolic extract of stalks of Senna martiana Benth. (Leguminoseae), native specie of northeast Brazil, resulted in the isolation and spectroscopic characterization of a new bianthrone glycoside, martianine 1 (10,10'-il-chrysophanol-10-oxi- 10,10'-bi-glucosyl). Its identification was established by HRMS, IR and 2D NMR experiments. The evaluation of martianine trypanocidal activity was carried out against gliceraldehyde 3-phosphate dehydrogenase enzyme from Trypanosoma cruzi. Its inhibitory constant (K{sub i}) is in the low micromolar concentration and it was determined by isothermal titration calorimetry to be 27.3 +-2.47 {mu}mol L{sup -1}. The non-competitive mechanism is asserted to be putative of the mode of action martianine displays against T. cruzi GAPDH. Results show that martianine has a great potential to become new lead molecule by inhibiting this key enzyme and for the development of new drugs against Chagas disease. (author)

  9. Alterations in Ascorbic Acid Levels by Transformation of the L-galactono-1,4-lactone Dehydrogenase Gene in Solanum tuberosum

    Directory of Open Access Journals (Sweden)

    Dong Yumei

    2015-09-01

    Full Text Available L-galactono-1, 4-lactone dehydrogenase (GLDH is an important enzyme that catalyzes the last step of the ascorbate biosynthetic pathways in plants. A full-length cDNA clone encoding GLDH was isolated from potato (Solanum tuberosum L. ‘Favorita’ leaf and subcloned into a binary vector, pBI121, to construct sense and antisense recombinant plant expression vectors. The recombinants were introduced into potato via Agrobacterium-mediated transformation, and plants were confirmed as transgenic using PCR and quantitative real-time PCR. Two anti-sense potato lines (G1 and G2 and three sense lines (G3, G4 and G5 were obtained. The GLDH activity of G4 and G5 were increased in vivo. Moreover, the ascorbic acid (AsA and dehydroascorbate (DHA contents were up-regulated in both leaves and tubers. However, the shoots of G1 were suppressed and its leaves were deformed. Additionally, the AsA contents in G1 leaves and tubers decreased by 28.8% and 10.3%, respectively. The GLDH activity in leaves treated with L-galactono-1,4-lactone (L-GalL increased in all lines, as did the AsA and DHA contents. These results indicate that GLDH activity plays an important role in regulating the AsA level as well as the growth and development of potato plants.

  10. Glutamate dehydrogenase (RocG) in Bacillus licheniformis WX-02: Enzymatic properties and specific functions in glutamic acid synthesis for poly-γ-glutamic acid production.

    Science.gov (United States)

    Tian, Guangming; Wang, Qin; Wei, Xuetuan; Ma, Xin; Chen, Shouwen

    2017-04-01

    Poly-γ-glutamic acid (γ-PGA), a natural biopolymer, is widely used in cosmetics, medicine, food, water treatment, and agriculture owing to its features of moisture sequestration, cation chelation, non-toxicity and biodegradability. Intracellular glutamic acid, the substrate of γ-PGA, is a limiting factor for high yield in γ-PGA production. Bacillus subtilis and Bacillus licheniformis are both important γ-PGA producing strains, and B. subtilis synthesizes glutamic acid in vivo using the unique GOGAT/GS pathway. However, little is known about the glutamate synthesis pathway in B. licheniformis. The aim of this work was to characterize the glutamate dehydrogenase (RocG) in glutamic acid synthesis from B. licheniformis with both in vivo and in vitro experiments. By re-directing the carbon flux distribution, the rocG gene deletion mutant WX-02ΔrocG produced intracellular glutamic acid with a concentration of 90ng/log(CFU), which was only 23.7% that of the wild-type WX-02 (380ng/log(CFU)). Furthermore, the γ-PGA yield of mutant WX-02ΔrocG was 5.37g/L, a decrease of 45.3% compared to the wild type (9.82g/L). In vitro enzymatic assays of RocG showed that RocG has higher affinity for 2-oxoglutarate than glutamate, and the glutamate synthesis rate was far above degradation. This is probably the first study to reveal the glutamic acid synthesis pathway and the specific functions of RocG in B. licheniformis. The results indicate that γ-PGA production can be enhanced through improving intracellular glutamic acid synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Effect of soil contamination with azadirachtin on dehydrogenase and catalase activity of soil

    Directory of Open Access Journals (Sweden)

    Rıdvan Kızılkaya

    2012-07-01

    Full Text Available nsecticides are used in modern agriculture in large quantities to control pests and increase crop yield. Their use, however, has resulted in the disruption of ecosystems because of the effects on non-target soil microorganisms, some environmental problems, and decreasing soil fertility. These negative effects of synthetic pesticides on the environment have led to the search for alternative means of pest control. One such alternative is use of natural plant products such as azadirachtin that have pesticidal activity. The aim of this experiment was to study the effect of soil contamination by azadirachtin (C35H44O16 on dehydrogenase (DHA and catalase activity (CA of soil under field conditions in Perm, Russia. The tests were conducted on loamy soil (pHH2O 6.7, ECH2O 0.213 dSm-1, organic carbon 0.99%, to which the following quantities of azadirachtin were added: 0, 15, 30 and 60 mL da-1 of soil. Experimental design was randomized plot design with three replications. The DHA and CA analyses were performed 7, 14 and 21 days after the field experiment was established. The results of field experiment showed that azadirachtin had a positive influence on the DHA and CA at different soil sampling times. The increased doses of azadirachtin applied resulted in the higher level of DHA and CA in soil. The soil DHA and CA showed the highest activity on the 21th day after 60 mL azadirachtin da-1 application doses.

  12. Reengineering of the human pyruvate dehydrogenase complex: from disintegration to highly active agglomerates.

    Science.gov (United States)

    Guo, Jin; Hezaveh, Samira; Tatur, Jana; Zeng, An-Ping; Jandt, Uwe

    2017-02-20

    The pyruvate dehydrogenase complex (PDC) plays a central role in cellular metabolism and regulation. As a metabolite-channeling multi-enzyme complex it acts as a complete nanomachine due to its unique geometry and by coupling a cascade of catalytic reactions using 'swinging arms'. Mammalian and specifically human PDC (hPDC) is assembled from multiple copies of E1 and E3 bound to a large E2/E3BP 60-meric core. A less restrictive and smaller catalytic core, which is still active, is highly desired for both fundamental research on channeling mechanisms and also to create a basis for further modification and engineering of new enzyme cascades. Here, we present the first experimental results of the successful disintegration of the E2/E3BP core while retaining its activity. This was achieved by C-terminal α-helixes double truncations (eight residues from E2 and seven residues from E3BP). Disintegration of the hPDC core via double truncations led to the formation of highly active (approximately 70% of wildtype) apparently unordered clusters or agglomerates and inactive non-agglomerated species (hexamer/trimer). After additional deletion of N-terminal 'swinging arms', the aforementioned C-terminal truncations also caused the formation of agglomerates of minimized E2/E3BP complexes. It is likely that these 'swinging arm' regions are not solely responsible for the formation of the large agglomerates. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  13. Kinetic analysis of adrenal 3 beta-hydroxysteroid dehydrogenase activity during human development.

    Science.gov (United States)

    Byrne, G C; Perry, Y S; Winter, J S

    1985-05-01

    Kinetic analyses of microsomal 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in adrenal glands from 11 individuals, aged 1-60 yr, were carried out to determine whether changes in substrate or cofactor affinity (Km) or cellular content, as reflected in maximal velocity, could explain the changes in adrenal delta 5-3 beta-hydroxysteroid secretion that occur in late childhood and puberty. The Km values for the cofactor NAD+ were similar regardless which substrate, dehydroepiandrosterone (DHA), pregnenolone, or 17-hydroxypregnenolone (17OH-delta 5P), was used. The Km values for DHA (0.3 microM), pregnenolone (0.4 microM), and 17OH-delta 5P (0.3 microM) were similar and within the intraadrenal concentration ranges for DHA and 17OH-delta 5P previously reported. Each substrate was a competitive inhibitor for the others, with close similarity between affinity and inhibition constants. These observations point to the presence of a single 3 beta-HSD, rather than several substrate-specific variants. There was no change in substrate Km with age; the maximal velocity was lower (0.1-0.6 nmol/mg X min) in a single 1-yr-old infant than in later life, but there was no significant change (mean, 2.9-4.6 nmol/mg X min for the three substrates) between values at 12 and 60 yr. This suggests that ACTH-mediated induction of 3 beta-HSD may be low in infancy and higher in adults, while in vivo studies point to a reduction in actual 3 beta-HSD activity during this period. The likely explanation for this paradox between enzyme levels and final activity is that 3 beta-HSD is progressively inhibited during late childhood and puberty by rising intraadrenal concentrations of various delta 4-3-ketosteroids.

  14. Enhanced pyruvate dehydrogenase activity improves cardiac outcomes in a murine model of cardiac arrest.

    Directory of Open Access Journals (Sweden)

    Lin Piao

    Full Text Available Post-ischemic changes in cellular metabolism alter myocardial and neurological function. Pyruvate dehydrogenase (PDH, the limiting step in mitochondrial glucose oxidation, is inhibited by increased expression of PDH kinase (PDK during ischemia/reperfusion injury. This results in decreased utilization of glucose to generate cellular ATP. Post-cardiac arrest (CA hypothermia improves outcomes and alters metabolism, but its influence on PDH and PDK activity following CA are unknown. We hypothesized that therapeutic hypothermia (TH following CA is associated with the inhibition of PDK activity and increased PDH activity. We further hypothesized that an inhibitor of PDK activity, dichloroacetate (DCA, would improve PDH activity and post-CA outcomes.Anesthetized and ventilated adult female C57BL/6 wild-type mice underwent a 12-minute KCl-induced CA followed by cardiopulmonary resuscitation. Compared to normothermic (37°C CA controls, administering TH (30°C improved overall survival (72-hour survival rate: 62.5% vs. 28.6%, P<0.001, post-resuscitation myocardial function (ejection fraction: 50.9±3.1% vs. 27.2±2.0%, P<0.001; aorta systolic pressure: 132.7±7.3 vs. 72.3±3.0 mmHg, P<0.001, and neurological scores at 72-hour post CA (9.5±1.3 vs. 5.4±1.3, P<0.05. In both heart and brain, CA increased lactate concentrations (1.9-fold and 3.1-fold increase, respectively, P<0.01, decreased PDH enzyme activity (24% and 50% reduction, respectively, P<0.01, and increased PDK protein expressions (1.2-fold and 1.9-fold, respectively, P<0.01. In contrast, post-CA treatment with TH normalized lactate concentrations (P<0.01 and P<0.05 and PDK expressions (P<0.001 and P<0.05, while increasing PDH activity (P<0.01 and P<0.01 in both the heart and brain. Additionally, treatment with DCA (0.2 mg/g body weight 30 min prior to CA improved both myocardial hemodynamics 2 hours post-CA (aortic systolic pressure: 123±3 vs. 96±4 mmHg, P<0.001 and 72-hour survival rates

  15. Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket

    Science.gov (United States)

    Malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our lab have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal g...

  16. Display of Bombyx mori alcohol dehydrogenases on the Bacillus subtilis spore surface to enhance enzymatic activity under adverse conditions.

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    Nan Wang

    Full Text Available Alcohol dehydrogenases (ADHs are oxidoreductases catalyzing the reversible oxidation of alcohols to corresponding aldehydes or ketones accompanied by nicotinamide adenine dinucleotide (NAD or nicotinamide adenine dinucleotide phosphate (NADP as coenzyme. ADHs attract major scientific and industrial interest for the evolutionary perspectives, afforded by their wide occurrence in nature, and for their use in industrial synthesis. However, the low activity of ADHs under extremes of pH and temperature often limits their application. To obtain ADH with high activity, in this study, we used Bombyx mori alcohol dehydrogenases (BmADH as foreign gene and constructed a recombinant integrative plasmid pJS700-BmADH. This pJS700-BmADH was transformed into Bacillus subtilis by double cross-over and produced an amylase inactivated mutant. The fusion protein containing BmADH was expressed on the spore surface and recognized by BmADH-specific antibody. We also assayed the alcohol dehydrogenase activity of the fusion protein together with the native BmADH at different pH and temperature levels, which indicated the recombinant enzyme exhibits activity over wider ranges of temperature and pH than its native form, perhaps due to the resistance properties of B. subtilis spores against adverse conditions.

  17. Enhancement of L-3-hydroxybutyryl-CoA dehydrogenase activity and circulating ketone body levels by pantethine. Relevance to dopaminergic injury

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    de Reggi Max

    2010-04-01

    Full Text Available Abstract Background The administration of the ketone bodies hydroxybutyrate and acetoacetate is known to exert a protective effect against metabolic disorders associated with cerebral pathologies. This suggests that the enhancement of their endogenous production might be a rational therapeutic approach. Ketone bodies are generated by fatty acid beta-oxidation, a process involving a mitochondrial oxido-reductase superfamily, with fatty acid-CoA thioesters as substrates. In this report, emphasis is on the penultimate step of the process, i.e. L-3-hydroxybutyryl-CoA dehydrogenase activity. We determined changes in enzyme activity and in circulating ketone body levels in the MPTP mouse model of Parkinson's disease. Since the active moiety of CoA is pantetheine, mice were treated with pantethine, its naturally-occurring form. Pantethine has the advantage of being known as an anti-inflammatory and hypolipidemic agent with very few side effects. Results We found that dehydrogenase activity and circulating ketone body levels were drastically reduced by the neurotoxin MPTP, whereas treatment with pantethine overcame these adverse effects. Pantethine prevented dopaminergic neuron loss and motility disorders. In vivo and in vitro experiments showed that the protection was associated with enhancement of glutathione (GSH production as well as restoration of respiratory chain complex I activity and mitochondrial ATP levels. Remarkably, pantethine treatment boosted the circulating ketone body levels in MPTP-intoxicated mice, but not in normal animals. Conclusions These finding demonstrate the feasibility of the enhancement of endogenous ketone body production and provide a promising therapeutic approach to Parkinson's disease as well as, conceivably, to other neurodegenerative disorders.

  18. Luteal 3beta-hydroxysteroid dehydrogenase and 20alpha-hydroxysteroid dehydrogenase activities in the rat corpus luteum of pseudopregnancy: Effect of the deciduoma reaction

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    Telleria Carlos M

    2004-05-01

    Full Text Available Abstract Background In the rat, the maintenance of gestation is dependent on progesterone production from the corpora lutea (CL, which are under the control of pituitary, decidual and placental hormones. The luteal metabolism of progesterone during gestation has been amply studied. However, the regulation of progesterone synthesis and degradation during pseudopregnancy (PSP, in which the CL are mainly under the control of pituitary prolactin (PRL, is not well known. The objectives of this investigation were: i to study the luteal metabolism of progesterone during PSP by measuring the activities of the enzymes 3beta-hydroxysteroid dehydrogenase (3betaHSD, involved in progesterone biosynthesis, and that of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD, involved in progesterone catabolism; and ii to determine the role of decidualization on progesterone metabolism in PSP. Methods PSP was induced mechanically at 10:00 h on the estrus of 4-day cycling Wistar rats, and the stimulus for decidualization was provided by scratching the uterus on day 4 of PSP. 3betaHSD and 20alphaHSD activities were measured in the CL isolated from ovaries of PSP rats using a spectrophotometric method. Serum concentrations of progesterone, PRL, androstenedione, and estradiol were measured by radioimmunoassay (RIA. Results The PSP stage induced mechanically in cycling rats lasted 11.3 ± 0.09 days (n = 14. Serum progesterone concentration was high until day 10 of PSP, and declined thereafter. Serum PRL concentration was high on the first days of PSP but decreased significantly from days 6 to 9, having minimal values on days 10 and 11. Luteal 3betaHSD activities were elevated until day 6 of PSP, after which they progressively declined, reaching minimal values at the end of PSP. Luteal 20alphaHSD activities were very low until day 9, but abruptly increased at the end of PSP. When the deciduoma was induced by scratching the uterus of pseudopregnant animals on day 4 (PSP

  19. CERTIFICATION REPORT The certification of the catalytic activity concentration of lactate dehydrogenase in ERM®-AD453k/IFCC

    OpenAIRE

    KUHLMANN JULIA; TOUSSAINT Brigitte; Schimmel, Heinz; Emons, Hendrik

    2016-01-01

    This report describes the production of ERM®-AD453k/IFCC, which is a material certified for the catalytic activity concentration of lactate dehydrogenase (LD). This material was produced in collaboration with the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) following ISO Guide 34:2009 [ ] and it is certified in accordance with ISO Guide 35:2006 [ ]. The starting material was a recombinant form of human LD isoenzyme 1 expressed in E. coli. It was produced, ...

  20. Characterization of Cardiac-Resident Progenitor Cells Expressing High Aldehyde Dehydrogenase Activity

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    Marc-Estienne Roehrich

    2013-01-01

    Full Text Available High aldehyde dehydrogenase (ALDH activity has been associated with stem and progenitor cells in various tissues. Human cord blood and bone marrow ALDH-bright (ALDHbr cells have displayed angiogenic activity in preclinical studies and have been shown to be safe in clinical trials in patients with ischemic cardiovascular disease. The presence of ALDHbr cells in the heart has not been evaluated so far. We have characterized ALDHbr cells isolated from mouse hearts. One percent of nonmyocytic cells from neonatal and adult hearts were ALDHbr. ALDHvery-br cells were more frequent in neonatal hearts than adult. ALDHbr cells were more frequent in atria than ventricles. Expression of ALDH1A1 isozyme transcripts was highest in ALDHvery-br cells, intermediate in ALDHbr cells, and lowest in ALDHdim cells. ALDH1A2 expression was highest in ALDHvery-br cells, intermediate in ALDHdim cells, and lowest in ALDHbr cells. ALDH1A3 and ALDH2 expression was detectable in ALDHvery-br and ALDHbr cells, unlike ALDHdim cells, albeit at lower levels compared with ALDH1A1 and ALDH1A2. Freshly isolated ALDHbr cells were enriched for cells expressing stem cell antigen-1, CD34, CD90, CD44, and CD106. ALDHbr cells, unlike ALDHdim cells, could be grown in culture for more than 40 passages. They expressed sarcomeric α-actinin and could be differentiated along multiple mesenchymal lineages. However, the proportion of ALDHbr cells declined with cell passage. In conclusion, the cardiac-derived ALDHbr population is enriched for progenitor cells that exhibit mesenchymal progenitor-like characteristics and can be expanded in culture. The regenerative potential of cardiac-derived ALDHbr cells remains to be evaluated.

  1. Plastidial glyceraldehyde-3-phosphate dehydrogenase deficiency leads to altered root development and affects the sugar and amino acid balance in Arabidopsis.

    Science.gov (United States)

    Muñoz-Bertomeu, Jesús; Cascales-Miñana, Borja; Mulet, Jose Miguel; Baroja-Fernández, Edurne; Pozueta-Romero, Javier; Kuhn, Josef M; Segura, Juan; Ros, Roc

    2009-10-01

    Glycolysis is a central metabolic pathway that, in plants, occurs in both the cytosol and the plastids. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with concomitant reduction of NAD(+) to NADH. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described. However, the in vivo functions of the plastidial isoforms remain unresolved. In this work, we have identified two Arabidopsis (Arabidopsis thaliana) chloroplast/plastid-localized GAPDH isoforms (GAPCp1 and GAPCp2). gapcp double mutants display a drastic phenotype of arrested root development, dwarfism, and sterility. In spite of their low gene expression level as compared with other GAPDHs, GAPCp down-regulation leads to altered gene expression and to drastic changes in the sugar and amino acid balance of the plant. We demonstrate that GAPCps are important for the synthesis of serine in roots. Serine supplementation to the growth medium rescues root developmental arrest and restores normal levels of carbohydrates and sugar biosynthetic activities in gapcp double mutants. We provide evidence that the phosphorylated pathway of Ser biosynthesis plays an important role in supplying serine to roots. Overall, these studies provide insights into the in vivo functions of the GAPCps in plants. Our results emphasize the importance of the plastidial glycolytic pathway, and specifically of GAPCps, in plant primary metabolism.

  2. High fat fed heart failure animals have enhanced mitochondrial function and acyl-coa dehydrogenase activities

    Science.gov (United States)

    We have previously shown that administration of high fat in heart failure (HF) increased mitochondrial respiration and did not alter left ventricular (LV) function. PPARalpha is a nuclear transcription factor that activates expression of genes involved in fatty acid uptake and utilization. We hypoth...

  3. Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase.

    Science.gov (United States)

    Li, Ming; Nie, Yao; Mu, Xiao Qing; Zhang, Rongzhen; Xu, Yan

    2016-07-03

    Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.

  4. Cryopreservation of glucose-6-phosphate dehydrogenase activity inside red blood cells: developing a specimen repository in support of development and evaluation of glucose-6-phosphate dehydrogenase deficiency tests.

    Science.gov (United States)

    Kahn, Maria; LaRue, Nicole; Bansil, Pooja; Kalnoky, Michael; McGray, Sarah; Domingo, Gonzalo J

    2013-08-20

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common human enzyme deficiency. It is characterized by abnormally low levels of G6PD activity. Individuals with G6PD deficiency are at risk of undergoing acute haemolysis when exposed to 8‒aminoquinoline-based drugs, such as primaquine. For this reason it is imperative to identify individuals with G6PD deficiency prior to administering these anti-malarial drugs. There is a need for the development and evaluation of point-of-care G6PD deficiency screening tests suitable for areas of the developing world where malarial treatments are frequently administered. The development and evaluation of new G6PD tests will be greatly assisted with the availability of specimen repositories. Cryopreservation of erythrocytes was evaluated as a means to preserve G6PD activity. Blood specimens from 31 patients including ten specimens with normal G6PD activity, three with intermediate activity, and 18 with deficient activity were cryopreserved for up to six months. Good correlation in G6PD activity between fresh and cryopreserved specimens (R2 = 0.95). The cryopreserved specimens show an overall small drop in mean G6PD activity of 0.23 U/g Hb (P=0.23). Cytochemical staining showed that intracellular G6PD activity distribution within the red blood cell populations is preserved during cryopreservation. Furthermore, the mosaic composition of red blood cells in heterozygous women is also preserved for six months or more. The fluorescent spot and the BinaxNOW qualitative tests for G6PD deficiency also showed high concordance in G6PD status determination between cryopreserved specimens and fresh specimens. A methodology for establishing a specimen panel for evaluation of G6PD tests is described. The approach is similar to that used in several malaria research facilities for the cryopreservation of parasites in clinical specimens and axenic cultures. Specimens stored in this manner will aid both the development and evaluation of

  5. Lactate dehydrogenase activity of rat epididymis and spermatozoa: Effect of constant light

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    RH Ponce

    2009-12-01

    Full Text Available During its passage through the epididymis, the gamete undergoes a process of “maturation” leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27 and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light: 10 h dark (group L:D. At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L. In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively. Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content and in spermatozoa, values of enzyme activities expressed per

  6. Electrochemical Studies of the Inhibition and Activation Effects of Al (III on the Activity of Bovine Liver Glutamate Dehydrogenase

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    Shuping Bi

    2005-04-01

    Full Text Available Since the study of Al3+ ion on the enzyme activity by using of electrochemical techniques was rarely found in available literatures, the differential-pulse polarography (DPP technique was applied to study the effects of Al3+ ion on the glutamate dehydrogenase (GDH activity in the catalytical reaction of α-KG +NADH+NH4 + ⇔ L-Glu+NAD++H2O by monitoring the DPP reduction current of NAD+. At the plant and animal physiologically relevant pH values (pH=6.5 and 7.5, the GDH enzyme activities were strongly depended on the concentrations of the metal ion in the assay mixture solutions. In the lower Al (III concentration solutions (80μM, the inhibition effects of Al (III were shown again. The cyclic voltammetry of NAD+ and NAD+-GDH in the presence of Al (III can help to explain some biological phenomena. According to the differential-pulse polarography and cyclic voltammetry experiments, the present research confirmed that the electrochemical technique is a convenient and reliable sensor for accurate determination of enzyme activity in biological and environmental samples.

  7. Analysis of Enzyme Activity of Alcohol Dehydrogenase and Alcohol Dehydrogenase 3 (ADH3 Gene Polymorphism of Alcoholics and Non-Alcoholics in Indonesia.

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    . Suhartini

    2016-12-01

    Full Text Available Alcohol is an addictive substance that is often misused worldwide, including in Indonesia. Ninety percent of the alcohol that enters the body will be metabolized in the liver using the alcohol dehydrogenase (ADH enzyme. It is important to determine the activity of ADH enzyme and ADH3 gene polymorphism on alcoholics and non-alcoholics in Yogyakarta, Indonesia. The aim of the study is  to determine ADH activity and identify ADH3 gene polymorphism of alcoholics and non-alcoholics in Yogyakarta, Indonesia. This study was an observational study with a cross-sectional design method. Blood samples were taken from 71 Javanese alcoholics and 71 non-alcoholics of Javanese descent in Yogyakarta, Indonesia. The participants were initially requested to sign an informed consent form. Examination of ADH enzyme activity used the spectrophotometry method and ADH3 gene polymorphism was assessed with PCR-RFLP using Ssp I restriction enzyme. The activity of ADH enzyme in all individuals appeared to be a slower type. The average of the ethanol value of alcoholics and non-alcoholics were 0.05554 mM and 0.0758 mM respectively. Gene type of alcoholics were ADH3*2(75.4%, ADH3*1/3*2(21.5%, and ADH3*1(3.1%, and non-alcoholics were ADH3*2(88.6%, ADH3*1/3*2(10.0%, and ADH3*1(1.4%. There were no significant differences between the activity of ADH with polymorphism of ADH3 gene in either alcoholics and non-alcoholics (p>0,05. Conclusion: The activity of ADH enzyme in all participants appeared to be a slower type. Most of the ADH3 gene polymorphism of alcoholics and non-alcoholics were both ADH3*2 (75.4% and 88.6%. There was no differences of ADH enzyme activity with ADH3 gene polymorphism between alcoholics and non-alcoholics of Javanese population in Yogyakarta, Indonesia.

  8. Escherichia coli Pyruvate Dehydrogenase Complex Is an Important Component of CXCL10-Mediated Antimicrobial Activity.

    Science.gov (United States)

    Schutte, Kirsten M; Fisher, Debra J; Burdick, Marie D; Mehrad, Borna; Mathers, Amy J; Mann, Barbara J; Nakamoto, Robert K; Hughes, Molly A

    2015-11-09

    Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains of Escherichia coli and report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enables E. coli to resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc, aceE. Resistance to CXCL10 also occurred following deletion of either aceF or lpdA, genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both the E. coli parent and aceE deletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine. Copyright © 2015 Schutte et al.

  9. Active site of Zn2+-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1

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    Jin-Suk Han

    2005-01-01

    Full Text Available The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261 is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/ H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.

  10. Active site of Zn(2+)-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1.

    Science.gov (United States)

    Han, Jin-Suk; Ishikawa, Kazuhiko

    2005-05-01

    The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261) is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A) of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.

  11. Active site of Zn2+-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1

    Science.gov (United States)

    Han, Jin-Suk; Ishikawa, Kazuhiko

    2005-01-01

    The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261) is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A) of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/ H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role. PMID:15876564

  12. γ-Aminobutyric Acid Pathway and Modified Tricarboxylic Acid Cycle Activity During Growth and Sporulation of Bacillus thuringiensis

    Science.gov (United States)

    Aronson, John N.; Borris, David P.; Doerner, Jeffrey F.; Akers, Evelyn

    1975-01-01

    Enzymatic analyses of Bacillus thuringiensis extracts suggested that a modified Krebs tricarboxylic acid cycle (without α-ketoglutarate dehydrogenase) can operate during sporulation in conjunction with the glyoxylic acid cycle and the γ-aminobutyric acid pathway. PMID:1180554

  13. Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3 Leads to Increase of the Fatty Acid Biotransformation Activity.

    Directory of Open Access Journals (Sweden)

    Ji-Min Woo

    Full Text Available The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid-induced stress. The metabolic and genomic responses of E. coli BL21(DE3 and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3. Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3 expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1 into n-heptanoic acid (5 and 11-hydroxyundec-9-enoic acid (4. This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass.

  14. Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3) Leads to Increase of the Fatty Acid Biotransformation Activity.

    Science.gov (United States)

    Woo, Ji-Min; Kim, Ji-Won; Song, Ji-Won; Blank, Lars M; Park, Jin-Byung

    The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid)-induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass.

  15. A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes. II. Further improvements of the staining procedure and some observations with glucose-6-phosphate dehydrogenase deficiency

    NARCIS (Netherlands)

    van Noorden, C. J.; Vogels, I. M.

    1985-01-01

    A cytochemical method for staining glucose-6-phosphate dehydrogenase (G6PD) activity in individual erythrocytes as reported previously has been optimized further by the incorporation of a number of technical improvements. Analysis of the enzyme content in erythrocytes of normal individuals as well

  16. Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties, activity, and expression during tuber dormancy progression

    Science.gov (United States)

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in meristems isolated from field-g...

  17. The changes in enzyme activities during citric acid fermentation by Aspergillus niger in solid culture.

    OpenAIRE

    桐村, 光太郎; 熊谷, 和夫; 森貞, 信也; "河邊, 誠一郎"; 宇佐美, 昭次; KOTARO, KIRIMURA; Kazuo, KUMAGAI; SHINYA, MORISADA; Seiichiro", "Kawabe; Shoji, Usami; 住友化学工業株式会社; 岡山理科大学基礎理学科; 早稲田大学理工学部応用化学科; Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University

    1984-01-01

    The changes of enzyme activities during citric acid fermentation in solid culture were studied with a high citric acidproducing strain, Aspergillus niger Yang no. 2,cultivated in synthetic medium containing 2 g/l of gelatin and glass wool as carrier.With fermentation time, the specific activity ratio of fructose-bisphosphate aldolase to glucose-6-phosphate dehydrogenase increased. Of the TCA cycle enzmes, the specific activities of citrate synthetase and aconitate hydratase remained almost co...

  18. Redox Status, Procoagulant Activity, and Metabolome of Fresh Frozen Plasma in Glucose 6-Phosphate Dehydrogenase Deficiency

    Directory of Open Access Journals (Sweden)

    Vassilis L. Tzounakas

    2018-02-01

    Full Text Available ObjectiveTransfusion of fresh frozen plasma (FFP helps in maintaining the coagulation parameters in patients with acquired multiple coagulation factor deficiencies and severe bleeding. However, along with coagulation factors and procoagulant extracellular vesicles (EVs, numerous bioactive and probably donor-related factors (metabolites, oxidized components, etc. are also carried to the recipient. The X-linked glucose 6-phosphate dehydrogenase deficiency (G6PD−, the most common human enzyme genetic defect, mainly affects males. By undermining the redox metabolism, the G6PD− cells are susceptible to the deleterious effects of oxidants. Considering the preferential transfusion of FFP from male donors, this study aimed at the assessment of FFP units derived from G6PD− males compared with control, to show whether they are comparable at physiological, metabolic and redox homeostasis levels.MethodsThe quality of n = 12 G6PD− and control FFP units was tested after 12 months of storage, by using hemolysis, redox, and procoagulant activity-targeted biochemical assays, flow cytometry for EV enumeration and phenotyping, untargeted metabolomics, in addition to statistical and bioinformatics tools.ResultsHigher procoagulant activity, phosphatidylserine positive EVs, RBC-vesiculation, and antioxidant capacity but lower oxidative modifications in lipids and proteins were detected in G6PD− FFP compared with controls. The FFP EVs varied in number, cell origin, and lipid/protein composition. Pathway analysis highlighted the riboflavin, purine, and glycerolipid/glycerophospholipid metabolisms as the most altered pathways with high impact in G6PD−. Multivariate and univariate analysis of FFP metabolomes showed excess of diacylglycerols, glycerophosphoinositol, aconitate, and ornithine but a deficiency in riboflavin, flavin mononucleotide, adenine, and arginine, among others, levels in G6PD− FFPs compared with control.ConclusionOur results point

  19. Structural and Functional Studies of WlbA: A Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Thoden, James B.; Holden, Hazel M. (UW)

    2010-09-08

    2,3-Diacetamido-2,3-dideoxy-D-mannuronic acid (ManNAc3NAcA) is an unusual dideoxy sugar first identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has since been observed in other organisms, including Bordetella pertussis, the causative agent of whooping cough. Five enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetyl-D-glucosamine. Here we describe a structural study of WlbA, the NAD-dependent dehydrogenase that catalyzes the second step in the pathway, namely, the oxidation of the C-3{prime} hydroxyl group on the UDP-linked sugar to a keto moiety and the reduction of NAD{sup +} to NADH. This enzyme has been shown to use {alpha}-ketoglutarate as an oxidant to regenerate the oxidized dinucleotide. For this investigation, three different crystal structures were determined: the enzyme with bound NAD(H), the enzyme in a complex with NAD(H) and {alpha}-ketoglutarate, and the enzyme in a complex with NAD(H) and its substrate (UDP-N-acetyl-D-glucosaminuronic acid). The tetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely to one another. Both {alpha}-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbon atoms (C-2 and C-3{prime}, respectively) abutting the re face of the cofactor. They are positioned {approx}3 {angstrom} from the nicotinamide C-4. The UDP-linked sugar substrate adopts a highly unusual curved conformation when bound in the WlbA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis.

  20. A Spontaneous Missense Mutation in Branched Chain Keto Acid Dehydrogenase Kinase in the Rat Affects Both the Central and Peripheral Nervous Systems.

    Directory of Open Access Journals (Sweden)

    J Samuel Zigler

    Full Text Available A novel mutation, causing a phenotype we named frogleg because its most obvious characteristic is a severe splaying of the hind limbs, arose spontaneously in a colony of Sprague-Dawley rats. Frogleg is a complex phenotype that includes abnormalities in hind limb function, reduced brain weight with dilated ventricles and infertility. Using micro-satellite markers spanning the entire rat genome, the mutation was mapped to a region of rat chromosome 1 between D1Rat131 and D1Rat287. Analysis of whole genome sequencing data within the linkage interval, identified a missense mutation in the branched-chain alpha-keto dehydrogenase kinase (Bckdk gene. The protein encoded by Bckdk is an integral part of an enzyme complex located in the mitochondrial matrix of many tissues which regulates the levels of the branched-chain amino acids (BCAAs, leucine, isoleucine and valine. BCAAs are essential amino acids (not synthesized by the body, and circulating levels must be tightly regulated; levels that are too high or too low are both deleterious. BCKDK phosphorylates Ser293 of the E1α subunit of the BCKDH protein, which catalyzes the rate-limiting step in the catabolism of the BCAAs, inhibiting BCKDH and thereby, limiting breakdown of the BCAAs. In contrast, when Ser293 is not phosphorylated, BCKDH activity is unchecked and the levels of the BCAAs will decrease dramatically. The mutation is located within the kinase domain of Bckdk and is predicted to be damaging. Consistent with this, we show that in rats homozygous for the mutation, phosphorylation of BCKDH in the brain is markedly decreased relative to wild type or heterozygous littermates. Further, circulating levels of the BCAAs are reduced by 70-80% in animals homozygous for the mutation. The frogleg phenotype shares important characteristics with a previously described Bckdk knockout mouse and with human subjects with Bckdk mutations. In addition, we report novel data regarding peripheral neuropathy of

  1. Lactate dehydrogenase-elevating virus

    Science.gov (United States)

    This book chapter describes the taxonomic classification of Lactate dehydrogenase-elevating virus (LDV). Included are: host, genome, classification, morphology, physicochemical and physical properties, nucleic acid, proteins, lipids, carbohydrates, geographic range, phylogenetic properties, biologic...

  2. Succinate Dehydrogenase Activity Assay in situ with Blue Tetrazolium Salt in Crabtree-Positive Saccharomyces cerevisiae Strain

    Directory of Open Access Journals (Sweden)

    Joanna Berlowska

    2008-01-01

    Full Text Available A spectrophotometric method for determining succinate dehydrogenase (SDH activity assay in azide-sensitive yeast Saccharomyces cerevisiae has been developed. The permeabilization of yeast cells by 0.05 % digitonin permitted to study yeast enzymatic activity in situ. The reduction of blue tetrazolium salt (BT to blue tetrazolium formazan (BTf was conducted in the presence of phenazine methosulphate (PMS as an exogenous electron carrier, and sodium azide (SA as an inhibitor of cytochrome oxidase (Cyt pathway. Various factors such as type of substrate, BT concentration, cell number, temperature and time of incubation, and different Cyt pathway blockers were optimized. In earlier studies, dimethyl sulfoxide (DMSO had been selected as the best solvent for extraction of BTf from yeast cells. The linear correlation between permeabilized yeast cell density and amount of formed formazan was evidenced in the range from 9·10^7 to 5·10^8 cells per sample solution. Below the yeast cell concentration of 10^7 the absorbance values were too low to detect formazans with good precision. This standarized procedure allows the estimation of SDH activity in whole cells, depending on vitality level of yeast populations. Significant increases of succinate dehydrogenase activities were observed in sequential passages as the result of the increase of activity of the strain and adaptation to cultivation conditions.

  3. Biological activity of pyrazole and imidazole-dehydroepiandrosterone derivatives on the activity of 17β-hydroxysteroid dehydrogenase.

    Science.gov (United States)

    Cabeza, Marisa; Posada, Alejandro; Sánchez-Márquez, Araceli; Heuze, Yvonne; Moreno, Isabel; Soriano, Juan; Garrido, Mariana; Cortés, Francisco; Bratoeff, Eugene

    2016-01-01

    The enzyme type 5 17β-hydroxysteroid dehydrogenase 5 (17β-HSD5) catalyzes the transformation of androstenedione (4-dione) to testosterone (T) in the prostate. This metabolic pathway remains active in cancer patients receiving androgen deprivation therapy. Since physicians seek to develop advantageous and better new treatments to increase the average survival of these patients, we synthesized several different dehydroepiandrosterone derivatives. These compounds have a pyrazole or imidazole function at C-17 and an ester moiety at C-3 and were studied as inhibitors of 17β-HSD5. The kinetic parameters of this enzyme were determined for use in inhibition assays. Their pharmacological effect was also determined on gonadectomized hamsters treated with Δ(4)-androstenedione (4-dione) or testosterone (T) and/or the novel compounds. The results indicated that the incorporation of a heterocycle at C-17 induced strong 17β-HSD5 inhibition. These derivatives decreased flank organ diameter and prostate weight in castrated hamsters treated with T or 4-dione. Inhibition of 17β-HSD5 by these compounds could have therapeutic potential for the treatment of prostate cancer and benign prostatic hyperplasia.

  4. [Malate dehydrogenase and lactate dehydrogenase in trematodes and turbellarians].

    Science.gov (United States)

    Vykhrestiuk, N P; Burenina, E A; Iarygina, G V

    1986-01-01

    Studies have been made on the activity and properties of malate and lactate dehydrogenases from the cattle rumen trematodes Eurytrema pancreaticum, Calicophoron ijimai and the turbellarian Phagocata sibirica which has a common free-living ancestor with the trematodes. All the species studied have a highly active malate dehydrogenase, its activity in the reaction of reducing oxaloacetate being 6-14 times higher than in the reaction of malate oxidation. The affinity of malate dehydrogenase to oxaloacetate was found to be higher than that to malate. The activity of lactate dehydrogenase (reducing the pyruvate) was lower than the activity of malate dehydrogenase, the difference being 50 times for C. ijimai, 4 times for E. pancreaticum and 10 times for P. sibirica.

  5. Triazolopyrimidine-based dihydroorotate dehydrogenase inhibitors with potent and selective activity against the malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Phillips, Margaret A; Gujjar, Ramesh; Malmquist, Nicholas A; White, John; El Mazouni, Farah; Baldwin, Jeffrey; Rathod, Pradipsinh K

    2008-06-26

    A Plasmodium falciparum dihydroorotate dehydrogenase ( PfDHODH) inhibitor that is potent ( KI = 15 nM) and species-selective (>5000-fold over the human enzyme) was identified by high-throughput screening. The substituted triazolopyrimidine and its structural analogues were produced by an inexpensive three-step synthesis, and the series showed good association between PfDHODH inhibition and parasite toxicity. This study has identified the first nanomolar PfDHODH inhibitor with potent antimalarial activity in whole cells (EC50 = 79 nM).

  6. Influence of cerebral global ischemia-reperfusion on succinate dehydrogenase activity in neurons of different neocortical layers

    Directory of Open Access Journals (Sweden)

    N. S. Shcherbak

    2014-01-01

    Full Text Available The aim of the study was to investigate changes in activity of succinate dehydrogenase (SDH in cytoplasm of neurons of different cortical layers in early and late reperfusion period after global cerebral ischemia in rats. Reversible global cerebral ischemia was modeled by occlusion of the brachiocephalic trunk, left subclavian artery and left common carotid artery for 10 minutes and following reperfusion during 2 or 7 days. The SDH activity in cytoplasm of neurons of II, III and V cortical layers was determined histoenzymatically. It is shown that the SDH activity in neurons of the studied cortical layers was characterized by the increased reperfusion period to the 2 days with a subsequent increased activity of the reperfusion period to the 7 days. The change in the SDH activity in cytoplasm of cortical neurons depends on the particular cerebral layer and duration of postischemic reperfusion.

  7. A single amino acid change (Y318F) in the L-arabitol dehydrogenase (LadA) from Aspergillus niger results in a significant increase in affinity for D-sorbitol

    NARCIS (Netherlands)

    Rutten, L.; Ribot, C.; Trejo-Aguilar, B.; Wosten, H.A.; De Vries, R.P.

    2009-01-01

    BACKGROUND: L-arabitol dehydrogenase (LAD) and xylitol dehydrogenase (XDH) are involved in the degradation of L-arabinose and D-xylose, which are among the most abundant monosaccharides on earth. Previous data demonstrated that LAD and XDH not only differ in the activity on their biological

  8. Persistence of myclobutanil and its impact on soil microbial biomass C and dehydrogenase enzyme activity in tea orchard soils

    Directory of Open Access Journals (Sweden)

    Dongdong Zhang

    2017-04-01

    Full Text Available Persistence of the fungicide myclobutanil in three tea orchard soils with different cultivating ages, neighboring wasteland and forest soils, and its influence on microbial activities in 2- and 50-year-oldtea orchard soils at three rates were studied in the laboratory. Dissipation data fitted well to first-order kinetic equation, except for sterilized treatments, in which neglected dissipation of myclobutanil was observed. At 25oC, the dissipation half-lives (DT50 at level of 1mg kg-1 were in the range of 15.07-69.32 days under non-flooded condition, significantly lower than flooded condition (p < 0.05, indicating that dissipation of myclobutanil was mainly driven by soil microorganisms under aerobic condition. Dissipation rate was significantly increased at 40oC compared to those at 4oC and 25oC for all five soils (p < 0.05. Under all incubation conditions, DT50 were lowest in 50-year-old tea orchard soil (p < 0.01. Correlation analysis between DT50 in tea orchard soils and soil properties showed that soil microbial biomass carbon was negatively correlated with DT50 under 25oC and 60% water holding capacity (p < 0.05. In general, soil microbial biomass carbon and dehydrogenase activity decreased as the concentration of myclobutanil and incubation time increased except 0.1 mg kg-1 spiked soils, in which soil dehydrogenase activity was stimulated after 10 days incubation.

  9. Purification of xylitol dehydrogenase and improved production of xylitol by increasing XDH activity and NADH supply in Gluconobacter oxydans.

    Science.gov (United States)

    Zhang, Jinliang; Li, Sha; Xu, Hong; Zhou, Peng; Zhang, Lujia; Ouyang, Pingkai

    2013-03-20

    Gluconobacter oxydans is known to be a suitable candidate for producing xylitol from d-arabitol. In this study, the enzyme responsible for reducing d-xylulose to xylitol was purified from G. oxydans NH-10 and characterized as xylitol dehydrogenase. It has been reported that XDH depends exclusively on NAD(+)/NADH as cofactors with a relatively low activity, which was proposed to be the direct reason for its limiting the overall conversion process. To better produce xylitol, an engineered G. oxydans PXPG was constructed to coexpress the XDH gene and a cofactor regeneration enzyme (glucose dehydrogenase) gene from Bacillus subtilis. Activities for both enzymes were more than twofold higher in the G. oxydans PXPG than in the wild strain. Approximately 12.23 g/L xylitol was obtained from 30 g/L d-arabitol by resting cells of the engineered strain with a conversion yield of 40.8%, whereas only 7.56 g/L xylitol was produced by the wild strain with a yield of 25.2%. These results demonstrated that increasing the XDH activity and the cofactor NADH supply could improve the xylitol productivity notably.

  10. Antimalarial Activity of Potential Inhibitors of Plasmodium falciparum Lactate Dehydrogenase Enzyme Selected by Docking Studies

    OpenAIRE

    Penna-Coutinho, Julia; Cortopassi, Wilian Augusto; Oliveira, Aline Alves; França, Tanos Celmar Costa; Krettli, Antoniana Ursine

    2011-01-01

    The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH) has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH) all exhibit ∼90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docki...

  11. Effect of thoracic x-irradiation on glucose-6-phosphate dehydrogenase activity of the pectoral muscle of guinea pig

    Energy Technology Data Exchange (ETDEWEB)

    Bhatavdekar, J.M.; Shah, V.C. (Gujarat Univ. School of Sciences, Ahmedabad (India))

    1981-01-01

    The histochemical distribution of glucose-6-phosphate dehydrogenase (G6PD) was observed in the major pectoral muscle of a guinea pig that had received 240 R thoracic X-irradiation. The irradiation effects were studied at 24, 48 and 72 hrs after X-irradiation. Type I fibres of the pectoral muscle were deeply stained showing high activity whereas type II fibres demonstrated minimum enzyme activity. The intermediate fibres had medium levels of G6PD activity. Type II fibres showed more staining at 24 and 48 hrs as compared with control muscle. However, at 72 hrs all three fibre types showed a marked inhibition of G6PD activity. The significance of these changes suggests that muscle G6PD metabolism generally altered after irradiation, but the specific nature of these changes and their causes still remain unclear.

  12. 3D-QSAR studies on a series of dihydroorotate dehydrogenase inhibitors: analogues of the active metabolite of leflunomide.

    Science.gov (United States)

    Li, Shun-Lai; He, Mao-Yu; Du, Hong-Guang

    2011-01-01

    The active metabolite of the novel immunosuppressive agent leflunomide has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. Self-organizing molecular field analysis (SOMFA), a simple three-dimensional quantitative structure-activity relationship (3D-QSAR) method is used to study the correlation between the molecular properties and the biological activities of a series of analogues of the active metabolite. The statistical results, cross-validated r(CV) (2) (0.664) and non cross-validated r(2) (0.687), show a good predictive ability. The final SOMFA model provides a better understanding of DHODH inhibitor-enzyme interactions, and may be useful for further modification and improvement of inhibitors of this important enzyme.

  13. 3D-QSAR Studies on a Series of Dihydroorotate Dehydrogenase Inhibitors: Analogues of the Active Metabolite of Leflunomide

    Directory of Open Access Journals (Sweden)

    Hong-Guang Du

    2011-05-01

    Full Text Available The active metabolite of the novel immunosuppressive agent leflunomide has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH. This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. Self-organizing molecular field analysis (SOMFA, a simple three-dimensional quantitative structure-activity relationship (3D-QSAR method is used to study the correlation between the molecular properties and the biological activities of a series of analogues of the active metabolite. The statistical results, cross-validated rCV2 (0.664 and non cross-validated r2 (0.687, show a good predictive ability. The final SOMFA model provides a better understanding of DHODH inhibitor-enzyme interactions, and may be useful for further modification and improvement of inhibitors of this important enzyme.

  14. 5,5'-Dithiobis-(2-nitrobenzoic acid) as a probe for a non-essential cysteine residue at the medium chain acyl-coenzyme A dehydrogenase binding site of the human 'electron transferring flavoprotein' (ETF).

    Science.gov (United States)

    Parker, A; Engel, P C

    1999-01-01

    Human 'electron transferring flavoprotein' (ETF) was inactivated by the thiol-specific reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The kinetic profile showed the reaction followed pseudo-first-order kinetics during the initial phase of inactivation. Monitoring the release of 5-thio-2-nitrobenzoate (TNB) showed that modification of 1 cysteine residue was responsible for the loss of activity. The inactivation of ETF by DTNB could be reversed upon incubation with thiol-containing reagents. The loss of activity was prevented by the inclusion of medium chain acyl-CoA dehydrogenase (MCAD) and octanoyl-CoA. Cyanolysis of the DTNB modified-ETF with KCN led to the release of TNB accompanied presumably by the formation of the thio-cyano enzyme and with almost full recovery of activity. Conservation studies and the lack of 100% inactivation, however, suggested that this cysteine residue is not essential for the interaction with MCAD.

  15. Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase

    NARCIS (Netherlands)

    Hengeveld, A.F.; Mierlo, van C.P.M.; Hooven, van den H.W.; Visser, A.J.W.G.; Kok, de A.

    2002-01-01

    A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies

  16. Antimalarial activity of Evolvulus alsinoids Linn.-an in vitro Plasmodium falciparum specific lactate dehydrogenase enzyme inhibition assay

    Directory of Open Access Journals (Sweden)

    Neeraj Sethiya

    2014-12-01

    Full Text Available Objective: To investigate the effect of standardized methanol extract of Evolvulus alsinoids Linn. (E. alsinoids on Plasmodium falciparum specific lactate dehydrogenase (PfLDH enzyme inhibition. Methods: To carry out enzyme inhibition studies, lactate dehydrogenase was cloned from Plasmodium falciparum 3D7 strain using expression vector pET28a and expressed in Escherichia coli BL21 (DE3. Protein purification was carried out by Ni-affinity chromatography. This protein was used for the inhibition study. Methanol extract of E. alsinoids was standardized by high performance thin layer chromatography described by previous literature and screened for PfLDH enzyme inhibitory activity and compared with gossypol (a known PfLDH enzyme inhibitor. Results: It was found that E. alsinoids possesses a compond scopoletin and potentially inhibits PfLDH [(25.04依0.51%]. Conclusions: Methanol extract of E. alsinoids showed significant PfLDH inhibition as evidenced from the experiments performed. The activity may be attributed to the presence of various polyphenolic and flavanoids compounds.

  17. Stimulation of glyceraldehyde-3-phosphate dehydrogenase mRNA levels by endogenous nitric oxide in cytokine-activated endothelium.

    Science.gov (United States)

    Bereta, J; Bereta, M

    1995-12-05

    Previous studies have shown that endogenous nitric oxide (NO) potentiates glycolysis in the cytokine-activated murine microvascular endothelial cells (MME). In the present study we investigate the influence of NO on the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme of the glycolytic pathway. Activation of MME with TNF-alpha and IFN-gamma resulted in a strong elevation of GAPDH mRNA levels. This effect was impaired in the presence of L-NMMA, the inhibitor of NO synthesis. We discuss the possibility that NO-mediated elevation of GAPDH mRNA levels may compensate for NO-mediated inhibition of GAPDH enzymatic activity, representing another adaptive mechanism which protects cells producing large amounts of NO against its cytotoxic effects.

  18. Stimulation of d- and l-lactate dehydrogenases transcriptional levels in presence of diammonium hydrogen phosphate resulting to enhanced lactic acid production by Lactobacillus strain.

    Science.gov (United States)

    Singhvi, Mamata; Zendo, Takeshi; Iida, Hiroshi; Gokhale, Digambar; Sonomoto, Kenji

    2017-12-01

    The present study revealed the effect of nitrogen sources on lactic acid production and stimulation of d- and l-lactate dehydrogenases (LDH) of parent Lactobacillus lactis NCIM 2368 and its mutant RM2-24 generated after UV mutagenesis. Both the parent and mutant strains were evaluated for d-lactic acid production in control and modified media. The modified media did not show remarkable effect on lactic acid production in case of parent whereas mutant exhibited significant enhancement in d-lactic acid production along with the appearance of l-lactic acid in the broth. Both LDH activities and specific activities were found to be higher in mutant than the parent strain. These results suggested that the diammonium hydrogen phosphate in modified media triggered the expression of LDH genes leading to enhanced lactic acid production. This observation has been proved by studying the expression levels of d- and l-LDH genes of parent and mutant in control and modified media using quantitative RT-PCR technique. In case of mutant, the transcriptional levels of d-LDH and l-LDH increased ∼17 fold and ∼1.38 fold respectively in modified medium compared to the values obtained with control medium. In case of parent, no significant change in transcriptional levels of d- and l-LDH was found when the cells were grown in either control medium or modified medium. This study suggested that the mutant, RM2-24 has l-LDH gene which is expressed in presence of (NH 4 ) 2 HPO 4 resulting in l-lactic acid production. Co-production of l-lactic acid in d-lactic acid fermentation may be detrimental in the PLA production. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. Alcohol Dehydrogenase-1B (rs1229984) and Aldehyde Dehydrogenase-2 (rs671) Genotypes and Alcoholic Ketosis Are Associated with the Serum Uric Acid Level in Japanese Alcoholic Men.

    Science.gov (United States)

    Yokoyama, Akira; Yokoyama, Tetsuji; Mizukami, Takeshi; Matsui, Toshifumi; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

    2016-05-01

    To identify determinants of hyperuricemia in alcoholics. The serum uric acid (UA) levels of 1759 Japanese alcoholic men (≥40 years) were measured on their first visit or within 3 days after admission; ADH1B and ALDH2 genotyping on blood DNA samples were performed. Dipstick urinalyses for ketonuria and serum UA measurements were simultaneously performed for 621 men on their first visit. Serum UA levels of >416 μmol/l (7.0 mg/dl) and ≥535 μmol/l (9.0 mg/dl) were observed in 30.4 and 7.8% of the subjects, respectively. Ketonuria was positive in 35.9% of the subjects, and a multivariate analysis revealed that the ketosis level was positively associated with the UA level. The presence of the ADH1B*2 allele and the ALDH2*1/*1 genotype increased the odds ratio (OR; 95% confidence interval) among subjects with a high UA level of >416 μmol/l (vs. ≤416 μmol/l; 2.04 [1.58-2.65] and 1.48 [1.09-2.01], respectively) and those with a high UA level of ≥535 μmol/l (vs. ≤416 μmol/l; 2.29 [1.42-3.71] and 3.03 [1.51-6.08], respectively). The ADH1B*2 plus ALDH2*1/*1 combination yielded the highest ORs (2.86 [1.61-5.10] and 6.21 [1.49-25.88] for a UA level of >416 μmol/l and ≥535 μmol/l, respectively), compared with the ADH1B*1/*1 plus ALDH2*1/*2 combination. The presence of diabetes and the consumption of Japanese sake rather than beer were negatively associated with the UA levels. The faster metabolism of ethanol and acetaldehyde by the ADH1B*2 allele and ALDH2*1/*1 genotype and higher ketosis levels were associated with higher UA levels in alcoholics, while diabetes and the consumption of sake were negative determinants. © The Author 2015. Medical Council on Alcohol and Oxford University Press. All rights reserved.

  20. Thermal destabilization of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans upon phosphate binding in the active site.

    Science.gov (United States)

    Rahuel-Clermont, Sophie; Arutyunov, Denis; Marchal, Stéphane; Orlov, Victor; Muronetz, Vladimir; Branlant, Guy

    2005-05-13

    Catalysis by the NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, a member of the aldehyde dehydrogenase (ALDH) family, relies on a local conformational reorganization of the active site. This rearrangement is promoted by the binding of NADP and is strongly kinetically favored by the formation of the ternary complex enzyme.NADP.substrate. Adiabatic differential scanning calorimetry was used to investigate the effect of ligands on the irreversible thermal denaturation of GAPN. We showed that phosphate binds to GAPN, resulting in the formation of a GAPN.phosphate binary complex characterized by a strongly decreased thermal stability, with a difference of at least 15 degrees C between the maximum temperatures of the thermal transition peaks. The kinetics of phosphate association and dissociation are slow, allowing both free and GAPN.phosphate complexes to be observed by differential scanning calorimetry and to be separated by native polyacrylamide electrophoresis run in phosphate buffer. Analysis of a set of mutants of GAPN strongly suggests that phosphate is bound to the substrate C-3 subsite. In addition, the substrate analog glycerol-3-phosphate has similar effects as does phosphate on the thermal behavior of GAPN. Based on the current knowledge on the catalytic mechanism of GAPN and other ALDHs, we propose that ligand-induced thermal destabilization is a mechanism that provides to ALDHs the required flexibility for an efficient catalysis.

  1. Taraxerone enhances alcohol oxidation via increases of alcohol dehyderogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities and gene expressions.

    Science.gov (United States)

    Sung, Chang-Keun; Kim, Seung-Mi; Oh, Chang-Jin; Yang, Sun-A; Han, Byung-Hee; Mo, Eun-Kyoung

    2012-07-01

    The present study, taraxerone (d-friedoolean-14-en-3-one) was isolated from Sedum sarmentosum with purity 96.383%, and its enhancing effects on alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities were determined: EC(50) values were 512.42 ± 3.12 and 500.16 ± 3.23 μM for ADH and ALDH, respectively. In order to obtain more information on taraxerone related with the alcohol metabolism, 40% ethanol (5 mL/kg body weight) with 0.5-1mM of taraxerone were administered to mice. The plasma alcohol and acetaldehyde concentrations of taraxerone-treated groups were significantly lowered than those of the control group (palcohol and acetaldehyde, respectively. Compare to the control group, the ADH and ALDH expressions in the liver tissues were abruptly increased in the taraxerone-treated groups after ethanol exposure. In addition, taraxerone prevented catalase, superoxide dismutase, and reduced glutathione concentrations from the decrease induced by ethanol administration with the concentration dependent manner. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Inactivation of the Kluyveromyces lactis KlPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobic alcoholic fermentation.

    Science.gov (United States)

    Zeeman, A M; Luttik, M A; Thiele, C; van Dijken, J P; Pronk, J T; Steensma, H Y

    1998-12-01

    The KlPDA1 gene, encoding the E1alpha subunit of the mitochondrial pyruvate-dehydrogenase (PDH) complex was isolated from a Kluyveromyces lactis genomic library by screening with a 1.1 kb internal fragment of the Saccharomyces cerevisiae PDA1 gene. The predicted amino acid sequence encoded by KlPDA1 showed 87% similarity and 79% identity to its S. cerevisiae counterpart. Disruption of KIPDA1 resulted in complete absence of PDH activity in cell extracts. The maximum specific growth rate on glucose of null mutants was 3.5-fold lower than that of the wild-type, whereas growth on ethanol was unaffected. Wild-type K. lactis CBS 2359 exhibits a Crabtree-negative phenotype, i.e. no ethanol was produced in aerobic batch cultures grown on glucose. In contrast, substantial amounts of ethanol and acetaldehyde were produced in aerobic cultures of an isogenic Klpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KlPDA1 gene but not, as previously found for S. cerevisiae pda1 mutants, by cultivation in the presence of leucine. The occurrence of aerobic fermentation and slow growth of the Klpda1 null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only at relatively low growth rates (D = 0.10 h(-1)) in aerobic, glucose-limited chemostat cultures, could the PDH bypass completely replace the PDH complex, thus allowing fully respiratory growth. This resulted in a lower biomass yield [g biomass (g glucose)-1] than in the wild-type due to a higher consumption of ATP in the PDH bypass compared to the formation of acetyl-CoA via the PDH complex.

  3. Effects of Macromolecular Crowding on Alcohol Dehydrogenase Activity Are Substrate-Dependent.

    Science.gov (United States)

    Wilcox, A E; LoConte, Micaela A; Slade, Kristin M

    2016-06-28

    Enzymes operate in a densely packed cellular environment that rarely matches the dilute conditions under which they are studied. To better understand the ramifications of this crowding, the Michaelis-Menten kinetics of yeast alcohol dehydrogenase (YADH) were monitored spectrophotometrically in the presence of high concentrations of dextran. Crowding decreased the maximal rate of the reaction by 40% for assays with ethanol, the primary substrate of YADH. This observation was attributed to slowed release of the reduced β-nicotinamide adenine dinucleotide product, which is rate-limiting. In contrast, when larger alcohols were used as the YADH substrate, the rate-limiting step becomes hydride transfer and crowding instead increased the maximal rate of the reaction by 20-40%. This work reveals the importance of considering enzyme mechanism when evaluating the ways in which crowding can alter kinetics.

  4. Application of reduced graphene oxide and carbon nanotube modified electrodes for measuring the enzymatic activity of alcohol dehydrogenase.

    Science.gov (United States)

    Wang, Xianlong; Li, Li; Wang, Yanping; Xu, Chongzheng; Zhao, Bo; Yang, Xiaodi

    2013-06-15

    An electrochemical method was developed to measure the enzymatic activity of alcohol dehydrogenase (ADH) by monitoring the amount of reduced nicotinamide adenine dinucleotide (NADH) generated in the catalysed oxidation of ethanol by ADH. The concentration of NADH was determined by amperometric measurements, which recorded the oxidation current of NADH versus time on reduced graphene oxide and functionalised multi-walled carbon nanotube modified electrodes. The initial reaction rates and the apparent Michaelis constants of the enzymatic reaction were obtained in the absence and presence of Al(3+) and nanometre-sized tridecameric aluminium polycationic (nano-Al(13)) species. The results showed that Al(3+) and nano-Al(13) exhibited inhibitory effect on the enzymatic activity of ADH. Fluorescence and circular dichroism spectra indicated the inhibitory effect was likely caused by the conformational changes of ADH and/or NADH induced by Al(3+) and nano-Al(13). Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Bioinformatics based structural characterization of glucose dehydrogenase (gdh gene and growth promoting activity of Leclercia sp. QAU-66

    Directory of Open Access Journals (Sweden)

    Muhammad Naveed

    2014-06-01

    Full Text Available Glucose dehydrogenase (GDH; EC 1.1. 5.2 is the member of quinoproteins group that use the redox cofactor pyrroloquinoline quinoine, calcium ions and glucose as substrate for its activity. In present study, Leclercia sp. QAU-66, isolated from rhizosphere of Vigna mungo, was characterized for phosphate solubilization and the role of GDH in plant growth promotion of Phaseolus vulgaris. The strain QAU-66 had ability to solubilize phosphorus and significantly (p < 0.05 promoted the shoot and root lengths of Phaseolus vulgaris. The structural determination of GDH protein was carried out using bioinformatics tools like Pfam, InterProScan, I-TASSER and COFACTOR. These tools predicted the structural based functional homology of pyrroloquinoline quinone domains in GDH. GDH of Leclercia sp. QAU-66 is one of the main factor that involved in plant growth promotion and provides a solid background for further research in plant growth promoting activities.

  6. Homo-D-lactic acid fermentation from arabinose by redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-lactate dehydrogenase gene-deficient Lactobacillus plantarum.

    Science.gov (United States)

    Okano, Kenji; Yoshida, Shogo; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2009-08-01

    Optically pure d-lactic acid fermentation from arabinose was achieved by using the Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase gene was substituted with a heterologous transketolase gene. After 27 h of fermentation, 38.6 g/liter of d-lactic acid was produced from 50 g/liter of arabinose.

  7. Influence of hexaconazole, carbofuran and ethion on soil microflora and dehydrogenase activities in soil and intact cell.

    Science.gov (United States)

    Kalam, A; Mukherjee, A K

    2001-01-01

    Total microbial count was highly affected (up to 61% at 1000 micrograms level) in presence of hexaconazole and persisted up to 21 days. Bacteria were more susceptible than actinomycetes. Carbofuran and ethion were moderately toxic to soil microflora. Inhibitory effects of all the three pesticides gradually decreased after 21 days as was evident by increase in total microbial count except in carbofuran. GDH activity in soil was also affected initially (up to 14 days) by all the three pesticides (60.3% in hexaconazole at 1000 micrograms level) and inhibition gradually decreased to zero except in carbofuran (15-20% toxicity persisted up to 35 days). GDH and LDH activity in presence of hexaconazole was strongly affected in intact cells of some standard culture of bacteria like Rhizobium sp. (host Dolichos sp., 32.1 and 72.5%), Bacillus subtilis Cohn (86.75 and 76.5%), Azotobacter sp. (36.9 and 55.4%) and B. sphaericus (67.6% GDH) respectively. Carbofuran inhibited the enzyme activity in B. subtilis (55.55 and 35.3%) and to some extent in B. sphaericus. Ethion moderately inhibited LDH activity in Rhodococcus sp. AK1 (17.1 and 33.3%), Rhizobium (27.6% LDH), E. coli HB 101 (34.2% LDH) as evidenced by formazan formation. From the result, it might be concluded that among the above three pesticides tested hexaconazole strongly inhibited the dehydrogenase system in bacteria including nitrogen fixing bacteria of soil and thus may affect soil fertility. It was concluded that hexaconazole was more toxic than ethion to dehydrogenase enzymes.

  8. Peroxisome proliferator-activated receptor-γ activation induces 11β-hydroxysteroid dehydrogenase type 1 activity in human alternative macrophages.

    Science.gov (United States)

    Chinetti-Gbaguidi, Giulia; Bouhlel, Mohamed Amine; Copin, Corinne; Duhem, Christian; Derudas, Bruno; Neve, Bernadette; Noel, Benoit; Eeckhoute, Jerome; Lefebvre, Philippe; Seckl, Jonathan R; Staels, Bart

    2012-03-01

    11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the intracellular reduction of inactive cortisone to active cortisol, the natural ligand activating the glucocorticoid receptor (GR). Peroxisome proliferator- activated receptor-γ (PPARγ) is a nuclear receptor controlling inflammation, lipid metabolism, and the macrophage polarization state. In this study, we investigated the impact of macrophage polarization on the expression and activity of 11β-HSD1 and the role of PPARγ therein. 11β-HSD1 gene expression is higher in proinflammatory M1 and anti-inflammatory M2 macrophages than in resting macrophages, whereas its activity is highest in M2 macrophages. Interestingly, PPARγ activation induces 11β-HSD1 enzyme activity in M2 macrophages but not in resting macrophages or M1 macrophages. Consequently, human M2 macrophages displayed enhanced responsiveness to the 11β-HSD1 substrate cortisone, an effect amplified by PPARγ induction of 11β-HSD1 activity, as illustrated by an increased expression of GR target genes. Our data identify a positive cross-talk between PPARγ and GR in human M2 macrophages via the induction of 11β-HSD1 expression and activity.

  9. Activation of the nickel-deficient carbon monoxide dehydrogenase from Rhodospirillum rubrum: Kinetic characterization and reductant requirement

    Energy Technology Data Exchange (ETDEWEB)

    Ensign, S.A.; Campbell, M.J.; Ludden, P.W. (Univ. of Wisconsin, Madison (USA))

    1990-02-27

    The requirements for and kinetics of the activation of the nickel-deficient (apo) CO dehydrogenase from Rhodospirillum rubrum by exogenous nickel have been investigated. The activation is strictly dependent upon the presence of a low-potential one-electron reductant. Sodium dithionite and reduced methylviologen are suitable reductants, whereas reduced indigo carmine and the two-electron reductants sodium borohydride, NADH, and dithiothreitol are ineffective in stimulating activation. The midpoint potential for activation was observed at approximately {minus}475 mV. The ability of a reductant to stimulate activation is correlated with the reduced state of the enzyme Fe{sub 4}-S{sub 4} centers. The activation follows apparent first-order kinetics in a saturable fashion, yielding a rate constant of 0.157 min{sup {minus}1} at saturating concentration of nickel. The initial rate at which the enzyme is activated by NiCl{sub 2} is also a saturable process, yielding a dissociation constant (K{sub D}) of 755 {mu}M for the initial association of nickel and enzyme. Cadmium(II), zinc(II), cobalt(II), and iron(II) are competitive inhibitors of nickel activation with inhibition constants of 2.44, 4.16, 175, and 349 {mu}M, respectively. Manganese(II), calcium(II), and magnesium(II) exhibit no inhibition of activation.

  10. Inhibitory action of marine algae extracts on the Trypanosoma cruzi dihydroorotate dehydrogenase activity and on the protozoan growth in mammalian cells.

    Science.gov (United States)

    Nara, Takeshi; Kamei, Yuto; Tsubouchi, Akiko; Annoura, Takeshi; Hirota, Kenichiro; Iizumi, Kyoichi; Dohmoto, Yuki; Ono, Takeaki; Aoki, Takashi

    2005-03-01

    Trypanosoma cruzi, the causative agent of Chagas' disease, replicates in mammalian cells and relies on the de novo pyrimidine biosynthetic pathway that supplies essential precursors for nucleic acid synthesis. The protozoan dihydroorotate dehydrogenase (DHOD), the fourth enzyme of the pathway catalyzing production of orotate from dihydroorotate, markedly differs from the human enzyme. This study was thus aimed to search for potent inhibitors against T. cruzi DHOD activity, and a number of methanol extracts prepared from green, brown, and red algae were assayed. The extracts from two brown algae, Fucus evanescens and Pelvetia babingtonii, yielded 59 and 58% decrease in the recombinant DHOD activity, respectively, at the concentration of 50 microg/ml. Inhibition by these extracts was noncompetitive with respect to dihydroorotate, with apparent Ki values of 35.3+/-5.9 and 10.3+/-4.4 microg/ml, respectively. Further, in an in vitro T. cruzi-HeLa cell infection system, ethanol-reconstituted F. evanescens and P. babingtonii extracts at the concentration of 1 microg/ml, respectively, decreased significantly the infection rate of host cells and the average parasite number per infected cell. These results imply that F. evanescens and P. babingtonii contain inhibitor(s) against the T. cruzi DHOD activity and against the protozoan infection and proliferation in mammalian cells. Identification of inhibitor(s) in these two brown algae and further screening of other marine algae may facilitate the discovery of new, anti-trypanosomal lead compounds.

  11. STUDIES ON THE DYNAMICS OF DEHYDROGENASES KREBS CYCLE ACTIVITY AT MONILINIA LAXA (ADERH. & RUHL. HONEY FUNGUS GROWN ON MEDIA WITH DIFFERENT CARBOHYDRATES

    Directory of Open Access Journals (Sweden)

    Elena Ciornea

    2011-11-01

    Full Text Available As ubiquitous organisms, fungi grow on a large number of organic substrate, alive or dead, confronting therefore with a wide variety of carbohydrates and various physical factors, and their versatility to adapt and be able to use a large number of these compounds could provide them the chance to survive. Given that, these fungi have a rich enzyme equipment that allows them to operate on different metabolic pathways, this study aims to monitor the dynamics activity of some Krebs cycle dehydrogenases in Monilinia laxa (Aderh & Ruhl. Honey species parasitic on various species of plum trees. To this end, the fungus was cultivated in vitro on media enriched with different carbohydrates and the isocitrate dehydrogenase, �-cetoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase activity in the fungus mycelium was followed, at 7, respectively, 14 days after the inoculation of the culture medium and determined using the spectrophotometric Sîsoev and Krasna method (Cojocaru, D.C., 2009. Data revealed obvious differences depending on the type of carbohydrate introduced into the medium and the age of the culture mycelia.

  12. STUDIES ON THE DYNAMICS OF DEHYDROGENASES KREBS CYCLE ACTIVITY AT MONILINIA LAXA (ADERH. & RUHL. HONEY FUNGUS GROWN ON MEDIA WITH DIFFERENT CARBOHYDRATES

    Directory of Open Access Journals (Sweden)

    Elena Ciornea

    2010-09-01

    Full Text Available As ubiquitous organisms, fungi grow on a large number of organic substrate, alive or dead, confronting therefore with a wide variety of carbohydrates and various physical factors, and their versatility to adapt and be able to use a large number of these compounds could provide them the chance to survive. Given that, these fungi have a rich enzyme equipment that allows them to operate on different metabolic pathways, this study aims to monitor the dynamics activity of some Krebs cycle dehydrogenases in Monilinia laxa (Aderh & Ruhl. Honey species parasitic on various species of plum trees. To this end, the fungus was cultivated in vitro on media enriched with different carbohydrates and the isocitrate dehydrogenase, �-cetoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase activity in the fungus mycelium was followed, at 7, respectively, 14 days after the inoculation of the culture medium and determined using the spectrophotometric Sîsoev and Krasna method (Cojocaru, D.C., 2009. Data revealed obvious differences depending on the type of carbohydrate introduced into the medium and the age of the culture mycelia.

  13. Adsorption of humic acid on acid-activated Greek bentonite.

    Science.gov (United States)

    Doulia, Danae; Leodopoulos, Ch; Gimouhopoulos, K; Rigas, F

    2009-12-15

    The adsorption of humic acid on bentonite from Milos Island (Greece) acid-treated with dilute H(2)SO(4) solutions over a concentration range between 0.25 and 13M has been studied. Bentonite activated with 3M sulfuric acid (AAS) showed a higher efficiency in removing humic acid from aqueous solutions and was selected for further investigation. The specific surface area of acid-activated bentonite was estimated using the methylene blue adsorption method. The morphology of untreated, activated, and HA-sorbed bentonite was studied under scanning electron microscope (SEM). The effects of contact time, adsorbate concentration, adsorbent dose, and temperature on the adsorption of humic acid onto bentonite activated with 3M H(2)SO(4) were studied using a batch adsorption technique. Acidic pH and high ionic strength proved to be favorable for the adsorption efficiency. Pseudo-first-order, pseudo-second-order, and intraparticle diffusion models were used to describe the kinetic data and the rate constants were evaluated. The experimental isotherm data were analyzed using Langmuir, Freundlich, and Temkin equations and the isotherm constants were determined. Thermodynamic parameters (DeltaH(o), DeltaS(o), and DeltaG(o)) of adsorption of humic acid onto acid-activated bentonite with 3M sulfuric acid were also evaluated.

  14. A new class of IMP dehydrogenase with a role in self-resistance of mycophenolic acid producing fungi

    DEFF Research Database (Denmark)

    Hansen, Bjarne Gram; Genee, Hans Jasper; Kaas, Christian Schrøder

    2011-01-01

    BACKGROUND: Many secondary metabolites produced by filamentous fungi have potent biological activities, to which the producer organism must be resistant. An example of pharmaceutical interest is mycophenolic acid (MPA), an immunosuppressant molecule produced by several Penicillium species...... not produce MPA. The growth of an A. nidulans strain expressing mpaF was only marginally affected by MPA at concentrations as high as 200 μg/ml. To further substantiate the role of mpaF in MPA resistance, we searched for mpaF orthologs in six MPA producer/non-producer strains from Penicillium subgenus...

  15. Conversion of L-sorbosone to L-ascorbic acid by a NADP-dependent dehydrogenase in bean and spinach leaf. [Phaseolus vulgaris L. ; Spinacia oleracea L

    Energy Technology Data Exchange (ETDEWEB)

    Loewus, M.W.; Bedgar, D.L.; Saito, Kazumi; Loewus, F.A. (Washington State Univ., Pullman (USA))

    1990-11-01

    An NADP-dependent dehydrogenase catalyzing the conversion of L-sorbosone to L-ascorbic acid has been isolated from Phaseolus vulgaris L. and Spinacia oleracea L. and partially purified. It is stable at {minus}20{degree}C for up to 8 months. Molecular masses, as determined by gel filtration, were 21 and 29 kilodaltons for bean and spinach enzymes, respectively. K{sub m} for sorbosone were 12 {plus minus} 2 and 18 {plus minus} 2 millimolar and for NADP{sup +}, 0.14 {plus minus} 0.05 and 1.2 {plus minus} 0.5 millimolar, for bean and spinach, respectively. Lycorine, a purported inhibitor of L-ascorbic acid biosynthesis, had no effect on the reaction.

  16. Aspirin inhibits glucose‑6‑phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites.

    Science.gov (United States)

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D Ramesh; Alfonso, Lloyd F; Marimuthu, Srinivasan; Bhat, G Jayarama

    2016-08-01

    Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT‑29 colorectal cancer cells, in order to compare aspirin‑mediated acetylation of G6PD and its activity between HCT 116 and HT‑29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT‑29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin‑acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH.

  17. Electrical activity of cellobiose dehydrogenase adsorbed on thiols: Influence of charge and hydrophobicity.

    Science.gov (United States)

    Lamberg, P; Hamit-Eminovski, J; Toscano, M D; Eicher-Lorka, O; Niaura, G; Arnebrant, T; Shleev, S; Ruzgas, T

    2017-06-01

    The interface between protein and material surface is of great research interest in applications varying from implants, tissue engineering to bioelectronics. Maintaining functionality of bioelements depends greatly on the immobilization process. In the present study direct electron transfer of cellobiose dehydrogenase from Humicola insolens (HiCDH), adsorbed on four different self-assembled monolayers (SAMs) formed by 5-6 chain length carbon thiols varying in terminal group structure was investigated. By using a combination of quartz crystal microbalance with dissipation, ellipsometry and electrochemistry the formation and function of the HiCDH film was studied. It was found that the presence of charged pyridinium groups was needed to successfully establish direct electron contact between the enzyme and electrode. SAMs formed from hydrophilic charged thiols achieved nearly two times higher current densities compared to hydrophobic charged thiols. Additionally, the results also indicated proportionality between HiCDH catalytic constant and water content of the enzyme film. Enzyme films on charged pyridine thiols had smaller variations in water content and viscoelastic properties than films adsorbed on the more hydrophobic thiols. This work highlights several perspectives on the underlying factors affecting performance of immobilized HiCDH. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Succinate-dependent energy generation and pyruvate dehydrogenase complex activity in isolated Ascaris suum mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, T.A.

    1988-01-01

    Body wall muscle from the parasitic nematode, Ascaris suum, contain unique anaerobic mitochondria that preferentially utilize fumarate and branched-chain enoyl CoA's as terminal electron acceptors instead of oxygen. While electron transport in these organelles is well characterized, the role of oxygen in succinate-dependent phosphorylation is still not clearly defined. Therefore, the present study was designed to more fully characterize succinate metabolism in these organelles as well as the in vitro regulation of a key mitochondrial enzyme, the pyruvate dehydrogenase complex (PDC). In the absence of added adenine nucleotides, incubations in succinate resulted in substantial elevations in intramitochrondrial ATP levels, but ATP/ADP ratios were considerably higher in incubations with malate. The stimulation of phosphorylation in aerobic incubations with succinate was rotenone sensitive and appears to be Site I dependent. Increase substrate level phosphorylation, coupled to propionate formation, or additional sites of electron-transport associated ATP synthesis were not significant. Under aerobic conditions, {sup 14}CO{sub 2} evolution from 1,4-({sup 14}C)succinate was stimulated and NADH/NAD{sup +} ratios were elevated, but the formation of {sup 14}C propionate was unchanged.

  19. Cloning, protein sequence clarification, and substrate specificity of a leucine dehydrogenase from Bacillus sphaericus ATCC4525.

    Science.gov (United States)

    Li, Hongmei; Zhu, Dunming; Hyatt, Brooke A; Malik, Fahad M; Biehl, Edward R; Hua, Ling

    2009-08-01

    Although an X-ray model sequence of a leucine dehydrogenase from Bacillus sphaericus ATCC4525 was reported, the amino acid sequence of this enzyme has not been confirmed. In the current study, this leucine dehydrogenase gene was cloned, sequenced, and over-expressed in Escherichia coli, and the protein sequence has been clarified. This leucine dehydrogenase is not identical with that of B. sphaericus IFO3525 because there are 16 different amino acid residues between these two proteins. Since the information on the catalytic properties of leucine dehydrogenase from B. sphaericus ATCC4525 has been limited, the recombinant enzyme was purified as His-tagged protein and further studied. This enzyme showed activity toward aliphatic substrates for both oxidative deamination and reductive amination and is an effective catalyst for the asymmetric synthesis of alpha-amino acids from the corresponding alpha-ketoacids.

  20. Pronounced between‐subject and circadian variability in thymidylate synthase and dihydropyrimidine dehydrogenase enzyme activity in human volunteers

    Science.gov (United States)

    Jacobs, Bart A. W.; Deenen, Maarten J.; Pluim, Dick; van Hasselt, J. G. Coen; Krähenbühl, Martin D.; van Geel, Robin M. J. M.; de Vries, Niels; Rosing, Hilde; Meulendijks, Didier; Burylo, Artur M.; Cats, Annemieke; Beijnen, Jos H.; Huitema, Alwin D. R.

    2016-01-01

    Abstract Aims The enzymatic activity of dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) are important for the tolerability and efficacy of the fluoropyrimidine drugs. In the present study, we explored between‐subject variability (BSV) and circadian rhythmicity in DPD and TS activity in human volunteers. Methods The BSVs in DPD activity (n = 20) in peripheral blood mononuclear cells (PBMCs) and in plasma, measured by means of the dihydrouracil (DHU) and uracil (U) plasma levels and DHU : U ratio (n = 40), and TS activity in PBMCs (n = 19), were examined. Samples were collected every 4 h throughout 1 day for assessment of circadian rhythmicity in DPD and TS activity in PBMCs (n = 12) and DHU : U plasma ratios (n = 23). In addition, the effects of genetic polymorphisms and gene expression on DPD and TS activity were explored. Results Population mean (± standard deviation) DPD activity in PBMCs and DHU : U plasma ratio were 9.2 (±2.1) nmol mg−1 h−1 and 10.6 (±2.4), respectively. Individual TS activity in PBMCs ranged from 0.024 nmol mg−1 h−1 to 0.596 nmol mg−1 h−1. Circadian rhythmicity was demonstrated for all phenotype markers. Between 00:30 h and 02:00 h, DPD activity in PBMCs peaked, while the DHU : U plasma ratio and TS activity in PBMCs showed trough activity. Peak‐to‐trough ratios for DPD and TS activity in PBMCs were 1.69 and 1.62, respectively. For the DHU : U plasma ratio, the peak‐to‐trough ratio was 1.43. Conclusions BSV and circadian variability in DPD and TS activity were demonstrated. Circadian rhythmicity in DPD might be tissue dependent. The results suggested an influence of circadian rhythms on phenotype‐guided fluoropyrimidine dosing and supported implications for chronotherapy with high‐dose fluoropyrimidine administration during the night. PMID:27161955

  1. Pronounced between-subject and circadian variability in thymidylate synthase and dihydropyrimidine dehydrogenase enzyme activity in human volunteers.

    Science.gov (United States)

    Jacobs, Bart A W; Deenen, Maarten J; Pluim, Dick; van Hasselt, J G Coen; Krähenbühl, Martin D; van Geel, Robin M J M; de Vries, Niels; Rosing, Hilde; Meulendijks, Didier; Burylo, Artur M; Cats, Annemieke; Beijnen, Jos H; Huitema, Alwin D R; Schellens, Jan H M

    2016-09-01

    The enzymatic activity of dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) are important for the tolerability and efficacy of the fluoropyrimidine drugs. In the present study, we explored between-subject variability (BSV) and circadian rhythmicity in DPD and TS activity in human volunteers. The BSVs in DPD activity (n = 20) in peripheral blood mononuclear cells (PBMCs) and in plasma, measured by means of the dihydrouracil (DHU) and uracil (U) plasma levels and DHU : U ratio (n = 40), and TS activity in PBMCs (n = 19), were examined. Samples were collected every 4 h throughout 1 day for assessment of circadian rhythmicity in DPD and TS activity in PBMCs (n = 12) and DHU : U plasma ratios (n = 23). In addition, the effects of genetic polymorphisms and gene expression on DPD and TS activity were explored. Population mean (± standard deviation) DPD activity in PBMCs and DHU : U plasma ratio were 9.2 (±2.1) nmol mg(-1) h(-1) and 10.6 (±2.4), respectively. Individual TS activity in PBMCs ranged from 0.024 nmol mg(-1) h(-1) to 0.596 nmol mg(-1) h(-1) . Circadian rhythmicity was demonstrated for all phenotype markers. Between 00:30 h and 02:00 h, DPD activity in PBMCs peaked, while the DHU : U plasma ratio and TS activity in PBMCs showed trough activity. Peak-to-trough ratios for DPD and TS activity in PBMCs were 1.69 and 1.62, respectively. For the DHU : U plasma ratio, the peak-to-trough ratio was 1.43. BSV and circadian variability in DPD and TS activity were demonstrated. Circadian rhythmicity in DPD might be tissue dependent. The results suggested an influence of circadian rhythms on phenotype-guided fluoropyrimidine dosing and supported implications for chronotherapy with high-dose fluoropyrimidine administration during the night. © 2016 The British Pharmacological Society.

  2. Carbon Flux Trapping: Highly Efficient Production of Polymer-Grade d-Lactic Acid with a Thermophilic d-Lactate Dehydrogenase.

    Science.gov (United States)

    Li, Chao; Tao, Fei; Xu, Ping

    2016-08-17

    High production of polymer-grade d-lactic acid is urgently required, particularly for the synthesis of polylactic acid. High-temperature fermentation has multiple advantages, such as lower equipment requirement and energy consumption, which are essential for lowering operating costs. We identified and introduced a unique d-lactate dehydrogenase into a thermotolerant butane-2,3-diol-producing strain. Carbon flux "trapping" was achieved by a "trapping point" created by combination of the introduced enzyme and the host efflux pump, which afforded irreversible transport of d-lactic acid. The overall carbon flux of the engineered strain was significantly enhanced and was redistributed predominantly to d-lactic acid. Under optimized conditions at 50 °C, d-lactic acid reached the highest titer (226.6 g L(-1) ) reported to date. This discovery allows us to extend the carbon flux trapping strategy to engineering complex metabolic networks. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Supplementation of medium with diammonium hydrogen phosphate enhanced the D-lactate dehydrogenase levels leading to increased D-lactic acid productivity.

    Science.gov (United States)

    Singhvi, Mamata; Jadhav, Akanksha; Gokhale, Digambar

    2013-10-01

    The production of D-lactic acid by Lactobacillus lactis RM2-24 was investigated using modified media to increase the efficiency of the fermentation process. The results indicated that the addition of 5 g/l peptone and 1 g/l (NH4)2HPO4 enhanced D-lactic acid production by 32%, as compared to that obtained from non supplemented media, with a productivity of 3.0 g/l/h. Lactate dehydrogenase (LDH) expression profile in these different media was studied which resulted in appearance of additional LDH isoform produced by cells when they were grown in HSYE supplemented with (NH4)2HPO4. The additional LDH appears to be L-LDH contributing to production of L-lactic acid in the fermented broth. This is totally new information in the lactic acid fermentation and could be very useful to industries engaged in D-lactic acid production. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Levels of the retinoic acid synthesizing enzyme aldehyde dehydrogenase-1A2 are lower in testicular tissue from men with infertility.

    Science.gov (United States)

    Amory, John K; Arnold, Samuel; Lardone, María C; Piottante, Antonio; Ebensperger, Mauricio; Isoherranen, Nina; Muller, Charles H; Walsh, Thomas; Castro, Andrea

    2014-04-01

    To determine whether decreased testicular levels of enzymes necessary for retinoic acid biosynthesis were associated with male infertility, as retinoic acid is known to be necessary for spermatogenesis. Observational analysis of testicular tissue samples, sperm indices, and serum hormone concentrations. Two infertility centers in Chile. 32 infertile men and 11 control men. Measurement of the three enzymes necessary for retinoic acid biosynthesis, aldehyde dehydrogenase (ALDH) 1A1, 1A2, and 1A3, in testicular tissue by a novel liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) peptide assay. ALDH isozyme levels compared by type of infertility and correlated with testicular germ cell numbers, sperm parameters, and serum and intratesticular hormone concentrations. Men with infertility had statistically significantly reduced levels of ALDH1A2 but not ALDH1A1 or ALDH1A3 in their testicular tissue compared with men with normal spermatogenesis. The ALDH1A2 protein levels were strongly correlated with the number of germ cells found via testicular biopsy. These findings suggest that ALDH1A2 is the enzyme involved in retinoic acid biosynthesis in human germ cells. Further study of the relationship between intratesticular ALDH1A2 and male infertility is warranted to determine whether men with infertility have a reduced ability to synthesize retinoic acid within their germ cells that could impair spermatogenesis. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  5. Chronic inhibition of 11 β -hydroxysteroid dehydrogenase type 1 activity decreases hypertension, insulin resistance, and hypertriglyceridemia in metabolic syndrome.

    Science.gov (United States)

    Schnackenberg, Christine G; Costell, Melissa H; Krosky, Daniel J; Cui, Jianqi; Wu, Charlene W; Hong, Victor S; Harpel, Mark R; Willette, Robert N; Yue, Tian-Li

    2013-01-01

    Metabolic syndrome is a constellation of risk factors including hypertension, dyslipidemia, insulin resistance, and obesity that promote the development of cardiovascular disease. Metabolic syndrome has been associated with changes in the secretion or metabolism of glucocorticoids, which have important functions in adipose, liver, kidney, and vasculature. Tissue concentrations of the active glucocorticoid cortisol are controlled by the conversion of cortisone to cortisol by 11 β -hydroxysteroid dehydrogenase type 1 (11 β -HSD1). Because of the various cardiovascular and metabolic activities of glucocorticoids, we tested the hypothesis that 11 β -HSD1 is a common mechanism in the hypertension, dyslipidemia, and insulin resistance in metabolic syndrome. In obese and lean SHR/NDmcr-cp (SHR-cp), cardiovascular, metabolic, and renal functions were measured before and during four weeks of administration of vehicle or compound 11 (10 mg/kg/d), a selective inhibitor of 11 β -HSD1. Compound 11 significantly decreased 11 β -HSD1 activity in adipose tissue and liver of SHR-cp. In obese SHR-cp, compound 11 significantly decreased mean arterial pressure, glucose intolerance, insulin resistance, hypertriglyceridemia, and plasma renin activity with no effect on heart rate, body weight gain, or microalbuminuria. These results suggest that 11 β -HSD1 activity in liver and adipose tissue is a common mediator of hypertension, hypertriglyceridemia, glucose intolerance, and insulin resistance in metabolic syndrome.

  6. In Silico Identification and in Vitro Activity of Novel Natural Inhibitors of Trypanosoma brucei Glyceraldehyde-3-phosphate-dehydrogenase

    Directory of Open Access Journals (Sweden)

    Fabian C. Herrmann

    2015-09-01

    Full Text Available As part of our ongoing efforts to identify natural products with activity against pathogens causing neglected tropical diseases, we are currently performing an extensive screening of natural product (NP databases against a multitude of protozoan parasite proteins. Within this project, we screened a database of NPs from a commercial supplier, AnalytiCon Discovery (Potsdam, Germany, against Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH, a glycolytic enzyme whose inhibition deprives the parasite of energy supply. NPs acting as potential inhibitors of the mentioned enzyme were identified using a pharmacophore-based virtual screening and subsequent docking of the identified hits into the active site of interest. In a set of 700 structures chosen for the screening, 13 (1.9% were predicted to possess significant affinity towards the enzyme and were therefore tested in an in vitro enzyme assay using recombinant TbGAPDH. Nine of these in silico hits (69% showed significant inhibitory activity at 50 µM, of which two geranylated benzophenone derivatives proved to be particularly active with IC50 values below 10 µM. These compounds also showed moderate in vitro activity against T. brucei rhodesiense and may thus represent interesting starting points for further optimization.

  7. Chronic Inhibition of 11β-Hydroxysteroid Dehydrogenase Type 1 Activity Decreases Hypertension, Insulin Resistance, and Hypertriglyceridemia in Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Christine G. Schnackenberg

    2013-01-01

    Full Text Available Metabolic syndrome is a constellation of risk factors including hypertension, dyslipidemia, insulin resistance, and obesity that promote the development of cardiovascular disease. Metabolic syndrome has been associated with changes in the secretion or metabolism of glucocorticoids, which have important functions in adipose, liver, kidney, and vasculature. Tissue concentrations of the active glucocorticoid cortisol are controlled by the conversion of cortisone to cortisol by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1. Because of the various cardiovascular and metabolic activities of glucocorticoids, we tested the hypothesis that 11β-HSD1 is a common mechanism in the hypertension, dyslipidemia, and insulin resistance in metabolic syndrome. In obese and lean SHR/NDmcr-cp (SHR-cp, cardiovascular, metabolic, and renal functions were measured before and during four weeks of administration of vehicle or compound 11 (10 mg/kg/d, a selective inhibitor of 11β-HSD1. Compound 11 significantly decreased 11β-HSD1 activity in adipose tissue and liver of SHR-cp. In obese SHR-cp, compound 11 significantly decreased mean arterial pressure, glucose intolerance, insulin resistance, hypertriglyceridemia, and plasma renin activity with no effect on heart rate, body weight gain, or microalbuminuria. These results suggest that 11β-HSD1 activity in liver and adipose tissue is a common mediator of hypertension, hypertriglyceridemia, glucose intolerance, and insulin resistance in metabolic syndrome.

  8. Differential activation of pregnane X receptor by carnosic acid, carnosol, ursolic acid, and rosmarinic acid.

    Science.gov (United States)

    Seow, Chun Ling; Lau, Aik Jiang

    2017-06-01

    Pregnane X receptor (PXR) regulates the expression of many genes, including those involved in drug metabolism and transport, and has been linked to various diseases, including inflammatory bowel disease. In the present study, we determined whether carnosic acid and other chemicals in rosemary extract (carnosol, ursolic acid, and rosmarinic acid) are PXR activators. As assessed in dual-luciferase reporter gene assays, carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, activated human PXR (hPXR) and mouse PXR (mPXR), whereas carnosol and ursolic acid, but not carnosic acid or rosmarinic acid, activated rat PXR (rPXR). Dose-response experiments indicated that carnosic acid, carnosol, and ursolic acid activated hPXR with EC50 values of 0.79, 2.22, and 10.77μM, respectively. Carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, transactivated the ligand-binding domain of hPXR and recruited steroid receptor coactivator-1 (SRC-1), SRC-2, and SRC-3 to the ligand-binding domain of hPXR. Carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, increased hPXR target gene expression, as shown by an increase in CYP3A4, UGT1A3, and ABCB1 mRNA expression in LS180 human colon adenocarcinoma cells. Rosmarinic acid did not attenuate the extent of hPXR activation by rifampicin, suggesting it is not an antagonist of hPXR. Overall, carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, are hPXR agonists, and carnosic acid shows species-dependent activation of hPXR and mPXR, but not rPXR. The findings provide new mechanistic insight on the effects of carnosic acid, carnosol, and ursolic acid on PXR-mediated biological effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Efficient production of L-Lactic acid by metabolically engineered Saccharomyces cerevisiae with a genome-integrated L-lactate dehydrogenase gene.

    Science.gov (United States)

    Ishida, Nobuhiro; Saitoh, Satoshi; Tokuhiro, Kenro; Nagamori, Eiji; Matsuyama, Takashi; Kitamoto, Katsuhiko; Takahashi, Haruo

    2005-04-01

    We developed a metabolically engineered yeast which produces lactic acid efficiently. In this recombinant strain, the coding region for pyruvate decarboxylase 1 (PDC1) on chromosome XII is substituted for that of the l-lactate dehydrogenase gene (LDH) through homologous recombination. The expression of mRNA for the genome-integrated LDH is regulated under the control of the native PDC1 promoter, while PDC1 is completely disrupted. Using this method, we constructed a diploid yeast transformant, with each haploid genome having a single insertion of bovine LDH. Yeast cells expressing LDH were observed to convert glucose to both lactate (55.6 g/liter) and ethanol (16.9 g/liter), with up to 62.2% of the glucose being transformed into lactic acid under neutralizing conditions. This transgenic strain, which expresses bovine LDH under the control of the PDC1 promoter, also showed high lactic acid production (50.2 g/liter) under nonneutralizing conditions. The differences in lactic acid production were compared among four different recombinants expressing a heterologous LDH gene (i.e., either the bovine LDH gene or the Bifidobacterium longum LDH gene): two transgenic strains with 2microm plasmid-based vectors and two genome-integrated strains.

  10. Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast

    DEFF Research Database (Denmark)

    Schifferdecker, Anna Judith; Siurkus, Juozas; Andersen, Mikael Rørdam

    2016-01-01

    Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we...

  11. Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat.

    Science.gov (United States)

    Hutson, N J; Kerbey, A L; Randle, P J; Sugden, P H

    1978-08-01

    1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with

  12. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lactate dehydrogenase isoenzymes test system. 862... Test Systems § 862.1445 Lactate dehydrogenase isoenzymes test system. (a) Identification. A lactate dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase...

  13. Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera, Tephritidae fruit-fly species and their hybrids

    Directory of Open Access Journals (Sweden)

    Eneas Carvalho

    2009-01-01

    Full Text Available The ADH (alcohol dehydrogenase system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae. Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits.

  14. Activity and electrophoretic profiles of liver aldehyde dehydrogenases from mice of inbred strains with different alcohol preference.

    Science.gov (United States)

    Yamazaki, H; Nishiguchi, K; Miyamoto, R; Ogita, Z I; Nakanishi, S

    1983-01-01

    1. The activity of low Km-aldehyde dehydrogenase (ALDH) in the liver mitochondrial fraction (MT-fraction) from male C57BL/6J strain mice (alcohol preferring) was significantly higher than that from DBA/2 mice (alcohol avoiding). The F1 hybrids (C57BL/6J X DBA/2) did not exhibit the intermediate activity to these two strains. 2. Strain differences in liver mitochondrial ALDH isozymes were observed by isoelectric focusing. C57BL/6J strain had two isozymes at pH 7.1 while DBA/2 had no band at this pH. F1 hybrid mice had similar two bands with lower density to those of C57BL/6J at pH 7.1. There was no difference in zymograms of the soluble fraction between C57BL/6J and DBA/2 strains. 3. The present results suggest that the difference in alcohol preference of mice may depend on some restricted ALDH isozymes with different pl or electric mobility rather than the enzymatic activity in the liver MT-fraction.

  15. Spaceflight exposure effects on transcription, activity, and localization of alcohol dehydrogenase in the roots of Arabidopsis thaliana

    Science.gov (United States)

    Porterfield, D. M.; Matthews, S. W.; Daugherty, C. J.; Musgrave, M. E.

    1997-01-01

    Although considerable research and speculation have been directed toward understanding a plant's perception of gravity and the resulting gravitropic responses, little is known about the role of gravity-dependent physical processes in normal physiological function. These studies were conducted to determine whether the roots of plants exposed to spaceflight conditions may be experiencing hypoxia. Arabidopsis thaliana (L.) Heynh. plants were grown in agar medium during 6 or 11 d of spaceflight exposure on shuttle missions STS-54 (CHROMEX-03) and STS-68 (CHROMEX-05), respectively. The analysis included measurement of agar redox potential and root alcohol dehydrogenase (ADH) activity, localization, and expression. ADH activity increased by 89% as a result of spaceflight exposure for both CHROMEX-03 and -05 experiments, and ADH RNase protection assays revealed a 136% increase in ADH mRNA. The increase in ADH activity associated with the spaceflight roots was realized by a 28% decrease in oxygen availability in a ground-based study; however, no reduction in redox potential was observed in measurements of the spaceflight bulk agar. Spaceflight exposure appears to effect a hypoxic response in the roots of agar-grown plants that may be caused by changes in gravity-mediated fluid and/or gas behavior.

  16. Field Trial of the CareStart Biosensor Analyzer for the Determination of Glucose-6-Phosphate Dehydrogenase Activity in Haiti.

    Science.gov (United States)

    Weppelmann, Thomas A; von Fricken, Michael E; Wilfong, Tara D; Aguenza, Elisa; Philippe, Taina T; Okech, Bernard A

    2017-10-01

    Throughout many developing and tropical countries around the world, malaria remains a significant threat to human health. One barrier to malaria elimination is the ability to safely administer primaquine chemotherapy for the radical cure of malaria infections in populations with a high prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency. In the current study, a field trial of the world's first quantitative, point-of-care assay for measuring G6PD activity was conducted in Haiti. The performance of the CareStart Biosensor Analyzer was compared with the gold standard spectrophotometric assay and genotyping of the G6PD allele in schoolchildren (N = 343) from the Ouest Department of Haiti. In this population, 19.5% of participants (67/343) had some form of G6PD deficiency (< 60% residual activity) and 9.9% (34/343) had moderate-to-severe G6PD deficiency (< 30% residual activity). Overall, 18.95% of participants had the presence of the A-allele (65/343) with 7.87% (27/343) considered at high risk for drug-induced hemolysis (hemizygous males and homozygous females). Compared with the spectrophotometric assay, the sensitivity and specificity to determine participants with < 60% residual activity were 53.7% and 94.6%, respectively; for participants with 30% residual activity, the sensitivity and specificity were 5.9% and 99.7%, respectively. The biosensor overestimated the activity in deficient individuals and underestimated it in participants with normal G6PD activity, indicating the potential for a systematic measurement error. Thus, we suggest that the current version of the biosensor lacks adequate sensitivity and should be improved prior to its use as a point-of-care diagnostic for G6PD deficiency.

  17. Role of isovaleryl-CoA dehydrogenase and short branched-chain acyl-CoA dehydrogenase in the metabolism of valproic acid: implications for the branched-chain amino acid oxidation pathway

    NARCIS (Netherlands)

    Luís, Paula B. M.; Ruiter, Jos P. N.; Ijlst, Lodewijk; Tavares de Almeida, Isabel; Duran, Marinus; Mohsen, Al-Walid; Vockley, Jerry; Wanders, Ronald J. A.; Silva, Margarida F. B.

    2011-01-01

    Many biological systems including the oxidative catabolic pathway for branched-chain amino acids (BCAAs) are affected in vivo by valproate therapy. In this study, we investigated the potential effect of valproic acid (VPA) and some of its metabolites on the metabolism of BCAAs. In vitro studies were

  18. Fibroblast Fatty-Acid Oxidation Flux Assays Stratify Risk in Newborns with Presumptive-Positive Results on Screening for Very-Long Chain Acyl-CoA Dehydrogenase Deficiency

    Directory of Open Access Journals (Sweden)

    Simon E. Olpin

    2017-02-01

    Full Text Available Very-long chain acyl-CoA dehydrogenase deficiency (VLCADD is a clinically heterogeneous disorder with three major phenotypes: severe neonatal/infantile, milder childhood and late onset myopathic. VLCADD is genetically heterogeneous with numerous pathogenic mutations and variants of uncertain significance. VLCADD is included in many newborn screening programs but these suffer from high false positive rates, primarily due to positive screens in heterozygotes. Separating these and newborns with two low-risk “mild” variants from clinically at risk patients can be problematic, as clinical and biochemical markers are often unreliable, particularly in stable neonates. We have measured fibroblast fatty acid oxidation flux using [9,10-H3]myristic acid and [9,10-H3]oleic acid from 69 clinically presenting VLCADD patients including myopathic and infantile phenotypes and 13 positive newborn screened patients. We also measured fibroblast VLCADD enzyme activity by UV-HPLC detection of product in a sub-set of patients and compared these results to oleate FAO-flux. Fibroblast enzyme assay by UV-HPLC detection failed to clearly discriminate between some clinically presenting VLCADD patient cell lines and cell lines from some simple heterozygotes. FAO-flux clearly discriminated between clinically presenting VLCADD patients and the false positive screened patients. FAO-flux at 37 °C provides information as to the likely clinical phenotype but FAO-flux at 41 °C is the best discriminator for identifying clinically at risk patients.

  19. Intratumoural thymidylate synthase and dihydropyrimidine dehydrogenase activities are good predictors of 5-fluorouracil sensitivity in colorectal cancer.

    Science.gov (United States)

    Ishibiki, Y; Kitajima, M; Sakamoto, K; Tomiki, Y; Sakamoto, S; Kamano, T

    2003-01-01

    To identify factors that influence the clinical response to 5-fluorouracil (5-FU), we studied the correlation between in vitro sensitivity to 5-FU and the expression of seven biological markers. The markers, thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), pyrimidine nucleoside phosphorylase, p53 (wild/mutant), p21, cyclo-oxygenase-2, and inducible nitric oxide synthase were measured in tumour tissues from 32 colorectal cancer patients. The activities of TS and DPD were significantly lower in the tumours sensitive to 5-FU compared with those that were not sensitive to 5-FU. In tumours with TS < 3.7 pmol/min per mg protein and DPD < 98 pmol/min per mg protein, the percentage of cases sensitive to 5-FU (67%) and the mean percentage inhibition of tumour cells by 5-FU (42.8%) were significantly higher than in the other tumours (0% and 13.1%, respectively). The other biological markers did not correlate with in vitro sensitivity to 5-FU. Tumour sensitivity to 5-FU can be more precisely predicted by taking the activities of both TS and DPD into consideration than by using either alone.

  20. Preventive effects of Chlorella on skeletal muscle atrophy in muscle-specific mitochondrial aldehyde dehydrogenase 2 activity-deficient mice.

    Science.gov (United States)

    Nakashima, Yuya; Ohsawa, Ikuroh; Nishimaki, Kiyomi; Kumamoto, Shoichiro; Maruyama, Isao; Suzuki, Yoshihiko; Ohta, Shigeo

    2014-10-11

    Oxidative stress is involved in age-related muscle atrophy, such as sarcopenia. Since Chlorella, a unicellular green alga, contains various antioxidant substances, we used a mouse model of enhanced oxidative stress to investigate whether Chlorella could prevent muscle atrophy. Aldehyde dehydrogenase 2 (ALDH2) is an anti-oxidative enzyme that detoxifies reactive aldehydes derived from lipid peroxides such as 4-hydroxy-2-nonenal (4-HNE). We therefore used transgenic mice expressing a dominant-negative form of ALDH2 (ALDH2*2 Tg mice) to selectively decrease ALDH2 activity in the muscles. To evaluate the effect of Chlorella, the mice were fed a Chlorella-supplemented diet (CSD) for 6 months. ALDH2*2 Tg mice exhibited small body size, muscle atrophy, decreased fat content, osteopenia, and kyphosis, accompanied by increased muscular 4-HNE levels. The CSD helped in recovery of body weight, enhanced oxidative stress, and increased levels of a muscle impairment marker, creatine phosphokinase (CPK) induced by ALDH2*2. Furthermore, histological and histochemical analyses revealed that the consumption of the CSD improved skeletal muscle atrophy and the activity of the mitochondrial cytochrome c oxidase. This study suggests that long-term consumption of Chlorella has the potential to prevent age-related muscle atrophy.

  1. Chaetomium thermophilum formate dehydrogenase has high activity in the reduction of hydrogen carbonate (HCO3 -) to formate.

    Science.gov (United States)

    Aslan, Aşkın Sevinç; Valjakka, Jarkko; Ruupunen, Jouni; Yildirim, Deniz; Turner, Nicholas J; Turunen, Ossi; Binay, Barış

    2017-01-01

    While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO2 could also be utilized in the conversion of CO2 to useful products via formate (HCOO-). In this study, we investigated the reduction of CO2 in the form of hydrogen carbonate (HCO3-) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO3- as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO3- to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7-8. However, the high concentrations of HCO3- reduced the reaction rate. CtFDH was modeled in the presence of HCO3- showing that it fits to the active site. The active site setting for hydride transfer in CO2 reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO3-, resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Carbohydrate ingestion prior to exercise augments the exercise-induced activation of the pyruvate dehydrogenase complex in human skeletal muscle.

    Science.gov (United States)

    Tsintzas, K; Williams, C; Constantin-Teodosiu, D; Hultman, E; Boobis, L; Greenhaff, P

    2000-09-01

    This study examined the effect of pre-exercise carbohydrate (CHO) ingestion on pyruvate dehydrogenase complex (PDC) activation, acetyl group availability and substrate level phosphorylation (glycogenolysis and phosphocreatine (PCr) hydrolysis) in human skeletal muscle during the transition from rest to steady-state exercise. Seven male subjects performed two 10 min treadmill runs at 70 % maximum oxygen uptake (VO2,max), 1 week apart. Each subject ingested 8 ml (kg body mass (BM))-1 of either a placebo solution (CON trial) or a 5.5 % CHO solution (CHO trial) 10 min before each run. Muscle biopsy samples were obtained from the vastus lateralis at rest and immediately after each trial. Muscle PDC activity was higher at the end of exercise in the CHO trial compared with the CON trial (1.78+/-0.18 and 1.27+/-0.16 mmol min(-1) (kg wet matter (WM))(-1), respectively; P 0.05) and this was accompanied by lower acetylcarnitine (7.1+/-1.2 and 9.1+/-1.1 mmol kg(-1) (dry matter (DM))(-1) in CHO and CON, respectively; Ptransition from rest to steady-state exercise. However, those changes did not affect the contribution of substrate level phosphorylation to ATP resynthesis.

  3. Pathways of Amino Acid Degradation in Nilaparvata lugens (Stål) with Special Reference to Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase (LKR/SDH).

    Science.gov (United States)

    Wan, Pin-Jun; Yuan, San-Yue; Tang, Yao-Hua; Li, Kai-Long; Yang, Lu; Fu, Qiang; Li, Guo-Qing

    2015-01-01

    Nilaparvata lugens harbors yeast-like symbionts (YLSs). In present paper, a genome-wide analysis found 115 genes from Ni. lugens and 90 genes from YLSs that were involved in the metabolic degradation of 20 proteinogenic amino acids. These 205 genes encoded for 77 enzymes. Accordingly, the degradation pathways for the 20 amino acids were manually constructed. It is postulated that Ni. lugens can independently degrade fourteen amino acids (threonine, alanine, glycine, serine, aspartate, asparagine, phenylalanine, tyrosine, glutamate, glutamine, proline, histidine, leucine and lysine). Ni. lugens and YLSs enzymes may work collaboratively to break down tryptophan, cysteine, arginine, isoleucine, methionine and valine. We cloned a lysine-ketoglutarate reductase/saccharopine dehydrogenase gene (Nllkr/sdh) that encoded a bifunctional enzyme catalyzing the first two steps of lysine catabolism. Nllkr/sdh is widely expressed in the first through fifth instar nymphs and adults, and is highly expressed in the fat body, ovary and gut in adults. Ingestion of dsNllkr/sdh by nymphs successfully knocked down the target gene, and caused nymphal/adult mortality, shortened nymphal development stage and reduced adult fresh weight. Moreover, Nllkr/sdh knockdown resulted in three defects: wings were shortened and thickened; cuticles were stretched and thinned; and old nymphal cuticles remained on the tips of legs and abdomen and were not completely shed. These data indicate that impaired lysine degradation negatively affects the survival and development of Ni. lugens.

  4. Change of glutamate dehydrogenase activity in the organs of ornamental flowering plants under the influence of fe2+ and cr3+ excess

    Directory of Open Access Journals (Sweden)

    V. P. Bessonova

    2007-03-01

    Full Text Available The influence of Fe2+ and Cr3+ excess on glutamate dehydrogenase activity in the organs of ornamental flowering plants has been studied. The increase of enzymatic activity in leaves and roots of Tagetes patula L. was ascertained during all experiment. This index in Lathyrus odoratus L. had enlarged to the 30th day, whereupon went down in relation to a control.

  5. Alpha-ketoglutarate reduces ethanol toxicity in Drosophila melanogaster by enhancing alcohol dehydrogenase activity and antioxidant capacity.

    Science.gov (United States)

    Bayliak, Maria M; Shmihel, Halyna V; Lylyk, Maria P; Storey, Kenneth B; Lushchak, Volodymyr I

    2016-09-01

    Ethanol at low concentrations (Drosophila melanogaster, whereas at higher concentrations it may be toxic. In this work, protective effects of dietary alpha-ketoglutarate (AKG) against ethanol toxicity were studied. Food supplementation with 10-mM AKG alleviated toxic effects of 8% ethanol added to food, and improved fly development. Two-day-old adult flies, reared on diet containing both AKG and ethanol, possessed higher alcohol dehydrogenase (ADH) activity as compared with those reared on control diet or diet with ethanol only. Native gel electrophoresis data suggested that this combination diet might promote post-translational modifications of ADH protein with the formation of a highly active ADH form. The ethanol-containing diet led to significantly higher levels of triacylglycerides stored in adult flies, and this parameter was not altered by AKG supplement. The influence of diet on antioxidant defenses was also assessed. In ethanol-fed flies, catalase activity was higher in males and the levels of low molecular mass thiols were unchanged in both sexes compared to control values. Feeding on a mixture of AKG and ethanol did not affect catalase activity but caused a higher level of low molecular mass thiols compared to ethanol-fed flies. It can be concluded that both a stimulation of some components of antioxidant defense and the increase in ADH activity may be responsible for the protective effects of AKG diet supplementation in combination with ethanol. The results suggest that AKG might be useful as a treatment option to neutralize toxic effects of excessive ethanol intake and to improve the physiological state of D. melanogaster and other animals, potentially including humans. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Antimalarial activity of Evolvulus alsinoids Linn.-an in vitro Plasmodium falciparum specific lactate dehydrogenase enzyme inhibition assay

    OpenAIRE

    Neeraj Sethiya; Priyadarshan Keluskar; Sanjay Ingle; Shrihari Mishra

    2014-01-01

    Objective: To investigate the effect of standardized methanol extract of Evolvulus alsinoids Linn. (E. alsinoids) on Plasmodium falciparum specific lactate dehydrogenase (PfLDH) enzyme inhibition. Methods: To carry out enzyme inhibition studies, lactate dehydrogenase was cloned from Plasmodium falciparum 3D7 strain using expression vector pET28a and expressed in Escherichia coli BL21 (DE3). Protein purification was carried out by Ni-affinity chromatography. This protein was ...

  7. Changes in succinate dehydrogenase (SDH) activity in Paramecium caudatum cells in culture as function of age.

    Science.gov (United States)

    Higashijima, Y; Chikamori, K; Sato, K; Araki, T

    2000-05-01

    SDH activity and its frequency distributions were determined cytophotometrically to investigate the change in respiratory oxidative energy metabolism in Paramecium caudatum (P. caudatum) cell populations during their growth in cell culture. Cells from 3 separate cultures were examined on day 2, 5 and 11 post-inoculation as measures of logarithmic, early and late stationary phases, respectively. SDH activity per individual cell is expressed as the increase in total absorbance (TA) at 590 nm of nitroblue tetrazolium (Nitro BT) formazans per min per cell area in a specimen (deltaTA/min/microm2) to exclude the influence of cell size on the data. On day 5, the mean SDH activity was higher, being approx 140% of that on day 2 and decreased significantly to only approx 11% at day 11. As the mean SDH activity rose, a certain portion of the cells demonstrated a wider range of activity than on day 2 leading to an increase in the width of the SDH activity-frequency distribution. Moreover, on day 11, approx 85% of cells shifted toward the lowest range of activity with further increase in width of the distribution as the mean activity declined. These findings suggest that the respiratory oxidative energy metabolism in P. caudatum cells rise while the cells change from logarithmic to early stationary phases and decays during late stationary phase with increase in its extent of variety within cell populations.

  8. Improved production of homo-D-lactic acid via xylose fermentation by introduction of xylose assimilation genes and redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-Lactate dehydrogenase gene-deficient Lactobacillus plantarum.

    Science.gov (United States)

    Okano, Kenji; Yoshida, Shogo; Yamada, Ryosuke; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

    2009-12-01

    The production of optically pure d-lactic acid via xylose fermentation was achieved by using a Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase genes were replaced with a heterologous transketolase gene. After 60 h of fermentation, 41.2 g/liter of d-lactic acid was produced from 50 g/liter of xylose.

  9. Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.

    Science.gov (United States)

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-03-01

    Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Identification of a metabolically stable triazolopyrimidine-based dihydroorotate dehydrogenase inhibitor with anti-malarial activity in mice

    Science.gov (United States)

    Gujjar, Ramesh; Marwaha, Alka; El Mazouni, Farah; White, John; White, Karen L.; Creason, Sharon; Shackleford, David M.; Baldwin, Jeffrey; Charman, William N.; Buckner, Frederick S; Charman, Susan; Rathod, Pradipsinh K.; Phillips, Margaret A.

    2009-01-01

    Plasmodium faliciparum causes 1–2 million deaths annually, yet current drug therapies are compromised by resistance. We previously described potent and selective triazolopyrimidine-based inhibitors of P. falciparum dihydroorotate dehydrogenase (PfDHODH) that inhibited parasite growth in vitro, however they showed no activity in vivo. Here we show that lack of efficacy against P. berghei in mice resulted from a combination of poor plasma exposure, and reduced potency against P. berghei DHODH. For compounds containing naphthyl (DSM1) or anthracenyl (DSM2), plasma exposure was reduced upon repeated dosing. Phenyl-substituted triazolopyrimidines were synthesized leading to identification of analogs with low predicted metabolism in human liver microsomes and which showed prolonged exposure in mice. Compound 21 (DSM74), containing p-trifluoromethylphenyl, suppressed growth of P. berghei in mice after oral administration. This study provides the first proof of concept that DHODH inhibitors can suppress Plasmodium growth in vivo, validating DHODH as a new target for anti-malarial chemotherapy. PMID:19296651

  11. A single amino acid change (Y318F) in the L-arabitol dehydrogenase (LadA) from Aspergillus niger results in a significant increase in affinity for D-sorbitol.

    Science.gov (United States)

    Rutten, Lucy; Ribot, Cecile; Trejo-Aguilar, Blanca; Wösten, Han A B; de Vries, Ronald P

    2009-08-12

    L-arabitol dehydrogenase (LAD) and xylitol dehydrogenase (XDH) are involved in the degradation of L-arabinose and D-xylose, which are among the most abundant monosaccharides on earth. Previous data demonstrated that LAD and XDH not only differ in the activity on their biological substrate, but also that only XDH has significant activity on D-sorbitol and may therefore be more closely related to D-sorbitol dehydrogenases (SDH). In this study we aimed to identify residues involved in the difference in substrate specificity. Phylogenetic analysis demonstrated that LAD, XDH and SDH form 3 distinct groups of the family of dehydrogenases containing an Alcohol dehydrogenase GroES-like domain (pfam08240) and likely have evolved from a common ancestor. Modelling of LadA and XdhA of the saprobic fungus Aspergillus niger on human SDH identified two residues in LadA (M70 and Y318), that may explain the absence of activity on D-sorbitol. While introduction of the mutation M70F in LadA of A. niger resulted in a nearly complete enzyme inactivation, the Y318F resulted in increased activity for L-arabitol and xylitol. Moreover, the affinity for D-sorbitol was increased in this mutant. These data demonstrates that Y318 of LadA contributes significantly to the substrate specificity difference between LAD and XDH/SDH.

  12. A single amino acid change (Y318F in the L-arabitol dehydrogenase (LadA from Aspergillus niger results in a significant increase in affinity for D-sorbitol

    Directory of Open Access Journals (Sweden)

    Ribot Cecile

    2009-08-01

    Full Text Available Abstract Background L-arabitol dehydrogenase (LAD and xylitol dehydrogenase (XDH are involved in the degradation of L-arabinose and D-xylose, which are among the most abundant monosaccharides on earth. Previous data demonstrated that LAD and XDH not only differ in the activity on their biological substrate, but also that only XDH has significant activity on D-sorbitol and may therefore be more closely related to D-sorbitol dehydrogenases (SDH. In this study we aimed to identify residues involved in the difference in substrate specificity. Results Phylogenetic analysis demonstrated that LAD, XDH and SDH form 3 distinct groups of the family of dehydrogenases containing an Alcohol dehydrogenase GroES-like domain (pfam08240 and likely have evolved from a common ancestor. Modelling of LadA and XdhA of the saprobic fungus Aspergillus niger on human SDH identified two residues in LadA (M70 and Y318, that may explain the absence of activity on D-sorbitol. While introduction of the mutation M70F in LadA of A. niger resulted in a nearly complete enzyme inactivation, the Y318F resulted in increased activity for L-arabitol and xylitol. Moreover, the affinity for D-sorbitol was increased in this mutant. Conclusion These data demonstrates that Y318 of LadA contributes significantly to the substrate specificity difference between LAD and XDH/SDH.

  13. Myricetin is a novel inhibitor of human inosine 5′-monophosphate dehydrogenase with anti-leukemia activity

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Huiling; Hu, Qian; Wang, Jingyuan; Liu, Zehui; Wu, Dang [Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237 (China); Lu, Weiqiang, E-mail: wqlu@bio.ecnu.edu.cn [Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241 (China); Huang, Jin, E-mail: huangjin@ecust.edu.cn [Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237 (China)

    2016-09-02

    Human inosine 5′-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC{sub 50} values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanning fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity. - Highlights: • Myricetin, a common dietary flavonoid, is a novel inhibitor of hIMPDH1/2. • Myricetin directly binds with hIMPDH1/2 and induces cell cycle arrest and apoptosis of leukemia cells. • The cytotoxicity of myricetin on K562 cells is markedly attenuated by exogenous addition of guanosine.

  14. Peroxisome proliferator-activated receptors (PPAR) and the mitochondrial aldehyde dehydrogenase (ALDH2) promoter in vitro and in vivo.

    Science.gov (United States)

    Crabb, D W; Pinaire, J; Chou, W Y; Sissom, S; Peters, J M; Harris, R A; Stewart, M

    2001-07-01

    The aldehyde dehydrogenase 2 (ALDH2) promoter contains a nuclear receptor response element (NRRE) that represents an overlapping direct repeat-1 (DR-1) and -5 (DR-5) element. Because DR-1 elements are preferred binding sites for peroxisome proliferator-activated receptors (PPARs), we tested the hypothesis that PPARs regulate ALDH2 expression. We examined the ability of PPAR isoforms to bind to the ALDH2 NRRE in electrophoretic mobility shift assays, their ability to activate the transcription of promoter-reporter constructs containing this NRRE, the effect of PPAR ligands on ALDH2 expression in liver, and the role of the PPARalpha on the expression of ALDH2 by using PPARalpha-null mice. In vitro translated PPARs bound the ALDH NRRE with high affinity. Mutation of the NRRE indicated that binding was mediated by the DR-1 element. Cotransfection of PPAR expression plasmids showed that PPARalpha had no effect on expression of heterologous promoter constructs containing the NRRE. PPARgamma slightly induced expression, whereas PPARdelta repressed basal activity of the promoter and blocked induction by hepatocyte nuclear factor 4. Treatment of rats with the PPAR ligand clofibrate repressed expression of ALDH2 in rats fed either stock rodent chow or a low-protein diet. Consistent with the transfection data, expression of ALDH2 protein was not different in PPARalpha-null mice. Treatment of the mice with the PPARalpha agonist WY14643 slightly decreased the level of ALDH2 protein in both wild-type and PPARalpha-null mice, suggesting that the effect of WY14643 was not mediated by the receptor. These data indicate that ALDH2 is not part of the battery of lipid metabolizing enzymes and proteins regulated by PPARalpha

  15. Effects of Betaine Aldehyde Dehydrogenase-Transgenic Soybean on Phosphatase Activities and Rhizospheric Bacterial Community of the Saline-Alkali Soil

    Directory of Open Access Journals (Sweden)

    Ying Nie

    2016-01-01

    Full Text Available The development of transgenic soybean has produced numerous economic benefits; however the potential impact of root exudates upon soil ecological systems and rhizospheric soil microbial diversity has also received intensive attention. In the present study, the influence of saline-alkali tolerant transgenic soybean of betaine aldehyde dehydrogenase on bacterial community structure and soil phosphatase during growth stages was investigated. The results showed that, compared with nontransgenic soybean as a control, the rhizospheric soil pH of transgenic soybean significantly decreased at the seedling stage. Compared to HN35, organic P content was 13.5% and 25.4% greater at the pod-filling stage and maturity, respectively. The acid phosphatase activity of SRTS was significantly better than HN35 by 12.74% at seedling, 14.03% at flowering, and 59.29% at podding, while alkaline phosphatase achieved maximum activity in the flowering stage and was markedly lower than HN35 by 13.25% at pod-filling. The 454 pyrosequencing technique was employed to investigate bacterial diversity, with a total of 25,499 operational taxonomic units (OTUs obtained from the 10 samples. Notably, the effect of SRTS on microbial richness and diversity of rhizospheric soil was marked at the stage of podding and pod-filling. Proteobacteria, Acidobacteria, and Actinobacteria were the dominant phyla among all samples. Compared with HN35, the relative abundance of Proteobacteria was lower by 2.01%, 2.06%, and 5.28% at the stage of seedling, at pod-bearing, and at maturity. In genus level, the relative abundance of Gp6, Sphingomonas sp., and GP4 was significantly inhibited by SRTS at the stage of pod-bearing and pod-filling.

  16. The activity of glucose-6-phosphate dehydrogenase (G6PD) in ...

    African Journals Online (AJOL)

    ... and ultraviolet (UV) spectrophotometric quantitative methods. The data obtained were statistically analyzed using student's t-test and analysis of variance. Results: There was statistically significant decrease in the G6PD activity from the third week of storage (p<0.05) at the blood bank, under optimum storage conditions.

  17. Developmental Defects of Caenorhabditis elegans Lacking Branched-chain α-Ketoacid Dehydrogenase Are Mainly Caused by Monomethyl Branched-chain Fatty Acid Deficiency.

    Science.gov (United States)

    Jia, Fan; Cui, Mingxue; Than, Minh T; Han, Min

    2016-02-05

    Branched-chain α-ketoacid dehydrogenase (BCKDH) catalyzes the critical step in the branched-chain amino acid (BCAA) catabolic pathway and has been the focus of extensive studies. Mutations in the complex disrupt many fundamental metabolic pathways and cause multiple human diseases including maple syrup urine disease (MSUD), autism, and other related neurological disorders. BCKDH may also be required for the synthesis of monomethyl branched-chain fatty acids (mmBCFAs) from BCAAs. The pathology of MSUD has been attributed mainly to BCAA accumulation, but the role of mmBCFA has not been evaluated. Here we show that disrupting BCKDH in Caenorhabditis elegans causes mmBCFA deficiency, in addition to BCAA accumulation. Worms with deficiency in BCKDH function manifest larval arrest and embryonic lethal phenotypes, and mmBCFA supplementation suppressed both without correcting BCAA levels. The majority of developmental defects caused by BCKDH deficiency may thus be attributed to lacking mmBCFAs in worms. Tissue-specific analysis shows that restoration of BCKDH function in multiple tissues can rescue the defects, but is especially effective in neurons. Taken together, we conclude that mmBCFA deficiency is largely responsible for the developmental defects in the worm and conceivably might also be a critical contributor to the pathology of human MSUD. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Novel strategy for phenyllactic acid biosynthesis from phenylalanine by whole cell recombinant Escherichia coli coexpressing L-phenylalanine oxidase and L-lactate dehydrogenase.

    Science.gov (United States)

    Zhang, Jianzhi; Li, Xi

    2018-01-01

    To enhance the efficiency of phenyllactic acid (PLA) production from L-phenylalanine (L-Phe) by introducing a novel artificial pathway into Escherichia coli RESULTS: The production of PLA from L-Phe by recombinant E. coli (ldh-lpox) coexpressing L-phenylalanine oxidase and L-lactate dehydrogenase was studied. The new PLA synthesis pathway was confirmed to be efficient in recombinant E. coli. Subsequently, two different biocatalyst processes were carried out and optimized for PLA production. In the whole cell biosynthesis process at high cell density using collected recombinant cells as catalyst, at optimal conditions (L-Phe 6 g/l, pH 7.5, 35 °C, CDW 24.5 g/l and 200 rpm), the recombinant E. coli (ldh-lpox) produced 1.62 g PLA/l with a conversion of 28% from L-Phe. Similarly, during the two-temperature-stage fermentation process in flasks using IPTG-induced cells, the temperature in the second stage was increased to 35 °C to benefit the biocatalyst process, and comparable phenyllactic acid production of 1.47 g/l was obtained from 12 g L-Phe/l. Recombinant E. coli (ldh-lpox) was efficient in PLA production realizing a high titer of several folds compared with studies using L-Phe as substrate.

  19. Dihydroorotate dehydrogenase inhibitor F901318 has potent in vitro activity against Scedosporium species and Lomentospora prolificans.

    Science.gov (United States)

    Wiederhold, Nathan P; Law, Derek; Birch, Michael

    2017-07-01

    Scedosporium species and Lomentospora prolificans are increasing causes of invasive infections in immunocompromised hosts and many isolates are resistant to available antifungals. Our objective was to assess the in vitro potency of F901318, a member of the orotomide class of antifungals, against Scedosporium species and L. prolificans . The in vitro potency of F901318 was evaluated against 66 Scedosporium and 7 L. prolificans clinical isolates using the CLSI M38-A2 reference standard. Scedosporium species included Scedosporium apiospermum ( n  =   43), Scedosporium aurantiacum ( n  =   6), Scedosporium dehoogii ( n  =   2) and Scedosporium boydii ( n  =   15). Positive comparators included amphotericin B, caspofungin, posaconazole and voriconazole. Against S. apiospermum and S. boydii F901318 geometric mean MICs/MECs (0.079 and 0.046 mg/L, respectively) were significantly lower than those observed with amphotericin (3.404 and 5.595 mg/L), posaconazole (1.937 and 1.823 mg/L), voriconazole (0.784 and 0.630 mg/L) and caspofungin (5.703 and 7.639 mg/L) ( P  8 mg/L). F901318 also maintained activity against L. prolificans isolates (range 0.12-0.25 mg/L) in contrast to other antifungals, of which none demonstrated in vitro activity. F901318 demonstrated potent in vitro activity against Scedosporium species and L. prolificans . This activity was maintained against isolates that had significantly reduced susceptibility to the other antifungals. Further studies are warranted to evaluate the in vivo efficacy of F901318 against Scedosporium species and L. prolificans .

  20. Dehydrogenase activity and quality of leachates in Technosols with gossan and sulfide materials from the São Domingos mine

    Science.gov (United States)

    Santos, Erika; Abreu, Manuela; Macías, Felipe; de Varennes, Amarílis

    2014-05-01

    Wastes produced by mining activity in São Domingos (Portuguese Iberian Pyrite Belt) were disposed over a large area. To speed up the ecological rehabilitation in this mine, an integrative strategy using different amendments+mine wastes was used to produce Technosols with enhanced soil functions. To evaluate the efficiency of these Technosols the dehydrogenase activity and chemical quality of leachates were monitored. Technosols were composed of different mine wastes (gossan and sulfide materials), collected at the São Domingos mine, and mixtures of amendments applied at 30 and 75 Mg/ha (rockwool+agriculture wastes+wastes from liquors distillation of strawberry tree fruits (Arbutus unedo L.) and/or carobs (Ceratonia siliqua L. fruits)). Three assays, under controlled conditions, were carried out: (1 and 2) Sulfide or gossan materials with/without amendments; (3) Sulfide wastes, with/without amendments, incubated during four months and then with application of an overlayer of gossan (~3 cm thick) with/without the same amendments. Dehydrogenase activity (DHA) and chemical characteristics of leachates (multielemental concentration, pH, and electric conductivity) were determined after four/seven/thirteen months of incubation. Sulfide wastes had more hazardous characteristics (pH~2 and total concentrations (g/kg) of Al (58.1), As (1.1), Cu (2.1), Fe (107.3), Pb (11.7), S (65.3) and Zn (1.1) than the gossan materials (pH=4.3; g/kg, Al: 24.8, As: 3.0, Cu: 0.2, Fe: 129, Pb: 9.2, S: 13.7, Zn: 0.04). Amendments application to gossan (assay 2) enhanced DHA in both sampling periods (µg TPF g dry weight 16 h-1, Control: 0,72-1,78; Amended treatments: 2.49-16.36 depending on mixture/application rate/sampling period). Greater application rates stimulated DHA (more than 1.5-fold with 75 Mg/ha). No differences were observed in DHA in the gossan layer with/without amendments (assay 3) suggesting a negative impact on gossan microrganisms from sulfide materials located below. In

  1. Biological activities of substituted trichostatic acid derivatives

    Indian Academy of Sciences (India)

    Administrator

    Abstract. New substituted trichostatic acid derivatives have been synthesized and evaluated for their biological activities towards the H661 non-small lung cancer cell line. These syntheses were achieved by alkylation of propiophenones to introduce the side chain with a terminal precursor of hydroxamic acid.

  2. Biological activities of substituted trichostatic acid derivatives

    Indian Academy of Sciences (India)

    New substituted trichostatic acid derivatives have been synthesized and evaluated for their biological activities towards the H661 non-small lung cancer cell line. These syntheses were achieved by alkylation of propiophenones to introduce the side chain with a terminal precursor of hydroxamic acid and aminobenzamide ...

  3. Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer

    Directory of Open Access Journals (Sweden)

    Iniesta Pilar

    2011-08-01

    Full Text Available Abstract Background Mortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312. Results The aldehyde dehydrogenase (ALDH positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect and through decrease in telomere length (long-term effect. Administration of this telomerase inhibitor (40 mg/kg in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls. Combination therapy consisting of irradiation (10Gy plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length in vivo. Conclusions We conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer.

  4. Effects of the cofactor binding sites on the activities of secondary alcohol dehydrogenase (SADH).

    Science.gov (United States)

    Wang, Tao; Chen, Xiangjun; Han, Jun; Ma, Sichun; Wang, Jianmei; Li, Xufeng; Zhang, Hui; Liu, Zhibin; Yang, Yi

    2016-07-01

    SADHs from Thermoanaerobacter ethanolicus are enzymes that, together with various cofactors, catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. To explore how cofactors bind to SADH, TeSADH was cloned in this study, and Ser(199) and Arg(200) were replaced by Tyr and Asp, respectively. Both sites were expected to be inside or adjacent to the cofactor-binding domain according to computational a prediction. Analysis of TeSADH activities revealed that the enzymatic efficiency (kcat/Km) of the S199Y mutant was noticeably enhanced using by NADH, NADPH as cofactors, and similar with that of wild-type using by NADP(+), NAD(+). Conversely, the activity of the R200D mutant significantly decreased with all cofactors. Furthermore, in yeast, the S199Y mutant substantially elevated the ethanol concentration compared with the wild type. Molecular dynamics simulation results indicated the H-bonding network between TeSADH and the cofactors was stronger for the S199Y mutant and the binding energy was simultaneously increased. Moreover, the fluorescence results indicated the S199Y mutant exhibited an increased preference for NAD(P)H, binding with NAD(P)H more compactly compared with wild type. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Neuropathology in Succinic Semialdehyde Dehydrogenase Deficiency

    NARCIS (Netherlands)

    Knerr, I.; Gibson, K.M.; Murdoch, G.; Salomons, G.S.; Jakobs, C.; Combs, S.; Pearl, P.L.

    2010-01-01

    Reported here is the novel finding of neuropathology in a patient with succinic semialdehyde dehydrogenase deficiency, an inherited disorder of γ-aminobutyric acid metabolism characterized by intellectual deficiency, hypotonia, and epilepsy, with 4-hydroxybutyric aciduria and abnormalities of the

  6. A quantitative cytochemical study of glucose-6-phosphate dehydrogenase and delta 5-3 beta-hydroxysteroid dehydrogenase activity in the membrana granulosa of the ovulable type of follicle of the rat.

    Science.gov (United States)

    Zoller, L C; Weisz, J

    1979-08-01

    During the last four days of follicular development prior to ovulation, the activities of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta OHD) and glucose-6-phosphate dehydrogenase (G-6-PD) were quantified in cryostat sections of the rat ovary. The product of the enzyme reactions were measured using a scanning and integrating microdensitometer. The enzyme activity was measured in the peripheral region, the antral region and the cumulus of the membrana granulosa (MG) of these follicles on the morning of each of the four days of the estrous cycle. G-6-PD activity was measured in the presence and absence of an intermediate hydrogen acceptor, phenazine methosulphate, to provide a measure of the quantity of Type I and Type II Hydrogen (H) generated: Type I H is considered to be related to hydroxylating reactions such as those of steroids and Type II H to other general biosynthetic activities of cells. In all three regions of the MG of follicles of the ovulable type, 3 beta OHD activity was lowest in estrus and diestrus-1, increased on diestrus-2 and peaked in proestrus. In estrus and diestrus-1, the level of 3 beta OHD activity in the three regions was comparable. However, by diestrus-2, and even more conspicuously in proestrus, enzyme activity was significantly greater in the peripheral region than in the antral region or in the cumulus. During the same period, the level of enzyme activity remained comparable in the last two regions. Throughout the estrous cycle, both Type I and Type II H generation from G-6-PD was greatest in the peripheral region, less in the antral region and least in the cumulus. In the eripheral region, Type I H generation increased progressively after diestrus-1, to reach a maximum in prestrus. In the antral region, Type I H generation increased between diestrus-1 and diestrus-2 and then remained unchanged through proestrus. In the cumulus, Type I H generation remained at levels seen in estrus throughout the remainder of the cycle. Generation

  7. Elevated lactate dehydrogenase activity and increased cardiovascular mortality in the arsenic-endemic areas of southwestern Taiwan

    Energy Technology Data Exchange (ETDEWEB)

    Liao, Ya-Tang [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan (China); Graduate Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taiwan (China); Genomics Research Center, Academia Sinica, Taiwan (China); Chen, Chien-Jen [Graduate Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taiwan (China); Genomics Research Center, Academia Sinica, Taiwan (China); Li, Wan-Fen [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan (China); Hsu, Ling-I [Genomics Research Center, Academia Sinica, Taiwan (China); Tsai, Li-Yu; Huang, Yeou-Lih [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Taiwan (China); Sun, Chien-Wen [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan (China); Chen, Wei J., E-mail: wjchen@ntu.edu.tw [Graduate Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taiwan (China); Genetic Epidemiology Core Laboratory, National Taiwan University Center for Genomic Medicine, Taiwan (China); Wang, Shu-Li, E-mail: slwang@nhri.org.tw [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan (China); Department of Public Health, College of Public Health, China Medical University, Taichung, Taiwan (China)

    2012-08-01

    Arsenic ingestion has been linked to increasing global prevalence of and mortality from cardiovascular disease (CVD); arsenic can be removed from drinking water to reduce related health effects. Lactate dehydrogenase (LDH) is used for the evaluation of acute arsenic toxicity in vivo and in vitro, but it is not validated for the evaluation of long-term, chronic arsenic exposure. The present study examined the long-term effect of chronic arsenic exposure on CVD and serum LDH levels, after consideration of arsenic metabolism capacity. A total of 380 subjects from an arseniasis-endemic area and 303 from a non-endemic area of southwestern Taiwan were recruited in 2002. Various urinary arsenic species were analyzed using high-performance liquid chromatography (HPLC) and hydride generation systems. Fasting serum was used for quantitative determination of the total LDH activity. A significant dose–response relationship was observed between arsenic exposure and LDH elevation, independent of urinary arsenic profiles (P < 0.001). Furthermore, abnormal LDH elevation was associated with CVD mortality after adjustment for Framingham risk scores for 10-year CVD and arsenic exposure (hazard ratio, 3.98; 95% confidence interval, 1.07–14.81). LDH was elevated in subjects with arsenic exposure in a dose-dependent manner. LDH is a marker of arsenic toxicity associated with CVD mortality. Results of this study have important implications for use in ascertaining long-term arsenic exposure risk of CVD. -- Highlights: ► We showed that arsenic exposure was correlated with LDH elevation. ► LDH elevation was related to arsenic methylation capacity. ► Abnormal LDH elevation can be a marker of susceptibility to CVD mortality.

  8. Effect of Follicular Fluid and Platelet-Activating Factor on Lactate Dehydrogenase C Expression in Human Asthenozoospermic Samples

    Directory of Open Access Journals (Sweden)

    Tahereh Esmaeilpour

    2014-01-01

    Full Text Available Background: Application of follicular fluid (FF and platelet-activating factor (PAF in artificial insemination improves sperm motility. Lactate dehydrogenase C (LDH-C is a key enzyme for sperm motility. In this study, the effects of FF and PAF on the sperm motility index and LDH-C expression were investigated. Moreover, LDH-C expression was compared between asthenozoospermic and normozoospermic samples. Methods: The expression of LDH-C was examined by quantitative real-time polymerase chain reaction (q-RT PCR and western blotting after it was treated with optimized concentrations of FF and PAF in twenty asthenozoospermic samples. Also, LDH-C expression was evaluated in five normozoospermic samples. Results: Samples with 75% FF and 100 nM of PAF had an increase in their percentages of progressive and slowly motile sperms and a decrease in their percentages of non-progressive and non-motile sperms. Moreover, LDH-C mRNA transcripts were not changed following PAF and FF treatment, and LDH-C protein was detected in highly progressive motile specimens treated with FF in the asthenozoospermic samples. Furthermore, LDH-C expression was more detectable in the normal sperms. Conclusion: Our results indicated that PAF had more beneficial effects than FF on sperm motility in the asthenozoospermic samples (P=0.0001, although the LDH-C expressions of the sperms were not changed significantly in both groups. We found no association between LDH-C expression and sperm motility after FF and PAF actions. This finding, however, requires further investigation. The fact that LDH-C protein was detected in the normozoospermic, but not asthenozoospermic, samples could be cited as a reason for the infertility in these patients.

  9. Differential impact of amino acids on OXPHOS system activity following carbohydrate starvation in Arabidopsis cell suspensions.

    Science.gov (United States)

    Cavalcanti, João Henrique F; Quinhones, Carla G S; Schertl, Peter; Brito, Danielle S; Eubel, Holger; Hildebrandt, Tatjana; Nunes-Nesi, Adriano; Braun, Hans-Peter; Araújo, Wagner L

    2017-12-01

    Plant respiration mostly depends on the activity of glycolysis and the oxidation of organic acids in the tricarboxylic acid cycle to synthesize ATP. However, during stress situations plant cells also use amino acids as alternative substrates to donate electrons through the electron-transfer flavoprotein (ETF)/ETF:ubiquinone oxidoreductase (ETF/ETFQO) complex to the mitochondrial electron transport chain (mETC). Given this, we investigated changes of the oxidative phosphorylation (OXPHOS) system in Arabidopsis thaliana cell culture under carbohydrate starvation supplied with a range of amino acids. Induction of isovaleryl-CoA dehydrogenase (IVDH) activity was observed under carbohydrate starvation which was associated with increased amounts of IVDH protein detected by immunoblotting. Furthermore, activities of the protein complexes of the mETC were reduced under carbohydrate starvation. We also observed that OXPHOS system activity behavior is differently affected by different amino acids and that proteins associated with amino acids catabolism are upregulated in cells following carbohydrate starvation. Collectively, our results support the contention that ETF/ETFQO is an essential pathway to donate electrons to the mETC and that amino acids are alternative substrates to maintain respiration under carbohydrate starvation. © 2017 Scandinavian Plant Physiology Society.

  10. The effects of chemical and radioactive properties of Tl-201 on human erythrocyte glucose 6-phosphate dehydrogenase activity

    Energy Technology Data Exchange (ETDEWEB)

    Sahin, Ali [Faculty of Medicine, Department of Nuclear Medicine, Ataturk University, Erzurum 25240 (Turkey)], E-mail: alibabam2001@yahoo.com; Senturk, Murat [Science Faculty, Department of Chemistry, Ataturk University, Erzurum 25240 (Turkey); Ciftci, Mehmet [Science and Arts Faculty, Department of Chemistry, Agri Ibrahim Cecen University, 04100, Agri (Turkey); Varoglu, Erhan [Faculty of Medicine, Department of Nuclear Medicine, Ataturk University, Erzurum 25240 (Turkey); Kufrevioglu, Omer Irfan [Science and Arts Faculty, Department of Chemistry, Agri Ibrahim Cecen University, 04100, Agri (Turkey)

    2010-04-15

    Aim: The inhibitory effects of thallium-201 ({sup 201}Tl) solution on human erythrocyte glucose 6-phosphate dehydrogenase (G6PD) activity were investigated. Methods: For this purpose, erythrocyte G6PD was initially purified 835-fold at a yield of 41.7% using 2',5'-Adenosine diphosphate sepharose 4B affinity gel chromatography. The purification was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed a single band for the final enzyme preparation. The in vitro and in vivo effects of the {sup 201}Tl solution including Tl{sup +}, Fe{sup +3} and Cu{sup +2} metals and the in vitro effects of the radiation effect of the {sup 201}Tl solution and non-radioactive Tl{sup +}, Fe{sup +3} and Cu{sup +2} metals on human erythrocyte G6PD enzyme were studied. Enzyme activity was determined with the Beutler method at 340 nm using a spectrophotometer. All purification procedures were carried out at +4 deg. C. Results: {sup 201}Tl solution and radiation exposure had inhibitory effects on the enzyme activity. IC{sub 50} value of {sup 201}Tl solution was 36.86 {mu}l ([Tl{sup +}]: 0.0036 {mu}M, [Cu{sup +2}]: 0.0116 {mu}M, [Fe{sup +3}]: 0.0132 {mu}M), of human erythrocytes G6PD. Seven human patients were also used for in vivo studies of {sup 201}Tl solution. Furthermore, non-radioactive Tl{sup +}, Fe{sup +3} and Cu{sup +2} were found not to have influenced the enzyme in vitro. Conclusion: Human erythrocyte G6PD activity was inhibited by exposure for up to 10 minutes to 0.057 mCi/kg {sup 201}Tl solution. It was detected in in vitro and in vivo studies that the human erythrocyte G6PD enzyme is inhibited due to the radiation effect of {sup 201}Tl solution.

  11. Comparison of endogenous cytokinins and cytokinin oxidase/dehydrogenase activity in germinating and thermoinhibited Tagetes minuta achenes.

    Science.gov (United States)

    Stirk, Wendy A; Novák, Ondřej; Zižková, Eva; Motyka, Vaclav; Strnad, Miroslav; van Staden, Johannes

    2012-05-01

    Tagetes minuta L. achenes are thermoinhibited at temperatures above 35°C and have accelerated radicle emergence (germination) when subsequently transferred to an optimal temperature (25°C). Endogenous cytokinins and cytokinin oxidase/dehydrogenase (CKX) activity were compared in normally germinating (25°C) and thermoinhibited (72h at 36°C then transferred to 25°C) T. minuta achenes. Following imbibition, endogenous cytokinin concentrations changed in normally germinating T. minuta achenes, with a gradual decrease in dihydrozeatin-type (DHZ) cytokinins, a large increase in cis-zeatin-type (cZ) cytokinins, a smaller increase in N⁶-(2-isopentenyl)adenine-type (iP) cytokinins and a peak of trans-zeatin-type (tZ) cytokinins at 13 h. These changes in the isoprenoid cytokinin profile were similar in the thermoinhibited achenes imbibed at 36°C, despite the thermal block preventing radicle emergence. The exception was the iP-type cytokinins that only increased when transferred to 25°C. Profiles of the physiologically active free bases showed an increase in tZ prior to radical emergence in both normally germinating (13 h) and thermoinhibited achenes. A large transient peak in aromatic cytokinins [N⁶-benzyladenine-type (BA)] occurred during early seedling establishment in normally germinating achenes (40 h) while a transient maximum in BA-type cytokinins was found prior to radicle emergence in the thermoinhibited achenes (24 h). The CKX activity was enhanced in normally germinating achenes as the cytokinin concentration increased following imbibition. In thermoinhibited achenes, an elevated temperature negatively affected the CKX activity that only increased when the achenes were transferred to 25°C, corresponding to an increase in iP-type cytokinins. However, the favored cytokinin deactivation pathway in T. minuta appears to be 9-glycosylation, as 9-glucosides accounted for over 50% of the total cytokinin pool in both normal and thermoinhibited achenes. Copyright

  12. Atividade da 6-fosfogliconato desidrogenase em deficientes de glicose-6-fosfato desidrogenase Activity of 6-phosphogluconate dehydrogenase in glucose-6-phosphate dehydrogenase deficiency

    Directory of Open Access Journals (Sweden)

    Daniela B. Nicolielo

    2006-06-01

    to know better the actuation of these enzymes. The goal of this study was to evaluate the 6PGD enzymatic activity on a population with G6PD deficiency, to verify if there is an elevation of the activity of this enzyme, and try to correlate to a possible increase on the number of reticulocytes or the presence of alterations on red series. The research with 2657 male individuals detected 97 deficient for G6PD, which determined a 3.65% prevalence for the Bauru (SP region, with mean enzymatic activity of 1.74 UI.g Hb-1. min-1 at 37ºC, 14,4% of the normal G6PD activity. Mean 6PGD enzymatic activity was 9.5 UI.g Hb-1. min-1 at 37ºC, and was elevated in 47.4% of the G6PD deficient individuals. The result obtained did not confirm the hypothesis that the elevation of the 6PGD enzymatic activity, in G6PD deficient individuals, was due to the presence of an increase of reticulocytes in blood stream, age or erythrocytometric alterations that could denote anemia. The most plausible theory is that the auto-limited hemolysis, imposed by oxidative processes, preserves young erythrocytes that have an elevated enzymatic activity, as naturally these enzymes lose activity with cellular aging.

  13. Structural characterization of a β-hydroxyacid dehydrogenase from Geobacter sulfurreducens and Geobacter metallireducens with succinic semialdehyde reductase activity

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Zheng, Yi; Qin, Ling; Wang, Shihua; Buchko, Garry W.; Garavito, Michael R.

    2014-07-30

    Beta-hydroxyacid dehydrogenase (β-HAD) genes have been identified in all sequenced genomes of eukaryotes and prokaryotes. Their gene products catalyze the NAD+- or NADP+-dependent oxidation of various β-hydroxy acid substrates into their corresponding semialdehyde. In many fungal and bacterial genomes, multiple β-HAD genes are observed leading to the hypothesis that these gene products may have unique, uncharacterized metabolic roles specific to their species. The genomes of Geobacter sulfurreducens and Geobacter metallireducens each contain two potential β-HAD genes. The protein sequences of one pair of these genes, Gs-βHAD (Q74DE4) and Gm-βHAD (Q39R98), have 65% sequence identity and 77% sequence similarity with each other. Both proteins reduce succinic semialdehyde, a metabolite of the GABA shunt. To further explore the structural and functional characteristics of these two β-HADs with a potentially unique substrate specificity, crystal structures for Gs-βHAD and Gm-βHAD in complex with NADP+ were determined to a resolution of 1.89 Å and 2.07 Å, respectively. The structure of both proteins are similar, composed of 14 α-helices and nine β-strands organized into two domains. Domain One (1-165) adopts a typical Rossmann fold composed of two α/β units: a six-strand parallel β-sheet surrounded by six α-helices (α1 – α6) followed by a mixed three-strand β-sheet surrounded by two α-helices (α7 and α8). Domain Two (166-287) is composed of a bundle of seven α-helices (α9 – α14). Four functional regions conserved in all β-HADs are spatially located near each other at the interdomain cleft in both Gs-βHAD and Gm-βHAD with a buried molecule of NADP+. The structural features of Gs-βHAD and Gm-βHAD are described in relation to the four conserved consensus sequences characteristic of β-HADs and the potential biochemical importance of these enzymes as an alternative pathway for the degradation of succinic semialdehyde.

  14. Selective inhibition of acyl-CoA dehydrogenases by a metabolite of hypoglycin.

    Science.gov (United States)

    Kean, E A

    1976-01-23

    Extracts of liver mitochondria from donor rats given hypoglycin, the toxic amino acid from the ackee plant (Blighia sapida) showed drastically reduced levels of acyl-CoA dehydrogenase activity with butyryl-CoA as substrate. Activity with octanoyl- and palmitoyl-CoA was unaffected. Evidence that the active agent is methylenecyclopropylacetyl-CoA, a hypoglycin metabolite, was obtained by observing effects of the compound on a partially purified enzyme mixture prepared from rabbit liver. At 13 muM concentration, it strongly inhibited butyryl-CoA dehydrogenase (EC 1.3.99.2) with butyryl-CoA as substrate; it was far less effective with palmitoyl-CoA as substrate for the other similar enzymes present in the preparation. Unlike normal substrates of the acyl-CoA dehydrogenases, the compound itself, and not a reaction product, is inhibitory. The observed effect is consistent with quite general inhibition of fatty acid beta-oxidation by hypoglycin.

  15. Inhibition of Coix seed extract on fatty acid synthase, a novel target for anticancer activity.

    Science.gov (United States)

    Yu, Fei; Gao, Jing; Zeng, Yong; Liu, Chang-Xiao

    2008-09-26

    Coix seed has been traditionally used to treat cancers in folk medicine. Study the anticancer action mechanism of Coix seed extract. After the treatment with Coix seed extract (10 microl/ml), the residual activity of fatty acid synthase (FAS) as overall reaction, beta-ketoacyl reduction, enoyl reduction, and acetyl acetyl coenzyme A (AcAcCoA) reduction was separately detected at 340 nm in the UV-190 spectrophotometer. After rats were administrated Coix seed extract (2.5, 5.0, and 10.0 ml/kg) intragastrically for 10 days consecutively, activities of FAS, malate dehydrogenase (MDH), lipid protein lipase (LPL), hepatic lipase (HL), triglyceride (TG), and glucose-6-phosphate dehydrogenase (G-6-PD) in the plasma, liver and fatty tissues were determined. Experiments in vitro showed that the inhibition of Coix seed extract on FAS activity was significant and dose dependent, and two active sites inhibited were beta-ketoacyl reductases (KR) and enoyl reductase (ER). Experiments in vivo showed that Coix seed extract inhibited FAS activity in the liver, and elevated LPL and HL activity in the plasma, and effected G-6-PD activity. The study supports that FAS is a novel target for anticancer activity, and provides a theoretical foundation for the wide application of Coix seed extract in traditional medicine.

  16. High yields of active Thermus thermophilus proline dehydrogenase are obtained using maltose-binding protein as a solubility tag.

    NARCIS (Netherlands)

    Huijbers, M.M.E.; Berkel, van W.J.H.

    2015-01-01

    Proline dehydrogenase (ProDH) catalyzes the FAD-dependent oxidation of proline to ¿1-pyrroline-5-carboxylate, the first step of proline catabolism in many organisms. Next to being involved in a number of physiological processes, ProDH is of interest for practical applications because the proline

  17. Loss of peroxisomes causes oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase activity to detect cancer cells

    NARCIS (Netherlands)

    Frederiks, Wilma M.; Vreeling-Sindelárová, Heleen; van Noorden, Cornelis J. F.

    2007-01-01

    Oxygen insensitivity of carcinoma cells and oxygen sensitivity of non-cancer cells in the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) enables detection of carcinoma cells in unfixed cell smears or cryostat sections of biopsies. The metabolic background of oxygen insensitivity is

  18. Determination of medium chain acyl-CoA dehydrogenase activity in cultured skin fibroblasts using mass spectrometry

    NARCIS (Netherlands)

    Niezen-Koning, K E; Chapman, T E; Mulder, I E; Smit, G P; Reijngoud, D J; Berger, R

    1991-01-01

    Medium chain acyl-CoA dehydrogenase deficiency, a defect of mitochondrial beta-oxidation, is one of the most frequently occurring among inborn errors of metabolism. We describe a rapid and sensitive gas chromatographic/mass spectrometric method allowing reliable assessment of medium chain acyl-CoA

  19. Characterization of 17α-hydroxysteroid dehydrogenase activity (17α-HSD and its involvement in the biosynthesis of epitestosterone

    Directory of Open Access Journals (Sweden)

    Breton Rock

    2005-07-01

    Full Text Available Abstract Background Epi-testosterone (epiT is the 17α-epimer of testosterone. It has been found at similar level as testosterone in human biological fluids. This steroid has thus been used as a natural internal standard for assessing testosterone abuse in sports. EpiT has been also shown to accumulate in mammary cyst fluid and in human prostate. It was found to possess antiandrogenic activity as well as neuroprotective effects. So far, the exact pathway leading to the formation of epiT has not been elucidated. Results In this report, we describe the isolation and characterization of the enzyme 17α-hydroxysteroid dehydrogenase. The name is given according to its most potent activity. Using cells stably expressing the enzyme, we show that 17α-HSD catalyzes efficienty the transformation of 4-androstenedione (4-dione, dehydroepiandrosterone (DHEA, 5α-androstane-3,17-dione (5α-dione and androsterone (ADT into their corresponding 17α-hydroxy-steroids : epiT, 5-androstene-3β,17α-diol (epi5diol, 5α-androstane-17α-ol-3-one (epiDHT and 5α-androstane-3α,17α-diol (epi3α-diol, respectively. Similar to other members of the aldo-keto reductase family that possess the ability to reduce the keto-group into hydroxyl-group at different position on the steroid nucleus, 17α-HSD could also catalyze the transformation of DHT, 5α-dione, and 5α-pregnane-3,20-dione (DHP into 3α-diol, ADT and 5α-pregnane-3α-ol-20-one (allopregnanolone through its less potent 3α-HSD activity. We also have over-expressed the 17α-HSD in Escherichia coli and have purified it by affinity chromatography. The purified enzyme exhibits the same catalytic properties that have been observed with cultured HEK-293 stably transfected cells. Using quantitative Realtime-PCR to study tissue distribution of this enzyme in the mouse, we observed that it is expressed at very high levels in the kidney. Conclusion The present study permits to clarify the biosynthesis pathway of epiT. It

  20. Influence of long-term hyper-gravity on the reactivity of succinic acid dehydrogenase and NADPH-diaphorase in the central nervous system of fish: a histochemical study

    Science.gov (United States)

    Anken, R. H.; Rahmann, H.

    In the course of a densitometric evaluation, the histochemically demonstrated reactivity of succinic acid dehydrogenase (SDH) and of NADPH-diaphorase (NADPHD) was determined in different brain nuclei of two teleost fish (cichlid fish Oreochromis mossambicus, swordtail fish Xiphophorus helleri), which had been kept under 3g hyper-gravity for 8 days. SDH was chosen since it is a rate limiting enzyme of the Krebs cycle and therefore it is regarded as a marker for metabolic and neuronal activity. NADPHD reactivity reflects the activity of nitric oxide synthase. Nitric oxide (NO) is a gaseous intercellular messenger that has been suggested to play a major role in several different in vivo models of neuronal plasticity including learning. Within particular vestibulum-connected brain centers, significant effects of hyper-gravity were obtained, e.g., in the magnocellular nucleus, a primary vestibular relay ganglion of the brain stem octavolateralis area, in the superior rectus subdivision of the oculomotoric nucleus and within cerebellar eurydendroid cells, which in teleosts possibly resemble the deep cerebellar nucleus of higher vertebrates. Non-vestibulum related nuclei did not respond to hypergravity in a significant way. The effect of hyper-gravity found was much less distinct in adult animals as compared to the circumstances seen in larval fish (Anken et al., Adv. Space Res. 17, 1996), possibly due to a development correlated loss of neuronal plasticity.

  1. Amino acid sequence of the nucleotide-binding site of D-(-)-beta-hydroxybutyrate dehydrogenase labeled with arylazido-beta-(3-/sup 3/H)alanylnicotinamide adenine dinucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Yamaguchi, M.; Chen, S.; Hatefi, Y.

    1986-08-26

    In the dark, arylazido-beta-alanylnicotinamide adenine dinucleotide (N3-NAD) can replace NAD as cofactor for D-(-)-beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria. When photoirradiated with visible light, N3-NAD forms a nitrene species that binds covalently to BDH and inhibits the enzyme. NAD(H) protects BDH against photolabeling and inhibition by N3-NAD (Yamaguchi, M., Chen, S., and Hatefi, Y. (1985) Biochemistry 24, 4912-4916). In the present study, a tryptic peptide of purified BDH photolabeled with arylazido-beta-(3-/sup 3/H) alanyl-NAD ((/sup 3/H)N3-NAD) was isolated and sequenced. The same tryptic peptide was also isolated from BDH not labeled with (/sup 3/H)N3-NAD and sequenced. Both peptides indicated the sequence Met-Glu-Ser-Tyr-Cys-Thr-Ser-Gly-Ser-Thr-Asp-Thr-Ser-Pro-Val-Ile-Lys. The residue labeled with (/sup 3/H)N3-NAD was Cys. This heptadecapeptide contains 14 uncharged residues and is marked by having in an undecapeptide segment 8 hydroxy amino acids located symmetrically around a central glycine.

  2. Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

    Directory of Open Access Journals (Sweden)

    Benedito Barraviera

    1988-10-01

    Full Text Available The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05 for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s., respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01 for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01 before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1

  3. Glucose-6-phosphate dehydrogenase

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a protein that ...

  4. Lactate dehydrogenase test

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003471.htm Lactate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Lactate dehydrogenase (LDH) is a protein that helps produce energy ...

  5. Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts.

    Science.gov (United States)

    White, Bronwyn E; Fenner, Caryn J; Smit, Martha S; Harrison, Susan T L

    2017-09-20

    The regeneration of cofactors and the supply of alkane substrate are key considerations for the biocatalytic activation of hydrocarbons by cytochrome P450s. This study focused on the biotransformation of n-octane to 1-octanol using resting Escherichia coli cells expressing the CYP153A6 operon, which includes the electron transport proteins ferredoxin and ferredoxin reductase. Glycerol dehydrogenase was co-expressed with the CYP153A6 operon to investigate the effects of boosting cofactor regeneration. In order to overcome the alkane supply bottleneck, various chemical and physical approaches to membrane permeabilisation were tested in strains with or without additional dehydrogenase expression. Dehydrogenase co-expression in whole cells did not improve product formation and reduced the stability of the system at high cell densities. Chemical permeabilisation resulted in initial hydroxylation rates that were up to two times higher than the whole cell system, but severely impacted biocatalyst stability. Mechanical cell breakage led to improved enzyme stability, but additional dehydrogenase expression was necessary to improve product formation. The best-performing system (in terms of final titres) consisted of mechanically ruptured cells expressing additional dehydrogenase. This system had an initial activity of 1.67 ± 0.12 U/gDCW (32% improvement on whole cells) and attained a product concentration of 34.8 ± 1.6 mM after 24 h (22% improvement on whole cells). Furthermore, the system was able to maintain activity when biotransformation was extended to 72 h, resulting in a final product titre of 60.9 ± 1.1 mM. This study suggests that CYP153A6 in whole cells is limited by coupling efficiencies rather than cofactor supply. However, the most significant limitation in the current system is hydrocarbon transport, with substrate import being the main determinant of hydroxylation rates, and product export playing a key role in system stability.

  6. Enzyme Architecture: The Role of a Flexible Loop in Activation of Glycerol-3-Phosphate Dehydrogenase for Catalysis of Hydride Transfer.

    Science.gov (United States)

    He, Rui; Reyes, Archie C; Amyes, Tina L; Richard, John

    2018-01-16

    The side chain of Q295 of glycerol-3-phosphate dehydrogenase from human liver (hlGPDH) lies in a flexible loop. This loop folds over the phosphodianion of substrate dihydroxyacetone phosphate (DHAP), where Q295 interacts with the side chain cation from R269, which is ion-paired to the substrate phosphodianion. Kinetic parameters kcat/Km (M-1 s-1) and kcat/KGAKHPi (M-2 s-1) were determined, respectively, for catalysis of the reduction of DHAP and for dianion activation of catalysis of reduction of glycolaldeyde (GA) catalyzed by wildtype, Q295G, Q295S, Q295A, and Q295N mutants of hlGPDH. The Q295 mutations result in up to a 150-fold decrease in (kcat/Km)DHAP and up to a 2.7 kcal/mol decrease in the intrinsic phosphodianion binding energy that is utilized in transition state stabilization. The data define a linear correlation with slope 1.1, between the intrinsic phosphodianion binding energy and the intrinsic phosphite dianion binding energy for activation of hlGPDH-catalyzed reduction of GA, which demonstrates a role for Q295 in optimizing the intrinsic dianion binding energy. The R269A mutation of wildtype GPDH results in a 9.1 kcal/mol destabilization of the transition state for reduction of DHAP [J. Am. Chem. Soc. (2015) 137, 5312-5315], but the same R269A mutation of N270A and Q295A mutants result in smaller 5.9 and 4.9 kcal/mol transition state destabilization. Similarly, the N270A or Q295A mutations of R269A GPDH each result in large falloffs in the efficiency of rescue of the R269A mutant by guanidine cation. We conclude that N270, which interacts for the substrate phosphodianion and Q295, which interacts with the guanidine side chain of R269, function cooperatively to optimize the apparent transition state stabilization provided by the cationic side chain of R269.

  7. Actividad Circadiana de la enzima succinato deshidrogenasa en Neurospora crassa Circadian rhythm in succinate dehydrogenase activity in Neurospora crassa

    Directory of Open Access Journals (Sweden)

    Álvarez Barón Claudia Patricia

    2004-12-01

    Full Text Available Neurospora crassa es un modelo de ritmicidad circadiana ampliamente estudiado. El metabolismo de este hongo es controlado por múltiples factores que incluyen el desarrollo, las características del medio y el reloj circadiano. El estudio de la regulación circadiana del metabolismo en este hongo podría estar enmascarado por el uso de medios restrictivos que suprimen su crecimiento y desarrollo. En el presente trabajo se estudió la variación en el tiempo de la actividad de la enzima Succinato Deshidrogenasa durante 24 horas en medio completo sin restricción para la conidiación en cepas de Neurospora
    crassa rítmica y arrítmica. Se encontró una variación circadiana en la actividad enzimática de la cepa rítmica con valores altos en las horas correspondientes a la noche y bajos durante el día. Este hallazgo resalta la importancia de profundizar en el estudio del control circadiano sobre el metabolismo de este hongo, con base en la existencia de vías de regulación múltiples sobre las enzimas del metabolismo y de mecanismos post-transcripcionales y transcripcionales de control circadiano.Neurospora crassa is a widely studied model of circadian rhythmicity. In this fungus, metabolism is controlled by multiple factors which include development, medium characteristics and the circadian clock. The study of the circadian control of metabolism in this fungus could be masked by the use of restrictive media that inhibit growth and development. In this report, the presence of a circadian rhythm in the activity of the enzyme Succinate Dehydrogenase in Neurospora crassa is demonstrated. Rhythmic and arrhythmic Neurospora strains were grown in complete medium without conidiation restriction. A circadian change in the enzymatic activity was found with high values in hours corresponding to the night and a low level during the day. This finding highlights the importance of deeper studies in the circadian control of metabolism in this fungus

  8. Expression pattern, ethanol-metabolizing activities, and cellular localization of alcohol and aldehyde dehydrogenases in human pancreas: implications for pathogenesis of alcohol-induced pancreatic injury.

    Science.gov (United States)

    Chiang, Chien-Ping; Wu, Chew-Wun; Lee, Shiao-Pieng; Chung, Chia-Chi; Wang, Chi-Wei; Lee, Shou-Lun; Nieh, Shin; Yin, Shih-Jiun

    2009-06-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are major enzymes responsible for metabolism of ethanol. Genetic polymorphisms of ADH1B, ADH1C, and ALDH2 occur among racial populations. The metabolic effect and metabolites contribute to pathogenesis of pancreatic injury. The goal of this study was to determine the functional expressions and cellular localization of ADH and ALDH families in human pancreas. Fifty five surgical specimens of normal pancreas as well as 15 samples each for chronic pancreatitis and pancreatic cancer from archival formalin-fixed paraffin-embedded tissue specimens were investigated. Class-specific antibodies were prepared by affinity chromatographies from rabbit antisera raised against recombinant human ADH1C1, ADH4, ADH5, ADH7, ALDH1A1, ALDH2, and ALDH3A1. The isozyme expression patterns of ADH/ALDH were identified by isoelectric focusing, and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting, and the cellular localizations were detected by immunohistochemistry and histochemistry. At 33 mM ethanol, pH 7.5, the activities were significantly different between allelic phenotypes of ADH1B. The activity of ALDH2-inactive phenotypes was slightly lower than ALDH2-active phenotypes at 200 microM acetaldehyde. The protein contents were in the following decreasing order: ALDH1A1, ALDH2, ADH1, and ADH5. ADH1B was detected in the acinar cells and ADH1C in the ductular, islet, and stellate cells. The expression of ADH1C appeared to be increased in the activated pancreatic stellate cells in chronic pancreatitis and pancreatic cancer. Alcohol dehydrogenase and ALDH family members are differentially expressed in the various cell types of pancreas. ADH1C may play an important role in modulation of activation of pancreatic stellate cells.

  9. Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase.

    OpenAIRE

    Lorowitz, W; CLARK, D.

    1982-01-01

    Mutants of Escherichia coli resistant to allyl alcohol were selected. Such mutants were found to lack alcohol dehydrogenase. In addition, mutants with temperature-sensitive alcohol dehydrogenase activity were obtained. These mutations, designated adhE, are all located at the previously described adh regulatory locus. Most adhE mutants were also defective in acetaldehyde dehydrogenase activity.

  10. Analysis of Isocitrate Dehydrogenase -2 (IDH-2) Activity in Human Serum as a Biomarker in Chemotherapy Patients of Breast Carcinoma: A Case-Control Study.

    Science.gov (United States)

    Gavel, Roshni; Mishra, S P; Khanna, Seema; Khanna, Rahul; Shah, Agni Gautam

    2017-05-01

    Breast cancer represents a major public health problem in women worldwide. For many cancers, serum tumour markers play an important role in patient treatment and monitoring. Isocitrate dehydrogenase enzyme is also used as a biomarker for various types of cancer. The purpose of this study was to determine serum Isocitrate dehydrogenase-2 (IDH-2) enzyme activity in breast cancer patients (pre and post chemotherapy) and also correlate the changes in enzyme activity with stages of cancer and control groups. In this case-control study, histologically confirmed 40 female patients aged 28-80 years who fulfilled the criteria for diagnosis of invasive breast cancer were selected in our study groups from surgery outpatient department of SS Hospital, BHU, Varanasi, India, and 40 healthy age matched females were selected between October 2013 to July 2015. The estimation of serum IDH-2 enzyme activity in before and after two cycles of neoadjuvant chemotherapy patients was performed by spectrophotometry assay. The mean serum IDH-2 activity in cases (Mean±SD) was significantly more than control group (pIDH-2 activity in cases was significantly decrease after neo-adjuvant chemotherapy (p=0.019). In stage II pre chemotherapy patients serum IDH-2 activity was higher than post chemotherapy (pIDH-2 activity was not significant (p-value>0.05). The serum IDH-2 can be a potential biomarker in breast carcinoma and can be used for prognosis and monitoring the chemotherapy response of the patients.

  11. Down-regulation of lactate dehydrogenase-A by siRNAs for reduced lactic acid formation of Chinese hamster ovary cells producing thrombopoietin.

    Science.gov (United States)

    Kim, Sung Hyun; Lee, Gyun Min

    2007-02-01

    Lactate, one of the major waste products in mammalian cell culture, can inhibit cell growth and affect cellular metabolism at high concentrations. To reduce lactate formation, lactate dehydrogenase-A (LDH-A), an enzyme catalyzing the conversion of glucose-derived pyruvate to lactate, was down-regulated by an expression vector of small interfering RNAs (siRNA) in recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Three clones expressing low levels of LDH-A, determined by reverse transcription-PCR and an enzyme activity test, were established in addition to a negative control cell line. LDH-A activities in the three clones were decreased by 75-89%, compared with that of the control CHO cell line, demonstrating that the effect of siRNA is more significant than that of other traditional methods such as homologous recombination (30%) and antisense mRNA (29%). The specific glucose consumption rates of the three clones were reduced to 54-87% when compared to the control cell line. Similarly, the specific lactate production rates were reduced to 45-79% of the control cell line level. In addition, reduction of LDH-A did not impair either cell proliferation or hTPO productivity. Taken together, these results show that the lactate formation rate in rCHO cell culture can be efficiently reduced through the down-regulation of LDH via siRNA.

  12. Effect of fermented sea tangle on the alcohol dehydrogenase and acetaldehyde dehydrogenase in Saccharomyces cerevisiae.

    Science.gov (United States)

    Cha, Jae-Young; Jeong, Jae-Jun; Yang, Hyun-Ju; Lee, Bae-Jin; Cho, Young-Su

    2011-08-01

    Sea tangle, a kind of brown seaweed, was fermented with Lactobacillus brevis BJ-20. The gamma-aminobutyric acid (GABA) content in fermented sea tangle (FST) was 5.56% (w/w) and GABA in total free amino acid of FST was 49.5%. The effect of FST on the enzyme activities and mRNA protein expression of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) involved in alcohol metabolism in Saccharomyces cerevisiae was investigated. Yeast was cultured in YPD medium supplemented with different concentrations of FST powder [0, 0.4, 0.8, and 1.0% (w/v)] for 18 h. FST had no cytotoxic effect on the yeast growth. The highest activities and protein expressions of ADH and ALDH from the cell-free extracts of S. cerevisiae were evident with the 0.4% and 0.8% (w/v) FST-supplemented concentrations, respectively. The highest concentrations of GABA as well as minerals (Zn, Ca, and Mg) were found in the cell-free extracts of S. cerevisiae cultured in medium supplemented with 0.4% (w/v) FST. The levels of GABA, Zn, Ca, and Mg in S. cerevisiae were strongly correlated with the enzyme activities of ADH and ALDH in yeast. These results indicate that FST can enhance the enzyme activities and protein expression of ADH and ALDH in S. cerevisiae.

  13. Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Bulfer, Stacie L.; Hendershot, Jenna M.; Trievel, Raymond C. (Michigan); (UCSF)

    2013-09-18

    Lysine biosynthesis in fungi, euglena, and certain archaebacteria occurs through the {alpha}-aminoadipate pathway. Enzymes in the first steps of this pathway have been proposed as potential targets for the development of antifungal therapies, as they are absent in animals but are conserved in several pathogenic fungi species, including Candida, Cryptococcus, and Aspergillus. One potential antifungal target in the {alpha}-aminoadipate pathway is the third enzyme in the pathway, homoisocitrate dehydrogenase (HICDH), which catalyzes the divalent metal-dependent conversion of homoisocitrate to 2-oxoadipate (2-OA) using nicotinamide adenine dinucleotide (NAD{sup +}) as a cofactor. HICDH belogns to a family of {beta}-hydroxyacid oxidative decarboxylases that includes malate dehydrogenase, tartrate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase (ICDH), and 3-isopropylmalte dehydrogenase (IPMDH). ICDH and IPMDH are well-characterized enzymes that catalyze the decarboxylation of isocitrate to yield 2-oxoglutarate (2-OG) in the citric acid cycle and the conversion of 3-isopropylmalate to 2-oxoisovalerate in the leucine biosynthetic pathway, respectively. Recent structural and biochemical studies of HICDH reveal that this enzyme shares sequence, structural, and mechanistic homology with ICDH and IPMDH. To date, the only published structures of HICDH are from the archaebacteria Thermus thermophilus (TtHICDH). Fungal HICDHs diverge from TtHICDH in several aspects, including their thermal stability, oligomerization state, and substrate specificity, thus warranting further characterization. To gain insights into these differences, they determined crystal structures of a fungal Schizosaccharomyces pombe HICDH (SpHICDH) as an apoenzyme and as a binary complex with additive tripeptide glycyl-glycyl-glycine (GGG) to 1.55 {angstrom} and 1.85 {angstrom} resolution, respectively. Finally, a comparison of the SpHICDH and TtHICDH structures reveal differences in

  14. Characterization and conservation of the inner E2 core domain structure of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Construction of a cDNA encoding the entire transacylase (E2b) precursor.

    Science.gov (United States)

    Griffin, T A; Lau, K S; Chuang, D T

    1988-10-05

    A cDNA clone encoding the entire transacylase (E2b) precursor of the bovine branched-chain alpha-keto acid dehydrogenase complex has been constructed from two overlapping incomplete cDNA clones which were isolated from a lambda ZAP library prepared from bovine liver poly(A)+ RNA. Nucleotide sequencing indicates that this bovine E2b cDNA insert (bE2-11) is 2701 base pairs in length with an open reading frame of 1446 base pairs. The bE2-11 cDNA insert encodes a leader peptide of 61 residues and a mature E2b polypeptide of 421 amino acid residues with a calculated monomeric molecular mass of 46,518 daltons. The molecular mass of the native E2b component isolated from bovine liver is 1,110,000 daltons as determined by sedimentation equilibrium. This value establishes the 24-subunit octahedral model for the quaternary structure of bovine E2b. The amino-terminal sequences of two tryptic fragments (A and B) of the E2b protein have been determined. Fragment A comprises residues 175 to 421 of the E2b protein and is the inner E2 core domain which contains the transacylase active site. Fragment B, produced by further tryptic cleavage of fragment, comprises residues 205 to 421, but does not have transacylase activity. Both fragments A and B confer the highly assembled 24-mer structure. The primary structure of the inner E2 core domain of bovine E2b (fragment A) is very similar to those of three other E2 proteins (human E2p, Escherichia coli E2p, and E. coli E2k). These similarities suggest that these E2 proteins are structurally and evolutionarily related.

  15. Antisense Inhibition of the Iron-Sulphur Subunit of Succinate Dehydrogenase Enhances Photosynthesis and Growth in Tomato via an Organic Acid-Mediated Effect on Stomatal Aperture

    National Research Council Canada - National Science Library

    Wagner L. Araújo; Adriano Nunes-Nesi; Sonia Osorio; Björn Usadel; Daniela Fuentes; Réka Nagy; Ilse Balbo; Martin Lehmann; Claudia Studart-Witkowski; Takayuki Tohge; Enrico Martinoia; Xavier Jordana; Fábio M. DaMatta; Alisdair R. Fernie

    2011-01-01

    Transgenic tomato [Solanum lycopersicum) plants expressing a fragment of the SI SDH2-2 gene encoding the iron sulfur subunit of the succinate dehydrogenase protein complex in the antisense orientation under the control of the 35S promoter...

  16. Fatty Acid Accumulation and Resulting PPARα Activation in Fibroblasts due to Trifunctional Protein Deficiency

    Directory of Open Access Journals (Sweden)

    Masato Wakabayashi

    2012-01-01

    Full Text Available To examine fatty acid accumulation and its toxic effects in cells, we analyzed skin fibroblasts from six patients with mitochondrial trifunctional protein deficiency, who had abnormalities in the second through fourth reactions in fatty acid β-oxidation system. We found free fatty acid accumulation, enhanced three acyl-CoA dehydrogenases, catalyzing the first reaction in the β-oxidation system and being assumed to have normal activities in these patients, and PPARα activation that was confirmed in the experiments using MK886, a PPARα specific antagonist and fenofibrate, a PPARα specific agonist. These novel findings suggest that the fatty acid accumulation and the resulting PPARα activation are major causes of the increase in the β-oxidation ability as probable compensation for fatty acid metabolism in the patients’ fibroblasts, and that enhanced cell proliferation and increased oxidative stress due to the PPARα activation relate to the development of specific clinical features such as hypertrophic cardiomyopathy, slight hepatomegaly, and skeletal myopathy. Additionally, significant suppression of the PPARα activation by means of MK886 treatment is assumed to provide a new method of treating this deficiency.

  17. Tetrahydro-2-naphthyl and 2-Indanyl Triazolopyrimidines Targeting Plasmodium falciparum Dihydroorotate Dehydrogenase Display Potent and Selective Antimalarial Activity

    OpenAIRE

    Kokkonda, Sreekanth; Deng, Xiaoyi; White, Karen L.; Coteron, Jose M.; Marco, Maria; de las Heras, Laura; White, John; El Mazouni, Farah; Tomchick, Diana R.; Manjalanagara, Krishne; Rudra, Kakali Rani; Chen, Gong; Morizzi, Julia; Ryan, Eileen; Kaminsky, Werner

    2016-01-01

    Malaria persists as one of the most devastating global infectious diseases. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) has been identified as a new malaria drug target, and a triazolopyrimidine-based DHODH inhibitor 1 (DSM265) is in clinical development. We sought to identify compounds with higher potency against Plasmodium DHODH while showing greater selectivity toward animal DHODHs. Herein we describe a series of novel triazolopyrimidines wherein the p-SF5-anili...

  18. Triazolopyrimidine-based dihydroorotate dehydrogenase inhibitors with potent and selective activity against the malaria parasite, Plasmodium falciparum

    OpenAIRE

    Phillips, Margaret A.; Gujjar, Ramesh; Malmquist, Nicholas A.; White, John; El Mazouni, Farah; Baldwin, Jeffrey; Rathod, Pradipsinh K.

    2008-01-01

    A Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitor that is potent (KI = 15 nM) and species-selective (>5,000-fold over the human enzyme) was identified by high-throughput screening. The substituted triazolopyrimidine and its structural analogs were produced by an inexpensive three-step synthesis and the series showed good association between PfDHODH inhibition and parasite toxicity. This study has identified the first nanomolar PfDHODH inhibitor with potent antimalarial ...

  19. Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays

    DEFF Research Database (Denmark)

    Wiggers, Henrik; Cheleski, J; Zottis, A

    2007-01-01

    OH), in the isothermal titration calorimetry (ITC) kinetic assays for the catalyzed reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi. The solvent effects on T. cruzi GAPDH had not yet been studied. This enzyme was shown here to be affected by the organic solvents content up to 5...... by standard spectrophotometric assays to establish a behavioral performance of T. cruzi GAPDH when used for screening of potential inhibitors. Udgivelsesdato: 2007-Nov-1...

  20. Polymorphisms in the promoter region of the human class II alcohol dehydrogenase (ADH4) gene affect both transcriptional activity and ethanol metabolism in Japanese subjects.

    Science.gov (United States)

    Kimura, Yukiko; Nishimura, Fusae T; Abe, Shuntaro; Fukunaga, Tatsushige; Tanii, Hideji; Saijoh, Kiyofumi

    2009-02-01

    Class II alcohol dehydrogenase (pi-ADH), encoded by alcohol dehydrogenase (ADH4), is considered to contribute to ethanol (EtOH) oxidation in the liver at high concentration. Four single nucleotide polymorphisms (SNPs) were found in the promoter region of this gene. Analysis of genotype distribution in 102 unrelated Japanese subjects revealed that four loci were in strong linkage disequilibrium and could be classified into three haplotypes. The effects of these polymorphisms on transcriptional activity were investigated in HepG2 cells. Transcriptional activity was significantly higher in cells with the -136A allele than in those with the -136C allele. To investigate whether this difference in transcriptional activity caused a difference in EtOH elimination, previous data on blood EtOH changes after 0.4 g/kg body weight alcohol ingestion were analyzed. When analyzed based on aldehyde dehydrogenase-2 gene (ALDH2) (487)Glu/Lys genotype, the significantly lower level of EtOH at peak in subjects with -136C/A and -136A/A genotype compared with subjects with -136C/C genotype indicated that -136 bp was a suggestive locus for differences in EtOH oxidation. This effect was observed only in subjects with ALDH2 (487)Glu/Glu. These results suggested that the SNP at -136bp in the ADH4 promoter had an effect on transcriptional regulation, and that the higher activity of the -136A allele compared with the -136C allele caused a lower level of blood EtOH after alcohol ingestion; that is, individuals with the -136A allele may consume more EtOH and might have a higher risk for development of alcohol dependence than those without the -136A allele.

  1. Fluoxetine elevates allopregnanolone in female rat brain but inhibits a steroid microsomal dehydrogenase rather than activating an aldo-keto reductase.

    Science.gov (United States)

    Fry, J P; Li, K Y; Devall, A J; Cockcroft, S; Honour, J W; Lovick, T A

    2014-12-01

    Fluoxetine, a selective serotonin reuptake inhibitor, elevates brain concentrations of the neuroactive progesterone metabolite allopregnanolone, an effect suggested to underlie its use in the treatment of premenstrual dysphoria. One report showed fluoxetine to activate the aldo-keto reductase (AKR) component of 3α-hydroxysteroid dehydrogenase (3α-HSD), which catalyses production of allopregnanolone from 5α-dihydroprogesterone. However, this action was not observed by others. The present study sought to clarify the site of action for fluoxetine in elevating brain allopregnanolone. Adult male rats and female rats in dioestrus were treated with fluoxetine and their brains assayed for allopregnanolone and its precursors, progesterone and 5α-dihydroprogesterone. Subcellular fractions of rat brain were also used to investigate the actions of fluoxetine on 3α-HSD activity in both the reductive direction, producing allopregnanolone from 5α-dihydroprogesterone, and the reverse oxidative direction. Fluoxetine was also tested on these recombinant enzyme activities expressed in HEK cells. Short-term treatment with fluoxetine increased brain allopregnanolone concentrations in female, but not male, rats. Enzyme assays on native rat brain fractions and on activities expressed in HEK cells showed fluoxetine did not affect the AKR producing allopregnanolone from 5α-dihydroprogesterone but did inhibit the microsomal dehydrogenase oxidizing allopregnanolone to 5α-dihydroprogesterone. Fluoxetine elevated allopregnanolone in female rat brain by inhibiting its oxidation to 5α-dihydroprogesterone by a microsomal dehydrogenase. This is a novel site of action for fluoxetine, with implications for the development of new agents and/or dosing regimens to raise brain allopregnanolone. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

  2. Eicosapentaenoic Acid Modulates Trichomonas vaginalis Activity.

    Science.gov (United States)

    Korosh, Travis; Jordan, Kelsey D; Wu, Ja-Shin; Yarlett, Nigel; Upmacis, Rita K

    2016-01-01

    Trichomonas vaginalis is a sexually transmitted parasite and, while it is often asymptomatic in males, the parasite is associated with disease in both sexes. Metronidazole is an effective treatment for trichomoniasis, but resistant strains have evolved and, thus, it has become necessary to investigate other possible therapies. In this study, we examined the effects of native and oxidized forms of the sodium salts of eicosapentaenoic, docosahexaenoic, and arachidonic acids on T. vaginalis activity. Eicosapentaenoic acid was the most toxic with 190 and 380 μM causing approximately 90% cell death in Casu2 and ATCC 50142 strains, respectively. In contrast, oxidized eicosapentaenoic acid was the least toxic, requiring > 3 mM to inhibit activity, while low levels (10 μM) were associated with increased parasite density. Mass spectrometric analysis of oxidized eicosapentaenoic acid revealed C20 products containing one to six additional oxygen atoms and various degrees of bond saturation. These results indicate that eicosapentaenoic acid has different effects on T. vaginalis survival, depending on whether it is present in the native or oxidized form. A better understanding of lipid metabolism in T. vaginalis may facilitate the design of synthetic fatty acids that are effective for the treatment of metronidazole-resistant T. vaginalis. © 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.

  3. Antioxidant activity, anti-proliferative activity, and amino acid profiles of ethanolic extracts of edible mushrooms.

    Science.gov (United States)

    Panthong, S; Boonsathorn, N; Chuchawankul, S

    2016-10-17

    Biological activities of various mushrooms have recently been discovered, particularly, immunomodulatory and antitumor activities. Herein, three edible mushrooms, Auricularia auricula-judae (AA), Pleurotus abalonus (PA) and Pleurotus sajor-caju (PS) extracted using Soxhlet ethanol extraction were evaluated for their antioxidative, anti-proliferative effects on leukemia cells. Using the Folin-Ciocalteau method and Trolox equivalent antioxidant capacity assay, phenolics and antioxidant activity were found in all sample mushrooms. Additionally, anti-proliferative activity of mushroom extracts against U937 leukemia cells was determined using a viability assay based on mitochondrial activity. PA (0.5 mg/mL) and AA (0.25-0.5 mg/mL) significantly reduced cell viability. Interestingly, PS caused a hormetic-like biphasic dose-response. Low doses (0-0.25 mg/L) of PS promoted cell proliferation up to 140% relative to control, whereas higher doses (0.50 mg/mL) inhibited cell proliferation. Against U937 cells, AA IC 50 was 0.28 ± 0.04 mg/mL, which was lower than PS or PA IC 50 (0.45 ± 0.01 and 0.49 ± 0.001 mg/mL, respectively). Furthermore, lactate dehydrogenase (LDH) leakage conferred cytotoxicity. PS and PA were not toxic to U937 cells at any tested concentration; AA (0.50 mg/mL) showed high LDH levels and caused 50% cytotoxicity. Additionally, UPLC-HRMS data indicated several phytochemicals known to support functional activities as either antioxidant or anti-proliferative. Glutamic acid was uniquely found in ethanolic extracts of AA, and was considered an anti-cancer amino acid with potent anti-proliferative effects on U937 cells. Collectively, all mushroom extracts exhibited antioxidant effects, but their anti-proliferative effects were dose-dependent. Nevertheless, the AA extract, with highest potency, is a promising candidate for future applications.

  4. Comprehensive analysis of 5-aminolevulinic acid dehydrogenase (ALAD variants and renal cell carcinoma risk among individuals exposed to lead.

    Directory of Open Access Journals (Sweden)

    Dana M van Bemmel

    Full Text Available BACKGROUND: Epidemiologic studies are reporting associations between lead exposure and human cancers. A polymorphism in the 5-aminolevulinic acid dehydratase (ALAD gene affects lead toxicokinetics and may modify the adverse effects of lead. METHODS: The objective of this study was to evaluate single-nucleotide polymorphisms (SNPs tagging the ALAD region among renal cancer cases and controls to determine whether genetic variation alters the relationship between lead and renal cancer. Occupational exposure to lead and risk of cancer was examined in a case-control study of renal cell carcinoma (RCC. Comprehensive analysis of variation across the ALAD gene was assessed using a tagging SNP approach among 987 cases and 1298 controls. Occupational lead exposure was estimated using questionnaire-based exposure assessment and expert review. Odds ratios (OR and 95% confidence intervals (CI were calculated using logistic regression. RESULTS: The adjusted risk associated with the ALAD variant rs8177796(CT/TT was increased (OR = 1.35, 95%CI = 1.05-1.73, p-value = 0.02 when compared to the major allele, regardless of lead exposure. Joint effects of lead and ALAD rs2761016 suggest an increased RCC risk for the homozygous wild-type and heterozygous alleles ((GGOR = 2.68, 95%CI = 1.17-6.12, p = 0.01; (GAOR = 1.79, 95%CI = 1.06-3.04 with an interaction approaching significance (p(int = 0.06. No significant modification in RCC risk was observed for the functional variant rs1800435(K68N. Haplotype analysis identified a region associated with risk supporting tagging SNP results. CONCLUSION: A common genetic variation in ALAD may alter the risk of RCC overall, and among individuals occupationally exposed to lead. Further work in larger exposed populations is warranted to determine if ALAD modifies RCC risk associated with lead exposure.

  5. Human dehydrogenase/reductase (SDR family) member 11 is a novel type of 17β-hydroxysteroid dehydrogenase.

    Science.gov (United States)

    Endo, Satoshi; Miyagi, Namiki; Matsunaga, Toshiyuki; Hara, Akira; Ikari, Akira

    2016-03-25

    We report characterization of a member of the short-chain dehydrogenase/reductase superfamily encoded in a human gene, DHRS11. The recombinant protein (DHRS11) efficiently catalyzed the conversion of the 17-keto group of estrone, 4- and 5-androstenes and 5α-androstanes into their 17β-hydroxyl metabolites with NADPH as a coenzyme. In contrast, it exhibited reductive 3β-hydroxysteroid dehydrogenase activity toward 5β-androstanes, 5β-pregnanes, 4-pregnenes and bile acids. Additionally, DHRS11 reduced α-dicarbonyls (such as diacetyl and methylglyoxal) and alicyclic ketones (such as 1-indanone and loxoprofen). The enzyme activity was inhibited in a mixed-type manner by flavonoids, and competitively by carbenoxolone, glycyrrhetinic acid, zearalenone, curcumin and flufenamic acid. The expression of DHRS11 mRNA was observed widely in human tissues, most abundantly in testis, small intestine, colon, kidney and cancer cell lines. Thus, DHRS11 represents a novel type of 17β-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. A Novel Caffeine Dehydrogenase in Pseudomonas sp. Strain CBB1 Oxidizes Caffeine to Trimethyluric Acid▿

    Science.gov (United States)

    Yu, Chi Li; Kale, Yogesh; Gopishetty, Sridhar; Louie, Tai Man; Subramanian, Mani

    2008-01-01

    A unique heterotrimeric caffeine dehydrogenase was purified from Pseudomonas sp. strain CBB1. This enzyme oxidized caffeine to trimethyluric acid stoichiometrically and hydrolytically, without producing hydrogen peroxide. The enzyme was not NAD(P)+ dependent; coenzyme Q0 was the preferred electron acceptor. The enzyme was specific for caffeine and theobromine and showed no activity with xanthine. PMID:17981969

  7. Identification of cysteine-319 as the target amino acid of 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate in bovine liver glutamate dehydrogenase.

    Science.gov (United States)

    Ozturk, D H; Colman, R F

    1991-07-23

    The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate (8-BDB-TA-5'-TP) has been shown to react with bovine liver glutamate dehydrogenase in the region of the GTP-dependent NADH inhibitory site with incorporation of about 1 mol of reagent/mol of subunit [Ozturk, D. H., Safer, D., & Colman, R. F. (1990) Biochemistry 29, 7112-7118]. The modified enzyme was shown to contain only 5 free sulfhydryl groups upon 5,5'-dithiobis (2-nitrobenzoate) titration as compared with 6 in the unmodified enzyme. In the unmodified enzyme digested with trypsin, 6 cysteinyl peptides were detected by high-performance liquid chromatography upon treatment with iodo [3H]acetic acid. In contrast, only 5 (carboxymethyl)cysteinyl peptides were detected in 8-BDB-TA-5'-TP-modified enzyme. When carboxymethylated modified and unmodified enzymes were digested with thermolysin, 6 peptide sequences containing (carboxymethyl)cysteine were obtained in the unmodified enzyme, but only 5 were observed in the modified enzyme. The (carboxymethyl)cysteine which was absent in the modified enzyme was determined to be Cys-319, leading to the conclusion that 8-BDB-TA-5'-TP reacts with Cys-319, thereby preventing it from subsequent reaction with radioactive iodoacetate. It was previously reported that 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate (6-BDB-TA-5'-DP) modifies Cys-319 in this enzyme [Batra, S. P., & Colman, R. F. (1986) Biochemistry 25, 3508-3515].(ABSTRACT TRUNCATED AT 250 WORDS)

  8. The Unusual Acid-Accumulating Behavior during Ripening of Cherimoya (Annona cherimola Mill.) is Linked to Changes in Transcription and Enzyme Activity Related to Citric and Malic Acid Metabolism.

    Science.gov (United States)

    González-Agüero, Mauricio; Tejerina Pardo, Luis; Zamudio, María Sofía; Contreras, Carolina; Undurraga, Pedro; Defilippi, Bruno G

    2016-04-25

    Cherimoya (Annona cherimola Mill.) is a subtropical fruit characterized by a significant increase in organic acid levels during ripening, making it an interesting model for studying the relationship between acidity and fruit flavor. In this work, we focused on understanding the balance between the concentration of organic acids and the gene expression and activity of enzymes involved in the synthesis and degradation of these metabolites during the development and ripening of cherimoya cv. "Concha Lisa". Our results showed an early accumulation of citric acid and other changes associated with the accumulation of transcripts encoding citrate catabolism enzymes. During ripening, a 2-fold increase in malic acid and a 6-fold increase in citric acid were detected. By comparing the contents of these compounds with gene expression and enzymatic activity levels, we determined that cytoplasmic NAD-dependent malate dehydrogenase (cyNAD-MDH) and mitochondrial citrate synthase (mCS) play important regulatory roles in the malic and citric acid biosynthetic pathways.

  9. Acid phosphatases activity in cheese and starters.

    Science.gov (United States)

    Andrews, A T; Alichanidis, E

    1975-06-01

    The acid phosphatase activity levels in a number of Greek cheeses and in Cheddar cheeses were found to be unaffected by storage for up to 18 months and 12 months respectively. In Cheddar cheese, starter organisms made an insignificant contribution to this activity. Studies of acid phosphatase prepared from Streptococcus cremoris-lactis NCDO 762 starter cultures showed that the enzyme was of high molecular weight and largely particle-bound. The pH of optimum activity was 5-2 and the enzyme was inhibited by F-minus,Al-3+, a number of heavy metals, oxidizing agents and sulphydryl-modifying reagents. Kinetic measurements at pH 5-2 gave a Km value for p-nitrophenyl phosphate of 1-2 mM. Orthophosphate, pyrophosphate and isoelectrically precipitated casein behaved as competitive inhibitors to the hydrolysis of p-nitrophenyl phosphate with Ki values of 1-2 mM, 1-0 mM, 1-0 MM and 1-1 mM respectively. In spite of this binding to the enzyme, casein provided a very poor substrate for the starter acid phosphatase. The properties of acid phosphatase present in Cheddar cheese made with Str. cremoris NCDO 924 starter were consistent with the enzyme being exclusively of milk origin and small differences between this and the acid phosphatase previously isolated from bovine milk were attributable to the binding of peptides produced during the cheese maturation to the enzyme molecules. It was concluded that in cheese, phosphatase action was due largely to the enzyme of milk origin, with that provided by the starter being of minor importance.

  10. Antidiabetic Activity from Gallic Acid Encapsulated Nanochitosan

    Science.gov (United States)

    Purbowatiningrum; Ngadiwiyana; Ismiyarto; Fachriyah, E.; Eviana, I.; Eldiana, O.; Amaliyah, N.; Sektianingrum, A. N.

    2017-02-01

    Diabetes mellitus (DM) has become a health problem in the world because it causes death. One of the phenolic compounds that have antidiabetic activity is gallic acid. However, the use of this compound still provides unsatisfactory results due to its degradation during the absorption process. The solution offered to solve the problem is by encapsulated it within chitosan nanoparticles that serve to protect the bioactive compound from degradation, increases of solubility and delivery of a bioactive compound to the target site by using freeze-drying technique. The result of chitosan nanoparticle’s Scanning Electron Microscopy (SEM) showed that chitosan nanoparticle’s size is uniform and it is smaller than chitosan. The value of encapsulation efficiency (EE) of gallic acid which encapsulated within chitosan nanoparticles is about 50.76%. Inhibition test result showed that gallic acid-chitosan nanoparticles at 50 ppm could inhibite α-glucosidase activity in 28.87% with 54.94 in IC50. So it can be concluded that gallic acid can be encapsulated in nanoparticles of chitosan and proved that it could inhibit α-glucosidase.

  11. Synthesis and anticonvulsant activity of novel bicyclic acidic amino acids

    DEFF Research Database (Denmark)

    Conti, Paola; De Amici, Marco; Joppolo Di Ventimiglia, Samuele

    2003-01-01

    Bicyclic acidic amino acids (+/-)-6 and (+/-)-7, which are conformationally constrained homologues of glutamic acid, were prepared via a strategy based on a 1,3-dipolar cycloaddition. The new amino acids were tested toward ionotropic and metabotropic glutamate receptor subtypes; both of them...

  12. The mobA gene is required for assimilatory and respiratory nitrate reduction but not xanthine dehydrogenase activity in Pseudomonas aeruginosa.

    Science.gov (United States)

    Noriega, Chris; Hassett, Daniel J; Rowe, John J

    2005-12-01

    The requirement for the mobA gene in key assimilatory and respiratory nitrogen metabolism of Pseudomonas aeruginosa PAO1 was investigated by mutational analysis of PA3030 (mobA; MoCo guanylating enzyme), PA1779 (nasA; assimilatory nitrate reductase), and PA3875 (narG; respiratory nitrate reductase). The mobA mutant was deficient in both assimilatory and respiratory nitrate reductase activities, whereas xanthine dehydrogenase activity remained unaffected. Thus, P. aeruginosa requires both the molybdopterin (MPT) and molybdopterin guanine dinucleotide (MGD) forms of the molybdenum cofactor for a complete spectrum of nitrogen metabolism, and one form cannot substitute for the other. Regulation studies using a Phi(PA3030-lacZGm) reporter strain suggest that expression of mobA is not influenced by the type of nitrogen source or by anaerobiosis, whereas assimilatory nitrate reductase activity was detected only in the presence of nitrate.

  13. 7α-hydroxylation of dehydroepiandrosterone does not interfere with the activation of glucocorticoids by 11β-hydroxysteroid dehydrogenase in E(t)C cerebellar neurons.

    Science.gov (United States)

    Gottfried-Blackmore, Andres; Jellinck, Peter H; Vecchiarelli, Haley A; Masheeb, Zahrah; Kaufmann, Martin; McEwen, Bruce S; Bulloch, Karen

    2013-11-01

    The neuroprotective action of dehydroepiandrosterone (DHEA) in the absence of a known specific receptor has been attributed to its metabolism by different cell types in the brain to various steroids, with a preference to its 7-hydroxylated products. The E(t)C cerebellar granule cell line converts DHEA almost exclusively to 7α-hydroxy-DHEA (7α-OH-DHEA). It has been postulated that DHEA's 7-OH and 7-oxo metabolites can decrease glucocorticoid levels by an interactive mechanism involving 11β-hydroxysteroid dehydrogenase (11β-HSD). In order to study the relationship of 7-hydroxylation of DHEA and glucocorticoid metabolism in intact brain cells, we examined whether E(t)C cerebellar neurons, which are avid producers of 7α-OH-DHEA, could also metabolize glucocorticoids. We report that E(t)C neuronal cells exhibit 11β-HSD1 reductase activity, and are able to convert 11-dehydrocorticosterone into corticosterone, whereas they do not demonstrate 11β-HSD2 dehydrogenase activity. Consequently, E(t)C cells incubated with DHEA did not yield 7-oxo- or 7β-OH-DHEA. Our findings are supported by the reductive environment of E(t)C cells through expression of hexose-6-phosphate dehydrogenase (H6PDH), which fosters 11β-HSD1 reductase activity. To further explore the role of 7α-OH-DHEA in E(t)C neuronal cells, we examined the effect of preventing its formation using the CYP450 inhibitor ketoconazole. Treatment of the cells with this drug decreased the yield of 7α-OH-DHEA by about 75% without the formation of alternate DHEA metabolites, and had minimal effects on glucocorticoid conversion. Likewise, elevated levels of corticosterone, the product of 11β-HSD1, had no effect on the metabolic profile of DHEA. This study shows that in a single population of whole-cells, with a highly reductive environment, 7α-OH-DHEA is unable to block the reducing activity of 11β-HSD1, and that 7-hydroxylation of DHEA does not interfere with the activation of glucocorticoids. Our investigation

  14. Control of the Quantum Efficiencies of Photosystems I and II, Electron Flow, and Enzyme Activation following Dark-to-Light Transitions in Pea Leaves: Relationship between NADP/NADPH Ratios and NADP-Malate Dehydrogenase Activation State.

    Science.gov (United States)

    Foyer, C H; Lelandais, M; Harbinson, J

    1992-07-01

    The quantum efficiencies of photosystems I and II (PSI and PSII), [NADP]/[NADPH] ratios, and the activities of chloroplastic fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were measured in intact pea (Pisum sativum L.) leaves in air following the transition from darkness to 750 microeinsteins per square meter per second irradiance. PSII efficiency declined from a low value to a minimum within the first 10 to 15 seconds of irradiance, after which it increased progressively to the steady-state value. The resistance of electron flow between the photosystems was high at this time, but it was not the principal factor limiting electron flow. Oxidation of P700 was restricted by acceptor side processes for approximately the first 60 seconds of illumination. Once the acceptor side limitation was relieved, the oxidation state of P700 was used to estimate the quantum efficiency of electron transport by PSI. This was observed to increase progressively with time. The quantum efficiencies of both photosystems increased in parallel, consistent with a predominant role for noncyclic electron transport. Fructose-1,6-bisphosphatase activity increased in an approximately sigmoidal fashion with time of irradiance, paralleling the changes in the quantum efficiencies of the photosystems. In contrast, the activation of NADP-malate dehydrogenase did not show a lag period but increased with time, reaching a maximum value at about 50 seconds of illumination, after which it declined. The NADP pool was not extensively reduced during the first 10 seconds of illumination, but became so subsequently. It remained in the reduced state until about 60 seconds of illumination and then became relatively oxidized. The empirical relationship between NADP-malate dehydrogenase activity and the reduction state of the NADP pool supports the suggestion that NADP-malate dehydrogenase activity is a useful estimate of the reduction state of the stroma.

  15. Oxyphil cell metaplasia in the parathyroids is characterized by somatic mitochondrial DNA mutations in NADH dehydrogenase genes and cytochrome c oxidase activity-impairing genes.

    Science.gov (United States)

    Müller-Höcker, Josef; Schäfer, Sabine; Krebs, Stefan; Blum, Helmut; Zsurka, Gábor; Kunz, Wolfram S; Prokisch, Holger; Seibel, Peter; Jung, Andreas

    2014-11-01

    Oxyphil cell transformation of epithelial cells due to the accumulation of mitochondria occurs often during cellular aging. To understand the pathogenic mechanisms, we studied mitochondrial DNA (mtDNA) alterations in the three cell types of the parathyroids using multiplex real-time PCR and next-generation sequencing. mtDNA was analyzed from cytochrome c oxidase (COX)-positive and COX-negative areas of 19 parathyroids. Mitochondria-rich pre-oxyphil/oxyphil cells were more prone to develop COX defects than the mitochondria-poor clear chief cells (P cells. In COX deficiency, the increase was even more pronounced, and COX-negative oxyphil cells had approximately two times more mtDNA than COX-positive oxyphil cells (P cells and, therefore, could be essential for inducing oxyphil cell transformation by increasing mtDNA/mitochondrial biogenesis. In contrast, COX-negative cells predominantly harbored mutations in the MT-CO1 and MT-CO3 genes and in regulatory mtDNA elements, but only rarely NADH dehydrogenase mutations. Thus, multiple hits in NADH dehydrogenase and COX activity-impairing genes represent the molecular basis of oxyphil cell transformation in the parathyroids.

  16. The influence of individualizing physical loads on speed, creatine kinase activity and lactate dehydrogenase in football players.

    Directory of Open Access Journals (Sweden)

    2008-06-01

    Full Text Available Introduction: One of the most important training problems in: contemporary football is speed preparation of a player for the season and the ability of keeping it on the same, relatively high level throughout the starting period [1]. The main process used for re-synthesis ATP during single, short-lasting efforts of maximal intensity, is decomposition of phospho-creatine under the influence of creatine kinase enzyme. Physical loads imposed during speed trainings often exceed the possibility of producing energy from phosphogenic reserve through oxygen - lactate free processes, because the supply of phospho-creatine is used very quickly. In such circumstances the lacking energy is refilled through processes called oxygen free glicolise with the help of lactate dehydrogenase enzyme. The aim of the work was to answer the question:

  17. Enhanced acyl-CoA dehydrogenase activity is associated with improved mitochondrial and contractile function in heart failure

    Science.gov (United States)

    Heart failure is associated with decreased myocardial fatty acid oxidation capacity and has been likened to energy starvation. Increased fatty acid availability results in an induction of genes promoting fatty acid oxidation. The aim of the present study was to investigate possible mechanisms by whi...

  18. Development of an amine dehydrogenase for synthesis of chiral amines.

    Science.gov (United States)

    Abrahamson, Michael J; Vázquez-Figueroa, Eduardo; Woodall, Nicholas B; Moore, Jeffrey C; Bommarius, Andreas S

    2012-04-16

    A leucine dehydrogenase has been successfully altered through several rounds of protein engineering to an enantioselective amine dehydrogenase. Instead of the wild-type α-keto acid, the new amine dehydrogenase now accepts the analogous ketone, methyl isobutyl ketone (MIBK), which corresponds to exchange of the carboxy group by a methyl group to produce chiral (R)-1,3-dimethylbutylamine. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Determination of the DNA and Amino Acid Sequences of the Lactate Dehydrogenase Gene from Plasmodium falciparum Strains K1 and PF FCBR: A Route to the Design of New Antimalarials

    OpenAIRE

    BALIK, Dilek TURGUT

    2014-01-01

    This paper describes cloning of the gene coding for the lactate dehydrogenase (LDH) gene from the two different strains of Plasmodium falciparum, K1 and PF FCBR. The DNA sequences of LDH genes of these two strains were found to be identical. Amino acid sequence alignment of LDH from P. falciparum strains K1 and PF FCBR (PfLDH) to some other known LDH sequences showed that PfLDH has 29% residue identities with Bacillus stearothermophilus (BsLDH) and 29%, 31%, 33% with dogfish, man-M4, and pig-...

  20. Lactate dehydrogenase has no control on lactate production but has a strong negative control on formate production in Lactococcus lactis

    DEFF Research Database (Denmark)

    Andersen, H.W.; Pedersen, M.B.; Hammer, Karin

    2001-01-01

    a homolactic pattern of fermentation. Only after lactate dehydrogenase activity was reduced ninefold compared to the wild-type was the growth rate significantly affected, and the ldh mutants started to produce mixed-acid products (formate, acetate, and ethanol in addition to lactate). Flux control coefficients...... were determined and it was found that lactate dehydrogenase exerted virtually no control on the glycolytic flux at the wild-type enzyme level and also not on the flux catalyzed by the enzyme itself, i.e. on the lactate production. As expected, the flux towards the mixed-acid products was strongly...

  1. Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Youngchang; Makowska-Grzyska, Magdalena; Gorla, Suresh Kumar; Gollapalli, Deviprasad R.; Cuny, Gregory D.; Joachimiak, Andrzej; Hedstrom, Lizbeth

    2015-04-21

    Inosine 5'-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment ofCryptosporidiuminfections. Here, the structure ofC. parvumIMPDH (CpIMPDH) in complex with inosine 5'-monophosphate (IMP) and P131, an inhibitor within vivoanticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO2moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compounds to replace these water molecules is a new strategy for the further optimization ofC. parvuminhibitors for both antiparasitic and antibacterial applications.

  2. Isocitrate dehydrogenase, lactate dehydrogenase and α-glycerophosphate dehydrogenase polymorphysm enzyme of Black Tiger Shrimp (Penaeus monodon Fab. which is hydrogen sulfide resistant

    Directory of Open Access Journals (Sweden)

    OKID PARAMA ASTIRIN

    2006-07-01

    Full Text Available The hydrogen sulfide is one of compound which very often found in the shrimp pond caused by anaerobic decomposition or as a natural condition of the sea water which have volcano activity. This research was obtaining information of the differences of genetic expression between black tiger shrimp which could resist to H2S and the one which could not survive in this H2S. This research also trying to obtain information the genetic variety which could resist to H2S. The genetic variety of black tiger shrimp which could resist to H2S has been analysis with allozyme electrophoresis technique, using specific tissue meat and buffer CAPM (Citric Acid Aminopolimorpholine pH 6. From the three enzymes analyzed it could be detected that IDH enzyme (Isocitrate dehydrogenase has locus polymorphic, whereas enzyme α-GDP (α-glycerophosphate dehydrogenase and LDH (lactate dehydrogenase with monomorphic locus. The average heterozygosity for the group which could resist to H2S is 0.070, whereas the group which could not survive in the H2S is 0.042. The control group has heterozygosity 0.041. The group which could resist to H2S with higher heterozygosity will have bigger chance to survive and have better adaptation ability in environmental changes. A high heterozygosity made possible for genetic population improvement by exploiting the good gene.

  3. A novel 3-hydroxysteroid dehydrogenase that regulates reproductive development and longevity.

    Directory of Open Access Journals (Sweden)

    Joshua Wollam

    Full Text Available Endogenous small molecule metabolites that regulate animal longevity are emerging as a novel means to influence health and life span. In C. elegans, bile acid-like steroids called the dafachronic acids (DAs regulate developmental timing and longevity through the conserved nuclear hormone receptor DAF-12, a homolog of mammalian sterol-regulated receptors LXR and FXR. Using metabolic genetics, mass spectrometry, and biochemical approaches, we identify new activities in DA biosynthesis and characterize an evolutionarily conserved short chain dehydrogenase, DHS-16, as a novel 3-hydroxysteroid dehydrogenase. Through regulation of DA production, DHS-16 controls DAF-12 activity governing longevity in response to signals from the gonad. Our elucidation of C. elegans bile acid biosynthetic pathways reveals the possibility of novel ligands as well as striking biochemical conservation to other animals, which could illuminate new targets for manipulating longevity in metazoans.

  4. Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids.

    Science.gov (United States)

    Zheng, Chang Ji; Yoo, Jung-Sung; Lee, Tae-Gyu; Cho, Hee-Young; Kim, Young-Ho; Kim, Won-Gon

    2005-09-26

    Long-chain unsaturated fatty acids, such as linoleic acid, show antibacterial activity and are the key ingredients of antimicrobial food additives and some antibacterial herbs. However, the precise mechanism for this antimicrobial activity remains unclear. We found that linoleic acid inhibited bacterial enoyl-acyl carrier protein reductase (FabI), an essential component of bacterial fatty acid synthesis, which has served as a promising target for antibacterial drugs. Additional unsaturated fatty acids including palmitoleic acid, oleic acid, linolenic acid, and arachidonic acid also exhibited the inhibition of FabI. However, neither the saturated form (stearic acid) nor the methyl ester of linoleic acid inhibited FabI. These FabI-inhibitory activities of various fatty acids and their derivatives very well correlated with the inhibition of fatty acid biosynthesis using [(14)C] acetate incorporation assay, and importantly, also correlated with antibacterial activity. Furthermore, the supplementation with exogenous fatty acids reversed the antibacterial effect of linoleic acid, which showing that it target fatty acid synthesis. Our data demonstrate for the first time that the antibacterial action of unsaturated fatty acids is mediated by the inhibition of fatty acid synthesis.

  5. Amino acid sequence of the tryptic peptide containing the catalytic-site thiol group of bovine liver uridine diphosphate glucose dehydrogenase.

    OpenAIRE

    Franzen, B.; Carrubba, C; Feingold, D S; Ashcom, J; Franzen, J S

    1981-01-01

    The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P...

  6. Tetrahydro-2-naphthyl and 2-Indanyl Triazolopyrimidines Targeting Plasmodium falciparum Dihydroorotate Dehydrogenase Display Potent and Selective Antimalarial Activity.

    Science.gov (United States)

    Kokkonda, Sreekanth; Deng, Xiaoyi; White, Karen L; Coteron, Jose M; Marco, Maria; de Las Heras, Laura; White, John; El Mazouni, Farah; Tomchick, Diana R; Manjalanagara, Krishne; Rudra, Kakali Rani; Chen, Gong; Morizzi, Julia; Ryan, Eileen; Kaminsky, Werner; Leroy, Didier; Martínez-Martínez, María Santos; Jimenez-Diaz, Maria Belen; Bazaga, Santiago Ferrer; Angulo-Barturen, Iñigo; Waterson, David; Burrows, Jeremy N; Matthews, Dave; Charman, Susan A; Phillips, Margaret A; Rathod, Pradipsinh K

    2016-06-09

    Malaria persists as one of the most devastating global infectious diseases. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) has been identified as a new malaria drug target, and a triazolopyrimidine-based DHODH inhibitor 1 (DSM265) is in clinical development. We sought to identify compounds with higher potency against Plasmodium DHODH while showing greater selectivity toward animal DHODHs. Herein we describe a series of novel triazolopyrimidines wherein the p-SF5-aniline was replaced with substituted 1,2,3,4-tetrahydro-2-naphthyl or 2-indanyl amines. These compounds showed strong species selectivity, and several highly potent tetrahydro-2-naphthyl derivatives were identified. Compounds with halogen substitutions displayed sustained plasma levels after oral dosing in rodents leading to efficacy in the P. falciparum SCID mouse malaria model. These data suggest that tetrahydro-2-naphthyl derivatives have the potential to be efficacious for the treatment of malaria, but due to higher metabolic clearance than 1, they most likely would need to be part of a multidose regimen.

  7. Dual loss of succinate dehydrogenase (SDH) and complex I activity is necessary to recapitulate the metabolic phenotype of SDH mutant tumors.

    Science.gov (United States)

    Lorendeau, Doriane; Rinaldi, Gianmarco; Boon, Ruben; Spincemaille, Pieter; Metzger, Kristine; Jäger, Christian; Christen, Stefan; Dong, Xiangyi; Kuenen, Sabine; Voordeckers, Karin; Verstreken, Patrik; Cassiman, David; Vermeersch, Pieter; Verfaillie, Catherine; Hiller, Karsten; Fendt, Sarah-Maria

    2017-09-01

    Mutations in succinate dehydrogenase (SDH) are associated with tumor development and neurodegenerative diseases. Only in tumors, loss of SDH activity is accompanied with the loss of complex I activity. Yet, it remains unknown whether the metabolic phenotype of SDH mutant tumors is driven by loss of complex I function, and whether this contributes to the peculiarity of tumor development versus neurodegeneration. We addressed this question by decoupling loss of SDH and complex I activity in cancer cells and neurons. We found that sole loss of SDH activity was not sufficient to recapitulate the metabolic phenotype of SDH mutant tumors, because it failed to decrease mitochondrial respiration and to activate reductive glutamine metabolism. These metabolic phenotypes were only induced upon the additional loss of complex I activity. Thus, we show that complex I function defines the metabolic differences between SDH mutation associated tumors and neurodegenerative diseases, which could open novel therapeutic options against both diseases. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  8. Preparation and some properties of L-3-glycerophosphate dehydrogenase from pig brain mitochondria.

    Science.gov (United States)

    Dawson, A P; Thorne, C J

    1969-01-01

    1. A method is described for extraction and partial purification of mitochondrial l-3-glycerophosphate dehydrogenase from pig brain. 2. By the criteria that have so far been applied, the extraction and purification procedures do not modify the activity of the enzyme towards artificial electron acceptors. 3. The amounts of acid-liberatable flavine and iron in the preparation were measured. 4. A study was made of the effects of various analogues of l-3-glycerophosphate on the activity of the enzyme.

  9. Formaldehyde dehydrogenase preparations from Methylococcus capsulatus (Bath) comprise methanol dehydrogenase and methylene tetrahydromethanopterin dehydrogenase.

    Science.gov (United States)

    Adeosun, Ekundayo K; Smith, Thomas J; Hoberg, Anne-Mette; Velarde, Giles; Ford, Robert; Dalton, Howard

    2004-03-01

    In methylotrophic bacteria, formaldehyde is an important but potentially toxic metabolic intermediate that can be assimilated into biomass or oxidized to yield energy. Previously reported was the purification of an NAD(P)(+)-dependent formaldehyde dehydrogenase (FDH) from the obligate methane-oxidizing methylotroph Methylococcus capsulatus (Bath), presumably important in formaldehyde oxidation, which required a heat-stable factor (known as the modifin) for FDH activity. Here, the major protein component of this FDH preparation was shown by biophysical techniques to comprise subunits of 64 and 8 kDa in an alpha(2)beta(2) arrangement. N-terminal sequencing of the subunits of FDH, together with enzymological characterization, showed that the alpha(2)beta(2) tetramer was a quinoprotein methanol dehydrogenase of the type found in other methylotrophs. The FDH preparations were shown to contain a highly active NAD(P)(+)-dependent methylene tetrahydromethanopterin dehydrogenase that was the probable source of the NAD(P)(+)-dependent formaldehyde oxidation activity. These results support previous findings that methylotrophs possess multiple pathways for formaldehyde dissimilation.

  10. Change in glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase activities in the organs of mice treated with 2-chloroethyl ethyl sulfide or its oxidation products.

    Science.gov (United States)

    Kim, Y B; Lee, Y S; Choi, D S; Cha, S H; Sok, D E

    1996-03-01

    Various organs or skin from male ICR mice treated intraperitoneally with 2-chloroethyl ethyl sulfide (CEES) or its oxidation derivatives 2-chloroethyl ethyl sulfoxide (CESSO) and 2-chloroethyl ethyl sulfone were analysed for changes in two thiol-containing enzymes, namely glutathione S-transferase (GST) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). CEES was more potent than its oxidation derivatives with respect to the decrease in organ weight and the loss in GAPDH activity, although the reverse was found in GST induction. Whereas the induction of GST was highest in the lung after multiple intraperitoneal intoxication with CEESO (8 and 32 mg/kg), the decrease in GAPDH activity after exposure to CEES (8 mg/kg body weight) was most remarkable in the spleen, the most susceptible organ to toxicity of CEES. GST and GAPDH activities in the skin of male hairless mice exposed subcutaneously to CEES (2 mg/kg body weight) were not altered significantly at 2-hr exposure, but decreased up to 60% of that of controls at 8 hr, when oedema formation was greatest. Taken together, it appears that GAPDH activity is a more sensitive biochemical parameter than GST activity in organs of mice treated with CEES or its oxidation products.

  11. Plant Formate Dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    John Markwell

    2005-01-10

    The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

  12. Efficient production of optically pure D-lactic acid from raw corn starch by using a genetically modified L-lactate dehydrogenase gene-deficient and alpha-amylase-secreting Lactobacillus plantarum strain.

    Science.gov (United States)

    Okano, Kenji; Zhang, Qiao; Shinkawa, Satoru; Yoshida, Shogo; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-01-01

    In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 alpha-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch.

  13. Improved localization of glucose-6-phosphate dehydrogenase activity in cells with 5-cyano-2,3-ditolyl-tetrazolium chloride as fluorescent redox dye reveals its cell cycle-dependent regulation

    NARCIS (Netherlands)

    Frederiks, Wilma M.; van Marle, Jan; van Oven, Carel; Comin-Anduix, Begonya; Cascante, Marta

    2006-01-01

    Since the introduction of cyano-ditolyl-tetrazolium chloride (CTC), a tetrazolium salt that gives rise to a fluorescent formazan after reduction, it has been applied to quantify activity of dehydrogenases in individual cells using flow cytometry. Confocal laser scanning microscopy (CLSM) showed that

  14. SERUM LACTATE-DEHYDROGENASE ISOENZYME-1 ACTIVITY IN PATIENTS WITH TESTICULAR GERM-CELL TUMORS CORRELATES WITH THE TOTAL NUMBER OF COPIES OF THE SHORT ARM OF CHROMOSOME-12 IN THE TUMOR

    NARCIS (Netherlands)

    VONEYBEN, FE; DEGRAAFF, WE; MARRINK, J; BLAABJERG, O; SLEIJFER, DT; KOOPS, HS; OOSTERHUIS, JW; PETERSEN, PH; VANECHTENARENDS, J; DEJONG, B

    1992-01-01

    The aim of our study was to assess the relationship between the serum lactate dehydrogenase isoenzyme 1 (S-LDH-1) activity in patients with testicular germ cell tumors and the number of copies of the short arm of chromosome 12 (12p) present in tumor. Twenty-seven adult patients with measurable tumor

  15. "JCE" Classroom Activity #109: My Acid Can Beat Up Your Acid!

    Science.gov (United States)

    Putti, Alice

    2011-01-01

    In this guided-inquiry activity, students investigate the ionization of strong and weak acids. Bead models are used to study acid ionization on a particulate level. Students analyze seven strong and weak acid models and make generalizations about the relationship between acid strength and dissociation. (Contains 1 table and 2 figures.)

  16. Characterization of human short chain dehydrogenase/reductase SDR16C family members related to retinol dehydrogenase 10.

    Science.gov (United States)

    Adams, Mark K; Lee, Seung-Ah; Belyaeva, Olga V; Wu, Lizhi; Kedishvili, Natalia Y

    2017-10-01

    All-trans-retinoic acid (RA) is a bioactive derivative of vitamin A that serves as an activating ligand for nuclear transcription factors, retinoic acid receptors. RA biosynthesis is initiated by the enzymes that oxidize retinol to retinaldehyde. It is well established that retinol dehydrogenase 10 (RDH10, SDR16C4), which belongs to the 16C family of the short chain dehydrogenase/reductase (SDR) superfamily of proteins, is the major enzyme responsible for the oxidation of retinol to retinaldehyde for RA biosynthesis during embryogenesis. However, several lines of evidence point towards the existence of additional retinol dehydrogenases that contribute to RA biosynthesis in vivo. In close proximity to RDH10 gene on human chromosome 8 are located two genes that are phylogenetically related to RDH10. The predicted protein products of these genes, retinol dehydrogenase epidermal 2 (RDHE2, SDR16C5) and retinol dehydrogenase epidermal 2-similar (RDHE2S, SDR16C6), share 59% and 56% sequence similarity with RDH10, respectively. Previously, we showed that the single ortholog of the human RDHE2 and RDHE2S in frogs, Xenopus laevis rdhe2, oxidizes retinol to retinaldehyde and is essential for frog embryonic development. In this study, we explored the potential of each of the two human proteins to contribute to RA biosynthesis. The results of this study demonstrate that human RDHE2 exhibits a relatively low but reproducible activity when expressed in either HepG2 or HEK293 cells. Expression of the native RDHE2 is downregulated in the presence of elevated levels of RA. On the other hand, the protein encoded by the human RDHE2S gene is unstable when expressed in HEK293 cells. RDHE2S protein produced in Sf9 cells is stable but has no detectable catalytic activity towards retinol. We conclude that the human RDHE2S does not contribute to RA biosynthesis, whereas the low-activity RA-sensitive human RDHE2 may have a role in adjusting the cellular levels of RA in accord with

  17. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120

    Science.gov (United States)

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F.; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

  18. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

    Science.gov (United States)

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

  19. Isolation and biochemical characterization of a glucose dehydrogenase from a hay infusion metagenome.

    Directory of Open Access Journals (Sweden)

    Alexander Basner

    Full Text Available Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different Bacillus species. The identified glucose dehydrogenase gene was expressed in E. coli, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P, the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein.

  20. Determining soil enzyme activities for the assessment of fungi and citric acid-assisted phytoextraction under cadmium and lead contamination.

    Science.gov (United States)

    Mao, Liang; Tang, Dong; Feng, Haiwei; Gao, Yang; Zhou, Pei; Xu, Lurong; Wang, Lumei

    2015-12-01

    Microorganism or chelate-assisted phytoextraction is an effective remediation tool for heavy metal polluted soil, but investigations into its impact on soil microbial activity are rarely reported. Consequently, cadmium (Cd)- and lead (Pb)-resistant fungi and citric acid (CA) were introduced to enhance phytoextraction by Solanum nigrum L. under varied Cd and Pb pollution levels in a greenhouse pot experiment. We then determined accumulation of Cd and Pb in S. nigrum and the soil enzyme activities of dehydrogenase, phosphatase, urease, catalase, sucrase, and amylase. Detrended canonical correspondence analysis (DCCA) was applied to assess the interactions between remediation strategies and soil enzyme activities. Results indicated that the addition of fungi, CA, or their combination enhanced the root biomass of S. nigrum, especially at the high-pollution level. The combined treatment of CA and fungi enhanced accumulation of Cd about 22-47 % and of Pb about 13-105 % in S. nigrum compared with the phytoextraction alone. However, S. nigrum was not shown to be a hyperaccumulator for Pb. Most enzyme activities were enhanced after remediation. The DCCA ordination graph showed increasing enzyme activity improvement by remediation in the order of phosphatase, amylase, catalase, dehydrogenase, and urease. Responses of soil enzyme activities were similar for both the addition of fungi and that of CA. In summary, results suggest that fungi and CA-assisted phytoextraction is a promising approach to restoring heavy metal polluted soil.

  1. Targeting isocitrate dehydrogenase (IDH) in cancer.

    Science.gov (United States)

    Fujii, Takeo; Khawaja, Muhammad Rizwan; DiNardo, Courtney D; Atkins, Johnique T; Janku, Filip

    2016-05-01

    Isocitrate dehydrogenase (IDH) is an essential enzyme for cellular respiration in the tricarboxylic acid (TCA) cycle. Recurrent mutations in IDH1 or IDH2 are prevalent in several cancers including glioma, acute myeloid leukemia (AML), cholangiocarcinoma and chondrosarcoma. The mutated IDH1 and IDH2 proteins have a gain-of-function, neomorphic activity, catalyzing the reduction of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG) by NADPH. Cancer-associated IDH mutations block normal cellular differentiation and promote tumorigenesis via the abnormal production of the oncometabolite 2-HG. High levels of 2-HG have been shown to inhibit α-KG dependent dioxygenases, including histone and deoxyribonucleic acid (DNA) demethylases, which play a key role in regulating the epigenetic state of cells. Current targeted inhibitors of IDH1 (AG120, IDH305), IDH2 (AG221), and pan-IDH1/2 (AG881) selectively inhibit mutant IDH protein and induce cell differentiation in in vitro and in vivo models. Preliminary results from phase I clinical trials with IDH inhibitors in patients with advanced hematologic malignancies have demonstrated an objective response rate ranging from 31% to 40% with durable responses (>1 year) observed. Furthermore, the IDH inhibitors have demonstrated early signals of activity in solid tumors with IDH mutations, including cholangiocarcinomas and low grade gliomas.

  2. Effects of temperature, pH-values and sodium chloride concentrations on the glucose-6-phosphate dehydrogenase activity by thermotolerant Bacillus strains

    Directory of Open Access Journals (Sweden)

    HAZEM AQEL

    2012-01-01

    Full Text Available Thirteen new isolated thermotolerant Bacillus strains and four known Bacillus species were used to evaluate the effect of growth temperature, pH-values and NaCl concentrations on the intracellular glucose-6-phosphate dehydrogenase (G6PDH activity. Results had shown a significant difference in G6PDH production among all species at all used temperatures (p<0.05. The response of individual new isolates and controls for production of G6PDH under growth conditions was variable. The optimal growth conditions did not correspond to the optimal cultivation conditions for maximum G6PDH production. The growth temperature showed the most significant effect on G6PDH activity. The combined effect of temperature and NaCl on the G6PDH activity was strongly pronounced in comparison with the combined effect of temperature and pH or pH and NaCl. Thermal stability at 53ºC and electrophoretic mobility were also investigated. G6PDH from HUTB41 was the most thermostable G6PDH enzyme with T50% of more than 360 minutes. Electrophoretic study demonstrated that G6PDH was composed of two isoenzymes for all strains except B. marinus and B. schlegelii that had three isoenzymes.

  3. Effect of in vivo ozone exposure to Dorset sheep, an animal model with low levels of erythrocyte glucose-6-phosphate dehydrogenase activity

    Energy Technology Data Exchange (ETDEWEB)

    Moore, G.S.; Calabrese, E.J.; Schulz, E.

    1981-02-01

    Considerable interest has recently been directed to the possible extrapulmonary effects caused by exposure to ambient ozone. As a result of ozone induced in vivo alteration of red cell function within human subjects, it has been hypothesized that individuals with an erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) deficiency would be at increased hemolytic risk to elevated ambient ozone exposure. In order to evaluate such an hypothesis in an experimental setting it would be of great value to have an appropriate animal model with erythrocyte G-6-PD activity similar to the absolute activity range found in the human population. While no such unique animal model is presently known, the literature has revealed that Dorset sheep have an erythrocyte G-6-PD activity comparable in absolute units to a human G-6-PD deficient. Based on this information, we evaluated the mechanisms by which sheep and human G-6-PD deficient red cells handle oxidant stress. We evaluated the effects of in vivo ozone exposure in Dorset sheep over a broad range of concentrations.

  4. Bioisosteric transformations and permutations in the triazolopyrimidine scaffold to identify the minimum pharmacophore required for inhibitory activity against Plasmodium falciparum dihydroorotate dehydrogenase

    Science.gov (United States)

    Marwaha, Alka; White, John; El_Mazouni, Farah; Creason, Sharon A; Kokkonda, Sreekanth; Buckner, Frederick S.; Charman, Susan A.; Phillips, Margaret A.; Rathod, Pradipsinh K.

    2012-01-01

    Plasmodium falciparum causes approximately 1 million deaths annually. However increasing resistance imposes a continuous threat to existing drug therapies. We previously reported a number of potent and selective triazolopyrimidine-based inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase that inhibit parasite in vitro growth with similar activity. Lead optimization of this series led to the recent identification of a preclinical candidate, showing good activity against P. falciparum in mice. As part of a backup program around this scaffold, we explored heteroatom rearrangement and substitution in the triazolopyrimidine ring and have identified several other ring configurations that are active as PfDHODH inhibitors. The imidazo[1,2-α]pyrimidines were shown to bind somewhat more potently than the triazolopyrimidines depending on the nature of the amino aniline substitution. DSM151, the best candidate in this series, binds with 4-fold better affinity (PfDHODH IC50 = 0.077 μM) than the equivalent triazolopyrimidine and suppresses parasites in vivo in the P. berghei model. PMID:22877245

  5. Stilbene Glucoside, a Putative Sleep Promoting Constituent from Polygonum multiflorum Affects Sleep Homeostasis by Affecting the Activities of Lactate Dehydrogenase and Salivary Alpha Amylase.

    Science.gov (United States)

    Wei, Qian; Ta, Guang; He, Wenjing; Wang, Wei; Wu, Qiucheng

    2017-01-01

    Chinese herbal medicine (CHM) has been used for treating insomnia for centuries. The most used CHM for insomnia was Polygonum multiflorum. However, the molecular mechanism for CHM preventing insomnia is unknown. Stilbene glucoside (THSG), an important active component of P. multiflorum, may play an important role for treating insomnia. To test the hypothesis, Kunming mice were treated with different dosages of THSG. To examine the sleep duration, a computer-controlled sleep-wake detection system was implemented. Electroencephalogram (EEG) and electromyogram (EMG) electrodes were implanted to determine sleep-wake state. RT-PCR and Western blot was used to measure the levels of lactate dehydrogenase (LDH) and saliva alpha amylase. Spearman's rank correlation coefficient was used to identify the strength of correlation between the variables. The results showed that THSG significantly prolonged the sleep time of the mice (p<0.01). THSG changed sleep profile by reducing wake and rapid eye movement (REM) period, and increasing non-REM period. RT-PCR and Western blot analysis showed that THSG could down-regulate the levels of LDH and saliva alpha amylase (p<0.05). The level of lactate and glucose was positively related with the activity of LDH and saliva alpha amylase (p<0.05), respectively. On the other hand, the activities of LDH and amylase were negatively associated with sleep duration (p<0.05). The levels of lactate and glucose affect sleep homeostasis. Thus, THSG may prevent insomnia by regulating sleep duration via LDH and salivary alpha amylase.

  6. Changes in pyruvate dehydrogenase complex (PDHc) activity and [3H]QNB-receptor binding in rat brain subsequent to intracerebroventricular injection of bromopyruvate.

    Science.gov (United States)

    Frölich, L; Strauss, M; Kornhuber, J; Hoyer, S; Sorbi, S; Riederer, P; Amaducci, L

    1990-01-01

    Pyruvate dehydrogenase complex (PDHc), a link between carbohydrate and acetylcholine metabolism, is a regulatory enzyme for glucose and neurotransmitter metabolism in the brain and is reduced in Alzheimer-diseased brain. To study functional consequences of an inhibition of PDHc on muscarinic receptor binding, bromopyruvate, a suicide inhibitor od PDHc, was injected intracerebroventricularly (icv) in rats. Bromopyruvate caused a reduction of PDHc activity in the 3 brain regions examined, however, reaching significance only in the cerebral cortex and the hippocampus and not in the striatum, 24 h after injection. 3, 6, and 12 weeks later, there was a normalization or transiently increased activity, respectively, of PDHc in these brain regions. No changes in concentrations of energy-rich phosphates could be demonstrated in the cerebral cortex 12 weeks after brompyruvate injection. The number of muscarinic receptors was significantly reduced in the cerebral cortex 12 weeks after injection. The data indicate that a transient reduction of brain PDHc activity in vivo is associated with a long-lasting reduction in muscarinic cholinergic receptors. Because comparable changes of PDHc and muscarinic receptors are found in dementia of Alzhemier type, the model of bromopyruvate inhibition of PDHc in rats is suggested to be useful for experimental dementia research.

  7. Effects of long-term oxygen treatment on α-ketoglutarate dehydrogenase activity and oxidative modifications in mitochondria of the guinea pig heart

    Directory of Open Access Journals (Sweden)

    Kaplan P

    2009-12-01

    Full Text Available Abstract Objective Oxygen therapy is used for the treatment of various diseases, but prolonged exposure to high concentrations of O2 is also associated with formation of free radicals and oxidative damage. Methods In the present study we compared α-ketoglutarate dehydrogenase (KGDH activity and mitochondrial oxidative damage in the hearts of guinea pigs after long-term (17 and 60 h oxygenation with 100% normobaric O2 and with partially negatively (O2 neg or positively (O2 posit ionized oxygen. Results Inhalation of O2 led to significant loss in KGDH activity and thiol group content and accumulation of bityrosines. Inhalation of O2 neg was accompanied by more pronounced KGDH inhibition, possibly due to additional formation of protein-lipid conjugates. In contrast, O2 posit prevented loss in KGDH activity and diminished mitochondrial oxidative damage. Conclusions These findings suggest that oxygen treatment is associated with impairment of heart energy metabolism and support the view that inhalation of O2 posit optimizes the beneficial effects of oxygen therapy.

  8. Suspended biofilm carrier and activated sludge removal of acidic pharmaceuticals

    DEFF Research Database (Denmark)

    Falås, Per; Baillon-Dhumez, Aude; Andersen, Henrik Rasmus

    2012-01-01

    Removal of seven active pharmaceutical substances (ibuprofen, ketoprofen, naproxen, diclofenac, clofibric acid, mefenamic acid, and gemfibrozil) was assessed by batch experiments, with suspended biofilm carriers and activated sludge from several full-scale wastewater treatment plants. A distinct...... and attached solids for the carriers) of diclofenac, ketoprofen, gemfibrozil, clofibric acid and mefenamic acid compared to the sludges. Among the target pharmaceuticals, only ibuprofen and naproxen showed similar removal rates per unit biomass for the sludges and biofilm carriers. In contrast...

  9. Inhibition of osteoblast activity by zoledronic acid

    Directory of Open Access Journals (Sweden)

    Fernanda Gonçalves Basso

    2013-10-01

    Full Text Available INTRODUCTION: Patients treated with nitrogen-containing bisphosphonates, such as zoledronic acid (ZA, have frequently shown oral bone exposure areas, termed osteonecrosis. In addition, these patients may also present low repair and regeneration potential, mainly after tooth extractions. These side-effects caused by bisphosphonates may be due to their inhibitory effects on oral mucosa and local bone cells. OBJECTIVE: To evaluate the effects of ZA on the mineralization capacity of cultured osteoblasts. MATERIALS AND METHODS: Human immortalized osteoblasts (SaOs-2 were grown in plain culture medium (Dulbecco's Modified Eagle Medium [DMEM] + 10% fetal bovine serum [FBS] in wells of 24-well plates. After 48-hour incubation, the plain DMEM was replaced by a solution with ZA at 5 µM which was maintained in contact with cells for seven, 14 or 21 days. After these periods, cells were evaluated regarding alkaline phosphatase (ALP activity and mineral nodule formation (alizarin red. Data were statistically analyzed by Mann-Whitney test, at 5% of significance level. RESULTS: ZA caused significant reduction on ALP activity and mineral nodules formation by cultured osteoblasts in all evaluated periods (p < 0.05. CONCLUSION: These data indicate that ZA causes inhibition on the osteogenic phenotype of cultured human osteoblasts, which, in turn, may reduce bone repair in patients subjected to ZA therapy.

  10. Some Properties of Glutamate Dehydrogenase from the Marine Red ...

    African Journals Online (AJOL)

    Daisy Ouya

    1) glutamate dehydrogenases (GDH) and (2) glutamine synthetase (GS)/ glutamate synthase (GOGAT). In the GS/ GOGAT route, ammonia is first incorporated into glutamine by the action of GS and subsequently into glutamic acid by GOGAT.

  11. Gastric alcohol dehydrogenase activity in man: influence of gender, age, alcohol consumption and smoking in a caucasian population

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Billinger, M. H.; Bode, C.

    2002-01-01

    potentially confounding factors (alcohol consumption, smoking, drug intake) on its activity in a Caucasian population. METHODS: ADH activity was assessed in endoscopic gastric biopsy specimens from 111 Caucasian subjects aged 20-80 years, of whom 51 were females. RESULTS: Highest ADH activity was measured......-80 years. In men aged 20-40 years, consumption of larger quantities of alcohol (>0.8 g/kg body weight/day) was associated with reduced ADH activity. H(2)-Receptor antagonist treatment also decreased gastric ADH activity. CONCLUSIONS: The results indicate that ADH activity in human gastric mucosa...... is negatively associated with consumption of larger quantities of alcohol. The question of whether ADH activity is higher in males or females can only be answered with respect to age. The gastric ADH activity in young men is distinctly higher compared to young women, but the opposite holds true in middle...

  12. An Ethanol Extract of Artemisia iwayomogi Activates PPARδ Leading to Activation of Fatty Acid Oxidation in Skeletal Muscle

    Science.gov (United States)

    Cho, Si Young; Jeong, Hyun Woo; Sohn, Jong Hee; Seo, Dae-Bang; Kim, Wan Gi; Lee, Sang-Jun

    2012-01-01

    Although Artemisia iwayomogi (AI) has been shown to improve the lipid metabolism, its mode of action is poorly understood. In this study, a 95% ethanol extract of AI (95EEAI) was identified as a potent ligand of peroxisome proliferator-activated receptorδ (PPARδ) using ligand binding analysis and cell-based reporter assay. In cultured primary human skeletal muscle cells, treatment of 95EEAI increased expression of two important PPARδ-regulated genes, carnitine palmitoyl-transferase-1 (CPT1) and pyruvate dehydrogenase kinase isozyme 4 (PDK4), and several genes acting in lipid efflux and energy expenditure. Furthermore, 95EEAI stimulated fatty acid oxidation in a PPARδ-dependent manner. High-fat diet-induced obese mice model further indicated that administration of 95EEAI attenuated diet-induced obesity through the activation of fatty acid oxidation in skeletal muscle. These results suggest that a 95% ethanol extract of AI may have a role as a new functional food material for the prevention and/or treatment of hyperlipidermia and obesity. PMID:22479450

  13. An ethanol extract of Artemisia iwayomogi activates PPARδ leading to activation of fatty acid oxidation in skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Si Young Cho

    Full Text Available Although Artemisia iwayomogi (AI has been shown to improve the lipid metabolism, its mode of action is poorly understood. In this study, a 95% ethanol extract of AI (95EEAI was identified as a potent ligand of peroxisome proliferator-activated receptorδ (PPARδ using ligand binding analysis and cell-based reporter assay. In cultured primary human skeletal muscle cells, treatment of 95EEAI increased expression of two important PPARδ-regulated genes, carnitine palmitoyl-transferase-1 (CPT1 and pyruvate dehydrogenase kinase isozyme 4 (PDK4, and several genes acting in lipid efflux and energy expenditure. Furthermore, 95EEAI stimulated fatty acid oxidation in a PPARδ-dependent manner. High-fat diet-induced obese mice model further indicated that administration of 95EEAI attenuated diet-induced obesity through the activation of fatty acid oxidation in skeletal muscle. These results suggest that a 95% ethanol extract of AI may have a role as a new functional food material for the prevention and/or treatment of hyperlipidermia and obesity.

  14. Alterations in mitochondrial DNA copy number and the activities of electron transport chain complexes and pyruvate dehydrogenase in the frontal cortex from subjects with autism.

    Science.gov (United States)

    Gu, F; Chauhan, V; Kaur, K; Brown, W T; LaFauci, G; Wegiel, J; Chauhan, A

    2013-09-03

    Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence suggests that mitochondrial dysfunction and oxidative stress may contribute to the etiology of autism. This is the first study to compare the activities of mitochondrial electron transport chain (ETC) complexes (I-V) and pyruvate dehydrogenase (PDH), as well as mitochondrial DNA (mtDNA) copy number in the frontal cortex tissues from autistic and age-matched control subjects. The activities of complexes I, V and PDH were most affected in autism (n=14) being significantly reduced by 31%, 36% and 35%, respectively. When 99% confidence interval (CI) of control group was taken as a reference range, impaired activities of complexes I, III and V were observed in 43%, 29% and 43% of autistic subjects, respectively. Reduced activities of all five ETC complexes were observed in 14% of autistic cases, and the activities of multiple complexes were decreased in 29% of autistic subjects. These results suggest that defects in complexes I and III (sites of mitochondrial free radical generation) and complex V (adenosine triphosphate synthase) are more prevalent in autism. PDH activity was also reduced in 57% of autistic subjects. The ratios of mtDNA of three mitochondrial genes ND1, ND4 and Cyt B (that encode for subunits of complexes I and III) to nuclear DNA were significantly increased in autism, suggesting a higher mtDNA copy number in autism. Compared with the 95% CI of the control group, 44% of autistic children showed higher copy numbers of all three mitochondrial genes examined. Furthermore, ND4 and Cyt B deletions were observed in 44% and 33% of autistic children, respectively. This study indicates that autism is associated with mitochondrial dysfunction in the brain.

  15. Effects of Metmyoglobin Reducing Activity and Thermal Stability of NADH-Dependent Reductase and Lactate Dehydrogenase on Premature Browning in Ground Beef.

    Science.gov (United States)

    Djimsa, Blanchefort A; Abraham, Anupam; Mafi, Gretchen G; VanOverbeke, Deborah L; Ramanathan, Ranjith

    2017-02-01

    Premature browning is a condition wherein ground beef exhibits a well-done appearance before reaching the USDA recommended internal cooked meat temperature of 71.1 °C; however, the mechanism is unclear. The objectives of this study were: (1) to determine the effects of packaging and temperature on metmyoglobin reducing activity (MRA) of cooked ground beef patties and (2) to assess the effects of temperature and pH on thermal stability of NADH-dependent reductase, lactate dehydrogenase (LDH), and oxymyoglobin (OxyMb) in-vitro. Beef patties (lean: fat = 85:15) were packaged in high-oxygen modified atmosphere (HiOX-MAP) or vacuum (VP) and cooked to either 65 or 71 °C. Internal meat color and MRA of both raw and cooked patties were determined. Purified NADH-dependent reductase and LDH were used to determine the effects of pH and temperature on enzyme activity. MRA of cooked patties was temperature and packaging dependent (P dependent reductase, and LDH were different and pH-dependent. LDH was able to generate NADH at 84 °C; whereas NADH-dependent reductase was least stable to heat. The results suggest that patties have MRA at cooking temperatures, which can influence cooked meat color. © 2017 Institute of Food Technologists®.

  16. Lead-optimization of aryl and aralkyl amine based triazolopyrimidine inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase with antimalarial activity in mice

    Science.gov (United States)

    Gujjar, Ramesh; Mazouni, Farah El; White, Karen L.; White, John; Creason, Sharon; Shackleford, David M.; Deng, Xiaoyi; Charman, William N.; Bathurst, Ian; Burrows, Jeremy; Floyd, David M.; Matthews, David; Buckner, Frederick S.; Charman, Susan A.; Phillips, Margaret A.; Rathod, Pradipsinh K.

    2011-01-01

    Malaria is one of the leading causes of severe infectious disease worldwide, yet our ability to maintain effective therapy to combat the illness is continually challenged by the emergence of drug resistance. We previously reported identification of a new class of triazolopyrimidine based P. falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors with antimalarial activity, leading to the discovery of a new lead series and novel target for drug development. Active compounds from the series contained a triazolopyrimidine ring attached to an aromatic group through a bridging nitrogen atom. Herein we describe systematic efforts to optimize the aromatic functionality with the goal of improving potency and in vivo properties of compounds from the series. These studies led to the identification of two new substituted aniline moieties (4-SF5-Ph and 3,5-Di-F-4-CF3-Ph) which, when coupled to the triazolopyrimidine ring showed good plasma exposure and better efficacy in the P. berghei mouse model of the disease, than previously reported compounds from the series. PMID:21517059

  17. Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis).

    Science.gov (United States)

    Guha, Anirban; Gera, Sandeep; Sharma, Anshu

    2012-03-01

    Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis (SCM) with the aim of developing suitable diagnostic kit for SCM. Trace elements and enzyme activity in milk were estimated with Atomic absorption Spectrophotometer, GBC 932 plus and biochemical methods, respectively. Somatic cell count (SCC) was done microscopically. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. A statistically significant (pmilk containing gram positive bacterial agents only. ALP was found to be elevated in milk infected by both gram positive and negative bacteria. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×10(5) cells/ml of milk as the benchmark. Only ALP and Zn, the former being superior, were found to be suitable for diagnosis of SCM irrespective of etiological agents. LDH, Co and Fe can be introduced in the screening programs where Gram positive bacteria are omnipresent. It is recommended that both ALP and Zn be measured together in milk to diagnose buffalo SCM, irrespective of etiology.

  18. Relevance of Frank's solvent classification as typically aqueous and typically non-aqueous to activities of firefly luciferase, alcohol dehydrogenase, and alpha-chymotrypsin in aqueous binaries.

    Science.gov (United States)

    Fadnavis, Nitin Wasantrao; Seshadri, Ramanujam; Sheelu, Gurrala; Madhuri, Kallakunta Vasantha

    2005-01-15

    Effects of cosolvent concentration on activity of fire fly luciferase, alpha-chymotrypsin, and alcohol dehydrogenase from baker's yeast (Saccharomyces cerevisiae) have been studied for several solvents with varying hydrophobicities (logP from +1.0 to -1.65) and polarities (dielectric constant from 7.4 to 109). The inhibitory effect of the cosolvent is examined in light of Frank's classification of solvents into 'typically aqueous (TA)' and 'typically non-aqueous (TNA).' The solvent concentration at which the enzyme activity decreases to half, the C(50) values, for TA solvents such as 1-cyclohexyl-2-pyrrolidinone, 2-butoxyethanol, 1-methyl-2-pyrrolidinone, tetrahydrofuran, t-butanol, and ethanol correlate quite well with their critical hydrophobic interaction concentration, rather than logP, while those for TNA solvents such as acetonitrile, dimethyl formamide, formamide, and dimethyl sulfoxide correlate well with logP. The interactions of TA solvents with proteins appear to be governed mainly by hydrophobic interactions while both hydrophobic and hydrophilic interactions play important role in case of TNA solvents.

  19. Isoniazid acetylating phenotype in patients with paracoccidioidomycosis and its relationship with serum sulfadoxin levels, glucose-6-phosphate dehydrogenase and glutathione reductase activities

    Directory of Open Access Journals (Sweden)

    Benedito Barraviera

    1991-06-01

    Full Text Available The authors evaluated the isoniazid acetylating phenotype and measured hematocrit, hemoglobin, glucose-6-phosphate dehydrogenase and glutathione reductase activities plus serum sulfadoxin levels in 39 patients with paracoccidioidomycosis (33 males and 6 females aged 17 to 58 years. Twenty one (53.84% of the patients presented a slow acetylatingphenotype and 18(46.16% a fast acetylating phenotype. Glucose-6-phosphate- dehydrogenase (G6PD acti vity was decreased in 5(23.80% slow acetylators and in 4(22.22% fast acetylators. Glutathione reductase activity was decreased in 14 (66.66% slow acetylators and in 12 (66.66% fast acetylators. Serum levels of free and total sulfadoxin Were higher in slow acetylator (p Os autores avaliaram o fenótipo acetilador da isoniazida, hematócrito, hemoglobina, atividade da glicose-6- fosfato desidrogenase, glutationa redutase e os níveis séricos de sulfadoxina de 39 doentes com paracoccidíoidomicose, senão 33 do sexo masculino e 6 do feminino, com idades compreendidas entre 17 e 58 anos. Vinte e um (53,84% doentes apresentaram fenótipo acetilador lento e 18 (46,16% rápido. A atividade da glicose-6-fosfato desidrogenase (G6PD esteve diminuída em 5 (23,80% acetiladores lentos e 4 (22,22% rápidos. A atividade da glutationa redutase esteve diminuída em 14 (66,66% acetiladores lentos e 12 (66,66% rápidos. Os níveis séricos de sulfadoxina livre e total foram maiores nos acetiladores lentos (p < 0,02. A análise dos resultados permite concluir que os níveis séricos de sulfadoxina relaciona-se com o fenótipo acetilador. Além disso, os níveis estiveram sempre acima de 50 µg/ml, níveis estes considerados terapêuticos. Por outro lado, a deficiência de glutationa redutase pode estar relacionada com a má absorção intestinal de nutrientes, entre eles riboflavina, vitamina precursora de FAD.

  20. Decreased 11β-Hydroxysteroid Dehydrogenase 1 Level and Activity in Murine Pancreatic Islets Caused by Insulin-Like Growth Factor I Overexpression.

    Directory of Open Access Journals (Sweden)

    Subrata Chowdhury

    Full Text Available We have reported a high expression of IGF-I in pancreatic islet β-cells of transgenic mice under the metallothionein promoter. cDNA microarray analysis of the islets revealed that the expression of 82 genes was significantly altered compared to wild-type mice. Of these, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1, which is responsible for the conversion of inert cortisone (11-dehydrocorticosterone, DHC in rodents to active cortisol (corticosterone in the liver and adipose tissues, has not been identified previously as an IGF-I target in pancreatic islets. We characterized the changes in its protein level, enzyme activity and glucose-stimulated insulin secretion. In freshly isolated islets, the level of 11β-HSD1 protein was significantly lower in MT-IGF mice. Using dual-labeled immunofluorescence, 11β-HSD1 was observed exclusively in glucagon-producing, islet α-cells but at a lower level in transgenic vs. wild-type animals. MT-IGF islets also exhibited reduced enzymatic activities. Dexamethasone (DEX and DHC inhibited glucose-stimulated insulin secretion from freshly isolated islets of wild-type mice. In the islets of MT-IGF mice, 48-h pre-incubation of DEX caused a significant decrease in insulin release, while the effect of DHC was largely blunted consistent with diminished 11β-HSD1 activity. In order to establish the function of intracrine glucocorticoids, we overexpressed 11β-HSD1 cDNA in MIN6 insulinoma cells, which together with DHC caused apoptosis and a significant decrease in proliferation. Both effects were abolished with the treatment of an 11β-HSD1 inhibitor. Our results demonstrate an inhibitory effect of IGF-I on 11β-HSD1 expression and activity within the pancreatic islets, which may mediate part of the IGF-I effects on cell proliferation, survival and insulin secretion.

  1. Elevation of 11β-hydroxysteroid dehydrogenase type 2 activity in Holocaust survivor offspring: evidence for an intergenerational effect of maternal trauma exposure.

    Science.gov (United States)

    Bierer, Linda M; Bader, Heather N; Daskalakis, Nikolaos P; Lehrner, Amy L; Makotkine, Iouri; Seckl, Jonathan R; Yehuda, Rachel

    2014-10-01

    Adult offspring of Holocaust survivors comprise an informative cohort in which to study intergenerational transmission of the effects of trauma exposure. Lower cortisol and enhanced glucocorticoid sensitivity have been previously demonstrated in Holocaust survivors with PTSD, and in offspring of Holocaust survivors in association with maternal PTSD. In other work, reduction in the activity of the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD-2), which inactivates cortisol, was identified in Holocaust survivors in comparison to age-matched, unexposed Jewish controls. Therefore, we investigated glucocorticoid metabolism in offspring of Holocaust survivors to evaluate if similar enzymatic decrements would be observed that might help to explain glucocorticoid alterations previously shown for Holocaust offspring. Holocaust offspring (n=85) and comparison subjects (n=27) were evaluated with clinical diagnostic interview and self-rating scales, and asked to collect a 24-h urine sample from which concentrations of cortisol and glucocorticoid metabolites were assayed by GCMS. 11β-HSD-2 activity was determined as the ratio of urinary cortisone to cortisol. Significantly reduced cortisol excretion was observed in Holocaust offspring compared to controls (p=.046), as had been shown for Holocaust survivors. However, 11β-HSD-2 activity was elevated for offspring compared to controls (p=.008), particularly among those whose mothers had been children, rather than adolescents or adults, during World War II (p=.032). The effect of paternal Holocaust exposure could not be reliably investigated in the current sample. The inverse association of offspring 11β-HSD-2 activity with maternal age at Holocaust exposure is consistent with the influence of glucocorticoid programming. Whereas a long standing reduction in 11β-HSD-2 activity among survivors is readily interpreted in the context of Holocaust related deprivation, understanding the directional effect on offspring will

  2. Epigallocatechin-3-gallate (EGCG) activates AMPK through the inhibition of glutamate dehydrogenase in muscle and pancreatic ß-cells: A potential beneficial effect in the pre-diabetic state?

    Science.gov (United States)

    Pournourmohammadi, Shirin; Grimaldi, Mariagrazia; Stridh, Malin H; Lavallard, Vanessa; Waagepetersen, Helle S; Wollheim, Claes B; Maechler, Pierre

    2017-07-01

    Glucose homeostasis is determined by insulin secretion from the ß-cells in pancreatic islets and by glucose uptake in skeletal muscle and other insulin target tissues. While glutamate dehydrogenase (GDH) senses mitochondrial energy supply and regulates insulin secretion, its role in the muscle has not been elucidated. Here we investigated the possible interplay between GDH and the cytosolic energy sensing enzyme 5'-AMP kinase (AMPK), in both isolated islets and myotubes from mice and humans. The green tea polyphenol epigallocatechin-3-gallate (EGCG) was used to inhibit GDH. Insulin secretion was reduced by EGCG upon glucose stimulation and blocked in response to glutamine combined with the allosteric GDH activator BCH (2-aminobicyclo-[2,2,1] heptane-2-carboxylic acid). Insulin secretion was similarly decreased in islets of mice with ß-cell-targeted deletion of GDH (ßGlud1 -/- ). EGCG did not further reduce insulin secretion in the mutant islets, validating its specificity. In human islets, EGCG attenuated both basal and nutrient-stimulated insulin secretion. Glutamine/BCH-induced lowering of AMPK phosphorylation did not operate in ßGlud1 -/- islets and was similarly prevented by EGCG in control islets, while high glucose systematically inactivated AMPK. In mouse C2C12 myotubes, like in islets, the inhibition of AMPK following GDH activation with glutamine/BCH was reversed by EGCG. Stimulation of GDH in primary human myotubes caused lowering of insulin-induced 2-deoxy-glucose uptake, partially counteracted by EGCG. Thus, mitochondrial energy provision through anaplerotic input via GDH influences the activity of the cytosolic energy sensor AMPK. EGCG may be useful in obesity by resensitizing insulin-resistant muscle while blunting hypersecretion of insulin in hypermetabolic states. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Identification and tissue-specific expression of a NADH-dependent activity of dihydropyrimidine dehydrogenase in man

    NARCIS (Netherlands)

    van Kuilenburg, A. B.; van Lenthe, H.; van Gennip, A. H.

    1996-01-01

    Homogenates of human liver and human fibroblasts were able to convert thymine into dihydrothymine in the presence of NADH whereas almost no NADH-dependent activity could be detected in human lymphocytes. The different tissue distribution of the NADH-dependent activity suggests that different types

  4. Very long chain acyl-coenzyme A dehydrogenase deficiency with adult onset

    DEFF Research Database (Denmark)

    Smelt, A H; Poorthuis, B J; Onkenhout, W

    1998-01-01

    Very long chain acyl-coenzyme A (acyl-CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial beta-oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified strongly elevated levels of tetradecenoic acid, 14:1(n-9), tetrade...... be due to residual enzyme activity as a consequence of the two missense mutations. Treatment with L-carnitine and medium chain triglycerides in the diet did not reduce the attacks of rhabdomyolysis....

  5. Amino acid sequence of the tryptic peptide containing the catalytic-site thiol group of bovine liver uridine diphosphate glucose dehydrogenase.

    Science.gov (United States)

    Franzen, B; Carrubba, C; Feingold, D S; Ashcom, J; Franzen, J S

    1981-01-01

    The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P-6. Homogeneous fluoresceincarboxamidomethylated peptide was prepared from a tryptic digest of fluoresceincarboxamidomethylated enzyme by specific adsorption--desorption from Sephadex G-25. The sequences of either peptide determined by the manual Edman dansyl procedure is: Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys. PMID:6896145

  6. Amino acid sequence of the tryptic peptide containing the catalytic-site thiol group of bovine liver uridine diphosphate glucose dehydrogenase.

    Science.gov (United States)

    Franzen, B; Carrubba, C; Feingold, D S; Ashcom, J; Franzen, J S

    1981-12-01

    The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P-6. Homogeneous fluoresceincarboxamidomethylated peptide was prepared from a tryptic digest of fluoresceincarboxamidomethylated enzyme by specific adsorption--desorption from Sephadex G-25. The sequences of either peptide determined by the manual Edman dansyl procedure is: Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys.

  7. Activity of earthworm in Latosol under simulated acid rain stress

    Science.gov (United States)

    Jia-En Zhang; Jiayu Yu; Ying Ouyang

    2015-01-01

    Acid rain is still an issue of environmental concerns. This study investigated the impacts of simulated acid rain (SAR) upon earthworm activity from the Latosol (acidic red soil). Laboratory experiment was performed by leaching the soil columns grown with earthworms (Eisenia fetida) at the SAR pH levels ranged from 2.0 to 6.5 over a 34-day period....

  8. The impact of hypoxia on the activity of lactate dehydrogenase in two different pre-clinical tumour models

    DEFF Research Database (Denmark)

    Lukacova, Slavka; Sørensen, Brita; Alsner, Jan

    2008-01-01

    squamous cell carcinoma, respectively. Low oxygen breathing for 1-72 h was used to increase tumour hypoxia. Ldh activity was measured in the serum and tumour cytosole with a colorimetric method. Tumour Ldha mRNA levels were assessed with RT-PCR. Results. The serum Ldh in non-tumour bearing CDF1 mice and C3...... carcinoma bearing mice. Reoxygenation for 4 or 24 hours had no additional effect on Ldh activity in any of the models. Discussion. Serum Ldh activity can be a marker for tumour burden in certain types of cancer. The relationship between serum and tumour Ldh and tumour hypoxia has not been confirmed. However...

  9. Detection of biologically active diterpenoic acids by Raman Spectroscopy

    DEFF Research Database (Denmark)

    Talian, Ivan; Orinak, Andrej; Efremov, Evtim V.

    2010-01-01

    Three poorly detectable, biologically active diterpenoic acids, kaurenoic, abietic, and gibberellic acid, were studied by using different modes of Raman spectroscopy. Because of their structural similarities, in the absence of strongly polarizable groups, conventional Raman spectroscopy is not su......Three poorly detectable, biologically active diterpenoic acids, kaurenoic, abietic, and gibberellic acid, were studied by using different modes of Raman spectroscopy. Because of their structural similarities, in the absence of strongly polarizable groups, conventional Raman spectroscopy......, and these 'fingerprints' would be suitable for the unambiguous identification of these diterpenoic acids....

  10. Effects of riboflavin deficiency and clofibrate treatment on the five acyl-CoA dehydrogenases in rat liver mitochondria.

    OpenAIRE

    Veitch, K; Draye, J P; Van Hoof, F; Sherratt, H S

    1988-01-01

    Rats were maintained on a riboflavin-deficient diet or on a diet containing clofibrate (0.5%, w/w). The activities of the mitochondrial FAD-dependent straight-chain acyl-CoA dehydrogenases (butyryl-CoA, octanoyl-CoA and palmitoyl-CoA) and the branched-chain acyl-CoA dehydrogenases (isovaleryl-CoA and isobutyryl-CoA) involved in the degradation of branched-chain acyl-CoA esters derived from branched-chain amino acids were assayed in liver mitochondrial extracts prepared in the absence and pres...

  11. Mode of action of lipoic acid in diabetes

    Indian Academy of Sciences (India)

    Administrator

    effect of these changes in the key enzymic activities is seen in the increased rates of oxidation of glucose and a reduction in fatty acid oxidation in diabetes following lipoic acid admini- stration. Keywords. Lipoic acid; diabetes; Coenzyme A; pyruvate dehydrogenase; lipid biosynthesis; acetoacetate metabolism. Introduction.

  12. The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains

    DEFF Research Database (Denmark)

    Jeppsson, M.; Johansson, B.; Jensen, Peter Ruhdal

    2003-01-01

    than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulose-derived inhibitors. Low G6PDH-activity strains were also more sensitive to H2O2 than the control strain TMB3001....... production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wildtype level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose...

  13. Antimicrobial activity of an Amazon medicinal plant (Chancapiedra) (Phyllanthus niruri L.) against Helicobacter pylori and lactic acid bacteria.

    Science.gov (United States)

    Ranilla, Lena Gálvez; Apostolidis, Emmanouil; Shetty, Kalidas

    2012-06-01

    The potential of water extracts of the Amazon medicinal plant Chancapiedra (Phyllanthus niruri L.) from Ecuador and Peru for antimicrobial activity against Helicobacter pylori and different strains of lactic acid bacteria such as Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus plantarum was investigated. H. pylori was inhibited by both water extracts in a dose dependent manner, whereas lactic acid bacterial growth was not affected. Both extracts contained ellagic acid and hydroxycinnamic acid derivatives and exhibited high free radical scavenging linked-antioxidant activities (89%). However, gallic acid was detected only in the Ecuadorian extract. Preliminary studies on the mode of action of Chancapiedra against H. pylori revealed that inhibition may not involve proline dehydrogenase-based oxidative phosphorylation inhibition associated with simple mono-phenolics and could involve ellagitannins or other non-phenolic compounds through a yet unknown mechanism. This study provides evidence about the potential of Chancapiedra for H. pylori inhibition without affecting beneficial lactic acid bacteria. Copyright © 2011 John Wiley & Sons, Ltd.

  14. The cortisol-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 in skeletal muscle in the pathogenesis of the metabolic syndrome.

    Science.gov (United States)

    Loerz, Christine; Maser, Edmund

    2017-11-01

    The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) contributes to intracellular glucocorticoid action by converting inactive cortisone to its receptor-active form cortisol (11-dehydrocorticosterone and corticosterone in mice and rats). The potential role of 11β-HSD1 in the pathogenesis of the metabolic syndrome has emerged over the past three decades. However, the precise impact of 11β-HSD1 in obesity-related diseases remains uncertain. Many studies from animal experiments to clinical studies have investigated liver and adipose tissue 11β-HSD1 in relation to obesity and its metabolic disorders including insulin resistance. But the relevance of 11β-HSD1 in skeletal muscle has been less extensively studied. On the other hand, skeletal muscle is assumed to be the main site of peripheral insulin resistance, but the biological relevance of 11β-HSD1 in skeletal muscle is unclear. This mini-review will focus on 11β-HSD1 in skeletal muscle and its postulated link to obesity and insulin-resistance. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Effect of sulfonated lignin on enzymatic activity of the ligninolytic enzymes Cα-dehydrogenase LigD and β-etherase LigF.

    Science.gov (United States)

    Wang, Chao; Ouyang, Xianhong; Su, Sisi; Liang, Xiao; Zhang, Chao; Wang, Wenya; Yuan, Qipeng; Li, Qiang

    2016-11-01

    NAD(+)-dependent Cα-dehydrogenase LigD and glutathione-dependent β-etherase LigF which selectively cleave the β-O-4 aryl ether linkage present in lignin, are key-enzymes for the biocatalytic depolymerization of lignin. However, the catalytic efficiency of the two enzymes is low when they are used to break down the β-aryl ether linkage in natural lignin. When sulfonated lignin was added to LigF hydrolysis reactions, the conversion rate of MPHPV decreased significantly from 99.5% to 32.6%. On the contrary, sulfonated lignin has little affection on LigD, which the conversion rate of GGE only decreased from 41.7% to 41%. The strong nonspecific interactions of enzymes onto sulfonated lignin detected by surface plasmon resonance (SPR) and isothermal titration calorimetric (ITC) was obvious and universal, which can reduce enzyme activity of many enzymes, including ligninolytic enzyme β-etherase LigF. To elucidate the exact mechanisms by which β-etherase LigF interact with lignin, molecular modeling was applied. Finally, analysis on catalytic efficiency of LigD and LigF in different concentrations and molecular weights of sulfonated lignin, solution ionic strength, pH, temperature and concentration of Tween 80 revealed that electrostatic interactions and hydrophobic interactions play important roles in absorption between LigF and sulfonated lignin. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Crassiflorone derivatives that inhibit Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and Trypanosoma cruzi trypanothione reductase (TcTR) and display trypanocidal activity.

    Science.gov (United States)

    Uliassi, Elisa; Fiorani, Giulia; Krauth-Siegel, R Luise; Bergamini, Christian; Fato, Romana; Bianchini, Giulia; Carlos Menéndez, J; Molina, Maria Teresa; López-Montero, Eulogio; Falchi, Federico; Cavalli, Andrea; Gul, Sheraz; Kuzikov, Maria; Ellinger, Bernhard; Witt, Gesa; Moraes, Carolina B; Freitas-Junior, Lucio H; Borsari, Chiara; Costi, Maria Paola; Bolognesi, Maria Laura

    2017-12-01

    Crassiflorone is a natural product with anti-mycobacterial and anti-gonorrhoeal properties, isolated from the stem bark of the African ebony tree Diospyros crassiflora. We noticed that its pentacyclic core possesses structural resemblance to the quinone-coumarin hybrid 3, which we reported to exhibit a dual-targeted inhibitory profile towards Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and Trypanosoma cruzi trypanothione reductase (TcTR). Following this basic idea, we synthesized a small library of crassiflorone derivatives 15-23 and investigated their potential as anti-trypanosomatid agents. 19 is the only compound of the series showing a balanced dual profile at 10 μM (% inhibition TbGAPDH  = 64% and % inhibition TcTR  = 65%). In phenotypic assay, the most active compounds were 18 and 21, which at 5 μM inhibited Tb bloodstream-form growth by 29% and 38%, respectively. Notably, all the newly synthesized compounds at 10 μM did not affect viability and the status of mitochondria in human A549 and 786-O cell lines, respectively. However, further optimization that addresses metabolic liabilities including solubility, as well as cytochromes P450 (CYP1A2, CYP2C9, CYP2C19, and CYP2D6) inhibition, is required before this class of natural product-derived compounds can be further progressed. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  17. Glucose-6-phosphate dehydrogenase deficiency

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/000528.htm Glucose-6-phosphate dehydrogenase deficiency To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a condition ...

  18. Characterization of a novel PQQ-dependent quinohemoprotein pyranose dehydrogenase from Coprinopsis cinerea classified into auxiliary activities family 12 in carbohydrate-active enzymes.

    Directory of Open Access Journals (Sweden)

    Kouta Takeda

    Full Text Available The basidiomycete Coprinopsis cinerea contains a quinohemoprotein (CcPDH named as CcSDH in our previous paper, which is a new type of pyrroloquinoline-quinone (PQQ-dependent pyranose dehydrogenase and is the first found among all eukaryotes. This enzyme has a three-domain structure consisting of an N-terminal heme b containing a cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase (CDH; EC 1.1.99.18 from the wood-rotting basidiomycete Phanerochaete chrysosporium, a C-terminal family 1-type carbohydrate-binding module, and a novel central catalytic domain containing PQQ as a cofactor. Here, we describe the biochemical and electrochemical characterization of recombinant CcPDH. UV-vis and resonance Raman spectroscopic studies clearly reveal characteristics of a 6-coordinated low-spin heme b in both the ferric and ferrous states, as well as intramolecular electron transfer from the PQQ to heme b. Moreover, the formal potential of the heme was evaluated to be 130 mV vs. NHE by cyclic voltammetry. These results indicate that the cytochrome domain of CcPDH possesses similar biophysical properties to that in CDH. A comparison of the conformations of monosaccharides as substrates and the associated catalytic efficiency (kcat/Km of CcPDH indicates that the enzyme prefers monosaccharides with equatorial C-2, C-3 hydroxyl groups and an axial C-4 hydroxyl group in the 1C4 chair conformation. Furthermore, a binding study shows a high binding affinity of CcPDH for cellulose, suggesting that CcPDH function is related to the enzymatic degradation of plant cell wall.

  19. Exercise training induces similar elevations in the activity of oxoglutarate dehydrogenase and peak oxygen uptake in the human quadriceps muscle

    DEFF Research Database (Denmark)

    Blomstrand, Eva; Krustrup, Peter; Søndergaard, Hans

    2011-01-01

    been shown to provide a quantitative measure of maximal oxidative metabolism, but it is not known whether the increase in this activity after a period of training reflects the elevation in peak oxygen consumption. Fourteen subjects performed one-legged knee extension exercise for 5-7 weeks, while...... the other leg remained untrained. Thereafter, the peak oxygen uptake by the quadriceps muscle was determined for both legs, and muscle biopsies were taken for assays of maximal enzyme activities (at 25°C). The peak oxygen uptake was 26% higher in the trained than in the untrained muscle (395 vs. 315 ml...

  20. Studies on lipoamide dehydrogenase

    NARCIS (Netherlands)

    Visser, J.

    1969-01-01

    Gel-filtration, ultracentrifugation and sucrose density gradient centrifugation demonstrated differences in physico-chemical properties of holoenzyme and apoenzyme of lipoamide dehydrogenase. The native apoenzyme has a mol.wt. of approx. 52,000 which is half that of the native holoenzyme. The

  1. Studies on lipoamide dehydrogenase

    NARCIS (Netherlands)

    Benen, J.A.E.

    1992-01-01

    At the onset of the investigations described in this thesis progress was being made on the elucidation of the crystal structure of the Azotobactervinelandii lipoamide dehydrogenase. Also the gene encoding this enzyme was cloned in our laboratory. By this, a

  2. Antibacterial activities of lactic acid bacteria isolated from cow ...

    African Journals Online (AJOL)

    26mm) against Klebsiella CB2. Conclusion: This study shows that LAB from cow faeces possess considerable antimicrobial activity against resistant Es- cherichia coli from the same environment. Keywords: Antibacterial activities, lactic acid ...

  3. Equine biochemical multiple acyl-CoA dehydrogenase deficiency (MADD) as a cause of rhabdomyolysis.

    Science.gov (United States)

    Westermann, C M; de Sain-van der Velden, M G M; van der Kolk, J H; Berger, R; Wijnberg, I D; Koeman, J P; Wanders, R J A; Lenstra, J A; Testerink, N; Vaandrager, A B; Vianey-Saban, C; Acquaviva-Bourdain, C; Dorland, L

    2007-08-01

    Two horses (a 7-year-old Groninger warmblood gelding and a six-month-old Trakehner mare) with pathologically confirmed rhabdomyolysis were diagnosed as suffering from multiple acyl-CoA dehydrogenase deficiency (MADD). This disorder has not been recognised in animals before. Clinical signs of both horses were a stiff, insecure gait, myoglobinuria, and finally recumbency. Urine, plasma, and muscle tissues were investigated. Analysis of plasma showed hyperglycemia, lactic acidemia, increased activity of muscle enzymes (ASAT, LDH, CK), and impaired kidney function (increased urea and creatinine). The most remarkable findings of organic acids in urine of both horses were increased lactic acid, ethylmalonic acid (EMA), 2-methylsuccinic acid, butyrylglycine (iso)valerylglycine, and hexanoylglycine. EMA was also increased in plasma of both animals. Furthermore, the profile of acylcarnitines in plasma from both animals showed a substantial elevation of C4-, C5-, C6-, C8-, and C5-DC-carnitine. Concentrations of acylcarnitines in urine of both animals revealed increased excretions of C2-, C3-, C4-, C5-, C6-, C5-OH-, C8-, C10:1-, C10-, and C5-DC-carnitine. In addition, concentrations of free carnitine were also increased. Quantitative biochemical measurement of enzyme activities in muscle tissue showed deficiencies of short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and isovaleryl-CoA dehydrogenase (IVD) also indicating MADD. Histology revealed extensive rhabdomyolysis with microvesicular lipidosis predominantly in type 1 muscle fibers and mitochondrial damage. However, the ETF and ETF-QO activities were within normal limits indicating the metabolic disorder to be acquired rather than inherited. To our knowledge, these are the first cases of biochemical MADD reported in equine medicine.

  4. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5’-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris, semiaquatic (Lontra longicaudis annectens and terrestrial (Sus scrofa

    Directory of Open Access Journals (Sweden)

    Myrna eBarjau Perez-Milicua

    2015-07-01

    Full Text Available Aquatic and semiaquatic mammals have the capacity of breath hold (apnea diving. Northern elephant seals (Mirounga angustirostris have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens can hold their breath for about 30 sec. Such periods of apnea may result in reduced oxygen concentration (hypoxia and reduced blood supply (ischemia to tissues. Production of adenosine 5’-triphosphate (ATP requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa, are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal (n=11, semiaquatic (neotropical river otter (n=4 and terrestrial (domestic pig (n=11. Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT was determined by spectrophotometry, and activity of inosine 5’-monophosphate dehydrogenase (IMPDH and the concentration of hypoxanthine (HX, inosine 5’-monophosphate (IMP, adenosine 5’-monophosphate (AMP, adenosine 5’-diphosphate (ADP, ATP, guanosine 5’-diphosphate (GDP, guanosine 5’-triphosphate (GTP, and xanthosine 5’-monophosphate (XMP were determined by high-performance liquid chromatography (HPLC. The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise, aquatic and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  5. The type I NADH dehydrogenase of Mycobacterium tuberculosis counters phagosomal NOX2 activity to inhibit TNF-alpha-mediated host cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Jessica L Miller

    2010-04-01

    Full Text Available The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persisting, intracellular pathogens such as the human pathogen Mycobacterium tuberculosis (Mtb to inhibit infection-induced apoptosis of macrophages is important for virulence. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase, NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis, but the molecular mechanism of this host pathogen interaction remains elusive. Here we show that the apoptogenic phenotype of MtbDeltanuoG was significantly reduced in human macrophages treated with caspase-3 and -8 inhibitors, TNF-alpha-neutralizing antibodies, and also after infection of murine TNF(-/- macrophages. Interestingly, incubation of macrophages with inhibitors of reactive oxygen species (ROS reduced not only the apoptosis induced by the nuoG mutant, but also its capacity to increase macrophage TNF-alpha secretion. The MtbDeltanuoG phagosomes showed increased ROS levels compared to Mtb phagosomes in primary murine and human alveolar macrophages. The increase in MtbDeltanuoG induced ROS and apoptosis was abolished in NOX-2 deficient (gp91(-/- macrophages. These results suggest that Mtb, via a NuoG-dependent mechanism, can neutralize NOX2-derived ROS in order to inhibit TNF-alpha-mediated host cell apoptosis. Consistently, an Mtb mutant deficient in secreted catalase induced increases in phagosomal ROS and host cell apoptosis, both of which were dependent upon macrophage NOX-2 activity. In conclusion, these results serendipitously reveal a novel connection between NOX2 activity, phagosomal ROS, and TNF-alpha signaling during infection-induced apoptosis in macrophages. Furthermore, our study reveals a novel function of NOX2 activity in innate immunity beyond the initial respiratory burst, which is the sensing of persistent intracellular pathogens

  6. Cinnamyl alcohol dehydrogenases in the mesocarp of ripening fruit of Prunus persica genotypes with different flesh characteristics: changes in activity and protein and transcript levels.

    Science.gov (United States)

    Gabotti, Damiano; Negrini, Noemi; Morgutti, Silvia; Nocito, Fabio F; Cocucci, Maurizio

    2015-07-01

    Development of fruit flesh texture quality traits may involve the metabolism of phenolic compounds. This study presents molecular and biochemical results on the possible role played by cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) during ripening [S3, S4 I (pre-climacteric) and S4 III (climacteric) stages] of peach [Prunus persica (L.) Batsch] fruit with different flesh firmness [non-melting flesh (NMF) 'Oro A'/melting flesh (MF) 'Springcrest' and 'Sanguinella'] and color (blood-flesh Sanguinella). A total of 24 putative full-length PRUPE_CAD genes were identified (in silico analysis) in the peach genome. The most abundant CAD isoforms, encoded by genes located on scaffolds 8 and 6, were probed by specifically developed anti-PRUPE_CAD sc8 and by anti-FaCAD (PRUPE_CAD sc6) polyclonal antibodies, respectively. PRUPE_CAD sc8 proteins (SDS-PAGE and native-PAGE/western blot) appeared responsible for the CAD activity (in vitro/in-gel assays) that increased with ripening (parallel to PRUPE_ACO1 transcripts accumulation and ethylene evolution) only in the mesocarp of Oro A and blood-flesh Sanguinella. Accumulation of PRUPE_CAD sc8 transcripts (semi-quantitative RT-PCR) occurred in all three cultivars, but in Oro A and Springcrest it was not always accompanied by that of the related proteins, suggesting possible post-transcriptional regulation. Flesh firmness, as well as levels of lignin, total phenolics and, where present (Sanguinella), anthocyanins, declined with ripening, suggesting that, at least in the studied peach cultivars, CAD activity is related to neither lignification nor differences in flesh firmness (NMF/MF). Further studies are necessary to clarify whether the high levels of CAD activity/expression in Sanguinella play a role in determining the characteristics of this blood-flesh fruit. © 2014 Scandinavian Plant Physiology Society.

  7. Reduced activity of 11beta-hydroxysteroid dehydrogenase type 2 is not responsible for sodium retention in nephrotic rats

    DEFF Research Database (Denmark)

    Bistrup, C; Thiesson, H C; Jensen, B L

    2005-01-01

    )] to suppress endogenous glucocorticoids in the proteinuric stage during active sodium retention. RESULTS: Nephrotic rats developed proteinuria, positive sodium balance, decreased plasma aldosterone concentration, and decreased urinary Na(+)/K(+) ratio. 11betaHSD2 mRNA expression was down-regulated but protein...

  8. Acid activated montmorillonite as catalysts in methyl esterification reactions of lauric acid.

    Science.gov (United States)

    Zatta, Leandro; Ramos, Luiz Pereira; Wypych, Fernando

    2012-01-01

    The catalytic activity of acid activated montmorillonite in the esterification of free fatty acids (FFA) is reported. Standard Montmorillonite (MMT) type STx-1 provided by the Clay Mineral Society repository was activated using phosphoric, nitric and sulphuric acids under different conditions and the resulting materials were characterized and evaluated as catalysts in the methyl esterification of lauric acid. Blank reactions carried out in the absence of any added catalyst presented conversions of 32.64, 69.79 and 79.23%, for alcohol:lauric acid molar ratios of 60:1, 12:1 and 6:1, respectively. In the presence of the untreated clay and using molar ratios of 12:1 and 6:1 with 12% of catalyst, conversions of 70.92 and 82.30% were obtained, respectively. For the acid activated clays, conversions up to 93.08% of lauric acid to methyl laurate were obtained, much higher than those observed for the thermal conversion or using untreated montmorillonite. Relative good correlations were observed between the catalytic activity and the development of acid sites and textural properties of the resulting materials. Therefore, a simple acid activation was able to improve the catalytic activity and produce clay catalysts that are environmental friendly, cost effective, noncorrosive and reusable.

  9. Biochemical Investigation on the activities of Acid and Alkaline ...

    African Journals Online (AJOL)

    The activities of acid phosphatase and alkaline phosphatase were investigated in two varieties of ripening Carica papaya fruit; Oblong-shaped variety which is also known as 'Agric pawpaw' and Pear-shaped variety which is also known as 'Local pawpaw'. Acid phosphatase activity decreased significantly (p < 0.01) ...

  10. Ovarian modulators of 11beta-hydroxysteroid dehydrogenase (11beta HSD) activity in follicular fluid from bovine and porcine large antral follicles and spontaneous ovarian cysts.

    Science.gov (United States)

    Thurston, Lisa M; Jonas, Kim C; Abayasekara, D Robert E; Michael, Anthony E

    2003-06-01

    In the ovary, cortisol is oxidized to cortisone by 11beta-hydroxysteroid dehydrogenase (11betaHSD). The present study investigated whether follicular fluid (FF) from large antral follicles and spontaneous ovarian cysts, isolated from bovine and porcine ovaries, contained modulators of 11betaHSD activity. Whereas FF from antral follicles had no significant effect over 1 h on NADP+-dependent 11betaHSD activity in rat kidney homogenates, enzyme activity was inhibited by FF from bovine and porcine ovarian cysts (80.5% +/- 2.3% and 72.8% +/- 3.4% of control, respectively). Following C18 reverse-phase chromatography, the hydrophilic fractions of FF from bovine and porcine antral follicles stimulated NADP+-dependent 11betaHSD activities (111.5% +/- 21.6% and 55.2% +/- 5.7% respectively). Hydrophobic compounds inhibited NADP+-dependent cortisol oxidation by 58.2% +/- 5.1% (bovine) and 45.7% +/- 2.0% (porcine). In both species, FF from ovarian cysts appeared to contain less of the hydrophilic stimuli to 11betaHSD activity and more of the hydrophobic inhibitors. The FF from antral follicles and ovarian cysts, and the C18 fractions thereof, had no significant effect on NAD+-dependent cortisol oxidation. The ovarian modulators of NADP+-dependent 11betaHSD activities did not coelute with cortisol, cortisone, estradiol, testosterone, progesterone, pregnenolone, and cholesterol. However, the 11betaHSD stimuli in porcine FF from both antral follicles and cysts coeluted with prostaglandin (PG) E2 and PGF2alpha. We conclude that large antral follicles and spontaneous ovarian cysts, in both the cow and the pig, contain ovarian modulators of the NADP+-dependent 11betaHSD activity. Moreover, FF from spontaneous ovarian cysts, because of decreased content of the 11betaHSD stimulus accompanied by increased content of the 11betaHSD inhibitors, exerts a net inhibitory effect on 11betaHSD activity.

  11. Natural Cinnamic Acids, Synthetic Derivatives and Hybrids with Antimicrobial Activity

    Directory of Open Access Journals (Sweden)

    Juan David Guzman

    2014-11-01

    Full Text Available Antimicrobial natural preparations involving cinnamon, storax and propolis have been long used topically for treating infections. Cinnamic acids and related molecules are partly responsible for the therapeutic effects observed in these preparations. Most of the cinnamic acids, their esters, amides, aldehydes and alcohols, show significant growth inhibition against one or several bacterial and fungal species. Of particular interest is the potent antitubercular activity observed for some of these cinnamic derivatives, which may be amenable as future drugs for treating tuberculosis. This review intends to summarize the literature data on the antimicrobial activity of the natural cinnamic acids and related derivatives. In addition, selected hybrids between cinnamic acids and biologically active scaffolds with antimicrobial activity were also included. A comprehensive literature search was performed collating the minimum inhibitory concentration (MIC of each cinnamic acid or derivative against the reported microorganisms. The MIC data allows the relative comparison between series of molecules and the derivation of structure-activity relationships.

  12. Cloning and expression analysis of alcohol dehydrogenase ( Adh ...

    African Journals Online (AJOL)

    Hybrid promoters are created by shuffling of DNA fragments while keeping intact regulatory regions crucial of promoter activity. Two fragments of alcohol dehydrogenase (Adh) promoter from Zea mays were selected to generate hybrid promoter. Sequence analysis of both alcohol dehydrogenase promoter fragments through ...

  13. Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (

    Directory of Open Access Journals (Sweden)

    Anirban Guha

    2012-03-01

    Full Text Available Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu, iron (Fe, zinc (Zn, cobalt (Co and manganese (Mn and enzyme activity of lactate dehydrogenase (LDH, alkaline phosphatase (ALP and aspartate aminotransferase (AST in riverine buffalo milk can be used as an indicator of subclinical mastitis (SCM with the aim of developing suitable diagnostic kit for SCM. Trace elements and enzyme activity in milk were estimated with Atomic absorption Spectrophotometer, GBC 932 plus and biochemical methods, respectively. Somatic cell count (SCC was done microscopically. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. A statistically significant (p<0.01 increase in SCC, Fe, Zn, Co and LDH occurred in SCM milk containing gram positive bacterial agents only. ALP was found to be elevated in milk infected by both gram positive and negative bacteria. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×105 cells/ml of milk as the benchmark. Only ALP and Zn, the former being superior, were found to be suitable for diagnosis of SCM irrespective of etiological agents. LDH, Co and Fe can be introduced in the screening programs where Gram positive bacteria are omnipresent. It is recommended that both ALP and Zn be measured together in milk to diagnose buffalo SCM, irrespective of etiology.

  14. Surface-displayed glyceraldehyde 3-phosphate dehydrogenase and galectin from Dirofilaria immitis enhance the activation of the fibrinolytic system of the host.

    Science.gov (United States)

    González-Miguel, Javier; Morchón, Rodrigo; Siles-Lucas, Mar; Oleaga, Ana; Simón, Fernando

    2015-05-01

    Cardiopulmonary dirofilariosis is a cosmopolitan disease caused by Dirofilaria immitis, a filaroid parasite whose adult worms live for years in the vascular system of its host. Previous studies have shown that D. immitis can use their excretory/secretory (ES) and surface antigens to enhance fibrinolysis, which could limit the formation of clots in its surrounding environment. Moreover, several isoforms of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and galectin (GAL) were identified in both antigenic extracts as plasminogen-binding proteins. The aim of this work is to study the interaction of the GAPDH and GAL of D. immitis with the fibrinolytic system of the host. This study includes the cloning, sequencing and expression of the recombinant forms of the GAPDH and GAL of D. immitis (rDiGAPDH and rDiGAL) and the analysis of their capacity as plasminogen-binding proteins. The results indicate that rDiGAPDH and rDiGAL are able to bind plasminogen and stimulate plasmin generation by tissue plasminogen activator (tPA). This interaction needs the involvement of lysine residues, many of which are located externally in both proteins as have been shown by the molecular modeling of their secondary structures. In addition, we show that rDiGAPDH and rDiGAL enhance the expression of the urokinase-type plasminogen activator (uPA) on canine endothelial cells in culture and that both proteins are expressed on the surface of D. immitis in close contact with the blood of the host. These data suggest that D. immitis could use the associated surface GAPDH and GAL as physiological plasminogen receptors to shift the fibrinolytic balance towards the generation of plasmin, which might constitute a survival mechanism to avoid the clot formation in its intravascular habitat. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Evidence for transcript-specific epigenetic regulation of glucocorticoid-stimulated skeletal muscle 11β-hydroxysteroid dehydrogenase-1 activity in type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Inder Warrick J

    2012-12-01

    Full Text Available Abstract Background The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1 converts inactive cortisone into active cortisol in insulin target tissues. In people with type 2 diabetes, skeletal muscle (SkM 11βHSD1 is upregulated by the potent glucocorticoid dexamethasone. The HSD11B1 gene has two promoters designated P1 and P2. CCAAT/enhancer-binding protein beta (C/EBPβ is known to regulate expression of 11βHSD1 via the P2 promoter. In this study, we investigated the potential role of altered DNA methylation of the P1 and P2 promoters in the observed dexamethasone-induced upregulation of SkM 11βHSD1 oxoreductase activity in human diabetic subjects. SkM biopsies from 15 people with type 2 diabetes were collected before and after treatment with oral dexamethasone 4 mg/day for 4 days and SkM 11βHSD1, C/EBPβ and P1 and P2 promoter region mRNA levels were measured by quantitative RT-PCR. 11βHSD1 oxoreductase activity was quantified by measuring the conversion of radiolabeled 3H-cortisone to cortisol by thin layer chromatography. Analysis of HSD11B1 promoter methylation (P1 and P2 was performed using Sequenom MassARRAY EpiTYPER analysis. Results Dexamethasone treatment resulted in a significant increase in 11βHSD1 mRNA levels (P = 0.003, oxoreductase activity (P = 0.017 and C/EBPβ mRNA (P = 0.015, and increased expression of both the P1 (P = 0.008 and P2 (P = 0.016 promoter regions . The distal P1 promoter region showed a significant reduction in methylation following dexamethasone (P = 0.026. There was a significant negative correlation between the change in methylation at this site and the increment in 11βHSD1 oxoreductase activity (r = −0.62, P = 0.014. Conclusions Our findings of reduced methylation in the HSD11B1 P1 promoter in association with increased 11βHSD1 oxoreductase activity implicate complex multi-promoter epigenetic mechanisms in the regulation of 11βHSD1 levels in SkM.

  16. Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry.

    Science.gov (United States)

    Idewaki, Yasuhiro; Iwase, Masanori; Fujii, Hiroki; Ohkuma, Toshiaki; Ide, Hitoshi; Kaizu, Shinako; Jodai, Tamaki; Kikuchi, Yohei; Hirano, Atsushi; Nakamura, Udai; Kubo, Michiaki; Kitazono, Takanari

    2015-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671) was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity) and drinking habits (lifetime abstainers vs. former or current drinkers) was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2). The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI)]: *1/*1 abstainers as the referent, 0.94 [0.76-1.16] in abstainers with *2, 1.00 [0.80-1.26] in *1/*1 drinkers, 0.71 [0.54-0.93] in drinkers with *2). Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28-6.13] in abstainers with *2, 1.89 [0.89-4.51] in *1/*1 drinkers, 2.35 [1.06-5.79] in drinkers with *2). In summary, patients with type 2 diabetes and ALDH2 *2 displayed a

  17. Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry.

    Directory of Open Access Journals (Sweden)

    Yasuhiro Idewaki

    Full Text Available Aldehyde dehydrogenase 2 (ALDH2 detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671 was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity and drinking habits (lifetime abstainers vs. former or current drinkers was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2. The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI]: *1/*1 abstainers as the referent, 0.94 [0.76-1.16] in abstainers with *2, 1.00 [0.80-1.26] in *1/*1 drinkers, 0.71 [0.54-0.93] in drinkers with *2. Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28-6.13] in abstainers with *2, 1.89 [0.89-4.51] in *1/*1 drinkers, 2.35 [1.06-5.79] in drinkers with *2. In summary, patients with type 2 diabetes and ALDH2 *2

  18. SDR-O: an orphan short-chain dehydrogenase/reductase localized at mouse chromosome 10/human chromosome 12.

    Science.gov (United States)

    Chen, Weiguo; Song, Min-Sun; Napoli, Joseph L

    2002-07-10

    We report cloning a cDNA that encodes a novel short-chain dehydrogenase/reductase, SDR-O, conserved in mouse, human and rat. Human and mouse liver express SDR-O (short-chain dehydrogenase/reductase-orphan) mRNA intensely. The mouse embryo expresses SDR-O mRNA as early as day seven. Human SDR-O localizes on chromosome 12; mouse SDR-O localizes on chromosome 10 with CRAD1, CRAD2 and RDH4. SDR-O shares highest amino acid similarity with rat RoDH1 and mouse RDH1 (69-70%), but does not have the retinol and 3alpha-hydroxysteroid dehydrogenase activity of either, nor is it active as a 17beta- or 11beta-hydroxysteroid dehydrogenase. Short-chain dehydrogenase/reductases catalyse the metabolism of ligands that bind with nuclear receptors: the occurrence of 'orphan' nuclear receptors may imply existence of 'orphan' SDR, suggesting that SDR-O may catalyse the metabolism of another class of nuclear receptor ligand. Alternatively, SDR-O may not have a catalytic function, but may regulate metabolism by binding substrates/products and/or by serving as a regulatory factor.

  19. Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yun-Hee [Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14214 (United States); Patel, Mulchand S., E-mail: mspatel@buffalo.edu [Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14214 (United States)

    2010-05-07

    Unlike pyruvate dehydrogenase complexes (PDCs) from prokaryotes, PDCs from higher eukaryotes have an additional structural component, E3-binding protein (BP), for binding of dihydrolipoamide dehydrogenase (E3) in the complex. Based on the 3D structure of the subcomplex of human (h) E3 with the di-domain (L3S1) of hBP, the amino acid residues (H348, D413, Y438, and R447) of hE3 for binding to hBP were substituted singly by alanine or other residues. These substitutions did not have large effects on hE3 activity when measured in its free form. However, when these hE3 mutants were reconstituted in the complex, the PDC activity was significantly reduced to 9% for Y438A, 20% for Y438H, and 18% for D413A. The binding of hE3 mutants with L3S1 determined by isothermal titration calorimetry revealed that the binding affinities of the Y438A, Y438H, and D413A mutants to L3S1 were severely reduced (1019-, 607-, and 402-fold, respectively). Unlike wild-type hE3 the binding of the Y438A mutant to L3S1 was accompanied by an unfavorable enthalpy change and a large positive entropy change. These results indicate that hE3-Y438 and hE3-D413 play important roles in binding of hE3 to hBP.

  20. Design, Synthesis, and Antimycobacterial Activity of Novel Theophylline-7-Acetic Acid Derivatives With Amino Acid Moieties.

    Science.gov (United States)

    Stavrakov, Georgi; Valcheva, Violeta; Voynikov, Yulian; Philipova, Irena; Atanasova, Mariyana; Konstantinov, Spiro; Peikov, Plamen; Doytchinova, Irini

    2016-03-01

    The theophylline-7-acetic acid (7-TAA) scaffold is a promising novel lead compound for antimycobacterial activity. Here, we derive a model for antitubercular activity prediction based on 14 7-TAA derivatives with amino acid moieties and their methyl esters. The model is applied to a combinatorial library, consisting of 40 amino acid and methyl ester derivatives of 7-TAA. The best three predicted compounds are synthesized and tested against Mycobacterium tuberculosis H37Rv. All of them are stable, non-toxic against human cells and show antimycobacterial activity in the nanomolar range being 60 times more active than ethambutol. © 2015 John Wiley & Sons A/S.

  1. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    Science.gov (United States)

    John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

    2016-10-25

    A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacylethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings. The subject matter disclosed herein relates to enhancers of amidohydrolase activity.

  2. Hibernation impact on the catalytic activities of the mitochondrial D-3-hydroxybutyrate dehydrogenase in liver and brain tissues of jerboa (Jaculus orientalis

    Directory of Open Access Journals (Sweden)

    Hafiani Assia

    2003-09-01

    Full Text Available Abstract Background Jerboa (Jaculus orientalis is a deep hibernating rodent native to subdesert highlands. During hibernation, a high level of ketone bodies i.e. acetoacetate (AcAc and D-3-hydroxybutyrate (BOH are produced in liver, which are used in brain as energetic fuel. These compounds are bioconverted by mitochondrial D-3-hydroxybutyrate dehydrogenase (BDH E.C. 1.1.1.30. Here we report, the function and the expression of BDH in terms of catalytic activities, kinetic parameters, levels of protein and mRNA in both tissues i.e brain and liver, in relation to the hibernating process. Results We found that: 1/ In euthemic jerboa the specific activity in liver is 2.4- and 6.4- fold higher than in brain, respectively for AcAc reduction and for BOH oxidation. The same differences were found in the hibernation state. 2/ In euthermic jerboa, the Michaelis constants, KM BOH and KM NAD+ are different in liver and in brain while KM AcAc, KM NADH and the dissociation constants, KD NAD+and KD NADH are similar. 3/ During prehibernating state, as compared to euthermic state, the liver BDH activity is reduced by half, while kinetic constants are strongly increased except KD NAD+. 4/ During hibernating state, BDH activity is significantly enhanced, moreover, kinetic constants (KM and KD are strongly modified as compared to the euthermic state; i.e. KD NAD+ in liver and KM AcAc in brain decrease 5 and 3 times respectively, while KD NADH in brain strongly increases up to 5.6 fold. 5/ Both protein content and mRNA level of BDH remain unchanged during the cold adaptation process. Conclusions These results cumulatively explained and are consistent with the existence of two BDH enzymatic forms in the liver and the brain. The apoenzyme would be subjected to differential conformational folding depending on the hibernation state. This regulation could be a result of either post-translational modifications and/or a modification of the mitochondrial membrane state

  3. A new, simple assay for long-chain acyl-CoA dehydrogenase in cultured skin fibroblasts using stable isotopes and GC-MS

    NARCIS (Netherlands)

    Niezen-Koning, K E; Wanders, R J; Nagel, G T; IJlst, L; Heijmans, Hugo

    1992-01-01

    In this paper, we present a new method for measurement of long-chain acyl-CoA dehydrogenase (LCAD) activities in cultured skin fibroblasts. The method is based upon gas chromatographic/mass spectrometric determination of 3-OH-hexadecanoic acid formed during incubation of fibroblasts in a medium

  4. Biological activity of sugarcane pyroligneous acid against ...

    African Journals Online (AJOL)

    aghomotsegin

    2013-10-23

    Oct 23, 2013 ... Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) annually cause enormous loss to the producers and their combat ... The fall armyworm, Spodoptera frugiperda (Smith, 1797). (Lepidoptera: Noctuidae), is a ... Tavares et al., 2009, 2011, 2013), and pyroligneous acid. (Azevedo et al., 2007; ...

  5. Hypochlorous Acid - Analytical Methods and Antimicrobial Activity

    African Journals Online (AJOL)

    HP

    Hypochlorous acid (HOCl) is produced by the human body's immune cells to fight infections. It is effective against a broad range of microorganisms. It is non-toxic, non-irritant and non-corrosive at proper usage concentrations. There are some available commercial products that contain HOCl. However, its low storage ...

  6. Hypochlorous Acid - Analytical Methods and Antimicrobial Activity ...

    African Journals Online (AJOL)

    Hypochlorous acid (HOCl) is produced by the human body's immune cells to fight infections. It is effective against a broad range of microorganisms. It is non-toxic, non-irritant and non-corrosive at proper usage concentrations. There are some available commercial products that contain HOCl. However, its low storage ...

  7. The Effect of EDTA and Citric acid on Soil Enzymes Activity, Substrate Induced Respiration and Pb Availability in a Contaminated Soil

    Directory of Open Access Journals (Sweden)

    seyed sajjad hosseini

    2017-03-01

    Full Text Available Introduction: Application of EDTA may increase the heavy metal availability and phytoextraction efficiency in contaminated soils. In spite of that, it might also have some adverse effects on soil biological properties. Metals as freeions are considered to be severely toxic, whereas the complexed form of these metalswith organic compounds or Fe/Mn oxides may be less available to soil microbes. However, apart from this fact, some of these compounds like EDTA and EDTA-metal complexes have low bio- chemo- and photo-degradablity and high solubility in their own characteristics andable to cause toxicity in soil environment. So more attentions have been paid to use of low molecular weight organic acids (LMWOAs such as Citric acid because of having less unfavorable effects to the environment. Citric acid increases heavy metals solubility in soils and it also improves soil microbial activity indirectly. Soil enzymes activity is a good indicator of soil quality, and it is more suitable for monitoring the soil quality compared to physical or chemical indicators. The aims of this research were to evaluate the changes of dehydrogenase, urease and alkaline phosphomonoesterase activities, substrate-induced respiration (SIR and Pb availability after EDTA and citric acid addition into a contaminated soil with PbCl2. Materials and Methods: An experiment was conducted in a completely randomized design with factorial arrangement and three replications in greenhouse condition. The soil samples collected from surface horizon (0-20 cm of the Typic haplocalsids, located in Mashhad, Iran. Soil samples were artificially contaminated with PbCl2 (500 mg Pb per kg of soil and incubated for one months in 70 % of water holding capacity at room temperature. The experimental treatments included control, 3 and 5 mmol EDTA (EDTA3 and EDTA5 and Citric acid (CA3 and CA5 per kg of soil. Soil enzymes activity, substrate-induced respiration and Pb availability of soil samples were

  8. Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties.

    OpenAIRE

    Allison, N; O'Donnell, M J; Fewson, C A

    1985-01-01

    Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydroge...

  9. Functional Role of Lanthanides in Enzymatic Activity and Transcriptional Regulation of Pyrroloquinoline Quinone-Dependent Alcohol Dehydrogenases in Pseudomonas putida KT2440

    Directory of Open Access Journals (Sweden)

    Matthias Wehrmann

    2017-06-01

    Full Text Available The oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs. Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs that are often functionally redundant. Here we report the first description and characterization of a lanthanide-dependent PQQ-ADH (PedH in a nonmethylotrophic bacterium based on the use of purified enzymes from the soil-dwelling model organism Pseudomonas putida KT2440. PedH (PP_2679 exhibits enzyme activity on a range of substrates similar to that of its Ca2+-dependent counterpart PedE (PP_2674, including linear and aromatic primary and secondary alcohols, as well as aldehydes, but only in the presence of lanthanide ions, including La3+, Ce3+, Pr3+, Sm3+, or Nd3+. Reporter assays revealed that PedH not only has a catalytic function but is also involved in the transcriptional regulation of pedE and pedH, most likely acting as a sensory module. Notably, the underlying regulatory network is responsive to as little as 1 to 10 nM lanthanum, a concentration assumed to be of ecological relevance. The present study further demonstrates that the PQQ-dependent oxidation system is crucial for efficient growth with a variety of volatile alcohols. From these results, we conclude that functional redundancy and inverse regulation of PedE and PedH represent an adaptive strategy of P. putida KT2440 to optimize growth with volatile alcohols in response to the availability of different lanthanides.

  10. S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase induces formation of C150-C154 intrasubunit disulfide bond in the active site of the enzyme.

    Science.gov (United States)

    Barinova, K V; Serebryakova, M V; Muronetz, V I; Schmalhausen, E V

    2017-12-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic protein involved in numerous non-glycolytic functions. S-glutathionylated GAPDH was revealed in plant and animal tissues. The role of GAPDH S-glutathionylation is not fully understood. Rabbit muscle GAPDH was S-glutathionylated in the presence of H 2 O 2 and reduced glutathione (GSH). The modified protein was assayed by MALDI-MS analysis, differential scanning calorimetry, dynamic light scattering, and ultracentrifugation. Incubation of GAPDH in the presence of H 2 O 2 together with GSH resulted in the complete inactivation of the enzyme. In contrast to irreversible oxidation of GAPDH by H 2 O 2 , this modification could be reversed in the excess of GSH or dithiothreitol. By data of MALDI-MS analysis, the modified protein contained both mixed disulfide between Cys150 and GSH and the intrasubunit disulfide bond between Cys150 and Cys154 (different subunits of tetrameric GAPDH may contain different products). S-glutathionylation results in loosening of the tertiary structure of GAPDH, decreases its affinity to NAD + and thermal stability. The mixed disulfide between Cys150 and GSH is an intermediate product of S-glutathionylation: its subsequent reaction with Cys154 results in the intrasubunit disulfide bond in the active site of GAPDH. The mixed disulfide and the C150-C154 disulfide bond protect GAPDH from irreversible oxidation and can be reduced in the excess of thiols. Conformational changes that were observed in S-glutathionylated GAPDH may affect interactions between GAPDH and other proteins (ligands), suggesting the role of S-glutathionylation in the redox signaling. The manuscript considers one of the possible mechanisms of redox regulation of cell functions. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Pristanic acid and phytanic acid: naturally occurring ligands for the nuclear receptor peroxisome proliferator-activated receptor alpha

    NARCIS (Netherlands)

    Zomer, A. W.; van der Burg, B.; Jansen, G. A.; Wanders, R. J.; Poll-The, B. T.; van der Saag, P. T.

    2000-01-01

    Phytanic acid and pristanic acid are branched-chain fatty acids, present at micromolar concentrations in the plasma of healthy individuals. Here we show that both phytanic acid and pristanic acid activate the peroxisome proliferator-activated receptor alpha (PPARalpha) in a concentration-dependent

  12. Production of optically pure L-lactic acid from lignocellulosic hydrolysate by using a newly isolated and D-lactate dehydrogenase gene-deficient Lactobacillus paracasei strain.

    Science.gov (United States)

    Kuo, Yang-Cheng; Yuan, Shuo-Fu; Wang, Chun-An; Huang, Yin-Jung; Guo, Gia-Luen; Hwang, Wen-Song

    2015-12-01

    The use of lignocellulosic feedstock for lactic acid production with a difficulty is that the release of inhibitory compounds during the pretreatment process which inhibit the growth of microorganism. Thus we report a novel lactic acid bacterium, Lactobacillus paracasei 7 BL, that has a high tolerance to inhibitors and produced optically pure l-lactic acid after the interruption of ldhD gene. The strain 7 BL fermented glucose efficiently and showed high titer of l-lactic acid (215 g/l) by fed-batch strategy. In addition, 99 g/l of l-lactic acid with high yield (0.96 g/g) and productivity (2.25-3.23 g/l/h) was obtained by using non-detoxified wood hydrolysate. Rice straw hydrolysate without detoxification was also tested and yielded a productivity rate as high as 5.27 g/l/h. Therefore, L. paracasei 7 BL represents a potential method of l-lactic acid production from lignocellulosic biomass and has attractive application for industries. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. [11 beta-hydroxysteroid-dehydrogenase: characteristics and the clinical significance of a key enzyme in cortisol metabolism].

    Science.gov (United States)

    Kerstens, M N; Dullaart, R P

    1999-03-06

    The enzyme 11 beta HSD catalyzes the interconversion of the biologically active cortisol and the biologically inactive cortisone. There are two distinct isozymes: 11 beta HSD type 1 is mainly expressed in liver and is a bidirectional enzyme, with both dehydrogenase and reductase activity. 11 beta HSD type 2 is mainly expressed in kidney and is a unidirectional enzyme with only dehydrogenase activity. 11 beta HSD type 2 protects the mineralocorticoid receptor from being activated by cortisol. Thus, specificity of this receptor in vivo is enzyme and not receptor mediated. The syndrome of apparent mineralocorticoid excess is caused by a congenital deficiency of 11 beta HSD type 2. Liquorice-induced hypertension is an example of an acquired defect in dehydrogenase activity of 11 beta HSD, caused by glycyrrhetinic acid. 11 beta HSD may play a role in the pathogenesis of 'essential' hypertension, obesity and type 1 diabetes mellitus. Angiotensin-converting enzyme inhibitors enhance dehydrogenase activity of 11 beta HSD, which may contribute to their natriuretic effect.

  14. Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

    Directory of Open Access Journals (Sweden)

    Mädje Katharina

    2012-01-01

    Full Text Available Abstract Background Enzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(PH. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH, presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S-1-(2-chlorophenylethanol from o-chloroacetophenone. Results As compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3 that gave an intracellular enzyme activity of 400 units/gCDW, co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/gCDW was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C brought up the FDH activity threefold to a level of 250 U/gCDW while reducing the XR activity by

  15. Retinoic acid signalling is activated in the postischemic heart and may influence remodelling.

    Directory of Open Access Journals (Sweden)

    Dusan Bilbija

    Full Text Available BACKGROUND: All-trans retinoic acid (atRA, an active derivative of vitamin A, regulates cell differentiation, proliferation and cardiac morphogenesis via transcriptional activation of retinoic acid receptors (RARs acting on retinoic acid response elements (RARE. We hypothesized that the retinoic acid (RA signalling pathway is activated in myocardial ischemia and postischemic remodelling. METHODS AND FINDINGS: Myocardial infarction was induced through ligating the left coronary artery in mice. In vivo cardiac activation of the RARs was measured by imaging RARE-luciferase reporter mice, and analysing expression of RAR target genes and proteins by real time RT-PCR and western blot. Endogenous retinoids in postinfarcted hearts were analysed by triple-stage liquid chromatography/tandem mass spectrometry. Cardiomyocytes (CM and cardiofibroblasts (CF were isolated from infarcted and sham operated RARE luciferase reporter hearts and monitored for RAR activity and expression of target genes. The effect of atRA on CF proliferation was evaluated by EdU incorporation. Myocardial infarction increased thoracic RAR activity in vivo (p<0.001, which was ascribed to the heart through ex vivo imaging (p = 0.002 with the largest signal 1 week postinfarct. This was accompanied by increased cardiac gene and protein expression of the RAR target genes retinol binding protein 1 (p = 0.01 for RNA, p = 0,006 for protein and aldehyde dehydrogenase 1A2 (p = 0.04 for RNA, p = 0,014 for protein, while gene expression of cytochrome P450 26B1 was downregulated (p = 0.007. Concomitantly, retinol accumulated in the infarcted zone (p = 0.02. CM and CF isolated from infarcted hearts had higher luminescence than those from sham operated hearts (p = 0.02 and p = 0.008. AtRA inhibited CF proliferation in vitro (p = 0.02. CONCLUSION: The RA signalling pathway is activated in postischemic hearts and may play a role in regulation of damage and

  16. Differential Activity of NADPH-Producing Dehydrogenases Renders Rodents Unsuitable Models to Study IDH1(R132) Mutation Effects in Human Glioblastoma

    NARCIS (Netherlands)

    Atai, Nadia A.; Renkema-Mills, Nynke A.; Bosman, Joost; Schmidt, Nadja; Rijkeboer, Denise; Tigchelaar, Wikky; Bosch, Klazien S.; Troost, Dirk; Jonker, Ard; Bleeker, Fonnet E.; Miletic, Hrvoje; Bjerkvig, Rolf; de Witt Hamer, Philip C.; van Noorden, Cornelis J. F.

    2011-01-01

    The somatic IDH1(R132) mutation in the isocitrate dehydrogenase 1 gene occurs in high frequency in glioma and in lower frequency in acute myeloid leukemia and thyroid cancer but not in other types of cancer. The mutation causes reduced NADPH production capacity in glioblastoma by 40% and is

  17. Quantitative cytochemical analysis of glucose-6-phosphate dehydrogenase activity in living isolated hepatocytes of European flounder for rapid analysis of xenobiotic effects

    NARCIS (Netherlands)

    Winzer, K.; van Noorden, C. J.; Köhler, A.

    2001-01-01

    There is a great need for rapid but reliable assays to determine quantitatively effects of xenobiotics on biological systems in environmental research. Hepatocytes of European flounder are sensitive to low-dose toxic stress. Glucose-6-phosphate dehydrogenase (G6PDH) is the major source of NADPH in

  18. The Immunomodulatory Activity of Jacaric Acid, a Conjugated Linolenic Acid Isomer, on Murine Peritoneal Macrophages

    OpenAIRE

    Liu, Wai Nam; Leung, Kwok Nam

    2015-01-01

    This study aims at demonstrating the immunomodulatory property of jacaric acid, a conjugated linolenic acid (CLNA) isomer that is present in jacaranda seed oil, on murine peritoneal macrophages. Our results showed that jacaric acid exhibited no significant cytotoxicity on the thioglycollate-elicited murine peritoneal macrophages as revealed by the neutral red uptake assay, but markedly increased their cytostatic activity on the T-cell lymphoma MBL-2 cells as measured by the fluorometric CyQua...

  19. Retinal pigment epithelial acid lipase activity and lipoprotein receptors: effects of dietary omega-3 fatty acids.

    OpenAIRE

    Elner, Victor M.

    2002-01-01

    PURPOSE: To show that fish oil-derived omega-3 polyunsaturated fatty acids, delivered to the retinal pigment epithelium (RPE) by circulating low-density lipoproteins (LDL), enhance already considerable RPE lysosomal acid lipase activity, providing for more efficient hydrolysis of intralysosomal RPE lipids, an effect that may help prevent development of age-related macular degeneration (ARMD). METHODS: Colorimetric biochemical and histochemical techniques were used to demonstrate RPE acid lipa...

  20. Antimicrobial activity of autochthonous lactic acid bacteria isolated ...

    African Journals Online (AJOL)

    Twenty samples of traditional fermented milk “Raib” were collected in eastern Algeria from individual household. They were evaluated for the presence of autochthonous bacteriocin-producing lactic acid bacteria. From 13 of these samples 52 strains of lactic acid bacteria were isolated, and shown to exhibit inhibitory activity ...

  1. Short Communication Antibacterial Activities of Lactic Acid Bacteria ...

    African Journals Online (AJOL)

    2011-12-20

    Dec 20, 2011 ... beneficial effects of lactic acid bacteria consumption include improving intestinal tract health (Parvez et ..... Gastrointestinal Microflora Exert Antimicrobial. Activity. Gut. 47:646–652. Ljungh A and Wadstrom T (2006). Lactic Acid. Bacteria as Probiotics. Curr. Issues Intest. Microbiol. 7:73–89. Marteau P, de ...

  2. Structure and Function of Lactate Dehydrogenase from Hagfish

    Directory of Open Access Journals (Sweden)

    Mitsumasa Okada

    2010-03-01

    Full Text Available The lactate dehydrogenases (LDHs in hagfish have been estimated to be the prototype of those in higher vertebrates. The effects of high hydrostatic pressure from 0.1 to 100 MPa on LDH activities from three hagfishes were examined. The LDH activities of Eptatretus burgeri, living at 45–60 m, were completely lost at 5 MPa. In contrast, LDH-A and -B in Eptatretus okinoseanus maintained 70% of their activities even at 100 MPa. These results show that the deeper the habitat, the higher the tolerance to pressure. To elucidate the molecular mechanisms for adaptation to high pressure, we compared the amino acid sequences and three-dimensional structures of LDHs in these hagfish. There were differences in six amino acids (6, 10, 20, 156, 269, and 341. These amino acidresidues are likely to contribute to the stability of the E. okinoseanus LDH under high-pressure conditions. The amino acids responsible for the pressure tolerance of hagfish are the same in both human and hagfish LDHs, and one substitution that occurred as an adaptation during evolution is coincident with that observed in a human disease. Mutation of these amino acids can cause anomalies that may be implicated in the development of human diseases.

  3. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

    2017-12-26

    A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacyl-ethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings.

  4. Proteolytic and Phytase Activity in Sourdough Lactic Acid Bacteria

    National Research Council Canada - National Science Library

    Valcheva, R; Yungareva, Ts; Kirilov, N; Dobreva, G; Minkov, I; Ivanova, I

    2009-01-01

    Twenty-six strains of lactic acid bacteria (LAB) isolated from French and Bulgarian sourdoughs were screened for their enzymatic activities, to elucidate their possible roles during the fermentation process...

  5. Research on Activators for Lead-Acid Batteries

    OpenAIRE

    Sugawara, Michio; Kozawa, Akiya

    2008-01-01

    Abstract : The ITE Battery Research group has developed a new organic battery activator for new and used lead-acid batteries. Ten years of investigation have established the validity of the ITE activator that prolongs the useful life of lead-acid batteries. It has been shown that the specific gravity of spent batteries can be restored to the original level in automotive, motive power; uninterruptible power supplies (UPS) and stationary energy storage batteries. Our results show that the disca...

  6. Tyrosine Phosphorylation of the UDP-Glucose Dehydrogenase of Escherichia coli Is at the Crossroads of Colanic Acid Synthesis and Polymyxin Resistance

    DEFF Research Database (Denmark)

    Lacour, S.; Bechet, E.; Cozzone, A.J.

    2008-01-01

    to participate in the regulation of the amount of the exopolysaccharide colanic acid, whereas Etk-mediated Ugd phosphorylation appeared to participate in the resistance of E. coli to the antibiotic polymyxin. Conclusions/Significance: Ugd phosphorylation seems to be at the junction between two distinct...