WorldWideScience

Sample records for acid dehydrogenase activity

  1. 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...

  2. 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...

  3. Abscisic acid effects on activity and expression of barley (Hordeum vulgare) plastidial glucose-6-phosphate dehydrogenase.

    Science.gov (United States)

    Cardi, Manuela; Chibani, Kamel; Cafasso, Donata; Rouhier, Nicolas; Jacquot, Jean-Pierre; Esposito, Sergio

    2011-07-01

    Total glucose-6-phosphate dehydrogenase (G6PDH) activity, protein abundance, and transcript levels of G6PDH isoforms were measured in response to exogenous abscisic acid (ABA) supply to barley (Hordeum vulgare cv Nure) hydroponic culture. Total G6PDH activity increased by 50% in roots treated for 12 h with exogenous 0.1 mM ABA. In roots, a considerable increase (35%) in plastidial P2-G6PDH transcript levels was observed during the first 3 h of ABA treatment. Similar protein variations were observed in immunoblotting analyses. In leaves, a 2-fold increase in total G6PDH activity was observed after ABA treatment, probably related to an increase in the mRNA level (increased by 50%) and amount of protein (increased by 85%) of P2-G6PDH. Together these results suggest that the plastidial P2-isoform plays an important role in ABA-treated barley plants.

  4. Regulation of pyruvate dehydrogenase activity and citric acid cycle intermediates during high cardiac power generation.

    Science.gov (United States)

    Sharma, Naveen; Okere, Isidore C; Brunengraber, Daniel Z; McElfresh, Tracy A; King, Kristen L; Sterk, Joseph P; Huang, Hazel; Chandler, Margaret P; Stanley, William C

    2005-01-15

    A high rate of cardiac work increases citric acid cycle (CAC) turnover and flux through pyruvate dehydrogenase (PDH); however, the mechanisms for these effects are poorly understood. We tested the hypotheses that an increase in cardiac energy expenditure: (1) activates PDH and reduces the product/substrate ratios ([NADH]/[NAD(+)] and [acetyl-CoA]/[CoA-SH]); and (2) increases the content of CAC intermediates. Measurements were made in anaesthetized pigs under control conditions and during 15 min of a high cardiac workload induced by dobutamine (Dob). A third group was made hyperglycaemic (14 mm) to stimulate flux through PDH during the high work state (Dob + Glu). Glucose and fatty acid oxidation were measured with (14)C-glucose and (3)H-oleate. Compared with control, the high workload groups had a similar increase in myocardial oxygen consumption ( and cardiac power. Dob increased PDH activity and glucose oxidation above control, but did not reduce the [NADH]/[NAD(+)] and [acetyl-CoA]/[CoA-SH] ratios, and there were no differences between the Dob and Dob + Glu groups. An additional group was treated with Dob + Glu and oxfenicine (Oxf) to inhibit fatty acid oxidation: this increased [CoA-SH] and glucose oxidation compared with Dob; however, there was no further activation of PDH or decrease in the [NADH]/[NAD(+)] ratio. Content of the 4-carbon CAC intermediates succinate, fumarate and malate increased 3-fold with Dob, but there was no change in citrate content, and the Dob + Glu and Dob + Glu + Oxf groups were not different from Dob. In conclusion, compared with normal conditions, at high myocardial energy expenditure (1) the increase in flux through PDH is regulated by activation of the enzyme complex and continues to be partially controlled through inhibition by fatty acid oxidation, and (2) there is expansion of the CAC pool size at the level of 4-carbon intermediates that is largely independent of myocardial fatty acid oxidation.

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

    Science.gov (United States)

    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.

  6. 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.

  7. L-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea.

    Science.gov (United States)

    Deutch, Charles E

    2013-11-01

    The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. L-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP(+)-dependent enzymes from chloroplasts and was separated from the NAD(+)-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis-Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K m for oxaloacetate was 20 μM; the K m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD(+) increased with pH but there was very little activity with NADP(+). At pH 7.0, the K m for L-malate was 5 mM and the K m for NAD(+) was 24 μM. The reductive activity was quite insensitive to inhibition by L-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10-20 times higher than the oxidative activity. These results indicate that the L-malate dehydrogenase in N. europaea is similar to other NAD(+)-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.

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

    DEFF Research Database (Denmark)

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

    1996-01-01

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

  9. Effect of sodium fluoride on adrenal gland of rabbit. I. Studies on ascorbic acid and delta 5-3beta hydroxysteroid dehydrogenase activity

    Energy Technology Data Exchange (ETDEWEB)

    Rao, K.; Susheela, A.K.

    1979-04-01

    Rabbits were given 50 mg sodium fluoride/kg body weight through the intragastric route every 24 hours for a total period of 200 days. The left adrenal gland was removed and its total weight recorded. Adrenal glands from rabbits sacrificed at varying intervals for other investigative purposes were also collected and their weights recorded. The data indicate a significant rise in the total weight of the gland. Both ascorbic acid and steroid dehydrogenase (Delta 5-3Beta hydroxysteroid dehydrogenase) were localized in the adrenal gland by histochemical methods. The results indicate that, in rabbits exposed to NaF, a reduction in ascorbic acid content as well as a depletion of steroid dehydrogenase activity occurs especially at the zona glomerulosa. The significance of the increase in weight of the gland to the reduction of the ascorbic acid content and steroid dehydrogenase activity is discussed. 15 references, 4 figures.

  10. Role of reduced lipoic acid in the redox regulation of mitochondrial aldehyde dehydrogenase (ALDH-2) activity. Implications for mitochondrial oxidative stress and nitrate tolerance.

    Science.gov (United States)

    Wenzel, Philip; Hink, Ulrich; Oelze, Matthias; Schuppan, Swaantje; Schaeuble, Karin; Schildknecht, Stefan; Ho, Kwok K; Weiner, Henry; Bachschmid, Markus; Münzel, Thomas; Daiber, Andreas

    2007-01-05

    Chronic therapy with nitroglycerin results in a rapid development of nitrate tolerance, which is associated with an increased production of reactive oxygen species. We have recently shown that mitochondria are an important source of nitroglycerin-induced oxidants and that the nitroglycerin-bioactivating mitochondrial aldehyde dehydrogenase is oxidatively inactivated in the setting of tolerance. Here we investigated the effect of various oxidants on aldehyde dehydrogenase activity and its restoration by dihydrolipoic acid. In vivo tolerance in Wistar rats was induced by infusion of nitroglycerin (6.6 microg/kg/min, 4 days). Vascular reactivity was measured by isometric tension studies of isolated aortic rings in response to nitroglycerin. Chronic nitroglycerin infusion lead to impaired vascular responses to nitroglycerin and decreased dehydrogenase activity, which was corrected by dihydrolipoic acid co-incubation. Superoxide, peroxynitrite, and nitroglycerin itself were highly efficient in inhibiting mitochondrial and yeast aldehyde dehydrogenase activity, which was restored by dithiol compounds such as dihydrolipoic acid and dithiothreitol. Hydrogen peroxide and nitric oxide were rather insensitive inhibitors. Our observations indicate that mitochondrial oxidative stress (especially superoxide and peroxynitrite) in response to organic nitrate treatment may inactivate aldehyde dehydrogenase thereby leading to nitrate tolerance. Glutathionylation obviously amplifies oxidative inactivation of the enzyme providing another regulatory pathway. Furthermore, the present data demonstrate that the mitochondrial dithiol compound dihydrolipoic acid restores mitochondrial aldehyde dehydrogenase activity via reduction of a disulfide at the active site and thereby improves nitrate tolerance.

  11. Effects of low molecular-weight organic acids and dehydrogenase activity in rhizosphere sediments of mangrove plants on phytoremediation of polycyclic aromatic hydrocarbons.

    Science.gov (United States)

    Wang, Yuanyuan; Fang, Ling; Lin, Li; Luan, Tiangang; Tam, Nora F Y

    2014-03-01

    This work evaluated the roles of the low-molecular-weight organic acids (LMWOAs) from root exudates and the dehydrogenase activity in the rhizosphere sediments of three mangrove plant species on the removal of mixed PAHs. The results showed that the concentrations of LMWOAs and dehydrogenase activity changed species-specifically with the levels of PAH contamination. In all plant species, the concentration of citric acid was the highest, followed by succinic acid. For these acids, succinic acid was positively related to the removal of all the PAHs except Chr. Positive correlations were also found between the removal percentages of 4-and 5-ring PAHs and all LMWOAs, except citric acid. LMWOAs enhanced dehydrogenase activity, which positively related to PAH removal percentages. These findings suggested that LMWOAs and dehydrogenase activity promoted the removal of PAHs. Among three mangrove plants, Bruguiera gymnorrhiza, the plant with the highest root biomass, dehydrogenase activity and concentrations of LMWOAs, was most efficient in removing PAHs. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. In vivo activity of 11beta-hydroxysteroid dehydrogenase type 1 and free fatty acid-induced insulin resistance.

    Science.gov (United States)

    Mai, K; Kullmann, V; Bobbert, T; Maser-Gluth, C; Möhlig, M; Bähr, V; Pfeiffer, A F H; Spranger, J; Diederich, S

    2005-10-01

    Free fatty acids (FFAs) induce hepatic insulin resistance and enhance hepatic gluconeogenesis. Glucocorticoids (GCs) also stimulate hepatic gluconeogenesis. The aim of this study was to investigate whether the FFA-induced hepatic insulin resistance is mediated by increased activity of hepatic 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), accompanied by elevated hepatic cortisol levels. Following a 10-h overnight fast, six healthy male volunteers were investigated. A euglycaemic hyperinsulinaemic clamp was performed during lipid or saline infusion. To assess hepatic 11beta-HSD1 activity, plasma cortisol levels were measured after oral administration of cortisone acetate during lipid or saline infusion. In addition, 11beta-HSD activities were determined in vivo by calculating the urinary ratios of GC metabolites. Lipid infusion increased FFAs (5.41 +/- 1.00 vs. 0.48 +/- 0.20 mmol/l; P < 0.005) and significantly increased insulin resistance [glucose infusion rate (GIR) 6.02 +/- 2.60 vs. 4.08 +/- 2.15 mg/kg/min; P < 0.005]. After lipid and saline infusions no changes in 11beta-HSD1 activity were found, neither by changes in cortisone acetate to cortisol conversion nor by differences in urinary free cortisol (UFF) or cortisone (UFE), 5beta-tetrahydrocortisol (THF), 5alpha-THF, cortisone (THE), UFF/UFE and (5alpha-THF + THF)/THE ratios. We found no change in hepatic and whole-body 11beta-HSD1 activity during acute FFA-induced insulin resistance. Further studies are necessary to clarify whether 11beta-HSD1 in muscle and adipose tissue is influenced by FFAs and whether 11beta-HSD1 is involved in other conditions of insulin resistance.

  15. Branched chain amino acid transaminase and branched chain alpha-ketoacid dehydrogenase activity in the brain, liver and skele­tal muscle of acute hepatic failure rats

    Directory of Open Access Journals (Sweden)

    Takei,Nobuyuki

    1985-02-01

    Full Text Available Branched chain amino acid (BCAA transaminase activity increased in both the mitochondrial and supernatant fractions of brain from hepatic failure rats, in which a partial hepatectomy was performed 24h following carbon tetrachloride (CCl4 administration, although the activity of liver and skeletal muscle was the same as in control rats. The elevation of mitochondrial BCAA transaminase activity in liver-injured rats was partly due to increased activity of brain specific Type III isozyme. Branched chain alpha-ketoacid (BCKA dehydrogenase in the brain homogenates was not significantly altered in acute hepatic failure rats, while the liver enzyme activity was markedly diminished. BCKA dehydrogenase activity in the brain homogenates was inhibited by adding ATP to the assay system, and was activated in vitro by preincubating the brain homogenate at 37 degrees C for 15 min. These findings suggest that brain BCAA catabolism is accelerated in acute hepatic failure rats.

  16. R-lipoic acid inhibits mammalian pyruvate dehydrogenase kinase.

    Science.gov (United States)

    Korotchkina, Lioubov G; Sidhu, Sukhdeep; Patel, Mulchand S

    2004-10-01

    The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects.

  17. A membrane-associated adenylate cyclase modulates lactate dehydrogenase and creatine kinase activities required for bull sperm capacitation induced by hyaluronic acid.

    Science.gov (United States)

    Fernández, Silvina; Córdoba, Mariana

    2017-04-01

    Hyaluronic acid, as well as heparin, is a glycosaminoglycan present in the female genital tract of cattle. The aim of this study was to evaluate oxidative metabolism and intracellular signals mediated by a membrane-associated adenylate cyclase (mAC), in sperm capacitation with hyaluronic acid and heparin, in cryopreserved bull sperm. The mAC inhibitor, 2',5'-dideoxyadenosine, was used in the present study. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities and lactate concentration were determined spectrophotometrically in the incubation medium. Capacitation and acrosome reaction were evaluated by chlortetracycline technique, while plasma membrane and acrosome integrity were determined by trypan blue stain/differential interference contrast microscopy. Heparin capacitated samples had a significant decrease in LDH and CK activities, while in hyaluronic acid capacitated samples LDH and CK activities both increased compared to control samples, in heparin and hyaluronic acid capacitation conditions, respectively. A significant increase in lactate concentration in the incubation medium occurred in hyaluronic acid-treated sperm samples compared to heparin treatment, indicating this energetic metabolite is produced during capacitation. The LDH and CK enzyme activities and lactate concentrations in the incubation medium were decreased with 2',5'-dideoxyadenosine treatment in hyaluronic acid samples. The mAC inhibitor significantly inhibited heparin-induced capacitation of sperm cells, but did not completely inhibit hyaluronic acid capacitation. Therefore, hyaluronic acid and heparin are physiological glycosaminoglycans capable of inducing in vitro capacitation in cryopreserved bull sperm, stimulating different enzymatic pathways and intracellular signals modulated by a mAC. Hyaluronic acid induces sperm capacitation involving LDH and CK activities, thereby reducing oxidative metabolism, and this process is mediated by mAC.

  18. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci.

    Science.gov (United States)

    Peralta, Guillermo Hugo; Bergamini, Carina Viviana; Hynes, Erica Rut

    2016-01-01

    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.

  19. Ebselen protects against behavioral and biochemical toxicities induced by 3-nitropropionic acid in rats: correlations between motor coordination, reactive species levels, and succinate dehydrogenase activity.

    Science.gov (United States)

    Wilhelm, Ethel A; Bortolatto, Cristiani F; Jesse, Cristiano R; Luchese, Cristiane

    2014-12-01

    The protective effect of ebselen was investigated against 3-nitropropionic acid (3-NP)-induced behavioral and biochemical toxicities in rats. Ebselen (10 or 25 mg/kg, intragastrically) was administered to rats 30 min before 3-NP (20 mg/kg, intraperitoneally) once a day for a period of 4 days. Locomotor activity, motor coordination, and body weight gain were determined. The striatal content of reactive oxygen species (ROS), reduced glutathione (GSH), ascorbic acid (AA), and protein carbonyl as well as catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) activities was determined 24 h after the last dose of 3-NP. Na(+)/ K(+)-ATPase, succinate dehydrogenase (SDH), and δ-aminolevulinic dehydratase (δ-ALA-D) activities were also determined. The results demonstrated that ebselen at a dose of 25 mg/kg, but not at 10 mg/kg, protected against (1) a decrease in locomotor activity, motor coordination impairment, and body weight loss; (2) striatal oxidative damage, which was characterized by an increase in ROS levels, protein carbonyl content, and GR activity, an inhibition of CAT and GPx activities, and a decrease in GSH levels; and (3) an inhibition of SDH and Na(+)/K(+)-ATPase activities, induced by 3-NP. GST activity and AA levels were not modified by ebselen or 3-NP. Ebselen was not effective against the inhibition of δ-ALA-D activity induced by 3-NP. The results revealed a significant correlation between SDH activity and ROS levels, and SDH activity and latency to fall (rotarod test). The present study highlighted the protective effect of ebselen against 3-NP-induced toxicity in rats.

  20. Analysis of amino acid residues involved in cold activity of monomeric isocitrate dehydrogenase from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea.

    Science.gov (United States)

    Yasuda, Wataru; Kobayashi, Miyuki; Takada, Yasuhiro

    2013-11-01

    Monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea (CmIDH-II and CpIDH-M, respectively) are cold-adapted enzymes and show a high degree of amino acid sequential identity to each other (77%). However, maximum activity of CpIDH-M at optimum temperature is much less than that of CmIDH-II. In the C-terminal region 3 of these enzymes, which was suggested from previous study to be responsible for their distinct catalytic ability, several sequential differences of amino acid residue are present. Among them, ten amino acid residues were exchanged between them by site-directed mutagenesis and several properties of the mutated enzymes were examined in this study. The mutated enzymes of CmIDH-II substituted its Gln671, Leu724 and Phe735 residues with the corresponding residues of CpIDH-M (termed Q671K, L724Q and F735L, respectively) showed lower specific activity and thermostability for activity than the wild-type enzyme. Furthermore, the decreased specific activity was also observed in L693F. In contrast, the corresponding mutants of CpIDH-M, F693L, Q724L and L735F, showed the increased specific activity and thermostability for activity. The catalytic efficiency (k(cat)/K(m)) values of these mutated CmIDH-II and CpIDH-M were lower and higher than those of their wild-type IDHs, respectively. These results suggest that the Gln671, Leu693, Leu724 and Phe735 residues of CmIDH-II are important for exerting its high catalytic ability.

  1. 3-nitropropionic acid inhibition of succinate dehydrogenase (complex II) activity in cultured Chinese hamster ovary cells: antagonism by L-carnitine.

    Science.gov (United States)

    Scallet, Andrew C; Haley, Raney L; Scallet, Dori M; Duhart, Helen M; Binienda, Zbigniew K

    2003-05-01

    3-Nitropropionic acid (3-NPA) is an inhibitor of the mitochondrial enzyme succinate dehydrogenase (SDH, a part of complex II) that links the tricarboxylic acid (TCA) cycle to the respiratory electron transport chain. 3-NPA inactivates SDH by covalently and irreversibly binding to its active site. We previously examined the effects of 3-NPA on the histochemical activity of SDH in vivo, by using the reduction of a yellow tetrazolium dye (nitro blue tetrazolium) to a blue formazan as an indicator. In studies of cultured cells, the related dye methylthiazoletetrazolium (MTT) has commonly been used as an indicator of the presence and number of viable cells; that is cells that are capable of producing energy via the TCA cycle. Here we observed that doses of 3-NPA as low as 10(-8) M inhibited formazan production in an in vitro model system using CHO cells. This effect was antagonized by l-carnitine, which greatly increased the production of formazan, indicating a considerable improvement in energy production by the cultured cells. CHO cells appear to be a convenient model for the evaluation of therapeutic compounds that may modulate cellular bioenergetics.

  2. INFLUENCE OF SELECTED PHARMACEUTICALS ON ACTIVATED SLUDGE DEHYDROGENASE ACTIVITY

    Directory of Open Access Journals (Sweden)

    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%.

  3. 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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  5. 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

    During prolonged exercise, carbohydrate oxidation may result from decreased pyruvate production and increased fatty acid supply and ultimately lead to reduced pyruvate dehydrogenase (PDH) activity. Pyruvate also interacts with the amino acids alanine, glutamine, and glutamate, whereby the decline...... 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...... peaked at 2 h of exercise, whereas pyruvate production peaked at 1 h of exercise and was reduced ( 30%) thereafter, suggesting that pyruvate availability primarily accounted for reduced carbohydrate oxidation. Increased free fatty acid uptake (P

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. Butachlor impact on protein, free amino acid and glutamine contents, and on activity levels of aminotransferases, glutamate dehydrogenase and glutamine synthetase in the fresh water snail, Pila globosa (Swainson).

    Science.gov (United States)

    Rajyalakshmi, T; Srinivas, T; Swamy, K V; Mohan, P M

    1996-08-01

    Biochemical changes followed in the freshwater snail Pila globosa (Swainson) during exposure to sublethal concentrations of the herbicide butachlor (26.6 ppm) in the ambient medium, at 3,6,12,24 and 48 h intervals, were marked by a significant decrease in total and soluble proteins, and an increase in free amino acids in foot and hepatopancreas up to 12 h before gradually recovering. Aminotransferase activities and glutamine content decreased during the early periods of exposure, while glutamate dehydrogenase activity increased. After an initial elevation, glutamate synthetase activity decreased at later intervals. Maximum effect of butachlor on the enzymes was seen after 12 h exposure. The extent of increase or decrease in different parameters examined varied between the two tissues studied. These changes are discussed in relation to the toxic stress of butachlor.

  9. 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.

  10. Cell wall-associated malate dehydrogenase activity from maize roots.

    Science.gov (United States)

    Hadži-Tašković Šukalović, Vesna; Vuletić, Mirjana; Marković, Ksenija; Vučinić, Zeljko

    2011-10-01

    Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn(2+) and Cu(2+) in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn(2+) and Cu(2+) in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.

  11. Malate dehydrogenase activity in human seminal plasma and spermatozoa homogenates

    Directory of Open Access Journals (Sweden)

    Hulya Leventerler

    2013-08-01

    Full Text Available Purpose: Malate Dehydrogenase is an important enzyme of the Krebs cycle, most cells require this enzyme for their metabolic activity. We evaluated the Malate Dehydrogenase (NAD/NADP activity in human seminal plasma and sperm homogenates in normozoospermic, fertile and infertile males. Also glucose and fructose concentrations were determined in the seminal plasma samples. Material and Methods: Malate Dehydrogenase (NAD/NADP activity in human seminal plasma and sperm homogenates of normozoospermic and infertile males was determined by spectrophotometric method. Semen analysis was considered according to the WHO Criteria. Results: Malat Dehydrogenase-NAD value in seminal plasma (the mean ± SD, mU/ml of asthenoteratospermic (40.0±25.7 and azospermic (38.0±43.6 groups were significantly lower than normozoospermic, (93.9±52.1 males. Malat Dehydrogenase-NAD value in sperm homogenates (the mean ± SD, mU/ 20x106 sperm of teratospermic group (136.8±61.8 was significantly higher compared to the normozoospermic (87.3±26.5 males. Glucose concentration (mg/dl in asthenoteratospermic (4.0±1.4 and azospermic (15.4±6.4 groups were significantly higher than fertile (2.0±2.1 males. Also fructose concentration (mg/dl in asthenoteratospermic (706.6±143.3 and azospermic (338.1±228.2 groups were significantly high compared to the normozoospermic (184.7±124.8 group. Conclusion: Sperm may be some part of the source of Malat Dehydrogenase activity in semen. Malat Dehydrogenase activity in seminal plasma has an important role on energy metabolism of sperm. Intermediate substrates of Krebs cycle might have been produced under the control of Malat Dehydrogenase and these substrates may be important for sperm motility and male infertility. [Cukurova Med J 2013; 38(4.000: 648-658

  12. 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

  13. 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

  14. Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis.

    Science.gov (United States)

    Long, Michael C; Nagegowda, Dinesh A; Kaminaga, Yasuhisa; Ho, Kwok Ki; Kish, Christine M; Schnepp, Jennifer; Sherman, Debra; Weiner, Henry; Rhodes, David; Dudareva, Natalia

    2009-07-01

    Benzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primary metabolites to secondary products. Benzoic acid biosynthesis from L-phenylalanine requires shortening of the propyl side chain by two carbons, which can occur via a beta-oxidative pathway or a non-beta-oxidative pathway, with benzaldehyde as a key intermediate. The non-beta-oxidative route requires benzaldehyde dehydrogenase (BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identified an Antirrhinum majus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript profiling, biochemical characterization of the purified recombinant protein, molecular homology modeling, in vivo stable isotope labeling, and transient expression in petunia flowers reveal that BALDH is capable of oxidizing benzaldehyde to BA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in the mitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.

  15. 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...

  16. 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

  17. 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.

  18. Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by tricarboxylic acid cycle metabolites.

    Science.gov (United States)

    Bartholomae, Maike; Meyer, Frederik M; Commichau, Fabian M; Burkovski, Andreas; Hillen, Wolfgang; Seidel, Gerald

    2014-02-01

    In Bacillus subtilis, recent in vivo studies revealed that particular enzymes of the tricarboxylic acid cycle form complexes that allow an efficient transfer of metabolites. Remarkably, a complex of the malate dehydrogenase (Mdh) (EC 1.1.1.37) with isocitrate dehydrogenase (Icd) (EC 1.1.1.42) was identified, although both enzymes do not catalyze subsequent reactions. In the present study, the interactions between these enzymes were characterized in vitro by surface plasmon resonance in the absence and presence of their substrates and cofactors. These analyses revealed a weak but specific interaction between Mdh and Icd, which was specifically stimulated by a mixture of substrates and cofactors of Icd: isocitrate, NADP(+) and Mg(2+). Wild-type Icd converted these substrates too fast, preventing any valid quantitative analysis of the interaction with Mdh. Therefore, binding of the IcdS104P mutant to Mdh was quantified because the mutation reduced the enzymatic activity by 174-fold but did not affect the stimulatory effect of substrates and cofactors on Icd-Mdh complex formation. The analysis of the unstimulated Mdh-IcdS104P interaction revealed kinetic constants of k(a) = 2.0 ± 0.2 × 10(2) m(-1) ·s(-1) and k(d) = 1.0 ± 0.1 × 10(-3) ·s(-1) and a K(D) value of 5.0 ± 0.1 μm. Addition of isocitrate, NADP(+) and Mg(2+) stimulated the affinity of IcdS104P to Mdh by 33-fold (K(D) = 0.15 ± 0.01 μm, k(a) = 1.7 ± 0.7 × 10(3) m(-1) ·s(-1), k(d) = 2.6 ± 0.6 × 10(-4) ·s(-1)). Analyses of the enzymatic activities of wild-type Icd and Mdh showed that Icd activity doubles in the presence of Mdh, whereas Mdh activity was slightly reduced by Icd. In summary, these data indicate substrate control of complex formation in the tricarboxylic acid cycle metabolon assembly and maintenance of the α-ketoglutarate supply for amino acid anabolism in vivo.

  19. 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.

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

    African Journals Online (AJOL)

    AJB SERVER

    2007-01-04

    Jan 4, 2007 ... Key words: Dehydrogenase activity, rhizosplane bacteria, atrazine, cypermethrin, ... resources for improved and sustainable agriculture ... Growth of cowpea and source of microbial community. The cowpea plant (Vigna unguiculata) was grown to maturity in an ..... stimulation of dehydrogenase activity.

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

    Science.gov (United States)

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

    2014-05-23

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

  2. 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.

  3. Coulometric bioelectrocatalytic reactions based on NAD-dependent dehydrogenases in tricarboxylic acid cycle

    Energy Technology Data Exchange (ETDEWEB)

    Fukuda, Jun [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan); Tsujimura, Seiya [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan)], E-mail: seiya@kais.kyoto-u.ac.jp; Kano, Kenji [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan)], E-mail: kkano@kais.kyoto-u.ac.jp

    2008-12-30

    This paper describes the characterization of mediated electro-enzymatic electrolysis systems based on NAD-dependent dehydrogenase reactions in the tricarboxylic acid (TCA) cycle. A micro-bulk electrolysis system with a carbon felt anode immersed in an electrolysis solution with a value of about 10 {mu}L was constructed for coulometric analysis of the substrate oxidation. Diaphorase (DI) was used to couple the NAD-dependent dehydrogenase reaction with the anode reaction of a suitable redox mediator. We focused on three types of NAD-dependant dehydrogenases reactions in this research: (1) isocitrate oxidation, in which the standard Gibbs energy change ({delta}G{sup o}') is negative; (2) {alpha}-ketoglutarate oxidation, which involves an electrochemically active coenzyme A (CoA); and (3) malate oxidation, which is thermodynamically unfavorable because of a large positive {delta}G{sup o}' value. The complete electrolysis of isocitrate was easily achieved, supporting the effective re-oxidation of NADH in the diaphorase-catalyzed electrochemical reaction. CoA was unfavorably oxidized at the electrodes in the presence of some mediators. The electrocatalytic oxidation of CoA was suppressed and the quantitative electrochemical oxidation of {alpha}-ketoglutarate was achieved by selecting a suitable mediator with negligibly slow electron transfer kinetics with CoA. The uphill malate oxidation was susceptible to product inhibition in the bioelectrochemical system, although NADH generated in the malate dehydrogenase reaction was immediately oxidized in the electrochemical system. The inhibition was successfully suppressed by linking citrate synthase to quench oxaloacetate and to make the total {delta}G{sup o}' value negative.

  4. The activity of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in the sera of patients with brain cancer.

    Science.gov (United States)

    Jelski, Wojciech; Laniewska-Dunaj, Magdalena; Orywal, Karolina; Kochanowicz, Jan; Rutkowski, Robert; Szmitkowski, Maciej

    2014-12-01

    Human brain tissue contains various alcohol dehydrogenase (ADH) isoenzymes and possess also aldehyde dehydrogenase (ALDH) activity. In our last experiments we have shown that ADH and ALDH are present also in the brain tumour cells. Moreover the activities of total ADH and class I isoenzymes were significantly higher in cancer tissue than healthy cells. It can suggests that these changes may be reflected by enzyme activity in the serum of patients with brain cancer. Serum samples were taken for routine biochemical investigation from 62 patients suffering from brain cancer (36 glioblastoma, 26 meningioma). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, the fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. A statistically significant increase of class I alcohol dehydrogenase isoenzymes was found in the sera of patients with brain cancer. The median activity of this class isoenzyme in the patients group increased about 24 % in the comparison to the control level. The total alcohol dehydrogenase activity was also significantly higher (26 %) among patients with brain tumour than healthy ones. The activities of other tested ADH isoenzymes and total ALDH were unchanged. The increase of the activity of total ADH and class I alcohol dehydrogenase isoenzyme in the sera of patients with brain cancer seems to be caused by the release of this isoenzyme from tumour's cells.

  5. 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.

  6. [Effect of overexpression of malate dehydrogenase on succinic acid production in Escherichia coli NZN111].

    Science.gov (United States)

    Liang, Liya; Ma, Jiangfeng; Liu, Rongming; Wang, Guangming; Xu, Bing; Zhang, Min; Jiang, Min

    2011-07-01

    Escherichia coli NZN111 is a double mutant with lactate dehydrogenase (ldhA) and pyruvate formate-lyase (pflB) inactivated. Under anaerobic conditions, disequilibrium of coenzyme NADH and NAD+ causes Escherichia coli NZN111 losing the glucose utilizing capability. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-mdh and overexpressed the mdh gene with 0.3 mmol/L of IPTG under anaerobic fermentation condition in sealed bottles. The specific malate dehydrogenase (MDH) activity in the recombinant strain was 14.8-fold higher than that in E. coli NZN111. The NADH/ NAD+ ratio decreased from 0.64 to 0.26 and the concentration of NAD+ and NADH increased 1.5-fold and 0.2-fold respectively. Under anaerobic conditions, the recombinant strain possessed the capability of growth and glucose absorption. We took dual-phase fermentation for succinate production. After the dry cell weight (DCW) reached 6.4 g/L under aerobic conditions, the cell culture was changed to anaerobic conditions. After 15 h, 14.75 g/L glucose was consumed and succinic acid reached 15.18 g/L. The yield of succinic acid was 1.03 g/g Glu and the productivity of succinic acid was 1.012 g/(L x h).

  7. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains.

    Science.gov (United States)

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  8. Determination of dehydrogenase activities involved in D-glucose oxidation in Gluconobacter and Acetobacter strains

    Directory of Open Access Journals (Sweden)

    Florencia Sainz

    2016-08-01

    Full Text Available Acetic acid bacteria (AAB are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane bound dehydrogenases. In the present study, the enzyme activity of the membrane bound dehydrogenases (membrane-bound PQQ-glucose dehydrogenase (mGDH, D-gluconate dehydrogenase (GADH and membrane-bound glycerol dehydrogenase (GLDH involved in the oxidation of D-glucose and D-gluconic acid (GA was determined in six strains of three different species of AAB (three natural and three type strains. Moreover, the effect of these activities on the production of related metabolites (GA, 2-keto-D-gluconic acid (2KGA and 5-keto-D-gluconic acid (5KGA was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the A. malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h, which coincided with glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of G. oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition.Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter were

  9. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains

    Science.gov (United States)

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  10. Mechanism of Hyperinsulinism in Short-chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency Involves Activation of Glutamate Dehydrogenase*

    Science.gov (United States)

    Li, Changhong; Chen, Pan; Palladino, Andrew; Narayan, Srinivas; Russell, Laurie K.; Sayed, Samir; Xiong, Guoxiang; Chen, Jie; Stokes, David; Butt, Yasmeen M.; Jones, Patricia M.; Collins, Heather W.; Cohen, Noam A.; Cohen, Akiva S.; Nissim, Itzhak; Smith, Thomas J.; Strauss, Arnold W.; Matschinsky, Franz M.; Bennett, Michael J.; Stanley, Charles A.

    2010-01-01

    The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh−/−). The hadh−/− mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh−/− mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh−/− mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh−/− islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh−/− islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh−/− islets also have increased [U-14C]glutamine oxidation. In contrast, hadh−/− mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh−/− islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh−/− islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD. PMID:20670938

  11. Mechanism of hyperinsulinism in short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency involves activation of glutamate dehydrogenase.

    Science.gov (United States)

    Li, Changhong; Chen, Pan; Palladino, Andrew; Narayan, Srinivas; Russell, Laurie K; Sayed, Samir; Xiong, Guoxiang; Chen, Jie; Stokes, David; Butt, Yasmeen M; Jones, Patricia M; Collins, Heather W; Cohen, Noam A; Cohen, Akiva S; Nissim, Itzhak; Smith, Thomas J; Strauss, Arnold W; Matschinsky, Franz M; Bennett, Michael J; Stanley, Charles A

    2010-10-01

    The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh(-/-)). The hadh(-/-) mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh(-/-) mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh(-/-) mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh(-/-) islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh(-/-) islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh(-/-) islets also have increased [U-(14)C]glutamine oxidation. In contrast, hadh(-/-) mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh(-/-) islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh(-/-) islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.

  12. In vitro interaction between psychotropic drugs and alcohol dehydrogenase activity.

    Science.gov (United States)

    Roig, M G; Bello, F; Burguillo, F J; Cachaza, J M; Kennedy, J F

    1991-03-01

    A series of CNS-stimulating and -depressant drugs have been studied for their in vitro interaction with horse liver alcohol dehydrogenase (ADH) activity. The depressant drugs studied included barbital, phenobarbital, thiopental, nitrazepam, chlorpromazine, sulpiride, clomethiazole, Li2CO3, diazepam, phenytoin, ethosuximide, morphine, and codeine. The stimulant drugs were theophylline, caffeine, amphetamine, imipramine, chlorimipramine, amitriptyline, and tranylcypromine. The results were as follows. First, ADH activity was inhibited by the action of chlorpromazine, tranylcypromine, imipramine, chlorimipramine, amitriptyline, sulpiride, amphetamine, codeine, ethosuximide, morphine, clomethiazole, nitrazepam, Li2CO3, theophylline, and phenobarbital, in descending order of inhibitory effect. Second, inhibition followed by activation of ADH activity was observed for imipramine and chlorimipramine. Third, activation of ADH activity was observed for phenytoin. Finally, the following drugs were not seen to exert any effect on ADH activity: barbital, thiopental, diazepam, and caffeine.

  13. 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.

  14. Toxicity of Nitrification Inhibitors on Dehydrogenase Activity in Soils

    Directory of Open Access Journals (Sweden)

    Ferisman Tindaon

    2011-01-01

    Full Text Available The objective of this research was to determine the effects of nitrification inhibitors (NIs such as 3,4-dimethylpyrazolephosphate=DMPP, 4-Chlor-methylpyrazole phosphate=ClMPP and dicyandiamide,DCD which might be expected to inhibit microbial activity, on dehydrogenase activity (DRA,in three different soils in laboratory conditions. Dehydrogenase activity were assessed via reduction of 2-p-Iodophenyl-3-p-nitrophenyl-5-phenyltetrazoliumchloride (INT. The toxicity and dose response curve of three NIs were quantified under laboratory conditions using a loamy clay, a sandy loam and a sandy soil. The quantitative determination of DHA was carried out spectrophotometrically. In all experiments, the influence of 5-1000 times the base concentration were examined. To evaluate the rate of inhibition with the increasing NI concentrations, dose reponse curves were presented and no observable effect level =NOEL, as well as effective dose ED10 and ED 50(10% and 50% inhibition were calculated. The NOEL for common microbial activity such as DHA was about 30–70 times higher than base concentration in all investigated soils. ClMPP exhibited the strongest influence on the non target microbial processes in the three soils if it compare to DMPP and DCD. The NOEL,ED10 and ED50 values higher in clay than in loamy or sandy soil. The NIs were generally most effective in sandy soils. The three NIs considered at the present state of knowledge as environmentally safe in use.

  15. 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.

  16. 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.

  17. Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia.

    Science.gov (United States)

    Santiago, Rocío; Alarcón, Borja; de Armas, Roberto; Vicente, Carlos; Legaz, María Estrella

    2012-06-01

    This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD.

  18. 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.

  19. Microbial metabolic activity in soil as measured by dehydrogenase determinations

    Science.gov (United States)

    Casida, L. E., Jr.

    1977-01-01

    The dehydrogenase technique for measuring the metabolic activity of microorganisms in soil was modified to use a 6-h, 37 C incubation with either glucose or yeast extract as the electron-donating substrate. The rate of formazan production remained constant during this time interval, and cellular multiplication apparently did not occur. The technique was used to follow changes in the overall metabolic activities of microorganisms in soil undergoing incubation with a limiting concentration of added nutrient. The sequence of events was similar to that obtained by using the Warburg respirometer to measure O2 consumption. However, the major peaks of activity occurred earlier with the respirometer. This possibly is due to the lack of atmospheric CO2 during the O2 consumption measurements.

  20. Histochemical investigations on the in vivo effects of fluoride on tricarboxylic acid cycle dehydrogenases from Pelargonium zonale. Part II

    Energy Technology Data Exchange (ETDEWEB)

    Lovelace, C.J.; Miller, G.W.

    1967-01-01

    In vivo effects of fluoride on tricarboxylic acid (TCA) cycle dehydrogenase enzymes of Pelargonium zonale were studied using p-nitro blue tetrazoleum chloride. Plants were exposed to 17 ppb HF, and enzyme activities in treated plants were compared to those in controls. Leaves of control plants were incubated in 5 x 10/sup -3/ M sodium fluoride. Injuries observed in fumigation and solution experiments were similar. Leaf tissue subjected to HF or sodium fluoride evidenced less succinic p-nitro blue tetrazoleum reductase activity than did control tissue. Other TCA cycle dehydrogenase enzymes were not observably affected by the fluoride concentrations used in these experiments. Excised leaves cultured in 5 x 10/sup -3/ M sodium fluoride exhibited less succinic p-nitro blue tetrazoleum reductase activity after 24 hr than did leaves cultured in 5 x 10/sup -3/ M sodium chloride. 8 references, 8 figures.

  1. Branched-chain amino acid metabolon: interaction of glutamate dehydrogenase with the mitochondrial branched-chain aminotransferase (BCATm).

    Science.gov (United States)

    Islam, Mohammad Mainul; Nautiyal, Manisha; Wynn, R Max; Mobley, James A; Chuang, David T; Hutson, Susan M

    2010-01-01

    The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain alpha-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain alpha-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5'-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5'-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5'-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.

  2. Activation of c-Jun-N-terminal kinase and decline of mitochondrial pyruvate dehydrogenase activity during brain aging.

    Science.gov (United States)

    Zhou, Qiongqiong; Lam, Philip Y; Han, Derick; Cadenas, Enrique

    2009-04-02

    Mitochondrial dysfunction is often associated with aging and neurodegeneration. c-Jun-N-terminal kinase (JNK) phosphorylation and its translocation to mitochondria increased as a function of age in rat brain. This was associated with a decrease of pyruvate dehydrogenase (PDH) activity upon phosphorylation of the E(1alpha) subunit of PDH. Phosphorylation of PDH is likely mediated by PDH kinase, the protein levels and activity of which increased with age. ATP levels were diminished, whereas lactic acid levels increased, thus indicating a shift toward anaerobic glycolysis. The energy transduction deficit due to impairment of PDH activity during aging may be associated with JNK signaling.

  3. 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...

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. NADP+-dependent glutamate dehydrogenase activity is impaired in mutants of Saccharomyces cerevisiae that lack aconitase.

    Science.gov (United States)

    González, A; Rodríguez, L; Olivera, H; Soberón, M

    1985-10-01

    A mutant of Saccharomyces cerevisiae lacking aconitase did not grow on minimal medium (MM) and had five- to tenfold less NADP+-dependent glutamate dehydrogenase (GDH) activity than the wild-type, although its glutamine synthetase (GS) activity was still inducible. When this mutant was incubated with glutamate as the sole nitrogen source, the 2-oxoglutarate content rose, and the NADP+-dependent GDH activity increased. Furthermore, carbon-limited cultures showed a direct relation between NADP+-dependent GDH activity and the intracellular 2-oxoglutarate content. We propose that the low NADP+-dependent GDH activity found in the mutant was due to the lack of 2-oxoglutarate or some other intermediate of the tricarboxylic acid cycle.

  9. Inhibitory effects of ionic liquids on the lactic dehydrogenase activity.

    Science.gov (United States)

    Dong, Xing; Fan, Yunchang; Zhang, Heng; Zhong, Yingying; Yang, Yang; Miao, Juan; Hua, Shaofeng

    2016-05-01

    Ionic liquids (ILs) were widely used in scientific and industrial application and have been reported to possess potential toxicity to the environment and human health. The effects of six typical N-methylimidazolium-based ILs ([Cnmim]X, n=4, 6, 8; X=Br(-), Cl(-), BF4(-), CF3SO3(-)) on the lactic dehydrogenase (LDH) activity and the molecular interaction mechanism of ILs and the LDH were investigated with the aid of spectroscopic techniques. Experimental results showed that the LDH activity was inhibited in the presence of ILs. For the ILs with the same anion but different cations, their inhibitory ability on the LDH activity increased with increasing the alkyl chain length on the IL cation. Thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of LDH with the addition of ILs. Both positive ΔH and ΔS suggested that hydrophobicity was the major driven force in the interaction process as expected.

  10. 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.

  11. 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.

  12. Acute in vivo regulation of 11beta-hydroxysteroid dehydrogenase type 1 activity by insulin and intralipid infusions in humans.

    Science.gov (United States)

    Wake, Deborah J; Homer, Natalie Z M; Andrew, Ruth; Walker, Brian R

    2006-11-01

    Extraadrenal regeneration of cortisol by 11beta-hydroxysteroid dehydrogenase type 1 (11HSD1) is increased after a mixed meal. It is unknown which tissue is responsible and whether this reflects the complex transcriptional control of 11HSD1 or posttranscriptional control exerted by supply of reduced nicotinamide adenine dinucleotide phosphate from hexose-6-phosphate dehydrogenase. The objective of this study was to test whether hyperinsulinemia and/or increased serum free fatty acids increase whole-body and intraadipose 11HSD1, and whether adipose 11HSD1 switches from dehydrogenase to reductase activity. In nine healthy men, we measured whole-body cortisol regeneration (by iv infusion of 9,11,12,12-[2H]4 -cortisol) and intra-adipose interconversion of cortisol and cortisone (by sc microdialysis infusion of [3H]4 -cortisol and [3H]2 -cortisone in separate cannulae) during: 1) a hyperinsulinemic euglycemic clamp; 2) iv lipid infusion (Intralipid 20% fat emulsion); and 3) saline infusion, each for 3.5 h. Hyperinsulinemia increased rate of appearance of 9,12,12-[2H]3 -cortisol (19.3 +/- 0.8 vs. 16.7 +/- 1.1 nmol/min with saline, P adipose, the predominant reaction was reductase conversion of cortisone to cortisol (after 3.5 h of saline infusion, reaching 11.0 +/- 2.7% per hour reductase vs. 5.2 +/- 1.3 dehydrogenase, P effects on whole-body deuterated cortisol metabolism, but increased both dehydrogenase and reductase (reaching 16.7 +/- 1.8, P adipose. Hyperinsulinemia and increased free fatty acids induce acute increases in 11HSD1 activity in adipose tissue that are not attributable to a switch from dehydrogenase to reductase. Hyperinsulinemia also increases systemic cortisol regeneration. These effects may enhance intracellular cortisol concentrations after a meal.

  13. c-Jun N-terminal kinase regulates mitochondrial bioenergetics by modulating pyruvate dehydrogenase activity in primary cortical neurons.

    Science.gov (United States)

    Zhou, Qiongqiong; Lam, Philip Y; Han, Derick; Cadenas, Enrique

    2008-01-01

    This study examines the role of c-jun N-terminal kinase (JNK) in mitochondrial signaling and bioenergetics in primary cortical neurons and isolated rat brain mitochondria. Exposure of neurons to either anisomycin (an activator of JNK/p38 mitogen-activated protein kinases) or H2O2 resulted in activation (phosphorylation) of JNK (mostly p46(JNK1)) and its translocation to mitochondria. Experiments with mitochondria isolated from either rat brain or primary cortical neurons and incubated with proteinase K revealed that phosphorylated JNK was associated with the outer mitochondrial membrane; this association resulted in the phosphorylation of the E(1alpha) subunit of pyruvate dehydrogenase, a key enzyme that catalyzes the oxidative decarboxylation of pyruvate and that links two major metabolic pathways: glycolysis and the tricarboxylic acid cycle. JNK-mediated phosphorylation of pyruvate dehydrogenase was not observed in experiments carried out with mitoplasts, thus suggesting the requirement of intact, functional mitochondria for this effect. JNK-mediated phosphorylation of pyruvate dehydrogenase was associated with a decline in its activity and, consequently, a shift to anaerobic pyruvate metabolism: the latter was confirmed by increased accumulation of lactic acid and decreased overall energy production (ATP levels). Pyruvate dehydrogenase appears to be a specific phosphorylation target for JNK, for other kinases, such as protein kinase A and protein kinase C did not elicit pyruvate dehydrogenase phosphorylation and did not decrease the activity of the complex. These results suggest that JNK mediates a signaling pathway that regulates metabolic functions in mitochondria as part of a network that coordinates cytosolic and mitochondrial processes relevant for cell function.

  14. 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.

  15. 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).

  16. Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine.

    Science.gov (United States)

    Bontpart, Thibaut; Marlin, Thérèse; Vialet, Sandrine; Guiraud, Jean-Luc; Pinasseau, Lucie; Meudec, Emmanuelle; Sommerer, Nicolas; Cheynier, Véronique; Terrier, Nancy

    2016-05-01

    In plants, the shikimate pathway provides aromatic amino acids that are used to generate numerous secondary metabolites, including phenolic compounds. In this pathway, shikimate dehydrogenases (SDH) 'classically' catalyse the reversible dehydrogenation of 3-dehydroshikimate to shikimate. The capacity of SDH to produce gallic acid from shikimate pathway metabolites has not been studied in depth. In grapevine berries, gallic acid mainly accumulates as galloylated flavan-3-ols. The four grapevine SDH proteins have been produced in Escherichia coli In vitro, VvSDH1 exhibited the highest 'classical' SDH activity. Two genes, VvSDH3 and VvSDH4, mainly expressed in immature berry tissues in which galloylated flavan-3-ols are accumulated, encoded enzymes with lower 'classical' activity but were able to produce gallic acid in vitro The over-expression of VvSDH3 in hairy-roots increased the content of aromatic amino acids and hydroxycinnamates, but had little or no effect on molecules more distant from the shikimate pathway (stilbenoids and flavan-3-ols). In parallel, the contents of gallic acid, β-glucogallin, and galloylated flavan-3-ols were increased, attesting to the influence of this gene on gallic acid metabolism. Phylogenetic analysis from dicotyledon SDHs opens the way for the examination of genes from other plants which accumulate gallic acid-based metabolites.

  17. Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity.

    Science.gov (United States)

    Kim, Eun Young; Kim, Won Kon; Kang, Hyo Jin; Kim, Jeong-Hoon; Chung, Sang J; Seo, Yeon Soo; Park, Sung Goo; Lee, Sang Chul; Bae, Kwang-Hee

    2012-09-01

    Acetylation is one of the most crucial post-translational modifications that affect protein function. Protein lysine acetylation is catalyzed by acetyltransferases, and acetyl-CoA functions as the source of the acetyl group. Additionally, acetyl-CoA plays critical roles in maintaining the balance between carbohydrate metabolism and fatty acid synthesis. Here, we sought to determine whether lysine acetylation is an important process for adipocyte differentiation. Based on an analysis of the acetylome during adipogenesis, various proteins displaying significant quantitative changes were identified by LC-MS/MS. Of these identified proteins, we focused on malate dehydrogenase 1 (MDH1). The acetylation level of MDH1 was increased up to 6-fold at the late stage of adipogenesis. Moreover, overexpression of MDH1 in 3T3-L1 preadipocytes induced a significant increase in the number of cells undergoing adipogenesis. The introduction of mutations to putative lysine acetylation sites showed a significant loss of the ability of cells to undergo adipogenic differentiation. Furthermore, the acetylation of MDH1 dramatically enhanced its enzymatic activity and subsequently increased the intracellular levels of NADPH. These results clearly suggest that adipogenic differentiation may be regulated by the acetylation of MDH1 and that the acetylation of MDH1 is one of the cross-talk mechanisms between adipogenesis and the intracellular energy level.

  18. Anilides of (R)-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase.

    Science.gov (United States)

    Bebernitz, G R; Aicher, T D; Stanton, J L; Gao, J; Shetty, S S; Knorr, D C; Strohschein, R J; Tan, J; Brand, L J; Liu, C; Wang, W H; Vinluan, C C; Kaplan, E L; Dragland, C J; DelGrande, D; Islam, A; Lozito, R J; Liu, X; Maniara, W M; Mann, W R

    2000-06-01

    The optimization of a series of anilide derivatives of (R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase (PDHK) is described that started from N-phenyl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide 1 (IC(50) = 35 +/- 1.4 microM). It was found that small electron-withdrawing groups on the ortho position of the anilide, i.e., chloro, acetyl, or bromo, increased potency 20-40-fold. The oral bioavailability of the compounds in this series is optimal (as measured by AUC) when the anilide is substituted at the 4-position with an electron-withdrawing group (i.e., carboxyl, carboxyamide, and sulfoxyamide). N-(2-Chloro-4-isobutylsulfamoylphenyl)-(R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropionamide (10a) inhibits PDHK in the primary enzymatic assay with an IC(50) of 13 +/- 1.5 nM, enhances the oxidation of [(14)C]lactate into (14)CO(2) in human fibroblasts, lowers blood lactate levels significantly 2.5 and 5 h after oral doses as low as 30 micromol/kg, and increases the ex vivo activity of PDH in muscle, kidney, liver, and heart tissues. However, in contrast to sodium dichloroacetate (DCA), these PDHK inhibitors did not lower blood glucose levels. Nevertheless, they are effective at increasing the utilization and disposal of lactate and could be of utility to ameliorate conditions of inappropriate blood lactate elevation.

  19. Glutamate 190 is a general acid catalyst in the 6-phosphogluconate-dehydrogenase-catalyzed reaction.

    Science.gov (United States)

    Karsten, W E; Chooback, L; Cook, P F

    1998-11-10

    Site-directed mutagenesis was used to change E190 of sheep liver 6-phosphogluconate dehydrogenase to A, D, H, K, Q, and R to probe its possible role as a general acid catalyst. Each of the mutant proteins was characterized with respect to the pH dependence of kinetic parameters. Mutations that eliminate a titrable group at position 190, result in pH-rate profiles with no observable pK on the basic side of the V/K6PG profile. Mutations that change the pK of the group at position 190 result in the expected pK perturbations in the V/K6PG profile. Kinetic parameters obtained at the pH optimum in the pH-rate profiles are consistent with a rate-limiting tautomerization of the 1,2-enediol of ribulose 5-phosphate consistent with the proposed role of E190. Data are also consistent with some participation of E190 in an isomerization required to form the active Michaelis complex.

  20. Equivalence between Pfr and Cyclic AMP in the Induction of d-Usnic Acid Dehydrogenase in the Lichen Evernia prunastri.

    Science.gov (United States)

    Avalos, A; Vicente, C

    1987-07-01

    d-Usnic acid dehydrogenase is induced in Evernia prunastri thalli by a supply of exogenous d-usnic acid in light. This effect is enhanced by red light pulses through a two step way: a very rapid increase of activity after the first 10 minutes of red light, which is not reversed by far-red light, and a slow enhancement following successive red light pulses at the beginning of each hour of incubation. The last response is completely reversed by far-red following red light. Although induction of the enzyme is not achieved in the dark, 0.1 and 0.5 millimolar cyclic AMP, or 0.1 millimolar dibutyryl cyclic AMP substitutes light action and, then, the enzyme is produced. In addition, phytochrome-far red-absorbing form-increases the amount of endogenously produced cyclic AMP and this effect is shown to be photoreversible when ethylenediaminetetraacetic acid is inhibiting adenylate cyclase.

  1. L(+) lactate dehydrogenase activity from the electric organ of Electrophorus electricus (L.).

    Science.gov (United States)

    Torres-da Matta, J; Nery da Matta, A; Hassón-Voloch, A

    1976-01-01

    Properties of L(+) lactate dehydrogenase (LDH) of Electrophorus electricus (L.) electric organ were studied, comparing the substrates pyruvate and lactate. Electric organ LDH is a soluble enzyme with a pH optimum of 7.4 for pyruvate and 9.0 for lactate. The apparent Km was lower for pyruvate (Km = 2.5 X 10(-4) M) than for lactate (Km = 1.5 X 10(-2) M). With lactate as a substrate at pH 7.4, malonate, oxalate and pyruvate inhibited competitively. For pyruvate as substrate at pH 9.0 malonate inhibited non-competitively and oxalate shiwed uncompetitive inhibition. The different effects of the carboxylic acids on LDH activity suggest different stereospecificities of the two enzyme-coenzyme complexes in the forward and reserve reactions. The reactions of electric organ LDH with substrates and inhibitors are consistent with electrophoretic analysis suggesting that the enzyme is of the M-type.

  2. Inhibition of Pyruvate Dehydrogenase Kinase 2 Protects Against Hepatic Steatosis Through Modulation of Tricarboxylic Acid Cycle Anaplerosis and Ketogenesis.

    Science.gov (United States)

    Go, Younghoon; Jeong, Ji Yun; Jeoung, Nam Ho; Jeon, Jae-Han; Park, Bo-Yoon; Kang, Hyeon-Ji; Ha, Chae-Myeong; Choi, Young-Keun; Lee, Sun Joo; Ham, Hye Jin; Kim, Byung-Gyu; Park, Keun-Gyu; Park, So Young; Lee, Chul-Ho; Choi, Cheol Soo; Park, Tae-Sik; Lee, W N Paul; Harris, Robert A; Lee, In-Kyu

    2016-10-01

    Hepatic steatosis is associated with increased insulin resistance and tricarboxylic acid (TCA) cycle flux, but decreased ketogenesis and pyruvate dehydrogenase complex (PDC) flux. This study examined whether hepatic PDC activation by inhibition of pyruvate dehydrogenase kinase 2 (PDK2) ameliorates these metabolic abnormalities. Wild-type mice fed a high-fat diet exhibited hepatic steatosis, insulin resistance, and increased levels of pyruvate, TCA cycle intermediates, and malonyl-CoA but reduced ketogenesis and PDC activity due to PDK2 induction. Hepatic PDC activation by PDK2 inhibition attenuated hepatic steatosis, improved hepatic insulin sensitivity, reduced hepatic glucose production, increased capacity for β-oxidation and ketogenesis, and decreased the capacity for lipogenesis. These results were attributed to altered enzymatic capacities and a reduction in TCA anaplerosis that limited the availability of oxaloacetate for the TCA cycle, which promoted ketogenesis. The current study reports that increasing hepatic PDC activity by inhibition of PDK2 ameliorates hepatic steatosis and insulin sensitivity by regulating TCA cycle anaplerosis and ketogenesis. The findings suggest PDK2 is a potential therapeutic target for nonalcoholic fatty liver disease.

  3. Enzymatic activity analysis and catalytic essential residues identification of Brucella abortus malate dehydrogenase.

    Science.gov (United States)

    Han, Xiangan; Tong, Yongliang; Tian, Mingxing; Zhang, Yuxi; Sun, Xiaoqing; Wang, Shaohui; Qiu, Xusheng; Ding, Chan; Yu, Shengqing

    2014-01-01

    Malate dehydrogenase (MDH) plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH) of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA) to L-malate (hereon referred to as MDH activity) was analyzed. Michaelis Constant (Km) and Maximum Reaction Velocity (Vmax) of the reaction were determined to be 6.45 × 10(-3) M and 0.87 mM L(-1)min(-1), respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu(2+), Zn(2+), and Pb(2+), respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH's roles in B. abortus metabolism.

  4. Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase

    Directory of Open Access Journals (Sweden)

    Xiangan Han

    2014-01-01

    Full Text Available Malate dehydrogenase (MDH plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA to L-malate (hereon referred to as MDH activity was analyzed. Michaelis Constant (Km and Maximum Reaction Velocity (Vmax of the reaction were determined to be 6.45×10−3 M and 0.87 mM L−1 min−1, respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu2+, Zn2+, and Pb2+, respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH’s roles in B. abortus metabolism.

  5. Salicylic Acid-Dependent Plant Stress Signaling via Mitochondrial Succinate Dehydrogenase1[OPEN

    Science.gov (United States)

    Thatcher, Louise F.

    2017-01-01

    Mitochondria are known for their role in ATP production and generation of reactive oxygen species, but little is known about the mechanism of their early involvement in plant stress signaling. The role of mitochondrial succinate dehydrogenase (SDH) in salicylic acid (SA) signaling was analyzed using two mutants: disrupted in stress response1 (dsr1), which is a point mutation in SDH1 identified in a loss of SA signaling screen, and a knockdown mutant (sdhaf2) for SDH assembly factor 2 that is required for FAD insertion into SDH1. Both mutants showed strongly decreased SA-inducible stress promoter responses and low SDH maximum capacity compared to wild type, while dsr1 also showed low succinate affinity, low catalytic efficiency, and increased resistance to SDH competitive inhibitors. The SA-induced promoter responses could be partially rescued in sdhaf2, but not in dsr1, by supplementing the plant growth media with succinate. Kinetic characterization showed that low concentrations of either SA or ubiquinone binding site inhibitors increased SDH activity and induced mitochondrial H2O2 production. Both dsr1 and sdhaf2 showed lower rates of SA-dependent H2O2 production in vitro in line with their low SA-dependent stress signaling responses in vivo. This provides quantitative and kinetic evidence that SA acts at or near the ubiquinone binding site of SDH to stimulate activity and contributes to plant stress signaling by increased rates of mitochondrial H2O2 production, leading to part of the SA-dependent transcriptional response in plant cells. PMID:28209841

  6. Medium-chain fatty acids and glutathione derivatives as inhibitors of S-nitrosoglutathione reduction mediated by alcohol dehydrogenase 3.

    Science.gov (United States)

    Staab, Claudia A; Hellgren, Mikko; Grafström, Roland C; Höög, Jan-Olov

    2009-06-15

    Alcohol dehydrogenase 3 (ADH3) has emerged as an important regulator of protein S-nitrosation in its function as S-nitrosoglutathione (GSNO) reductase. GSNO depletion is associated with various disease conditions, emphasizing the potential value of a specific ADH3 inhibitor. The present study investigated inhibition of ADH3-mediated GSNO reduction by various substrate analogues, including medium-chain fatty acids and glutathione derivatives. The observed inhibition type was non-competitive. Similar to the Michaelis constants for the corresponding omega-hydroxy fatty acids, the inhibition constants for fatty acids were in the micromolar range and showed a clear dependency on chain length with optimal inhibitory capacity for eleven and twelve carbons. The most efficient inhibitors found were undecanoic acid, dodecanoic acid and dodecanedioic acid, with no significant difference in inhibition constant. All glutathione-derived inhibitors displayed inhibition constants in the millimolar range, at least three orders of magnitudes higher than the Michaelis constants of the high-affinity substrates GSNO and S-hydroxymethylglutathione. The experimental results as well as docking simulations with GSNO and S-methylglutathione suggest that for ADH3 ligands with a glutathione scaffold, in contrast to fatty acids, a zinc-binding moiety is imperative for correct orientation and stabilization of the hydrophilic glutathione scaffold within a predominantly hydrophobic active site.

  7. 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.

  8. Regulation of glutamate dehydrogenase activity in relation to carbon limitation and protein catabolism in carrot cell suspension cultures.

    Science.gov (United States)

    Robinson, S A; Stewart, G R; Phillips, R

    1992-03-01

    Glutamate dehydrogenase (GDH) specific activity and function have been studied in cell suspension cultures of carrot (Daucus carota L. cv Chantenay) in response to carbon and nitrogen supply in the culture medium. The specific activity of GDH was derepressed in sucrose-starved cells concomitant with protein catabolism, ammonium excretion, and the accumulation of metabolically active amino acids. The addition of sucrose led to a rapid decrease in GDH specific activity, an uptake of ammonium from the medium, and a decrease in amino acid levels. The extent of GDH derepression was correlated positively with cellular glutamate concentration. These findings strengthen the view that the function of GDH is the catabolism of glutamate, which under conditions of carbon stress provides carbon skeletons for tricarboxylic acid cycle activity.

  9. A survey for isoenzymes of glucosephosphate isomerase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and 6-Phosphogluconate dehydrogenase in C3-, C 4-and crassulacean-acid-metabolism plants, and green algae.

    Science.gov (United States)

    Herbert, M; Burkhard, C; Schnarrenberger, C

    1979-01-01

    Two isoenzymes each of glucosephosphate isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.43) were separated by (NH4)2SO4 gradient solubilization and DEAE-cellulose ion-exchange chromatography from green leaves of the C3-plants spinach (Spinacia oleracea L.), tobacco (Nicotiana tabacum L.) and wheat (Triticum aestivum L.), of the Crassulacean-acid-metabolism plants Crassula lycopodioides Lam., Bryophyllum calycinum Salisb. and Sedum rubrotinctum R.T. Clausen, and from the green algae Chlorella vulgaris and Chlamydomonas reinhardii. After isolation of cell organelles from spinach leaves by isopyenic centrifugation in sucrose gradients one of two isoenzymes of each of the four enzymes was found to be associated with whole chloroplasts while the other was restricted to the soluble cell fraction, implying the same intracellular distribution of these isoenzymes also in the other species.Among C4-plants, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were found in only one form in corn (Zea mays L.), sugar cane (Saccharum officinarum L.) and Coix lacrymajobi L., but as two isoenzymes in Atriplex spongiosa L. and Portulaca oleracea L. In corn, the two dehydrogenases were mainly associated with isolated mesophyll protoplasts while in Atriplex spongiosa they were of similar specific activity in both mesophyll protoplasts and bundle-sheath strands. In all five C4-plants three isoenzymes of glucosephosphate isomerase and phosphoglucomutase were found. In corn two were localized in the bundle-sheath strands and the third one in the mesophyll protoplasts. The amount of activity of the enzymes was similar in each of the two cell fractions. Apparently, C4 plants have isoenzymes not only in two cell compartments, but also in physiologically closely linked cell types such as mesophyll and bundle-sheath cells.

  10. Requirement of succinate dehydrogenase activity for symbiotic bacteroid differentiation of Rhizobium meliloti in alfalfa nodules.

    OpenAIRE

    Gardiol, A E; Truchet, G L; Dazzo, F. B.

    1987-01-01

    Transmission electron microscopy was used to study the cellular morphologies of a wild-type Rhizobium meliloti strain (L5-30), a nitrogen fixation-ineffective (Fix-) succinate dehydrogenase mutant (Sdh-) strain, and a Fix+ Sdh+ revertant strain within alfalfa nodules and after free-living growth in a minimal medium containing 27 mM mannitol plus 20 mM succinate. The results showed a requirement of succinate dehydrogenase activity for symbiotic differentiation and maintenance of R. meliloti ba...

  11. Brain pyruvate and 2-oxoglutarate dehydrogenase complexes are mitochondrial targets of the CoA ester of the Refsum disease marker phytanic acid.

    Science.gov (United States)

    Bunik, Victoria I; Raddatz, Günter; Wanders, Ronald J A; Reiser, Georg

    2006-06-12

    Pyruvate and 2-oxoglutarate dehydrogenase complexes are strongly inhibited by phytanoyl-CoA (IC(50) approximately 10(-6)-10(-7) M). Palmitoyl-CoA is 10-fold less potent. Phytanic or palmitic acids have no inhibitory effect up to 0.3 mM. At the substrate saturation, the acyl-CoA's affect the first and second enzymatic components of the 2-oxoglutarate dehydrogenase complex, while the third component is inhibited only at a low saturation with its substrate dihydrolipoamide. Thus, key regulatory branch points of mitochondrial metabolism are targets of a cellular derivative of phytanic acid. Decreased activity of the complexes might therefore contribute to neurological symptoms upon accumulation of phytanic acid in Refsum disease.

  12. Dye-linked D-amino acid dehydrogenases: biochemical characteristics and applications in biotechnology.

    Science.gov (United States)

    Satomura, Takenori; Sakuraba, Haruhiko; Suye, Shin-Ichiro; Ohshima, Toshihisa

    2015-11-01

    Dye-linked D-amino acid dehydrogenases (Dye-DADHs) catalyze the dehydrogenation of free D-amino acids in the presence of an artificial electron acceptor. Although Dye-DADHs functioning in catabolism of L-alanine and as primary enzymes in electron transport chains are widely distributed in mesophilic Gram-negative bacteria, biochemical and biotechnological information on these enzymes remains scanty. This is in large part due to their instability after isolation. On the other hand, in the last decade, several novel types of Dye-DADH have been found in thermophilic bacteria and hyperthermophilic archaea, where they contribute not only to L-alanine catabolism but also to the catabolism of other amino acids, including D-arginine and L-hydroxyproline. In this minireview, we summarize recent developments in our understanding of the biochemical characteristics of Dye-DADHs and their specific application to electrochemical biosensors.

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

    NARCIS (Netherlands)

    Schakenraad, J.M.; Hardonk, M.J.; Feijen, J.; 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

  14. Inhibitory effects of Aphanizomenon flos-aquae constituents on human UDP-glucose dehydrogenase activity.

    Science.gov (United States)

    Scoglio, Stefano; Lo Curcio, Valeria; Catalani, Simona; Palma, Francesco; Battistelli, Serafina; Benedetti, Serena

    2016-12-01

    The purpose of this study was to investigate the in vitro inhibitory effects of the edible microalga Aphanizomenon flos-aquae (AFA) on human UDP-α-d-glucose 6-dehydrogenase (UGDH) activity, a cytosolic enzyme involved both in tumor progression and in phytochemical bioavailability. Both the hydrophilic and ethanolic AFA extracts as well as the constitutive active principles phycocyanin (PC), phycocyanobilin (PCB) and mycosporine-like amino acids (MAAs) were tested. Among AFA components, PCB presented the strongest inhibitory effect on UGDH activity, acting as a competitive inhibitor with respect to UDP-glucose and a non-competitive inhibitor with respect to NAD(+). In preliminary experiments, AFA PCB was also effective in reducing the colony formation capacity of PC-3 prostate cancer cells and FTC-133 thyroid cancer cells. Overall, these findings confirmed that AFA and its active principles are natural compounds with high biological activity. Further studies evaluating the effects of AFA PCB in reducing tumor cell growth and phytochemical glucuronidation are encouraged.

  15. 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

  16. 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.

  17. Crystal structure of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase, an NAD⁺-dependent dismutase from Mesorhizobium loti.

    Science.gov (United States)

    Mugo, Andrew Njagi; Kobayashi, Jun; Mikami, Bunzo; Yoshikane, Yu; Yagi, Toshiharu; Ohnishi, Kouhei

    2015-01-02

    5-Formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase (FHMPCDH) from Mesorhizobium loti is the fifth enzyme in degradation pathway I for pyridoxine. The enzyme catalyzes a dismutation reaction: the oxidation of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid (FHMPC) to 3-hydroxy-2-methylpyridine 4,5-dicarboxylic acid with NAD(+) and reduction of FHMPC to 4-pyridoxic acid with NADH. FHMPCDH belongs to the l-3-hydroxyacyl-CoA dehydrogenase (HAD) family. The crystal structure was determined by molecular replacement and refined to a resolution of 1.55Å (R-factor of 16.4%, Rfree=19.4%). There were two monomers in the asymmetric unit. The overall structure of the monomer consisted of N- and C-terminal domains connected by a short linker loop. The monomer was similar to members of the HAD family (RMSD=1.9Å). The active site was located between the domains and highly conserved to that of human heart l-3-hydroxyacyl-CoA dehydrogenase (HhHAD). His-Glu catalytic dyad, a serine and two asparagine residues of HhHAD were conserved. Ser116, His137 and Glu149 in FHMPCDH are connected by a hydrogen bonding network forming a catalytic triad. The functions of the active site residues in the reaction mechanism are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Gene clone,expression and enzyme activity assay of a cytosolic malate dehydrogenase from apple fruits

    Institute of Scientific and Technical Information of China (English)

    Yuxin YAO; Yujin HAO; Ming LI; Mingli PANG; Zhi LIU; Heng ZHAI

    2008-01-01

    Malate dehydrogenase (MDH) ubiquitously exists in animals,plants and microoganisms,and catalyzes the interconversion from oxaloacetate to malate.Cytosolic NAD-dependent MDH gene (cyMDH)encodes a key enzyme crucial for malic acid synthesis in the cytosol which has not been extensively characterized in plants.In this study,a full-length cDNA of cyMDH was isolated from apple fruits with RT-PCR as well as 3' and 5' rapid amplification of cDNA ends,and designated as Mal-cyMDH (GenBank accession No.DQ221207).It contained a 996-bp ORF and its sequence analysis shows a high similarity to other plant cyMDHs.Phylogenetic analysis indicated that almost all the cyMDHs could be clustered into the same group and it was likely to represent the original MDH.A roughly 37-kDa fused protein was obtained by the recombinant prokaryotic expression and its enzyme activity assay showed that it mainly catalyzed oxaloacetate to malate.It was also discovered that the enzyme activity of cyMDH exhibited remarkable difference between the high- and low-acid apple germplasm.

  19. Dehydrogenase and Oxoreductase Activities of Porcine Placental 11Beta-Hydroxysteroid Dehydrogenase

    Science.gov (United States)

    2016-06-07

    Subsequently, samples were thawed and extracted with ethyl acetate to obtain tritiated steroids. These extracts were sub- jected to thin layer chromatography...containing .02 M EDTA (PBS) to remove most radio- activity. Tissue fragments were then homogenized in PBS and aliquots removed for DNA analysis (32). In...either 3H-cortisol or 3H-cortisone were used as substrate. Incubations were conducted as above-noted and DNA measures conducted. Statistical

  20. Role of alcohol dehydrogenase activity and the acetaldehyde in ethanol- induced ethane and pentane production by isolated perfused rat liver.

    Science.gov (United States)

    Müller, A; Sies, H

    1982-01-01

    The volatile hydrocarbons ethane and n-pentane are produced at increased rates by isolated perfused rat liver during the metabolism of acutely ethanol. The effect is half-maximal at 0.5 mM-ethanol, and its is not observed when inhibitors of alcohol dehydrogenase such as 4-methyl- or 4-propyl-pyrazole are also present. Propanol, another substrate for the dehydrogenase, is also active. Increased alkane production can be initiated by adding acetaldehyde in the presence of 4-methyl- or 4-propyl-pyrazole. An antioxidant, cyanidanol, suppresses the ethanol-induced alkane production. The data obtained with the isolated organ demonstrate that products known to arise from the peroxidation of polyunsaturated fatty acids are formed in the presence of ethanol and that the activity of alcohol dehydrogenase is required for the generation of the active radical species. The mere presence of ethanol, e.g. at binding sites of special form(s) of cytochrome P-450, it not sufficient to elicit an increased production of volatile hydrocarbons by rat liver. PMID:6751324

  1. The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity.

    Science.gov (United States)

    Love, Lorenzo K; LeBlanc, Paul J; Inglis, J Greig; Bradley, Nicolette S; Choptiany, Jon; Heigenhauser, George J F; Peters, Sandra J

    2011-08-01

    Pyruvate dehydrogenase (PDH) is a mitochondrial enzyme responsible for regulating the conversion of pyruvate to acetyl-CoA for use in the tricarboxylic acid cycle. PDH is regulated through phosphorylation and inactivation by PDH kinase (PDK) and dephosphorylation and activation by PDH phosphatase (PDP). The effect of endurance training on PDK in humans has been investigated; however, to date no study has examined the effect of endurance training on PDP in humans. Therefore, the purpose of this study was to examine differences in PDP activity and PDP1 protein content in human skeletal muscle across a range of muscle aerobic capacities. This association is important as higher PDP activity and protein content will allow for increased activation of PDH, and carbohydrate oxidation. The main findings of this study were that 1) PDP activity (r(2) = 0.399, P = 0.001) and PDP1 protein expression (r(2) = 0.153, P = 0.039) were positively correlated with citrate synthase (CS) activity as a marker for muscle aerobic capacity; 2) E1α (r(2) = 0.310, P = 0.002) and PDK2 protein (r(2) = 0.229, P =0.012) are positively correlated with muscle CS activity; and 3) although it is the most abundant isoform, PDP1 protein content only explained ∼ 18% of the variance in PDP activity (r(2) = 0.184, P = 0.033). In addition, PDP1 in combination with E1α explained ∼ 38% of the variance in PDP activity (r(2) = 0.383, P = 0.005), suggesting that there may be alternative regulatory mechanisms of this enzyme other than protein content. These data suggest that with higher muscle aerobic capacity (CS activity) there is a greater capacity for carbohydrate oxidation (E1α), in concert with higher potential for PDH activation (PDP activity).

  2. 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.

  3. Single amino-acid substitution in the N-terminal arm altered the tetramer stability of rat muscle lactate dehydrogenase A

    Institute of Scientific and Technical Information of China (English)

    YUAN; Chong; (

    2001-01-01

    [1]Price, N. C., Assembly of multi-subunit structures, in Mechanisms of Protein Folding (ed. Pain, R. H.), New York: Oxford University Press, 1994, 160-193.[2]Casal, J. I., Ahern, T. J., Davenport, R. C. et al., Subunit interface of triosephosphate isomerase: Site-directed mutagenesis and characterization of the altered enzyme, Biochemistry, 1987, 26: 1258-1264.[3]Chakerian, A. E., Matthews, K. S., Characterization of mutations in oligomerization domain of lac repressor protein, J. Biol. Chem., 1991, 266: 22206-22214.[4]Mandelman, D., Schwarz, F. P., Li, H. Y. et al., The role of quaternary interactions on the stability and activity of ascorbate peroxidase, Protein Sci., 1998, 7: 2089-2098.[5]Thomas, M. C., Ballantine, S. P., Bethell, S. S. et al., Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii, Biochemistry, 1998, 37: 11629-11636.[6]Holbrook, J. J., Liljas, A., Steindel, S. J. et al., Lactate dehydrogenase, in The Enzymes (ed. Boyer, P. D.), Vol. 11, 3rd ed., New York: Academic Press, 1975, 191-292.[7]Zettlmeissl, G., Rudolph, R., Jaenicke, R., Reconstitution of lactic dehydrogenase after acid dissociation, Eur. J. Biochem., 1981, 121: 169-175.[8]Zettlmeissl, G., Rudolph, R., Jaenicke, R., Rate-determining folding and association reactions on the reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride, Biochemistry, 1982, 21: 3946-3950.[9]Hermann, R., Jaenicke, R., Rudolph, R., Analysis of the reconstitution of oligomeric enzymes by cross-linking with glutaraldehyde: Kinetics of reassociation of lactic dehydrogenase, Biochemistry, 1981, 20: 5195-5201.[10]Jaenicke, R., Folding and association of protein, Prog. Biophys. Mol. Biol., 1987, 49: 117-237.[11]Opitz, U., Rudolph, R., Jaenicke, R. et al., Proteolytic dimeric of porcine muscle lactate dehydrogenase: Characterization, folding, and

  4. 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.

  5. Group III alcohol dehydrogenase from Pectobacterium atrosepticum: insights into enzymatic activity and organization of the metal ion-containing region.

    Science.gov (United States)

    Elleuche, Skander; Fodor, Krisztian; von der Heyde, Amélie; Klippel, Barbara; Wilmanns, Matthias; Antranikian, Garabed

    2014-05-01

    NAD(P)(+)-dependent alcohol dehydrogenases (ADH) are widely distributed in all phyla. These proteins can be assigned to three nonhomologous groups of isozymes, with group III being highly diverse with regards to catalytic activity and primary structure. Members of group III ADHs share a conserved stretch of amino acid residues important for cofactor binding and metal ion coordination, while sequence identities for complete proteins are highly diverse (90 %). A putative group III ADH PaYqhD has been identified in BLAST analysis from the plant pathogenic enterobacterium Pectobacterium atrosepticum. The PaYqhD gene was expressed in the heterologous host Escherichia coli, and the recombinant protein was purified in a two-step purification procedure to homogeneity indicating an obligate dimerization of monomers. Four conserved amino acid residues involved in metal ion coordination were substituted with alanine, and their importance for catalytic activity was confirmed by circular dichroism spectrum determination, in vitro, and growth experiments. PaYqhD exhibits optimal activity at 40 °C with short carbon chain aldehyde compounds and NADPH as cofactor indicating the enzyme to be an aldehyde reductase. No oxidative activities towards alcoholic compounds were detectable. EDTA completely inhibited catalytic activity and was fully restored by the addition of Co(2+). Activity measurements together with sequence alignments and structure analysis confirmed that PaYqhD belongs to the butanol dehydrogenase-like enzymes within group III of ADHs.

  6. Purification and characterization of xylitol dehydrogenase with l-arabitol dehydrogenase activity from the newly isolated pentose-fermenting yeast Meyerozyma caribbica 5XY2.

    Science.gov (United States)

    Sukpipat, Wiphat; Komeda, Hidenobu; Prasertsan, Poonsuk; Asano, Yasuhisa

    2017-01-01

    Meyerozyma caribbica strain 5XY2, which was isolated from an alcohol fermentation starter in Thailand, was found to catabolize l-arabinose as well as d-glucose and d-xylose. The highest production amounts of ethanol from d-glucose, xylitol from d-xylose, and l-arabitol from l-arabinose were 0.45 g/g d-glucose, 0.60 g/g d-xylose, and 0.61 g/g l-arabinose with 21.7 g/L ethanol, 20.2 g/L xylitol, and 30.3 g/l l-arabitol, respectively. The enzyme with l-arabitol dehydrogenase (LAD) activity was purified from the strain and found to exhibit broad specificity to polyols, such as xylitol, d-sorbitol, ribitol, and l-arabitol. Xylitol was the preferred substrate with Km=16.1 mM and kcat/Km=67.0 min(-1)mM(-1), while l-arabitol was also a substrate for the enzyme with Km=31.1 mM and kcat/Km=6.5 min(-1) mM(-1). Therefore, this enzyme from M. caribbica was named xylitol dehydrogenase (McXDH). McXDH had an optimum temperature and pH at 40°C and 9.5, respectively. The McXDH gene included a coding sequence of 1086 bp encoding a putative 362 amino acid protein of 39 kDa with an apparent homopentamer structure. Native McXDH and recombinant McXDH exhibited relative activities toward l-arabitol of approximately 20% that toward xylitol, suggesting the applicability of this enzyme with the functions of XDH and LAD to the development of pentose-fermenting Saccharomyces cerevisiae.

  7. Growth hormone-induced insulin resistance in human subjects involves reduced pyruvate dehydrogenase activity

    DEFF Research Database (Denmark)

    Nellemann, Birgitte; Vendelbo, Mikkel H; Nielsen, Thomas S

    2014-01-01

    Insulin resistance induced by growth hormone (GH) is linked to promotion of lipolysis by unknown mechanisms. We hypothesized that suppression of the activity of pyruvate dehydrogenase in the active form (PDHa) underlies GH-induced insulin resistance similar to what is observed during fasting....

  8. Relationship of lactate dehydrogenase activity with body measeurements of Angus x Charolais cows and calves

    Science.gov (United States)

    Angus x Charolais cows (n = 87) and their Angus-sired, spring-born calves (n = 86) were utilized to examine relationships between lactate dehydrogenase (LDH) activity and body measurements of beef cows; and the relationship between maternal LDH activity in late gestation and subsequent calf birth we...

  9. Saturated fatty acids in human visceral adipose tissue are associated with increased 11- β-hydroxysteroid-dehydrogenase type 1 expression.

    Science.gov (United States)

    Petrus, Paul; Rosqvist, Fredrik; Edholm, David; Mejhert, Niklas; Arner, Peter; Dahlman, Ingrid; Rydén, Mikael; Sundbom, Magnus; Risérus, Ulf

    2015-05-02

    Visceral fat accumulation is associated with metabolic disease. It is therefore relevant to study factors that regulate adipose tissue distribution. Recent data shows that overeating saturated fatty acids promotes greater visceral fat storage than overeating unsaturated fatty acids. Visceral adiposity is observed in states of hypercortisolism, and the enzyme 11-β-hydroxysteroid-dehydrogenase type 1 (11β-hsd1) is a major regulator of cortisol activity by converting inactive cortisone to cortisol in adipose tissue. We hypothesized that tissue fatty acid composition regulates body fat distribution through local effects on the expression of 11β-hsd1 and its corresponding gene (HSD11B1) resulting in altered cortisol activity. Visceral- and subcutaneous adipose tissue biopsies were collected during Roux-en-Y gastric bypass surgery from 45 obese women (BMI; 41±4 kg/m2). The fatty acid composition of each biopsy was measured and correlated to the mRNA levels of HSD11B1. 11β-hsd1 protein levels were determined in a subgroup (n=12) by western blot analysis. Our main finding was that tissue saturated fatty acids (e.g. palmitate) were associated with increased 11β-hsd1 gene- and protein-expression in visceral but not subcutaneous adipose tissue. The present study proposes a link between HSD11B1 and saturated fatty acids in visceral, but not subcutaneous adipose tissue. Nutritional regulation of visceral fat mass through HSD11B1 is of interest for the modulation of metabolic risk and warrants further investigation.

  10. Evolution of a transition state: role of Lys100 in the active site of isocitrate dehydrogenase.

    Science.gov (United States)

    Miller, Stephen P; Gonçalves, Susana; Matias, Pedro M; Dean, Antony M

    2014-05-26

    An active site lysine essential to catalysis in isocitrate dehydrogenase (IDH) is absent from related enzymes. As all family members catalyze the same oxidative β-decarboxylation at the (2R)-malate core common to their substrates, it seems odd that an amino acid essential to one is not found in all. Ordinarily, hydride transfer to a nicotinamide C4 neutralizes the positive charge at N1 directly. In IDH, the negatively charged C4-carboxylate of isocitrate stabilizes the ground state positive charge on the adjacent nicotinamide N1, opposing hydride transfer. The critical lysine is poised to stabilize-and perhaps even protonate-an oxyanion formed on the nicotinamide 3-carboxamide, thereby enabling the hydride to be transferred while the positive charge at N1 is maintained. IDH might catalyze the same overall reaction as other family members, but dehydrogenation proceeds through a distinct, though related, transition state. Partial activation of lysine mutants by K(+) and NH4 (+) represents a throwback to the primordial state of the first promiscuous substrate family member.

  11. Mitochondrial Dihydrolipoyl Dehydrogenase Activity Shapes Photosynthesis and Photorespiration of Arabidopsis thaliana.

    Science.gov (United States)

    Timm, Stefan; Wittmiß, Maria; Gamlien, Sabine; Ewald, Ralph; Florian, Alexandra; Frank, Marcus; Wirtz, Markus; Hell, Rüdiger; Fernie, Alisdair R; Bauwe, Hermann

    2015-07-01

    Mitochondrial dihydrolipoyl dehydrogenase (mtLPD; L-protein) is an integral component of several multienzyme systems involved in the tricarboxylic acid (TCA) cycle, photorespiration, and the degradation of branched-chain α-ketoacids. The majority of the mtLPD present in photosynthesizing tissue is used for glycine decarboxylase (GDC), necessary for the high-flux photorespiratory glycine-into-serine conversion. We previously suggested that GDC activity could be a signal in a regulatory network that adjusts carbon flux through the Calvin-Benson cycle in response to photorespiration. Here, we show that elevated GDC L-protein activity significantly alters several diagnostic parameters of cellular metabolism and leaf gas exchange in Arabidopsis thaliana. Overexpressor lines displayed markedly decreased steady state contents of TCA cycle and photorespiratory intermediates as well as elevated NAD(P)(+)-to-NAD(P)H ratios. Additionally, increased rates of CO2 assimilation, photorespiration, and plant growth were observed. Intriguingly, however, day respiration rates remained unaffected. By contrast, respiration was enhanced in the first half of the dark phase but depressed in the second. We also observed enhanced sucrose biosynthesis in the light in combination with a lower diel magnitude of starch accumulation and breakdown. These data thus substantiate our prior hypothesis that facilitating flux through the photorespiratory pathway stimulates photosynthetic CO2 assimilation in the Calvin-Benson cycle. They furthermore suggest that this regulation is, at least in part, dependent on increased light-capture/use efficiency.

  12. Equilibrium concentrations for pyruvate dehydrogenase and the citric acid cycle at specified concentrations of certain coenzymes.

    Science.gov (United States)

    Alberty, Robert A

    2004-04-01

    It is of interest to calculate equilibrium compositions of systems of biochemical reactions at specified concentrations of coenzymes because these reactants tend to be in steady states. Thermodynamic calculations under these conditions require the definition of a further transformed Gibbs energy G" by use of a Legendre transform. These calculations are applied to the pyruvate dehydrogenase reaction plus the citric acid cycle, but steady-state concentrations of CoA, acetyl-CoA and succinyl-CoA cannot be specified because they are involved in the conservation of carbon atoms. These calculations require the use of linear algebra to obtain further transformed Gibbs energies of formation of reactants and computer programs to calculate equilibrium compositions. At specified temperature, pH, ionic strength and specified concentrations of several coenzymes, the equilibrium composition depends on the specified concentrations of the coenzymes and the initial amounts of reactants.

  13. Highly stereoselective biosynthesis of (R)-α-hydroxy carboxylic acids through rationally re-designed mutation of D-lactate dehydrogenase.

    Science.gov (United States)

    Zheng, Zhaojuan; Sheng, Binbin; Gao, Chao; Zhang, Haiwei; Qin, Tong; Ma, Cuiqing; Xu, Ping

    2013-12-02

    An NAD-dependent D-lactate dehydrogenase (D-nLDH) of Lactobacillus bulgaricus ATCC 11842 was rationally re-designed for asymmetric reduction of a homologous series of α-keto carboxylic acids such as phenylpyruvic acid (PPA), α-ketobutyric acid, α-ketovaleric acid, β-hydroxypyruvate. Compared with wild-type D-nLDH, the Y52L mutant D-nLDH showed elevated activities toward unnatural substrates especially with large substitutes at C-3. By the biocatalysis combined with a formate dehydrogenase for in situ generation of NADH, the corresponding (R)-α-hydroxy carboxylic acids could be produced at high yields and highly optical purities. Taking the production of chiral (R)-phenyllactic acid (PLA) from PPA for example, 50 mM PPA was completely reduced to (R)-PLA in 90 min with a high yield of 99.0% and a highly optical purity (>99.9% e.e.) by the coupling system. The results presented in this work suggest a promising alternative for the production of chiral α-hydroxy carboxylic acids.

  14. 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.

  15. 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

  16. The content and distribution of troponin I, troponin T, myoglobin, and alpha-hydroxybutyric acid dehydrogenase in the human heart

    NARCIS (Netherlands)

    Swaanenburg, JCJM; Visser-VanBrummen, PJ; DeJongste, MJL; Tiebosch, ATHM

    2001-01-01

    We studied the content and distribution of heart-specific markers troponin I and troponin T in relation to conventional non-heart specific myoglobin and alpha-hydroxybutyric acid dehydrogenase (HBD) in the hearts of 34 patients who died of various causes. Tissue was obtained from the right and left

  17. 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.

  18. Effects of synthetic detergents on in vivo activity of tissue phosphatases and succinic dehydrogenase from Mystus vittatus.

    Science.gov (United States)

    Mohan, D; Verma, S R

    1981-05-01

    African catfish (Mystus vittatus) were exposed to three sub-lethal concentrations of Swascofix E45 (13.8, 9.2 and 4.6 mg/l) and Swascol 3L (69.3, 46.2 and 23.1 mg/l) for 15 and 30 days, and their effects on alkaline and acid phosphatase, and succinic dehydrogenase in liver, kidney and intestine were measured. The enzymes were found to be inhibited in all the tissues. Maximum inhibition (38.44%) was observed in liver alkaline phosphatase activity after 30 days with the highest concentration of Swascofix E45 and the lowest inhibition (0.118%) was found in kidney acid phosphatase activity with the lowest concentration of Swascol 3L after 15 days. Insignificant enzyme stimulation in some cases was also observed.

  19. Effects of synthetic detergents on in vivo activity of tissue phosphatases and succinic dehydrogenase from Mystus vittatus

    Energy Technology Data Exchange (ETDEWEB)

    Mohan, D.; Verma, S.R.

    1981-05-01

    African catfish (Mystus vittatus) were exposed to three sub-lethal concentrations of Swascofix E45 (13.8, 9.2 and 4.6 mg/l) and Swascol 3L (69.3, 46.2 and 23.1 mg/l) for 15 and 30 days, and their effects on alkaline and acid phosphatase, and succinic dehydrogenase in liver, kidney and intestine were measured. The enzymes were found to be inhibited in all the tissues. Maximum inhibition (38.44%) was observed in liver alkaline phosphatase activity after 30 days with the highest concentration of Swascofix E45 and the lowest inhibition (0.118%) was found in kidney acid phosphatase activity with the lowest concentration of Swascol 3L after 15 days. Insignificant enzyme stimulation in some cases was also observed.

  20. 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

  1. Activity of the mitochondrial pyruvate dehydrogenase complex in plants is stimulated in the presence of malate.

    Science.gov (United States)

    Igamberdiev, Abir U; Lernmark, Ulrikа; Gardeström, Per

    2014-11-01

    The effect of malate on the steady-state activity of the pea (Pisum sativum L.) and barley (Hordeum vulgare L.) leaf pyruvate dehydrogenase complex (PDC) has been studied in isolated mitochondria. The addition of malate was found to be stimulatory for the mitochondrial PDC, however there was no stimulation of chloroplast PDC. The stimulation was saturated below 1mM malate and was apparently related to а partially activated complex, which activity increased in the presence of malate by about twofold. Malate also reversed the reduction of PDC activity in the presence of glycine. Based on the obtained kinetic data, we suggest that the effect of malate is rather not a direct activation of PDC but involves the establishment of NAD-malate dehydrogenase equilibrium, decreasing concentration of NADH and relieving its inhibitory effect of PDC.

  2. The activity of class I, II, III and IV of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in brain cancer.

    Science.gov (United States)

    Laniewska-Dunaj, Magdalena; Jelski, Wojciech; Orywal, Karolina; Kochanowicz, Jan; Rutkowski, Robert; Szmitkowski, Maciej

    2013-07-01

    The brain being highly sensitive to the action of alcohol is potentially susceptible to its carcinogenic effects. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the main enzymes involved in ethanol metabolism, which leads to the generation of carcinogenic acetaldehyde. Human brain tissue contains various ADH isoenzymes and possess also ALDH activity. The purpose of this study was to compare the capacity for ethanol metabolism measured by ADH isoenzymes and ALDH activity in cancer tissues and healthy brain cells. The samples were taken from 62 brain cancer patients (36 glioblastoma, 26 meningioma). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, the fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. The total activity of ADH, and activity of class I ADH were significantly higher in cancer cells than in healthy tissues. The other tested classes of ADH and ALDH did not show statistically significant differences of activity in cancer and in normal cells. Analysis of the enzymes activity did not show significant differences depending on the location of the tumor. The differences in the activity of total alcohol dehydrogenase, and class I isoenzyme between cancer tissues and healthy brain cells might be a factor for metabolic changes and disturbances in low mature cancer cells and additionally might be a reason for higher level of acetaldehyde which can intensify the carcinogenesis.

  3. Cytokinin Dehydrogenase activity in primary roots and characterization of primary metabolites from leaves and rootlets of Ricinus Communis

    Directory of Open Access Journals (Sweden)

    Maria Elizabeth da Costa Marques

    2013-12-01

    Full Text Available The aim of this study was to determine the activity of cytokinin dehydrogenase (CKX and to measure other biochemical components in the primary leaves and radicles of castor seedlings (BRS Energia in the initial phase of growth. The crude protein extract obtained after a 1-h extraction from the root tissues of seedlings showed no detectable CKX enzymatic activity when incubated with the substrate isopentenyl adenine for 1 h. However, after precipitation with ammonium sulfate at 70% saturation, the pellet showed CKX activity. The peroxidase enzyme activity was higher in the leaves than in the radicles. The total and reducing sugar content was 1.5 times higher in the leaves than in the radicles. The amino acid and protein contents were 6.4 and 9.2 times higher in the leaves than in the radicles, respectively.

  4. 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.

  5. Circulating purine compounds, uric acid, and xanthine oxidase/dehydrogenase relationship in essential hypertension and end stage renal disease.

    Science.gov (United States)

    Boban, Milojkovic; Kocic, Gordana; Radenkovic, Sonja; Pavlovic, Radmila; Cvetkovic, Tatjana; Deljanin-Ilic, Marina; Ilic, Stevan; Bobana, Milojkovic D; Djindjic, Boris; Stojanovic, Dijana; Sokolovic, Dusan; Jevtovic-Stoimenov, Tatjana

    2014-05-01

    Purine nucleotide liberation and their metabolic rate of interconversion may be important in the development of hypertension and its renal consequences. In the present study, blood triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) breakdown pathway was evaluated in relation to uric acid concentration and xanthine dehydrogenase/xanthine oxidase (XDH/XO) in patients with essential hypertension, patients with chronic renal diseases on dialysis, and control individuals. The pattern of nucleotide catabolism was significantly shifted toward catabolic compounds, including ADP, AMP, and uric acid in patients on dialysis program. A significant fall of ATP was more expressed in a group of patients on dialysis program, compared with the control value (p<0.001), while ADP and AMP were significantly increased in both groups of patients compared with control healthy individuals (p<0.001), together with their final degradation product, uric acid (p<0.001). The index of ATP/ADP and ATP/uric acid showed gradual significant fall in both the groups, compared with the control value (p<0.001), near five times in a group on dialysis. Total XOD was up-regulated significantly in a group with essential hypertension, more than in a group on dialysis. The activity of XO, which dominantly contributes reactive oxygen species (ROS) production, significantly increased in dialysis group, more than in a group with essential hypertension. In conclusion, the examination of the role of circulating purine nucleotides and uric acid in pathogenesis of hypertension and possible development of renal disease, together with XO role in ROS production, may help in modulating their liberation and ROS production in slowing progression from hypertension to renal failure.

  6. Branched-chain Amino Acid Metabolon: INTERACTION OF GLUTAMATE DEHYDROGENASE WITH THE MITOCHONDRIAL BRANCHED-CHAIN AMINOTRANSFERASE (BCATm)*

    OpenAIRE

    Islam, Mohammad Mainul; Nautiyal, Manisha; Wynn, R. Max; Mobley, James A.; Chuang, David T.; Hutson, Susan M.

    2009-01-01

    The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain α-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain α-keto acids from BCATm to E1. The protein complex also contains glut...

  7. assessment of the activity of glucose-6-phosphate dehydrogenase ...

    African Journals Online (AJOL)

    Uwaifoh

    2012-10-31

    Oct 31, 2012 ... activity of G-6-PD was determined in type 2 diabetes mellitus patients and control subjects ... that reason, monitoring of G-6-PD activity may be an important tool in preventing diabetic injury due to ... ingestion of certain drugs or food (eg fava beans) or .... Festus OO., supervised this study with the assistance.

  8. Enumeration and confirmation of Clostridium tyrobutyricum in silages using neutral red, D-cycloserine, and lactate dehydrogenase activity.

    Science.gov (United States)

    Jonsson, A

    1990-03-01

    Spores of clostridia in big bale silages, manure, and dairy products were enumerated and distinguished from other spore formers by using Reinforced Clostridium Agar containing .005% neutral red. Spores of Clostridium tyrobutyricum predominated, but spores of Clostridium butyricum, Clostridium sporogenes, Clostridium bifermentans, Clostridium putrificum, and Clostridium sphenoides occurred to a lesser extent. In samples with high bacterial spore counts, growth of Bacillus spp., but not C. tyrobutyricum, was retarded by the addition of 200 ppm D-cycloserine. Clostridia isolated from silages and milk products were identified and tested on lactate dehydrogenase activity. Of 275 investigated strains, only strains identified as C. tyrobutyricum tested positively. Only 65% of the tested strains of C. tyrobutyricum grew in the confirmatory substrate containing minerals, lactic acid, and acetic acid. Tyrobutyricum Broth was not selective for C. tyrobutyricum, since C. butyricum and C. sporogenes also grew in this medium.

  9. 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.

  10. Multichannel Simultaneous Determination of Activities of Lactate Dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Lianjia [Iowa State Univ., Ames, IA (United States)

    2000-09-12

    It is very important to find the best conditions for some enzymes to do the best catalysis in current pharmaceutical industries. Based on the results above, we could say that this set-up could be widely used in finding the optimal condition for best enzyme activity of a certain enzyme. Instead of looking for the best condition for enzyme activity by doing many similar reactions repeatedly, we can complete this assignment with just one run if we could apply enough conditions.

  11. 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.

  12. 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

  13. α -Ketoglutarate accumulation is not dependent on isocitrate dehydrogenase activity during tellurite detoxification in Escherichia coli.

    Science.gov (United States)

    Reinoso, Claudia A; Appanna, Vasu D; Vásquez, Claudio C

    2013-01-01

    Tellurite is toxic to most microorganisms because of its ability to generate oxidative stress. However, the way in which tellurite interferes with cellular processes is not fully understood to date. In this line, it was previously shown that tellurite-exposed cells displayed reduced activity of the α-ketoglutarate dehydrogenase complex (α-KGDH), which resulted in α-ketoglutarate (α-KG) accumulation. In this work, we assessed if α-KG accumulation in tellurite-exposed E. coli could also result from increased isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) activities, both enzymes involved in α-KG synthesis. Unexpectedly both activities were found to decrease in the presence of the toxicant, an observation that seems to result from the decreased transcription of icdA and gdhA genes (encoding ICDH and GDH, resp.). Accordingly, isocitrate levels were found to increase in tellurite-exposed E. coli. In the presence of the toxicant, cells lacking icdA or gdhA exhibited decreased reactive oxygen species (ROS) levels and higher tellurite sensitivity as compared to the wild type strain. Finally, a novel branch activity of ICDH as tellurite reductase is presented.

  14. 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.

  15. 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.

  16. Differential pulse voltammetric studies on the effects of Al(Ⅲ) on the lactate dehydrogenase activity

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In this paper, differential pulse voltammetry (DPV) was applied to study the effects of aluminum Al(Ⅲ) on the lactate dehydrogenase (LDH) activity. Michaelis-Menten constant (KNADHm) and maximum velocity (vmax) in the enzyme promoting catalytic reaction of "pyruvate(Pyr) + NADH + H+ LDH(=) lactate + NAD+" under different conditions by monitoring DPV reduction current of NAD+ were reported.(C) 2007 Shu Ping Bi. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

  17. In vitro effect of some anthelmintics on lactate dehydrogenase activity of Cotylophoron cotylophorum (Digenea: paramphistomidae).

    Science.gov (United States)

    Veerakumari, L; Munuswamy, N

    2000-07-24

    Effects of praziquantel (PZQ), levamisole (LEV), mebendazole (MBZ), fenbendazole (FBZ) and albendazole (ABZ) on the lactate dehydrogenase (LDH) activity of Cotylophoron cotylophorum were studied in vitro. Maximum levels of inhibition of LDH catalysing both oxidation and reduction reactions were observed in PZQ- and LEV-treated worms. Similarly, benzimidazoles - MBZ, FBZ and ABZ - have also significantly inhibited the activity of LDH catalysing the oxidation of lactate; whereas the activity of LDH catalysing the reduction of pyruvate was accelerated. This affects the mitochondrial energy generating process which ultimately proves fatal to the parasite. Therefore, the mode of action of benzimidazoles is primarily on the activation of LDH catalysing the conversion of pyruvate to lactate.

  18. 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

  19. 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

    at 3 h 23 min ± 11 min). Femoral arterial and venous blood, blood flow measurements, and muscle samples were obtained hourly during exercise and recovery (3 h). Carbohydrate oxidation peaked at 30 min of exercise and subsequently decreased for the remainder of the exercise bout (P ... with pyruvate metabolism, and they comprised 68% of total amino-acid release during exercise and recovery. Thus reduced pyruvate production was primarily associated with reduced carbohydrate oxidation, whereas the greatest production of pyruvate was related to glutamate, glutamine, and alanine metabolism...

  20. Localization of the nucleic acid channel regulatory subunit, cytosolic malate dehydrogenase.

    Science.gov (United States)

    Hanss, Basil; Leal-Pinto, Edgar; Teixeira, Avelino; Tran, Baohuong; Lee, Chun-Hui; Henderson, Scott C; Klotman, Paul E

    2008-01-01

    NACh is a nucleic acid-conducting channel found in apical membrane of rat kidney proximal tubules. It is a heteromultimeric complex consisting of at least two proteins: a 45-kDa pore-forming subunit and a 36-kDa regulatory subunit. The regulatory subunit confers ion selectivity and influences gating kinetics. The regulatory subunit has been identified as cytosolic malate dehydrogenase (cMDH). cMDH is described in the literature as a soluble protein that is not associated with plasma membrane. Yet a role for cMDH as the regulatory subunit of NACh requires that it be present at the plasma membrane. To resolve this conflict, studies were initiated to determine whether cMDH could be found at the plasma membrane. Before performing localization studies, a suitable model system that expressed NACh was identified. A channel was identified in LLC-PK(1) cells, a line derived from pig proximal tubule, that is selective for nucleic acid and has a conductance of approximately 10 pS. It exhibits dose-dependent blockade by heparan sulfate or L-malate. These characteristics are similar to what has been reported for NACh from rat kidney and indicate that NACh is present in LLC-PK(1) cells. LLC-PK(1) cells were therefore used as a model system for immunolocalization of cMDH. Both immunofluorescence and immunoelectron microscopy demonstrated cMDH at the plasma membrane of LLC-PK(1) cells. This finding supports prior functional data that describe a role for cMDH as the regulatory subunit of NACh.

  1. Identification and Validation of Aspartic Acid Semialdehyde Dehydrogenase as a New Anti-Mycobacterium Tuberculosis Target.

    Science.gov (United States)

    Meng, Jianzhou; Yang, Yanhui; Xiao, Chunling; Guan, Yan; Hao, Xueqin; Deng, Qi; Lu, Zhongyang

    2015-09-30

    Aspartic acid semialdehyde dehydrogenase (ASADH) lies at the first branch point in the essential aspartic acid biosynthetic pathway that is found in bacteria and plants but is absent from animals. Mutations in the asadh gene encoding ASADH produce an inactive enzyme, which is lethal. Therefore, in this study, we investigated the hypothesis that ASADH represents a new anti-Mycobacterium tuberculosis (MTB) target. An asadh promoter-replacement mutant MTB, designated MTB::asadh, in which asadh gene expression is regulated by pristinamycin, was constructed to investigate the physiological functions of ASADH in the host bacteria. Bacterial growth was evaluated by monitoring OD600 and ASADH expression was analyzed by Western blotting. The results showed that the growth and survival of MTB::asadh was completely inhibited in the absence of the inducer pristinamycin. Furthermore, the growth of the mutant was rigorously dependent on the presence of the inducer in the medium. The starved mutant exhibited a marked reduction (approximately 80%) in the cell wall materials compared to the wild-type, in addition to obvious morphological differences that were apparent in scanning electron microscopy studies; however, with the addition of pristinamycin, the cell wall contents and morphology similar to those of the wild-type strain were recovered. The starved mutant also exhibited almost no pathogenicity in an in vitro model of infection using mouse macrophage J774A.1 cells. The mutant showed a concentration-dependent recovery of pathogenicity with the addition of the inducer. These findings implicate ASADH as a promising target for the development of novel anti-MTB drugs.

  2. Identification and Validation of Aspartic Acid Semialdehyde Dehydrogenase as a New Anti-Mycobacterium Tuberculosis Target

    Directory of Open Access Journals (Sweden)

    Jianzhou Meng

    2015-09-01

    Full Text Available Aspartic acid semialdehyde dehydrogenase (ASADH lies at the first branch point in the essential aspartic acid biosynthetic pathway that is found in bacteria and plants but is absent from animals. Mutations in the asadh gene encoding ASADH produce an inactive enzyme, which is lethal. Therefore, in this study, we investigated the hypothesis that ASADH represents a new anti-Mycobacterium tuberculosis (MTB target. An asadh promoter-replacement mutant MTB, designated MTB::asadh, in which asadh gene expression is regulated by pristinamycin, was constructed to investigate the physiological functions of ASADH in the host bacteria. Bacterial growth was evaluated by monitoring OD600 and ASADH expression was analyzed by Western blotting. The results showed that the growth and survival of MTB::asadh was completely inhibited in the absence of the inducer pristinamycin. Furthermore, the growth of the mutant was rigorously dependent on the presence of the inducer in the medium. The starved mutant exhibited a marked reduction (approximately 80% in the cell wall materials compared to the wild-type, in addition to obvious morphological differences that were apparent in scanning electron microscopy studies; however, with the addition of pristinamycin, the cell wall contents and morphology similar to those of the wild-type strain were recovered. The starved mutant also exhibited almost no pathogenicity in an in vitro model of infection using mouse macrophage J774A.1 cells. The mutant showed a concentration-dependent recovery of pathogenicity with the addition of the inducer. These findings implicate ASADH as a promising target for the development of novel anti-MTB drugs.

  3. 11-Oxoeicosatetraenoic acid is a cyclooxygenase-2/15-hydroxyprostaglandin dehydrogenase-derived antiproliferative eicosanoid.

    Science.gov (United States)

    Liu, Xiaojing; Zhang, Suhong; Arora, Jasbir S; Snyder, Nathaniel W; Shah, Sumit J; Blair, Ian A

    2011-12-19

    Previously, we established that 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (HETE) was a significant cyclooxygenase (COX)-2-derived arachidonic acid (AA) metabolite in epithelial cells. Stable isotope dilution chiral liquid chromatography (LC)-electron capture atmospheric pressure chemical ionization (ECAPCI)/mass spectrometry (MS) was used to quantify COX-2-derived eicosanoids in the human colorectal adenocarcinoma (LoVo) epithelial cell line, which expresses both COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH). 11(R)-HETE secretion reached peak concentrations within minutes after AA addition before rapidly diminishing, suggesting further metabolism had occurred. Surprisingly, recombinant 15-PGDH, which is normally specific for oxidation of eicosanoid 15(S)-hydroxyl groups, was found to convert 11(R)-HETE to 11-oxo-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (ETE). Furthermore, LoVo cell lysates converted 11(R)-HETE to 11-oxo-ETE and inhibition of 15-PGDH with 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (CAY10397) (50 μM) significantly suppressed endogenous 11-oxo-ETE production with a corresponding increase in 11(R)-HETE. These data confirmed COX-2 and 15-PGDH as enzymes responsible for 11-oxo-ETE biosynthesis. Finally, addition of AA to the LoVo cells resulted in rapid secretion of 11-oxo-ETE into the media, reaching peak levels within 20 min of starting the incubation. This was followed by a sharp decrease in 11-oxo-ETE levels. Glutathione (GSH) S-transferase (GST) was found to metabolize 11-oxo-ETE to the 11-oxo-ETE-GSH (OEG)-adduct in LoVo cells, as confirmed by LC-MS/MS analysis. Bromodeoxyuridine (BrdU)-based cell proliferation assays in human umbilical vein endothelial cells (HUVECs) revealed that the half-maximal inhibitory concentration (IC(50)) of 11-oxo-ETE for inhibition of HUVEC proliferation was 2.1 μM. These results show that 11-oxo-ETE is a novel COX-2/15-PGDH-derived eicosanoid, which inhibits endothelial

  4. Expression in Escherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation.

    Science.gov (United States)

    Höög, J O; Weis, M; Zeppezauer, M; Jörnvall, H; von Bahr-Lindström, H

    1987-12-01

    Human alcohol dehydrogenase (ADH, beta beta isozyme of class I) was expressed in Escherichia coli, purified to homogeneity, and characterized regarding N-terminal processing. The expression system was obtained by ligation of a cDNA fragment corresponding to the beta-subunit of human liver alcohol dehydrogenase into the vector pKK 223-3 containing the tac promoter. The enzyme, detected by Western-blot analysis and ethanol oxidizing activity, constituted up to 3% of the total amount of protein. Recombinant ADH was separated from E. coli ADH by ion-exchange chromatography and the isolated enzyme was essentially pure as judged by SDS-polyacrylamide gel electrophoresis and sequence analysis. The N-terminal sequence was identical to that of the authentic beta-subunit except that the N-terminus was non-acetylated, indicating a correct removal of the initiator methionine, but lack of further processing.

  5. Alpha-hydroxybutyrate dehydrogenase activity in sex-linked muscular dystrophy.

    Science.gov (United States)

    Johnston, H A; Wilkinson, J H; Withycombe, W A; Raymond, S

    1966-05-01

    In two families with severe sex-linked muscular dystrophy, high levels of alpha-hydroxybutyrate dehydrogenase (HBD), lactate dehydrogenase (LD), aspartate transaminase (AspT), aldolase, and creatine phosphokinase (CPK) were found in the sera of three young affected males. In both families the mother had a raised level of HBD activity. Four sisters of the three affected boys had raised serum enzyme levels, and they are regarded as presumptive carriers of the disease. Biopsy specimens of dystrophic muscle had LD and HBD contents which were significantly lower than those of control specimens, while the HBD/LD ratios were markedly greater. Muscle from two unaffected members of the same family also exhibited high ratios, indicating the presence of the electrophoretically fast LD isoenzymes, and this was confirmed by acrylamide-gel electrophoresis.

  6. Escherichia coli D-malate dehydrogenase, a generalist enzyme active in the leucine biosynthesis pathway.

    Science.gov (United States)

    Vorobieva, Anastassia A; Khan, Mohammad Shahneawz; Soumillion, Patrice

    2014-10-17

    The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of D-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on D-malate as a carbon source, the D-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB(-) strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (L(+)-tartrate, D-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution.

  7. 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.

  8. Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid.

    Science.gov (United States)

    Ishitani, M; Nakamura, T; Han, S Y; Takabe, T

    1995-01-01

    When subjected to salt stress or drought, some vascular plants such as barley respond with an increased accumulation of the osmoprotectant glycine betaine (betaine), being the last step of betaine synthesis catalyzed by betaine aldehyde dehydrogenase (BADH). We report here cloning and characterization of BADH cDNA from barley, a monocot, and the expression pattern of a BADH transcript. An open reading frame of 1515 bp encoded a protein which showed high homology to BADH enzymes present in other plants (spinach and sugar-beet) and in Escherichia coli. Transgenic tobacco plants harboring the clone expressed high levels of both BADH protein and its enzymatic activity. Northern blot analyses indicated that BADH mRNA levels increased almost 8-fold and 2-fold, respectively, in leaves and roots of barley plants grown in high-salt conditions, and that these levels decreased upon release of the stress, whereas they did not decrease under continuous salt stress. BADH transcripts also accumulate in response to water stress or drought, indicating a common response of the plant to osmotic changes that affect its water status. The addition of abscisic acid (ABA) to plants during growth also increased the levels of BADH transcripts dramatically, although the response was delayed when compared to that found for salt-stressed plants. Removal of plant roots before transferring the plants to high-salt conditions reduced only slightly the accumulation of BADH transcripts in the leaves.

  9. Equivalence between Pfr and Cyclic AMP in the Induction of d-Usnic Acid Dehydrogenase in the Lichen Evernia prunastri1

    Science.gov (United States)

    Avalos, A.; Vicente, C.

    1987-01-01

    d-Usnic acid dehydrogenase is induced in Evernia prunastri thalli by a supply of exogenous d-usnic acid in light. This effect is enhanced by red light pulses through a two step way: a very rapid increase of activity after the first 10 minutes of red light, which is not reversed by far-red light, and a slow enhancement following successive red light pulses at the beginning of each hour of incubation. The last response is completely reversed by far-red following red light. Although induction of the enzyme is not achieved in the dark, 0.1 and 0.5 millimolar cyclic AMP, or 0.1 millimolar dibutyryl cyclic AMP substitutes light action and, then, the enzyme is produced. In addition, phytochrome—far red-absorbing form—increases the amount of endogenously produced cyclic AMP and this effect is shown to be photoreversible when ethylenediaminetetraacetic acid is inhibiting adenylate cyclase. PMID:16665525

  10. Effect of dehydrogenase, phosphatase and urease activity in cotton soil after applying thiamethoxam as seed treatment.

    Science.gov (United States)

    Jyot, Gagan; Mandal, Kousik; Singh, Balwinder

    2015-05-01

    Soil enzymes are indicators of microbial activities in soil and are often considered as an indicator of soil health and fertility. They are very sensitive to the agricultural practices, pH of the soil, nutrients, inhibitors and weather conditions. To understand the effect of an insecticide, thiamethoxam, on different soil enzyme activities, the experiments were conducted at cotton experimental fields of Punjab Agricultural University, Ludhiana. The results here were presented to understand the impact of thiamethoxam on soil enzyme activities. Thiamethoxam was applied as seed treatment to control the pest. Soil from three localities, i.e. soil in which seed was treated with recommended dose at 2.1 g a.i. kg(-1), soil in which seed was treated with four times recommended dose at 8.4 g a.i. kg(-1) and from the control field, were tested for different enzyme activities. Phosphatase and dehydrogenase activities were high in control soil in comparison to control soil while no effect of this insecticide on urease activity. Thiamethoxam had inhibitory effects on dehydrogenase and phosphatase activities. Therefore, it can be attributed that agricultural practices, weather conditions and use of thiamethoxam might be responsible for the different level of enzyme activities in soil.

  11. The separate roles of PQQ and apo-enzyme syntheses in the regulation of glucose dehydrogenase activity in Klebsiella pneumoniae NCTC 418.

    Science.gov (United States)

    Hommes, R W; Herman, P T; Postma, P W; Tempest, D W; Neijssel, O M

    1989-01-01

    No holoenzyme pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and only very low apoenzyme levels could be detected in cells of Klebsiella pneumoniae, growing anaerobically, or carrying out a fumarate or nitrate respiration. Low glucose dehydrogenase activity in some aerobic glucose-excess cultures of K. pneumoniae (ammonia or sulphate limitation) was increased significantly by addition of PQQ, whereas in cells already possessing a high glucose dehydrogenase activity (phosphate or potassium limitation) extra PQQ had almost no effect. These observations indicate that the glucose dehydrogenase activity in K. pneumoniae is modulated by both PQQ synthesis and synthesis of the glucose dehydrogenase apo-enzyme.

  12. Glycerol-3-phosphate dehydrogenase 1 deficiency induces compensatory amino acid metabolism during fasting in mice.

    Science.gov (United States)

    Sato, Tomoki; Yoshida, Yuma; Morita, Akihito; Mori, Nobuko; Miura, Shinji

    2016-11-01

    Glucose is used as an energy source in many organs and obtained from dietary carbohydrates. However, when the external energy supply is interrupted, e.g., during fasting, carbohydrates preserved in the liver and glycogenic precursors derived from other organs are used to maintain blood glucose levels. Glycerol and glycogenic amino acids derived from adipocytes and skeletal muscles are utilized as glycogenic precursors. Glycerol-3-phosphate dehydrogenase 1 (GPD1), an NAD(+)/NADH-dependent enzyme present in the cytosol, catalyzes the reversible conversion of glycerol-3-phosphate (G3P) to dihydroxyacetone phosphate (DHAP). Since G3P is one of the substrates utilized for gluconeogenesis in the liver, the conversion of G3P to DHAP by GPD1 is essential for maintaining blood glucose levels during fasting. We focused on GPD1 and examined its roles in gluconeogenesis during fasting. Using GPD1 null model BALB/cHeA mice (HeA mice), we measured gluconeogenesis from glycerol and the change of blood glucose levels under fasting conditions. We also measured gene expression related to gluconeogenesis in the liver and protein metabolism in skeletal muscle. BALB/cBy mice (By mice) were used as a control. The blood glucose levels in the HeA mice were lower than that in the By mice after glycerol administration. Although lack of GPD1 inhibited gluconeogenesis from glycerol, blood glucose levels in the HeA mice after 1-4h of fasting were significantly higher than that in the By mice. Muscle protein synthesis in HeA mice was significantly lower than that in the By mice. Moreover, blood alanine levels and usage of alanine for gluconeogenesis in the liver were significantly higher in the HeA mice than that in the By mice. Although these data indicate that a lack of GPD1 inhibits gluconeogenesis from glycerol, chronic GPD1 deficiency may induce an adaptation that enhances gluconeogenesis from glycogenic amino acids. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Human placental glucose dehydrogenase: IEF polymorphism in two Italian populations and enzyme activity in the six common phenotypes.

    Science.gov (United States)

    Scacchi, R; Corbo, R M; Calzolari, E; Laconi, G; Palmarino, R; Lucarelli, P

    1985-01-01

    Glucose dehydrogenase (hexose-6-phosphate dehydrogenase) has been assayed qualitatively and quantitatively in more than 600 human placentae collected in two Italian populations. The gene frequencies for GDH1, GDH2 and GDH3 were, respectively, 0.66, 0.21 and 0.12 in Continental Italy and 0.65, 0.23 and 0.12 in Sardinia. Among the six common phenotypes there was no difference in catalytic activity.

  14. 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.

  15. Exercise-induced pyruvate dehydrogenase activation is not affected by 7 days of bed rest

    DEFF Research Database (Denmark)

    Kiilerich, Kristian; Jørgensen, Stine Ringholm; Biensø, Rasmus Sjørup

    2011-01-01

    To test the hypothesis that physical inactivity impairs the exercise-induced modulation of pyruvate dehydrogenase (PDH), 6 healthy normally physically active male subjects completed 7 days of bed rest. Before and immediately after the bed rest, the subjects completed an OGTT and a one-legged knee...... after bed rest than before, indicating glucose intolerance. There were no differences in lactate release/uptake across the exercising muscle before and after bed rest, but glucose uptake after 40min of exercise was larger (P=0.05) before bed rest than after. Muscle glycogen content tended to be higher...

  16. Effects of Al(III and Nano-Al13 Species on Malate Dehydrogenase Activity

    Directory of Open Access Journals (Sweden)

    Rong Fu Chen

    2011-05-01

    Full Text Available The effects of different aluminum species on malate dehydrogenase (MDH activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT modified glass carbon electrode (GCE. The results showed that Al(III and Al13 can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III and Al13 concentration increase. Our study also found that the effects of Al(III and Al13 on the activity of MDH depended on the pH value and aluminum speciation. Electrochemical and circular dichroism spectra methods were applied to study the effects of nano-sized aluminum compounds on biomolecules.

  17. Effects of Al(III) and nano-Al13 species on malate dehydrogenase activity.

    Science.gov (United States)

    Yang, Xiaodi; Cai, Ling; Peng, Yu; Li, Huihui; Chen, Rong Fu; Shen, Ren Fang

    2011-01-01

    The effects of different aluminum species on malate dehydrogenase (MDH) activity were investigated by monitoring amperometric i-t curves for the oxidation of NADH at low overpotential using a functionalized multi-wall nanotube (MWNT) modified glass carbon electrode (GCE). The results showed that Al(III) and Al(13) can activate the enzymatic activity of MDH, and the activation reaches maximum levels as the Al(III) and Al(13) concentration increase. Our study also found that the effects of Al(III) and Al(13) on the activity of MDH depended on the pH value and aluminum speciation. Electrochemical and circular dichroism spectra methods were applied to study the effects of nano-sized aluminum compounds on biomolecules.

  18. [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.

  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. 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.

  1. Which way does the citric acid cycle turn during hypoxia? The critical role of α-ketoglutarate dehydrogenase complex.

    Science.gov (United States)

    Chinopoulos, Christos

    2013-08-01

    The citric acid cycle forms a major metabolic hub and as such it is involved in many disease states involving energetic imbalance. In spite of the fact that it is being branded as a "cycle", during hypoxia, when the electron transport chain does not oxidize reducing equivalents, segments of this metabolic pathway remain operational but exhibit opposing directionalities. This serves the purpose of harnessing high-energy phosphates through matrix substrate-level phosphorylation in the absence of oxidative phosphorylation. In this Mini-Review, these segments are appraised, pointing to the critical importance of the α-ketoglutarate dehydrogenase complex dictating their directionalities. Copyright © 2013 Wiley Periodicals, Inc.

  2. Secondary amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase.

    Science.gov (United States)

    Aicher, T D; Anderson, R C; Gao, J; Shetty, S S; Coppola, G M; Stanton, J L; Knorr, D C; Sperbeck, D M; Brand, L J; Vinluan, C C; Kaplan, E L; Dragland, C J; Tomaselli, H C; Islam, A; Lozito, R J; Liu, X; Maniara, W M; Fillers, W S; DelGrande, D; Walter, R E; Mann, W R

    2000-01-27

    N'-methyl-N-(4-tert-butyl-1,2,5,6-tetrahydropyridine)thiourea, SDZ048-619 (1), is a modest inhibitor (IC(50) = 180 microM) of pyruvate dehydrogenase kinase (PDHK). In an optimization of the N-methylcarbothioamide moiety of 1, it was discovered that amides with a small acyl group, in particular appropriately substituted amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, are inhibitors of PDHK. Utilizing this acyl moiety, herein is reported the rationale leading to the optimization of a series of acylated piperazine derivatives. Methyl substitution of the piperazine at the 2- and 5-positions (with S and R absolute stereochemistry) markedly increased the potency of the lead compound (>1,000-fold). Oral bioavailability of the compounds in this series is good and is optimal (as measured by AUC) when the 4-position of the piperazine is substituted with an electron-poor benzoyl moiety. (+)-1-N-[2,5-(S, R)-Dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropanamide (14e) inhibits PDHK in the primary enzymatic assay with an IC(50) of 16 +/- 2 nM, enhances the oxidation of [(14)C]lactate into (14)CO(2) in human fibroblasts with an EC(50) of 57 +/- 13 nM, diminishes lactate significantly 2.5 h post-oral-dose at doses as low as 1 micromol/kg, and increases the ex vivo activity of PDH in muscle, liver, and fat tissues in normal Sprague-Dawley rats. These PDHK inhibitors, however, do not lower glucose in diabetic animal models.

  3. Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives.

    Science.gov (United States)

    Su, Xiangdong; Vicker, Nigel; Lawrence, Harshani; Smith, Andrew; Purohit, Atul; Reed, Michael J; Potter, Barry V L

    2007-05-01

    11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11beta-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11beta-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18beta-glycyrrhetinic acid (18beta-GA) derivatives (2-5) and their inhibitory activities against rat hepatic11beta-HSD1 and rat renal 11beta-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds' ability to inhibit human 11beta-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18beta-GA derivatives 2 and 3 with apparent selectivity for rat 11beta-HSD1 showed a high percentage inhibition for human microsomal 11beta-HSD1 at 10 microM and exhibited IC50 values of 400 and 1100 nM, respectively. The side chain modified 18beta-GA derivatives 4 and 5, although showing selectivity for rat 11beta-HSD1 inhibited human microsomal 11beta-HSD1 with IC50 values in the low micromolar range.

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

    Directory of Open Access Journals (Sweden)

    Mortensen Uffe H

    2011-09-01

    Full Text Available Abstract 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. The target of MPA is inosine-5'-monophosphate dehydrogenase (IMPDH, which catalyses the rate limiting step in the synthesis of guanine nucleotides. The recent discovery of the MPA biosynthetic gene cluster from Penicillium brevicompactum revealed an extra copy of the IMPDH-encoding gene (mpaF embedded within the cluster. This finding suggests that the key component of MPA self resistance is likely based on the IMPDH encoded by mpaF. Results In accordance with our hypothesis, heterologous expression of mpaF dramatically increased MPA resistance in a model fungus, Aspergillus nidulans, which does 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 Penicillium. All six strains were found to hold two copies of IMPDH. A cladistic analysis based on the corresponding cDNA sequences revealed a novel group constituting mpaF homologs. Interestingly, a conserved tyrosine residue in the original class of IMPDHs is replaced by a phenylalanine residue in the new IMPDH class. Conclusions We identified a novel variant of the IMPDH-encoding gene in six different strains from Penicillium subgenus Penicillium. The novel IMPDH variant from MPA producer P. brevicompactum was shown to confer a high degree of MPA resistance when expressed in a non-producer fungus. Our study provides a basis for understanding the molecular mechanism of MPA resistance and has relevance for biotechnological and pharmaceutical applications.

  5. 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.

  6. 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...... 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...

  7. Dynamic expression of the retinoic acid-synthesizing enzyme retinol dehydrogenase 10 (rdh10) in the developing mouse brain and sensory organs.

    Science.gov (United States)

    Romand, Raymond; Kondo, Takako; Cammas, Laura; Hashino, Eri; Dollé, Pascal

    2008-06-20

    Organs develop through many tissue interactions during embryogenesis, involving numerous signaling cascades and gene products. One of these signaling molecules is retinoic acid (RA), an active vitamin A derivative, which in mammalian embryos is synthesized from maternal retinol by two oxidative reactions involving alcohol/retinol dehydrogenases (ADH/RDHs) and retinaldehyde dehydrogenases (RALDHs), respectively. The activity of RALDHs is known to be crucial for RA synthesis; however, recently a retinol dehydrogenase (RDH10) has been shown to represent a new limiting factor in this synthesis. We investigated the spatiotemporal distribution of Rdh10 gene transcripts by in situ hybridization and quantitative polymerase chain reaction (PCR) during development of the brain and sensory organs. Although Rdh10 relative mRNA levels decline throughout brain development, we show a strong and lasting expression in the meninges and choroid plexuses. Rdh10 expression is also specifically seen in the striatum, a known site of retinoid signaling. In the eye, regional expression is observed both in the prospective pigmented epithelium and neural retina. In the inner ear Rdh10 expression is specific to the endolymphatic system and later the stria vascularis, both organs being involved in endolymph homeostasis. Furthermore, in the peripheral olfactory system and the vibrissae follicles, expression is present from early stages in regions where sensory receptors appear and mesenchymal/epithelial interactions take place. The distribution of Rdh10 transcripts during brain and sensory organ development is consistent with a role of this enzyme in generating region-specific pools of retinaldehyde that will be used by the various RALDHs to refine the patterns of RA synthesis.

  8. 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.

  9. [Succinic semialdehyde dehydrogenase deficiency: decrease in 4-OH-butyric acid levels with low doses of vigabatrin].

    Science.gov (United States)

    Escalera, G Iglesias; Ferrer, I; Marina, Ll Carrasco; Sala, P Ruiz; Salomons, G S; Jakobs, C; Pérez-Cerdá, C

    2010-02-01

    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 variety of neurological manifestations and psychiatric symptoms. The treatment usually used is vigabatrin, but its clinical efficacy is under discussion. We present two affected siblings. The older brother was examined when he was 2.5 years old due to psychomotor and developmental delay, disturbances in motor coordination, axial hypotonia and language disability. His younger brother had mild axial hypotonia when 5 months old. Metabolic studies demonstrated a high plasma and urine concentration of gamma-hydroxybutyric acid. Mutation analysis of the gene ALDH5A1 confirmed the disease. After 1 year of treatment with low-doses of vigabatrin of the older patient, a decrease in gamma-hydroxybutyric acid plasma levels and a slow clinical improvement were observed.

  10. Resveratrol inhibits 11β-hydroxysteroid dehydrogenase type 1 activity in rat adipose microsomes.

    Science.gov (United States)

    Tagawa, Noriko; Kubota, Sayaka; Kato, Ikuo; Kobayashi, Yoshiharu

    2013-09-01

    It has been suggested that resveratrol, a polyphenol in wine, can regulate adiposity because it decreases adipose deposition in mice and rats; however, the mechanism underlying this effect has not been fully clarified. In humans and rodents, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is expressed in liver and adipose tissue. 11β-HSD1 converts inactive glucocorticoid into active glucocorticoid in adipocytes. Activated glucocorticoid plays an important role in the pathogenesis of central obesity. The objective of this study was to investigate the effects of resveratrol on 11β-HSD1 activity in rodent adipose tissue. 11β-HSD1 activity in microsomes from rat mesenteric adipose depots and 3T3-L1 adipocytes was determined in the presence of 11-dehydrocorticosterone with or without varying concentrations of resveratrol. Significant inhibition of 11β-HSD1 by resveratrol was observed in rat adipose microsomes and 3T3-L1 adipocytes within 10 min. Time- and dose-dependent effects were also observed. The 11β-HSD1 activity by resveratrol was also inhibited in rat epididymal adipose tissue, and this inhibition was not recovered by estrogen receptor blockers. The kinetic study revealed that resveratrol acted as a non-competitive inhibitor of 11β-HSD1. Ki and IC50 values of resveratrol were 39.6 and 35.2 μM respectively. Further, resveratrol did not affect the activities of 11β-HSD2 and hexose-6-phosphate dehydrogenase. These results suggest that the most likely mechanism of 11β-HSD1 inhibition by resveratrol is via interaction between resveratrol and 11β-HSD1 enzyme, rather than via a transcriptional pathway. We demonstrated that the antiobesity effects of resveratrol may partially be attributed to the inhibition of 11β-HSD1 activity in adipocytes.

  11. Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1.

    Science.gov (United States)

    Wu, Tung-Yun; Chen, Chang-Ting; Liu, Jessica Tse-Jin; Bogorad, Igor W; Damoiseaux, Robert; Liao, James C

    2016-06-01

    Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.

  12. Glutathione metabolism and glucose 6-phosphate dehydrogenase activity in experimental liver injury.

    Directory of Open Access Journals (Sweden)

    Watanabe,Akiharu

    1983-12-01

    Full Text Available Increased activities of liver glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49 and 6-phosphogluconate dehydrogenase (6PGD, EC 1.1.1.44 in the pentose phosphate cycle were accompanied with a depletion of reduced glutathione (GSH following an intragastric administration of carbon tetrachloride (CCl4 to rats. Oxidized glutathione (GSSG also decreased remarkably, keeping the GSSG: GSH ratio constant. No significant alteration of glutathione reductase (EC 1.6.4.2., glutathione peroxidase (EC 1.11.1.9 and malic enzyme (EC 1.1.1.40 activities in the supernatant and gamma-glutamyl transpeptidase (gamma-GTP, EC 2.3.2.2 activity in the homogenate of the injured liver were observed. Furthermore, no marked difference in the GSH-synthesizing activity was found between control and CCl4-intoxicated liver. An intraperitoneal injection of GSH produced a significant increase in liver GSH content in control rats but not in CCl4-treated rats; G6PD activity was not affected. Intraperitoneal injections of diethylmaleate resulted in continuously diminished levels of liver GSH without any alteration of liver G6PD activity. In vitro disappearance of GSH added to the liver homogenate from CCl4-treated rats occurred enzymatically and could not be prevented by the addition of a NADPH-generating system. The results suggest that increased G6PD activity in CCl4-injured liver does not play an important role in the maintenance of glutathione in the reduced form and that the decreased GSH content in the injured liver might be caused by enhanced GSH catabolism not due to gamma-GTP.

  13. 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.

  14. 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.

  15. Activity of formaldehyde dehydrogenase on titanium dioxide films with different crystallinities

    Science.gov (United States)

    Nakamura, Hitomi; Kato, Katsuya; Masuda, Yoshitake; Kato, Kazumi

    2015-02-01

    Many biosensors have been developed and used in recent years, and to enhance the sensitivity and stability of enzyme biosensors, immobilization of the enzymes on material surfaces is a necessary and important step. Therefore, there has been considerable interest in understanding how material interfaces affect enzyme adsorption. In this study, the influence of the crystallinity of titanium dioxide (TiO2) films on the quantity and activity of the immobilized enzyme, i.e., formaldehyde dehydrogenase (FDH), was investigated. It was found that TiO2 films with high crystallinity, which were annealed at 550 °C, showed higher enzyme immobilization and activity compared with the non-annealed TiO2 film. These results suggest that the activity of enzymes could be affected by the crystallinity of surface materials.

  16. Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase

    Directory of Open Access Journals (Sweden)

    Tripty A. Hirani

    2011-01-01

    Full Text Available We have developed an in vitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thaliana α2β2-heterotetrameric pyruvate dehydrogenase (E1 plus A. thaliana E1-kinase (AtPDK. Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1α, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1α active-site loop sequences. Mutation of Asp295 to Ala, Asn, or Leu greatly reduced phosphorylation of Ser292, while mutation of Gly297 had relatively little effect. Quantitative two-hybrid analysis was used to show that mutation of Asp295 did not substantially affect binding of AtPDK to E1α. When using pyruvate as a variable substrate, the Asp295 mutant proteins had modest changes in kcat, Km, and kcat/Km values. Therefore, we propose that Asp295 plays an important role in stabilizing the active-site loop structure, facilitating transfer of the γ-phosphate from ATP to the Ser residue at regulatory site one of E1α.

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

    NARCIS (Netherlands)

    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

    2016-01-01

    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 h

  18. Acute and chronic effects of diazinon on the activities of three dehydrogenases in the digestive system of a freshwater teleost fish Channa punctatus.

    Science.gov (United States)

    Sastry, K V; Malik, P V

    1982-01-01

    The effect of acute exposure to LC50 for 96 h (3.1 mg/l) and chronic exposure to a sublethal concentration (0.31 mg/l) of diazinon has been studied in the liver, stomach, intestine and pyloric ceca of a freshwater teleost fish, Channa punctatus. In acute exposure succinate dehydrogenase (SDH) activity was elevated in intestine and pyloric ceca. No alteration was noted in lactate dehydrogenase activity but pyruvate dehydrogenase was inhibited in pyloric ceca. Chronic exposure resulted in inhibition of the activities of the three dehydrogenases in all the four parts at both intervals.

  19. Increment of antioxidase activity of transgenic tobacco with betaine aldehyde dehydrogenase

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Superoxide dismutase (SOD) activity in the leaves of transgenic tobacco plants with betaine aldehyde dehydrogenase (BADH) gene was about 36% higher than that in the control plants (parent plants),activities of peroxi-dase (POD) and catalase (Cat) increased by about 62% and 88% respectively. Activities of ascorbate peroxidase (AsSPOD),dehydroascorbate redutase (DAsAR) and gluta-thione reductase (GR) in ascorbate-glutothion pathway lo-cated at chloroplasts increased by 67.7%,47.9% and 38.8% respectively. These results indicated that the H2O2 produced by SOD catalyzing superoxide anion radicals (O2- ) could be fully decomposed,and could not derive to form the strongest toxicant radicals ·OH. This is the first report to elucidate quantitatively that the activities of two kinds of antioxidative enzymes decomposed radicals and active oxygen were matched. Photoinhibition tolerant capacity of the transgenic tobacco plants was 35% higher than that in the parent plants. Increment of photoinhibition tolerant capacity in the trans-genic tobacco plants might be due to increment of antioxida-tive enzymes activities,in turn being able to more effectively scavenge active oxygen and radicals,protect organization and function of chloroplasts. These results showed that the increment of antioxidative enzymes activities in the trans-genic tobacco might be one of the reasons for the increment of resistance in the transgenic tobacco.

  20. Glutaric acid and its metabolites cause apoptosis in immature oligodendrocytes: a novel mechanism of white matter degeneration in glutaryl-CoA dehydrogenase deficiency.

    Science.gov (United States)

    Gerstner, Bettina; Gratopp, Alexander; Marcinkowski, Monika; Sifringer, Marco; Obladen, Michael; Bührer, Christoph

    2005-06-01

    Glutaryl-CoA dehydrogenase deficiency is an inherited metabolic disease characterized by elevated concentrations of glutaric acid (GA) and its metabolites glutaconic acid (GC) and 3-hydroxy-glutaric acid (3-OH-GA). Its hallmarks are striatal and cortical degeneration, which have been linked to excitotoxic neuronal cell death. However, magnetic resonance imaging studies have also revealed widespread white matter disease. Correspondingly, we decided to investigate the effects of GA, GC, and 3-OH-GA on the rat immature oligodendroglia cell line, OLN-93. For comparison, we also exposed the neuroblastoma line SH-SY5Y and the microglia line BV-2 to GA, GC, and 3-OH-GA. Cell viability was measured by metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium. Flow cytometry was used to assess apoptosis via annexin-V, anti-active caspase-3 antibody, and propidium iodide staining. GA, GC, and 3-OH-GA reduced OLN-93 oligodendroglia cell viability in a dose-dependent manner. Toxicity of GA, GC, and 3-OH-GA was abrogated by preincubation with the pan-caspase inhibitor z-VAD-fmk. Apoptosis but not necrosis was detected at various stages (early: annexin-V; effector: caspase-3) after 24-48 h of incubation with GA, GC, or 3-OH-GA in OLN-93 but not in neuroblastoma or microglia cells. OLN-93 lacked expression of N-methyl-d-aspartate receptors, making classical glutamatergic excitotoxicity an unlikely explanation for the selective toxicity of GA, GC, and 3-OH-GA for OLN-93 cells. GA, GC, and 3-OH-GA directly initiate the apoptotic cascade in oligodendroglia cells. This mechanism may contribute to the white matter damage observed in glutaryl-CoA dehydrogenase deficiency.

  1. Crystal structure of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase complexed with an analogue of 1,3-bisphospho-d-glyceric acid.

    Science.gov (United States)

    Ladame, Sylvain; Castilho, Marcelo S; Silva, Carlos H T P; Denier, Colette; Hannaert, Véronique; Périé, Jacques; Oliva, Glaucius; Willson, Michèle

    2003-11-01

    We report here the first crystal structure of a stable isosteric analogue of 1,3-bisphospho-d-glyceric acid (1,3-BPGA) bound to the catalytic domain of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) in which the two phosphoryl moieties interact with Arg249. This complex possibly illustrates a step of the catalytic process by which Arg249 may induce compression of the product formed, allowing its expulsion from the active site. Structural modifications were introduced into this isosteric analogue and the respective inhibitory effects of the resulting diphosphorylated compounds on T. cruzi and Trypanosoma brucei gGAPDHs were investigated by enzymatic inhibition studies, fluorescence spectroscopy, site-directed mutagenesis, and molecular modelling. Despite the high homology between the two trypanomastid gGAPDHs (> 95%), we have identified specific interactions that could be used to design selective irreversible inhibitors against T. cruzi gGAPDH.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. Urinary Lactate Dehydrogenase Activity and Its Isozyme Patterns in Kawasaki Disease.

    Science.gov (United States)

    Kawamura, Yoichi; Takeshita, Seiichiro; Kanai, Takashi; Takizawa, Mari; Yoshida, Yusuke; Tsujita, Yuki; Nonoyama, Shigeaki

    2017-01-01

    Abnormal urinary findings, such as sterile pyuria, proteinuria, and microscopic hematuria, are often seen in the acute phase of Kawasaki disease (KD). We investigated the potential significance of urinary lactate dehydrogenase (U-LDH) activity and its isozyme patterns in KD. Total U-LDH activity and its isozymes (U-LDH1-5) levels were compared among 120 patients with KD, 18 patients with viral infection (VI), and 43 patients with upper urinary tract infection (UTI) and additionally compared between intravenous immunoglobulin (IVIG) responders (n = 89) and nonresponders (n = 31) with KD. Total U-LDH activity was higher in KD (35.4 ± 4.8 IU/L, P IVIG nonresponders of KD had significantly higher levels of total U-LDH activity (45.1 ± 4.7 IU/L, P IVIG responders (32.0 ± 2.8 IU/L). KD patients have increased levels of total U-LDH activity, especially U-LDH-1 and U-LDH2, indicating a unique pattern of U-LDH isozymes different from that in UTI patients.

  7. 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.

  8. In Vitro and In Vivo Effects and Safety Assessment of Corn Peptides on Alcohol Dehydrogenase Activities

    Institute of Scientific and Technical Information of China (English)

    LI Hong-mei; WEN Lian-kui; LI Shi-jun; ZHANG Da-li; LIN Bai-song

    2011-01-01

    The in vitro and in vivo effects of corn peptides(CPs) prepared from corn gluten meal by proteolysis with an alkaline protease and fractions of CPs from Sephadex G-15 and G-10 columns on activities of alcohol dehydrogenase(ADH) were studied.The results show that CPs and fraction 3 of CPs from Sephadex G-10 column enhance in vitro ADH activity.Furthermore,the in vitro accelerating effect of the fraction 3 of CPs on ADH activity was superior to that of glutathione,which was also found even in the presence of ADH inhibitor,such as pyrazole.In the in vivo experiments,the animals were fed with different dosages of CPs and with a dose of Chinese distilled spirit orally,and sacrificed for the measurement of ADH activity.In vivo experimental results indicate that CPS enhanced hepatic ADH activities.To test the safety of CPs as health food,30 d feeding test was performed.No obvious toxic effects were detected in treated Wistar rats.

  9. 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

    2017-05-31

    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.

  10. A rapid procedure for eliminating chromatofocusing buffer and concentrating minor active subforms of mitochondrial malate dehydrogenase.

    Science.gov (United States)

    Gelpí, J L; Gracia, V; Imperial, S; Mazo, A; Cortés, A

    1990-11-01

    Mitochondrial malate dehydrogenase from several sources contains different molecular forms whose origin is still under discussion. Separation of these subforms has been achieved by chromatofocusing. A simple and rapid method, based on 5' AMP Sepharose chromatography, has been developed to concentrate mitochondrial malate dehydrogenase subforms and simultaneously remove chromatofocusing buffer.

  11. 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.

  12. 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

  13. 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

    During exercise involving a small muscle mass, peak oxygen uptake is thought to be limited by peripheral factors, such as the degree of oxygen extraction from the blood and/or mitochondrial oxidative capacity. Previously, the maximal activity of the Krebs cycle enzyme oxoglutarate dehydrogenase has...

  14. Xanthine dehydrogenase-1 silencing in Aedes aegypti mosquitoes 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

    2017-02-08

    Aedesaegypti 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.

  15. Efficient synthesis of D-branched-chain amino acids and their labeled compounds with stable isotopes using D-amino acid dehydrogenase.

    Science.gov (United States)

    Akita, Hironaga; Suzuki, Hirokazu; Doi, Katsumi; Ohshima, Toshihisa

    2014-02-01

    D-Branched-chain amino acids (D-BCAAs) such as D-leucine, D-isoleucine, and D-valine are known to be peptide antibiotic intermediates and to exhibit a variety of bioactivities. Consequently, much effort is going into achieving simple stereospecific synthesis of D-BCAAs, especially analogs labeled with stable isotopes. Up to now, however, no effective method has been reported. Here, we report the establishment of an efficient system for enantioselective synthesis of D-BCAAs and production of D-BCAAs labeled with stable isotopes. This system is based on two thermostable enzymes: D-amino acid dehydrogenase, catalyzing NADPH-dependent enantioselective amination of 2-oxo acids to produce the corresponding D-amino acids, and glucose dehydrogenase, catalyzing NADPH regeneration from NADP(+) and D-glucose. After incubation with the enzymes for 2 h at 65°C and pH 10.5, 2-oxo-4-methylvaleric acid was converted to D-leucine with an excellent yield (>99 %) and optical purity (>99 %). Using this system, we produced five different D-BCAAs labeled with stable isotopes: D-[1-(13)C,(15)N]leucine, D-[1-(13)C]leucine, D-[(15)N]leucine, D-[(15)N]isoleucine, and D-[(15)N]valine. The structure of each labeled D-amino acid was confirmed using time-of-flight mass spectrometry and nuclear magnetic resonance analysis. These analyses confirmed that the developed system was highly useful for production of D-BCAAs labeled with stable isotopes, making this the first reported enzymatic production of D-BCAAs labeled with stable isotopes. Our findings facilitate tracer studies investigating D-BCAAs and their derivatives.

  16. Alcohol dehydrogenase (ADH activity in soybean (Glycine max [L.] Merr. under flooding stress

    Directory of Open Access Journals (Sweden)

    Govinda Rizal and Shanta Karki

    2011-03-01

    Full Text Available Sowing time of soybean (Glycine max [L.] Merr. often coincides with the early onset of rainy season. Germinating seedsencounter a transient to prolonged period of water-logging that causes anoxia (absence of oxygen and hypoxia (insufficientoxygen resulting in poor germination. This reduces crop stability and yield. One of the factors responsible for flood tolerance isactivity of alcohol dehydrogenase (ADH during flood. The effect of ADH activity during flooding and difference in floodtolerance level were investigated using two soybean cultivars, Peking and Tamahomare, and their F9 recombinant inbred lines(RILs. Tamahomare showed higher ADH activity than Peking. There was a great variation in ADH activity among the RILs.QTL analysis detected five QTLs for ADH activity (qAas1-5 on five linkage groups, LG_A2, D1a, F, K and L. The QTL qAas4was close to a QTL for shoot damage and conductivity of germinating seeds after flooding treatment.

  17. 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.

  18. 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.

  19. Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana.

    Science.gov (United States)

    Yoshida, Keisuke; Hisabori, Toru

    2016-06-01

    Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower K(I). Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance.

  20. 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.

  1. 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.

  2. Green tea polyphenols control dysregulated glutamate dehydrogenase in transgenic mice by hijacking the ADP activation site.

    Science.gov (United States)

    Li, Changhong; Li, Ming; Chen, Pan; Narayan, Srinivas; Matschinsky, Franz M; Bennett, Michael J; Stanley, Charles A; Smith, Thomas J

    2011-09-30

    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 β-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.

  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. Inhibition of aldehyde dehydrogenase 2 activity enhances antimycin-induced rat cardiomyocytes apoptosis through activation of MAPK signaling pathway.

    Science.gov (United States)

    Zhang, Peng; Xu, Danling; Wang, Shijun; Fu, Han; Wang, Keqiang; Zou, Yunzeng; Sun, Aijun; Ge, Junbo

    2011-12-01

    Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial-specific enzyme, has been proved to be involved in oxidative stress-induced cell apoptosis, while little is known in cardiomyocytes. This study was aimed at investigating the role of ALDH2 in antimycin A-induced cardiomyocytes apoptosis by suppressing ALDH2 activity with a specific ALDH2 inhibitor Daidzin. Antimycin A (40μg/ml) was used to induce neonatal cardiomyocytes apoptosis. Daidzin (60μM) effectively inhibited ALDH2 activity by 50% without own effect on cell apoptosis, and significantly enhanced antimycin A-induced cardiomyocytes apoptosis from 33.5±4.4 to 56.5±6.4% (Hochest method, pdaidzin treated cardiomyocytes compared to the cells treated with antimycin A alone. These findings indicated that modifying mitochondrial ALDH2 activity/expression might be a potential therapeutic option on reducing oxidative insults induced cardiomyocytes apoptosis.

  5. Reduced 11beta-hydroxysteroid dehydrogenase activity in patients with the nephrotic syndrome.

    Science.gov (United States)

    Vogt, B; Dick, B; N'Gankam, V; Frey, F J; Frey, B M

    1999-02-01

    Patients with the nephrotic syndrome (NS) exhibit abnormal renal sodium retention which cannot completely explained by a secondary hyperaldosteronism due to reduced renal perfusion. As an alternative mechanism to explain this phenomenon we postulate a cortisol-mediated mineralocorticoid effect as a consequence of a reduced activity of 11beta-hydroxysteroid dehydrogenase (11beta-HSD). A down-regulation of 11beta-HSD, i.e. of the shuttle of active to inactive glucocorticosteroids, has been shown to cause mineralocorticoid effects. Therefore we investigated the activity of 11beta-HSD by measuring the urinary ratio of (tetrahydrocortisol + 5alpha-tetrahydrocortisol)/tetrahydrocortisone [(THF+5alpha-THF)/THE] by gas-chromatography in 29 NS patients with biopsy-proven glomerulonephritis and 29 healthy control subjects. The ratio of (THF+5alpha-THF)/THE was higher in NS patients (median 1.49, range 0.45-4.07) than in the control subjects (0.98, 0.60-1.36; pnew mechanism contributing to the exaggerated sodium retention in patients with the NS.

  6. 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.

  7. 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.

  8. Identification of Tumor Endothelial Cells with High Aldehyde Dehydrogenase Activity and a Highly Angiogenic Phenotype

    Science.gov (United States)

    Maishi, Nako; Ohga, Noritaka; Hida, Yasuhiro; Kawamoto, Taisuke; Iida, Junichiro; Shindoh, Masanobu; Tsuchiya, Kunihiko; Shinohara, Nobuo; Hida, Kyoko

    2014-01-01

    Tumor blood vessels play an important role in tumor progression and metastasis. It has been reported that tumor endothelial cells (TECs) exhibit highly angiogenic phenotypes compared with those of normal endothelial cells (NECs). TECs show higher proliferative and migratory abilities than those NECs, together with upregulation of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Furthermore, compared with NECs, stem cell markers such as Sca-1, CD90, and multidrug resistance 1 are upregulated in TECs, suggesting that stem-like cells exist in tumor blood vessels. In this study, to reveal the biological role of stem-like TECs, we analyzed expression of the stem cell marker aldehyde dehydrogenase (ALDH) in TECs and characterized ALDHhigh TECs. TECs and NECs were isolated from melanoma-xenografted nude mice and normal dermis, respectively. ALDH mRNA expression and activity were higher in TECs than those in NECs. Next, ALDHhigh/low TECs were isolated by fluorescence-activated cell sorting to compare their characteristics. Compared with ALDHlow TECs, ALDHhigh TECs formed more tubes on Matrigel-coated plates and sustained the tubular networks longer. Furthermore, VEGFR2 expression was higher in ALDHhigh TECs than that in ALDHlow TECs. In addition, ALDH was expressed in the tumor blood vessels of in vivo mouse models of melanoma and oral carcinoma, but not in normal blood vessels. These findings indicate that ALDHhigh TECs exhibit an angiogenic phenotype. Stem-like TECs may have an essential role in tumor angiogenesis. PMID:25437864

  9. Identification of tumor endothelial cells with high aldehyde dehydrogenase activity and a highly angiogenic phenotype.

    Directory of Open Access Journals (Sweden)

    Hitomi Ohmura-Kakutani

    Full Text Available Tumor blood vessels play an important role in tumor progression and metastasis. It has been reported that tumor endothelial cells (TECs exhibit highly angiogenic phenotypes compared with those of normal endothelial cells (NECs. TECs show higher proliferative and migratory abilities than those NECs, together with upregulation of vascular endothelial growth factor (VEGF and VEGF receptor 2 (VEGFR2. Furthermore, compared with NECs, stem cell markers such as Sca-1, CD90, and multidrug resistance 1 are upregulated in TECs, suggesting that stem-like cells exist in tumor blood vessels. In this study, to reveal the biological role of stem-like TECs, we analyzed expression of the stem cell marker aldehyde dehydrogenase (ALDH in TECs and characterized ALDHhigh TECs. TECs and NECs were isolated from melanoma-xenografted nude mice and normal dermis, respectively. ALDH mRNA expression and activity were higher in TECs than those in NECs. Next, ALDHhigh/low TECs were isolated by fluorescence-activated cell sorting to compare their characteristics. Compared with ALDHlow TECs, ALDHhigh TECs formed more tubes on Matrigel-coated plates and sustained the tubular networks longer. Furthermore, VEGFR2 expression was higher in ALDHhigh TECs than that in ALDHlow TECs. In addition, ALDH was expressed in the tumor blood vessels of in vivo mouse models of melanoma and oral carcinoma, but not in normal blood vessels. These findings indicate that ALDHhigh TECs exhibit an angiogenic phenotype. Stem-like TECs may have an essential role in tumor angiogenesis.

  10. 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.

  11. Optimization of enzyme assisted extraction of Fructus Mori polysaccharides and its activities on antioxidant and alcohol dehydrogenase.

    Science.gov (United States)

    Deng, Qingfang; Zhou, Xin; Chen, Huaguo

    2014-10-13

    In the present study, enzyme assisted extraction of Fructus Mori polysaccharides (FMPS) from F. mori using four kinds of enzymes and three compound enzymes were examined. Research found that glucose oxidase offered a better performance in enhancement of the extraction yields of FMPS, antioxidant and activate alcohol dehydrogenase activities. The glucose oxidase assisted extraction process was further optimized by using response surface method (RSM) to obtain maximum yield of crude FMPS. The results showed that optimized extraction conditions were ratio of enzyme amount 0.40%, enzyme treated time 38 min, treated temperature 58 °C and liquid-solid radio 11.0. Under these conditions, the mean experimental value of extraction yield (16.16 ± 0.14%) corresponded well with the predicted values and increased 160% than none enzyme treated ones. Pharmacological verification tests showed that F. mori crude polysaccharides had good antioxidant and activate alcohol dehydrogenase activities in vitro. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. 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.

  13. 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

  14. Synthesis and biological evaluation of phosphate prodrugs of 4-phospho-D-erythronohydroxamic acid, an inhibitor of 6-phosphogluconate dehydrogenase.

    Science.gov (United States)

    Ruda, Gian Filippo; Alibu, Vincent P; Mitsos, Christos; Bidet, Olivier; Kaiser, Marcel; Brun, Reto; Barrett, Michael P; Gilbert, Ian H

    2007-08-01

    We have previously reported the discovery of potent and selective inhibitors of 6-phosphogluconate dehydrogenase, the third enzyme of the phosphate pentose pathway, from Trypanosoma brucei, the causative organism of human African trypanosomiasis. These inhibitors were charged phosphate derivatives with restricted capacity to enter cells. Herein, we report the synthesis of five different classes of prodrugs: phosphoramidate; bis-S-acyl thioethyl esters (bis-SATE); bis-pivaloxymethyl (bis-POM); CycloSaligenyl; and phenyl, S-acyl thioethyl mixed phosphate esters (mix-SATE). Prodrugs were studied for stability and activity against the intact parasites. Most prodrugs caused inhibition of the growth of the parasites. The activity of the prodrugs against the parasites appeared to be related to their stability in aqueous buffer.

  15. Urinary Lactate Dehydrogenase Activity and Its Isozyme Patterns in Kawasaki Disease

    Science.gov (United States)

    Kawamura, Yoichi; Kanai, Takashi; Takizawa, Mari; Yoshida, Yusuke; Tsujita, Yuki; Nonoyama, Shigeaki

    2017-01-01

    Abnormal urinary findings, such as sterile pyuria, proteinuria, and microscopic hematuria, are often seen in the acute phase of Kawasaki disease (KD). We investigated the potential significance of urinary lactate dehydrogenase (U-LDH) activity and its isozyme patterns in KD. Total U-LDH activity and its isozymes (U-LDH1-5) levels were compared among 120 patients with KD, 18 patients with viral infection (VI), and 43 patients with upper urinary tract infection (UTI) and additionally compared between intravenous immunoglobulin (IVIG) responders (n = 89) and nonresponders (n = 31) with KD. Total U-LDH activity was higher in KD (35.4 ± 4.8 IU/L, P < 0.05) and UTI patients (66.0 ± 8.0 IU/L, P < 0.01) than in VI patients (17.0 ± 6.2 IU/L). In the isozyme pattern analysis, KD patients had high levels of U-LDH1 and U-LDH2, while UTI patients had high levels of U-LDH3, U-LDH4, and U-LDH5. Furthermore, IVIG nonresponders of KD had significantly higher levels of total U-LDH activity (45.1 ± 4.7 IU/L, P < 0.05), especially U-LDH1 and U-LDH2 (P < 0.05), than IVIG responders (32.0 ± 2.8 IU/L). KD patients have increased levels of total U-LDH activity, especially U-LDH-1 and U-LDH2, indicating a unique pattern of U-LDH isozymes different from that in UTI patients. PMID:28348604

  16. Expanded Hematopoietic Progenitor Cells Reselected for High Aldehyde Dehydrogenase Activity Demonstrate Islet Regenerative Functions.

    Science.gov (United States)

    Seneviratne, Ayesh K; Bell, Gillian I; Sherman, Stephen E; Cooper, Tyler T; Putman, David M; Hess, David A

    2016-04-01

    Human umbilical cord blood (UCB) hematopoietic progenitor cells (HPC) purified for high aldehyde dehydrogenase activity (ALDH(hi) ) stimulate islet regeneration after transplantation into mice with streptozotocin-induced β cell deletion. However, ALDH(hi) cells represent a rare progenitor subset and widespread use of UCB ALDH(hi) cells to stimulate islet regeneration will require progenitor cell expansion without loss of islet regenerative functions. Here we demonstrate that prospectively purified UCB ALDH(hi) cells expand efficiently under serum-free, xeno-free conditions with minimal growth factor supplementation. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, kinetic analyses over 9 days revealed the frequency of ALDH(hi) cells diminished as culture time progressed such that total ALDH(hi) cell number was maximal (increased 3-fold) at day 6. Subsequently, day 6 expanded cells (bulk cells) were sorted after culture to reselect differentiated progeny with low ALDH-activity (ALDH(lo) subset) from less differentiated progeny with high ALDH-activity (ALDH(hi) subset). The ALDH(hi) subset retained primitive cell surface marker coexpression (32.0% ± 7.0% CD34(+) /CD38(-) cells, 37.0% ± 6.9% CD34(+) /CD133(+) cells), and demonstrated increased hematopoietic colony forming cell function compared with the ALDH(lo) subset. Notably, bulk cells or ALDH(lo) cells did not possess the functional capacity to lower hyperglycemia after transplantation into streptozotocin-treated NOD/SCID mice. However, transplantation of the repurified ALDH(hi) subset significantly reduced hyperglycemia, improved glucose tolerance, and increased islet-associated cell proliferation and capillary formation. Thus, expansion and delivery of reselected UCB cells that retain high ALDH-activity after short-term culture represents an improved strategy for the development of cellular therapies to enhance islet regeneration in situ.

  17. Urinary Lactate Dehydrogenase Activity and Its Isozyme Patterns in Kawasaki Disease

    Directory of Open Access Journals (Sweden)

    Yoichi Kawamura

    2017-01-01

    Full Text Available Abnormal urinary findings, such as sterile pyuria, proteinuria, and microscopic hematuria, are often seen in the acute phase of Kawasaki disease (KD. We investigated the potential significance of urinary lactate dehydrogenase (U-LDH activity and its isozyme patterns in KD. Total U-LDH activity and its isozymes (U-LDH1-5 levels were compared among 120 patients with KD, 18 patients with viral infection (VI, and 43 patients with upper urinary tract infection (UTI and additionally compared between intravenous immunoglobulin (IVIG responders (n=89 and nonresponders (n=31 with KD. Total U-LDH activity was higher in KD (35.4±4.8 IU/L, P<0.05 and UTI patients (66.0±8.0 IU/L, P<0.01 than in VI patients (17.0±6.2 IU/L. In the isozyme pattern analysis, KD patients had high levels of U-LDH1 and U-LDH2, while UTI patients had high levels of U-LDH3, U-LDH4, and U-LDH5. Furthermore, IVIG nonresponders of KD had significantly higher levels of total U-LDH activity (45.1±4.7 IU/L, P<0.05, especially U-LDH1 and U-LDH2 (P<0.05, than IVIG responders (32.0±2.8 IU/L. KD patients have increased levels of total U-LDH activity, especially U-LDH-1 and U-LDH2, indicating a unique pattern of U-LDH isozymes different from that in UTI patients.

  18. Effects of DNA on immunoglobulin production stimulating activity of alcohol dehydrogenase.

    Science.gov (United States)

    Okamoto, T; Furutani, H; Sasaki, T; Sugahara, T

    1999-09-01

    Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells and lymphocytes. The IPSF activity of ADH-I was suppressed by coexistence of short DNA whose chain length is less than 200 base pairs (bp) and fibrous DNA in a dose-dependent manner. These DNA preparations completely inhibited the IPSF activity at the concentration of 250 mug/ml and 1.0 mg/ml, respectively. DNA sample termed long DNA whose average chain length is 400-7000 bp slightly stimulated IPSF activity at 0.06 mug/ml. However, long DNA suppressed IPSF activity by half at 1.0 mg/ml. The laser confocal microscopic analysis had revealed that ADH-I was incorporated by HB4C5 cells. The uptake of ADH-I was strongly inhibited by short DNA and fibrous DNA. However, long DNA did not suppress the internalization of ADH-I into HB4C5 cells. These findings indicate that short DNA and fibrous DNA depress IPSF activity of ADH-I by inhibiting the internalization of this enzyme. According to the gel-filtration analysis using HPLC, ADH-I did not directly interact with short DNA. It is expected from these findings that short DNA influences HB4C5 cells to suppress the internalization of ADH-I. Moreover, these facts also strongly suggest that ADH-I acts as IPSF after internalization into the cell.

  19. 5年生人参出苗期几种脱氢酶活力比较%Comparison of Dehydrogenase Activity in 5-year Ginseng during Seedling Stage

    Institute of Scientific and Technical Information of China (English)

    刘宏; 赵雨; 邢楠楠; 张惠; 刘海龙

    2012-01-01

    OBJECTIVE To compare the activities of malate dehydrogenase (MDH), lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PDH) and α-Glycerophosphate dehydrogenase (α-GPD) in 5-year Ginseng Radix in seedling stage. METHODS Adopt neutral buffer solution to extract the coarse enzyme. Use spectrophotometry to test the activities of MDH, LDH, ADH, G6PDH, α-GPD. RESULTS In seedling stage of Ginseng Radix, there were different changes trends of these five dehydrogenase in root and sprout. And there were peak values in different stages. CONCLUSION The activities of MDH, LDH, ADH, G6PDH, a-GPD can be used as the evaluation indicators of quality of Ginseng Radix.%目的 对5年生人参出苗期参根和参苗中苹果酸脱氢酶(malate dehydrogenase,MDH)、乳酸脱氢酶(lactate dehydrogenase,LDH)、乙醇脱氢酶(Alcohol dehydrogenase,ADH)、葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PDH)、α-磷酸甘油脱氢酶(α-glycerophosphate dehydrogenase,α-GPD)5种脱氢酶的活力进行比较.方法 采用中性缓冲液提取粗酶液,应用分光光度法测定MDH,LDH,ADH,G6PDH,α-GPD的活力.结果 在人参出苗期,参根和参苗中的5种脱氢酶活力变化趋势有所不同,并在不同时期出现峰值.结论 MDH,LDH,ADH,G6PDH,α-GPD的活力可以作为人参生长过程中长势优劣的评价指标.

  20. 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.

  1. Reduced mitochondrial malate dehydrogenase activity has a strong effect on photorespiratory metabolism as revealed by 13C labelling.

    Science.gov (United States)

    Lindén, Pernilla; Keech, Olivier; Stenlund, Hans; Gardeström, Per; Moritz, Thomas

    2016-05-01

    Mitochondrial malate dehydrogenase (mMDH) catalyses the interconversion of malate and oxaloacetate (OAA) in the tricarboxylic acid (TCA) cycle. Its activity is important for redox control of the mitochondrial matrix, through which it may participate in regulation of TCA cycle turnover. In Arabidopsis, there are two isoforms of mMDH. Here, we investigated to which extent the lack of the major isoform, mMDH1 accounting for about 60% of the activity, affected leaf metabolism. In air, rosettes of mmdh1 plants were only slightly smaller than wild type plants although the fresh weight was decreased by about 50%. In low CO2 the difference was much bigger, with mutant plants accumulating only 14% of fresh weight as compared to wild type. To investigate the metabolic background to the differences in growth, we developed a (13)CO2 labelling method, using a custom-built chamber that enabled simultaneous treatment of sets of plants under controlled conditions. The metabolic profiles were analysed by gas- and liquid- chromatography coupled to mass spectrometry to investigate the metabolic adjustments between wild type and mmdh1 The genotypes responded similarly to high CO2 treatment both with respect to metabolite pools and (13)C incorporation during a 2-h treatment. However, under low CO2 several metabolites differed between the two genotypes and, interestingly most of these were closely associated with photorespiration. We found that while the glycine/serine ratio increased, a concomitant altered glutamine/glutamate/α-ketoglutarate relation occurred. Taken together, our results indicate that adequate mMDH activity is essential to shuttle reductants out from the mitochondria to support the photorespiratory flux, and strengthen the idea that photorespiration is tightly intertwined with peripheral metabolic reactions.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Activation of Human Salivary Aldehyde Dehydrogenase by Sulforaphane: Mechanism and Significance

    Science.gov (United States)

    Alam, Md. Fazle; Laskar, Amaj Ahmed; Maryam, Lubna

    2016-01-01

    Cruciferous vegetables contain the bio-active compound sulforaphane (SF) which has been reported to protect individuals against various diseases by a number of mechanisms, including activation of the phase II detoxification enzymes. In this study, we show that the extracts of five cruciferous vegetables that we commonly consume and SF activate human salivary aldehyde dehydrogenase (hsALDH), which is a very important detoxifying enzyme in the mouth. Maximum activation was observed at 1 μg/ml of cabbage extract with 2.6 fold increase in the activity. There was a ~1.9 fold increase in the activity of hsALDH at SF concentration of ≥ 100 nM. The concentration of SF at half the maximum response (EC50 value) was determined to be 52 ± 2 nM. There was an increase in the Vmax and a decrease in the Km of the enzyme in the presence of SF. Hence, SF interacts with the enzyme and increases its affinity for the substrate. UV absorbance, fluorescence and CD studies revealed that SF binds to hsALDH and does not disrupt its native structure. SF binds with the enzyme with a binding constant of 1.23 x 107 M-1. There is one binding site on hsALDH for SF, and the thermodynamic parameters indicate the formation of a spontaneous strong complex between the two. Molecular docking analysis depicted that SF fits into the active site of ALDH3A1, and facilitates the catalytic mechanism of the enzyme. SF being an antioxidant, is very likely to protect the catalytic Cys 243 residue from oxidation, which leads to the increase in the catalytic efficiency and hence the activation of the enzyme. Further, hsALDH which is virtually inactive towards acetaldehyde exhibited significant activity towards it in the presence of SF. It is therefore very likely that consumption of large quantities of cruciferous vegetables or SF supplements, through their activating effect on hsALDH can protect individuals who are alcohol intolerant against acetaldehyde toxicity and also lower the risk of oral cancer

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

    Directory of Open Access Journals (Sweden)

    Delphine Mirebeau-Prunier

    Full Text Available 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.

  5. 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)

  6. Effects of silver nanoparticle on lactate dehydrogenase activity and histological changes of heart tissue in male wistar rats

    Directory of Open Access Journals (Sweden)

    Noushin Naghsh

    2013-03-01

    Full Text Available Background & Objective: The silver nanoparticles are important in many applications of nanoparticles on human health . The toxicity of silver nanoparticles are not well documented yet. The aim of this study was to investigate the effect of silver nanoparticles on lactate dehydrogenase activity and histological changes in heart tissue.   Materials &Methods: In this study, 40 adult male wistar rats of 220±20gr were divided in to five groups including control and four experimental groups. The latter groups were injected intraperitoneally spherical nano silver particles of 50, 100, 200 and 400 ppm respectively for five consecutive days. Then three, eight and twelve days after the last injection, blood samples were collected and lactate dehydrogenase (LDH activity was assayed . Also, tissue samples from the heart muscle were prepared and studied after staining with Hematoxiline-Eosine. Data of LDH activity was analyzed by One way- ANOVA- test and P-value of ≤ 0.05 were considered as significant.   Results : The result showed that different concentrations of silver nanoparticles have no significant effect on the lactate dehydrogenase (p=0.192 . T he histological study of the tissue after exposure to 400 ppm concentration of silver nanoparticles showed the start of primary apoptosis in heart tissue.   Conclusion: The LDH activity was not changed significantly after exposure to different concentration of silver nanoparticles, which shows the safety of these particles on LDH activity.

  7. 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.

  8. Benzofuran derivatives inhibit 11β-hydroxysteroid dehydrogenase type 1 activity in rat adipose tissue.

    Science.gov (United States)

    Kiyonaga, Daisuke; Tagawa, Noriko; Yamaguchi, Yuko; Wakabayashi, Midori; Kogure, Toshiaki; Ueda, Masafumi; Miyata, Okiko; Kobayashi, Yoshiharu

    2012-01-01

    Excess glucocorticoids promote visceral obesity and insulin resistance. The main regulator of intracellular glucocorticoid levels are 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts inactive glucocorticoid into bioactive glucocorticoid such as cortisol in humans and corticosterone in rodents; therefore, the inhibition of 11β-HSD1 has considerable therapeutic potential for metabolic diseases including obesity and diabetes. Benzofuran is a key structure in many biologically active compounds such as benzbromarone, malibatol A and (+)-liphagal. The aim of this study was to investigate the inhibitory effect of benzofuran derivatives on 11β-HSD1 in mesenteric adipose tissue from rodents. 11β-HSD1 activity was determined by incubation of rat mesenteric adipose tissue microsomes in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) with and without benzofuran derivatives (Compounds 1-14). The corticosterone produced was measured by HPLC. More than 40% of 11β-HSD1 inhibition was observed in Compounds 1, 5, 7 and 8. Moreover, Compounds 7 and 8 inhibited the 11β-HSD1 activity in adipose microsomes dose- and time-dependently, as well as in 3T3-L1 adipocytes. Compounds 7 and 8 did not inhibit 11β-HSD type 2 (11β-HSD2), whereas Compounds 1 and 5 inhibited 11β-HSD2 by 18.7% and 56.3%, respectively. Further, a kinetic study revealed that Compounds 7 and 8 acted as non-competitive inhibitors of 11β-HSD1. Ki (nmol/h/mg protein) values of Compounds 7 and 8 were 17.5 and 24.0, respectively, with IC50 (µM) of 10.2 and 25.6, respectively. These data indicate that Compounds 7 and 8 are convincing candidates for seed compounds as specific inhibitors of 11β-HSD1 and have the potential to be developed as anti-obesity drugs.

  9. Death mode-dependent reduction in succinate dehydrogenase activity in hair cells of aging rat cochleae

    Institute of Scientific and Technical Information of China (English)

    YANG Wei-ping; HU Bo-hua; SUN Jian-he; ZHAI Suo-qiang; Donald Henderson

    2010-01-01

    Background Our previous studies have shown that both apoptosis and necrosis are involved in hair cell (HC) pathogenesis in aging cochleae. To better understand the biological mechanisms responsible for the regulation of HC death, we examined the activity of succinate dehydrogenase (SDH), a mitochondrial bioenergetic enzyme, in the HCs of aging cochleae.Methods The auditory brainstem response thresholds elicited by tone bursts at 4, 10 and 20 kHz were measured in both young (2-3 months) and aging (22-23 months) Wistar rats. SDH activity was evaluated with a colorimetric assay using nitroblue tetrazolium monosodium salt. The SDH-labeled organs of Corti were double stained with propidium iodide, a DNA intercalating fluorescent probe for illustration of HC nuclei. All the specimens were examined with fluorescence microscopy and confocal microscopy.Results Aging rats exhibited a significant elevation of ABR thresholds with threshold shifts being 34 dB at 20 kHz, 28 dB at 10 kHz, and 25 dB at 4 kHz. Consistent with the reduction in the cochlear function, aging cochleae exhibited the reduction of SDH staining intensity in the apical and the basal ends of the cochleae, where a large number of apoptotic, necrotic, and missing HCs were evident. The reduction in SDH staining appeared in a cell-death-mode dependent fashion. Specifically, SDH labeling remained in apoptotic HCs. In contrast, SDH staining was markedly reduced or absent in necrotic HCs.Conclusions In the aging cochlea, SDH activity is preserved in HCs undergoing apoptosis, but is substantially reduced in necrosis. These results suggest that mitochondrial energetic function is involved in the regulation of cell death pathways in the pathogenesis of aging cochleae.

  10. Effects of high-fat diet and physical activity on pyruvate dehydrogenase kinase-4 in mouse skeletal muscle

    Directory of Open Access Journals (Sweden)

    Rinnankoski-Tuikka Rita

    2012-06-01

    Full Text Available Abstract Background The expression of PDK4 is elevated by diabetes, fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. It is previously shown that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α, a master regulator of energy metabolism, coactivates in cell lines pyruvate dehydrogenase kinase-4 (PDK4 gene expression via the estrogen-related receptor α (ERRα. We investigated the effects of long-term high-fat diet and physical activity on the expression of PDK4, PGC-1α and ERRα and the amount and function of mitochondria in skeletal muscle. Methods Insulin resistance was induced by a high-fat (HF diet for 19 weeks in C57BL/6 J mice, which were either sedentary or with access to running wheels. The skeletal muscle expression levels of PDK4, PGC-1α and ERRα were measured and the quality and quantity of mitochondrial function was assessed. Results The HF mice were more insulin-resistant than the low-fat (LF -fed mice. Upregulation of PDK4 and ERRα mRNA and protein levels were seen after the HF diet, and when combined with running even more profound effects on the mRNA expression levels were observed. Chronic HF feeding and voluntary running did not have significant effects on PGC-1α mRNA or protein levels. No remarkable difference was found in the amount or function of mitochondria. Conclusions Our results support the view that insulin resistance is not mediated by the decreased qualitative or quantitative properties of mitochondria. Instead, the role of PDK4 should be contemplated as a possible contributor to high-fat diet-induced insulin resistance.

  11. Studies on Electrolyte Conductivity and Activity of Dehydrogenase of Chinese Fir and Masson Pine Bare-Root Seedling under Water and Cold Stress

    Institute of Scientific and Technical Information of China (English)

    Yu Fangyuan; Xu Xizeng; Guo Xinbao

    2003-01-01

    The electrolyte conductivity and activity of dehydrogenase of bare-root seedlings of both Chinese fir (Cunningha-mia lanceolata (Lamb.) Hook.) and Masson pine (Pinus massoniana Lamb.) under freezing and desiccation treatments were studied.The results showed that needle electrolyte conductivity of both species increase significantly after freezing treatment and there are nosignificant differences in needle electrolyte conductivity between the two species. The dehydrogenase activity (ARD) of fine roots ofboth Chinese fir and Masson pine was negatively correlated with increasing freezing and desiccation. The results suggest that bothelectrolyte conductivity and dehydrogenase activity could be used as quick indicators of Chinese fir and Masson pine bare-root seed-ling quality.

  12. α-Ketoglutarate Accumulation Is Not Dependent on Isocitrate Dehydrogenase Activity during Tellurite Detoxification in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Claudia A. Reinoso

    2013-01-01

    Full Text Available Tellurite is toxic to most microorganisms because of its ability to generate oxidative stress. However, the way in which tellurite interferes with cellular processes is not fully understood to date. In this line, it was previously shown that tellurite-exposed cells displayed reduced activity of the α-ketoglutarate dehydrogenase complex (α-KGDH, which resulted in α-ketoglutarate (α-KG accumulation. In this work, we assessed if α-KG accumulation in tellurite-exposed E. coli could also result from increased isocitrate dehydrogenase (ICDH and glutamate dehydrogenase (GDH activities, both enzymes involved in α-KG synthesis. Unexpectedly both activities were found to decrease in the presence of the toxicant, an observation that seems to result from the decreased transcription of icdA and gdhA genes (encoding ICDH and GDH, resp.. Accordingly, isocitrate levels were found to increase in tellurite-exposed E. coli. In the presence of the toxicant, cells lacking icdA or gdhA exhibited decreased reactive oxygen species (ROS levels and higher tellurite sensitivity as compared to the wild type strain. Finally, a novel branch activity of ICDH as tellurite reductase is presented.

  13. Cloning of a Serratia marcescens DNA fragment that induces quinoprotein glucose dehydrogenase-mediated gluconic acid production in Escherichia coli in the presence of stationary phase Serratia marcescens.

    Science.gov (United States)

    Krishnaraj, P U; Goldstein, A H

    2001-12-18

    Serratia marcescens ER2 was isolated from an endorhizosphere sample based on its high level of mineral phosphate solubilizing (MPS) activity. This phenotype was correlated with expression of the direct oxidation pathway. An ER2 plasmid library constructed in Escherichia coli strain DH5alpha was screened for MPS activity. A recombinant clone DH5alpha (pKG3791) was capable of gluconic acid (GA) production and tricalcium phosphate solubilization but only in the presence of stationary phase ER2 cells. GA production in DH5alpha (pKG3791) was apparently the result of the quinoprotein glucose dehydrogenase activity because AG121 (a Tn5 knockout of gcd) carrying pKG3791 did not produce GA under the same conditions. GA production by DH5alpha (pKG3791) was not observed when ER2 was replaced by another PQQ-producing strain bacterium. These data add to a growing body of evidence that E. coli contains some type of PQQ biosynthesis pathway distinct from those previously characterized in Gram-negative bacteria and that these genes may be induced under appropriate conditions.

  14. Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling.

    Science.gov (United States)

    Sullivan, James P; Spinola, Monica; Dodge, Michael; Raso, Maria G; Behrens, Carmen; Gao, Boning; Schuster, Katja; Shao, Chunli; Larsen, Jill E; Sullivan, Laura A; Honorio, Sofia; Xie, Yang; Scaglioni, Pier P; DiMaio, J Michael; Gazdar, Adi F; Shay, Jerry W; Wistuba, Ignacio I; Minna, John D

    2010-12-01

    Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for ALDH1A1, ALDH3A1, and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity, and that this activity is associated with ALDH1A1 expression. Isolated ALDH(+) lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared with their ALDH(-) counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH(+) cells. Suppression of the Notch pathway by treatment with either a γ-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH(+) lung cancer cells, commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together, these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential, that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis, and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH(+) component, implicating Notch signaling in lung cancer stem cell maintenance.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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

  17. [Effect of sectioning the celiac and vagus nerves on the activity and isoenzymatic composition of rat liver lactate dehydrogenase].

    Science.gov (United States)

    Shanygina, K I; Parfernova, N S

    1977-01-01

    Total activity of lactate dehydrogenase (LDH) was increased in rat liver cytoplasm after dissection of nervus celiacus; the enzyme activity was, however, decreased after section of nervus vagus. As showen by polyacrylamide gel electrophoresis the alterations in the enzyme activity occurred mainly due to changes in the isoenzyme LDH5, prevailing in this liver fraction. Administration of insulin did not restore the LDH activity, decreased after vagotomy. The suggestion is corroborated that the regulatory effects of sympathetic and parasympathetic nervous systems on glycolysis are of oppositely directed character.

  18. 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.

  19. 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...

  20. 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.

  1. 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...

  2. Estrogen modification of human glutamate dehydrogenases is linked to enzyme activation state.

    Science.gov (United States)

    Borompokas, Nikolas; Papachatzaki, Maria-Martha; Kanavouras, Konstantinos; Mastorodemos, Vasileios; Zaganas, Ioannis; Spanaki, Cleanthe; Plaitakis, Andreas

    2010-10-08

    Mammalian glutamate dehydrogenase (GDH) is a housekeeping enzyme central to the metabolism of glutamate. Its activity is potently inhibited by GTP (IC(50) = 0.1-0.3 μM) and thought to be controlled by the need of the cell in ATP. Estrogens are also known to inhibit mammalian GDH, but at relatively high concentrations. Because, in addition to this housekeeping human (h) GDH1, humans have acquired via a duplication event an hGDH2 isoform expressed in human cortical astrocytes, we tested here the interaction of estrogens with the two human isoenzymes. The results showed that, under base-line conditions, diethylstilbestrol potently inhibited hGDH2 (IC(50) = 0.08 ± 0.01 μM) and with ∼18-fold lower affinity hGDH1 (IC(50) = 1.67 ± 0.06 μM; p < 0.001). Similarly, 17β-estradiol showed a ∼18-fold higher affinity for hGDH2 (IC(50) = 1.53 ± 0.24 μM) than for hGDH1 (IC(50) = 26.94 ± 1.07 μM; p < 0.001). Also, estriol and progesterone were more potent inhibitors of hGDH2 than hGDH1. Structure/function analyses revealed that the evolutionary R443S substitution, which confers low basal activity, was largely responsible for sensitivity of hGDH2 to estrogens. Inhibition of both human GDHs by estrogens was inversely related to their state of activation induced by ADP, with the slope of this correlation being steeper for hGDH2 than for hGDH1. Also, the study of hGDH1 and hGDH2 mutants displaying different states of activation revealed that the affinity of estrogen for these enzymes correlated inversely (R = 0.99; p = 0.0001) with basal catalytic activity. Because astrocytes are known to synthesize estrogens, these hormones, by interacting potently with hGDH2 in its closed state, may contribute to regulation of glutamate metabolism in brain.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    . 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.

  7. 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...

  8. Isocitrate dehydrogenase 1 mutations prime the all-trans retinoic acid myeloid differentiation pathway in acute myeloid leukemia.

    Science.gov (United States)

    Boutzen, Héléna; Saland, Estelle; Larrue, Clément; de Toni, Fabienne; Gales, Lara; Castelli, Florence A; Cathebas, Mathilde; Zaghdoudi, Sonia; Stuani, Lucille; Kaoma, Tony; Riscal, Romain; Yang, Guangli; Hirsch, Pierre; David, Marion; De Mas-Mansat, Véronique; Delabesse, Eric; Vallar, Laurent; Delhommeau, François; Jouanin, Isabelle; Ouerfelli, Ouathek; Le Cam, Laurent; Linares, Laetitia K; Junot, Christophe; Portais, Jean-Charles; Vergez, François; Récher, Christian; Sarry, Jean-Emmanuel

    2016-04-04

    Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs caused by recurrent mutations, such as the isocitrate dehydrogenase (IDH) mutations found in 15% of AML patients. These mutations result in the production of the oncometabolite (R)-2-hydroxyglutarate (2-HG), leading to a hypermethylation phenotype that dysregulates hematopoietic differentiation. In this study, we identified mutant R132H IDH1-specific gene signatures regulated by key transcription factors, particularly CEBPα, involved in myeloid differentiation and retinoid responsiveness. We show that treatment with all-trans retinoic acid (ATRA) at clinically achievable doses markedly enhanced terminal granulocytic differentiation in AML cell lines, primary patient samples, and a xenograft mouse model carrying mutant IDH1. Moreover, treatment with a cell-permeable form of 2-HG sensitized wild-type IDH1 AML cells to ATRA-induced myeloid differentiation, whereas inhibition of 2-HG production significantly reduced ATRA effects in mutant IDH1 cells. ATRA treatment specifically decreased cell viability and induced apoptosis of mutant IDH1 blasts in vitro. ATRA also reduced tumor burden of mutant IDH1 AML cells xenografted in NOD-Scid-IL2rγ(null)mice and markedly increased overall survival, revealing a potent antileukemic effect of ATRA in the presence of IDH1 mutation. This therapeutic strategy holds promise for this AML patient subgroup in future clinical studies.

  9. Changes in succinate dehydrogenase activity in various parts of the brain during combined exposure to vibration and licorice root.

    Science.gov (United States)

    Oganisyan, A O; Oganesyan, K R; Minasyan, S M

    2005-06-01

    Data obtained in the studies reported here provide evidence that during exposure to vibration for 30 days, feeding with licorice root significantly increases the activity of the anaerobic respiration enzyme succinate dehydrogenase (SDH) in the cerebral cortex, while activity in the subcortex, conversely, decreases. Combined treatment for 30 days with licorice root and vibration after a preliminary 30-day period of feeding with licorice root resulted in high SDH activity in all the structures studied, improving brain energy supply and metabolism and ameliorating the effect of vibration.

  10. Purification, characterization, and cloning of a bifunctional molybdoenzyme with hydratase and alcohol dehydrogenase activity

    NARCIS (Netherlands)

    Jin, J.; Straathof, A.J.J.; Pinkse, M.W.H.; Hanefeld, U.

    2010-01-01

    A bifunctional hydratase/alcohol dehydrogenase was isolated from the cyclohexanol degrading bacterium Alicycliphilus denitrificans DSMZ 14773. The enzyme catalyzes the addition of water to α,β-unsaturated carbonyl compounds and the subsequent alcohol oxidation. The purified enzyme showed three

  11. Purification, characterization, and cloning of a bifunctional molybdoenzyme with hydratase and alcohol dehydrogenase activity

    NARCIS (Netherlands)

    Jin, J.; Straathof, A.J.J.; Pinkse, M.W.H.; Hanefeld, U.

    2010-01-01

    A bifunctional hydratase/alcohol dehydrogenase was isolated from the cyclohexanol degrading bacterium Alicycliphilus denitrificans DSMZ 14773. The enzyme catalyzes the addition of water to α,β-unsaturated carbonyl compounds and the subsequent alcohol oxidation. The purified enzyme showed three subun

  12. Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Doherty, R.E.; Haywood-Small, S.L. [Biomedical Research Centre, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom); Sisley, K. [Department of Oncology, Academic Unit of Ophthalmology and Orthopties, University of Sheffield, Sheffield S10 2RX (United Kingdom); Cross, N.A., E-mail: n.cross@shu.ac.uk [Biomedical Research Centre, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Isolated ALDH{sup Hi} PC3 cells preferentially form primitive holoclone-type colonies. Black-Right-Pointing-Pointer Primitive holoclone colonies are predominantly ALDH{sup Lo} but contain rare ALDH{sup Hi} cells. Black-Right-Pointing-Pointer Holoclone-forming cells are not restricted to the ALDH{sup Hi} population. Black-Right-Pointing-Pointer ALDH phenotypic plasticity occurs in PC3 cells (ALDH{sup Lo} to ALDH{sup Hi} and vice versa). Black-Right-Pointing-Pointer ALDH{sup Hi} cells are observed but very rare in PC3 spheroids grown in stem cell medium. -- Abstract: Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH{sup Lo} cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH{sup Hi} population, or whether all ALDH{sup Hi} cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH{sup Hi} cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH{sup Hi} cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH{sup Lo} population can develop ALDH{sup Hi} populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH{sup Hi} cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in

  13. Selective modification of the pyruvate dehydrogenase kinase isoform profile in skeletal muscle in hyperthyroidism: implications for the regulatory impact of glucose on fatty acid oxidation.

    Science.gov (United States)

    Sugden, M C; Lall, H S; Harris, R A; Holness, M J

    2000-11-01

    The pyruvate dehydrogenase kinases (PDK1-4) regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Immunoblot analysis with antibodies raised against recombinant PDK isoforms demonstrated changes in PDK isoform expression in response to experimental hyperthyroidism (100 microg/100 g body weight; 3 days) that was selective for fast-twitch vs slow-twitch skeletal muscle in that PDK2 expression was increased in the fast-twitch skeletal muscle (the anterior tibialis) (by 1. 6-fold; P lactate --> glucose (Cori) and glucose --> alanine --> glucose cycles. We also propose that enhanced relative expression of the pyruvate-insensitive PDK isoform (PDK4) in skeletal muscle in hyperthyroidism uncouples glycolytic flux from pyruvate oxidation, sparing pyruvate for non-oxidative entry into the tricarboxylic acid (TCA) cycle, and thereby supporting entry of acetyl-CoA (derived from fatty acid oxidation) into the TCA cycle.

  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. Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

    Science.gov (United States)

    Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

    2011-06-01

    Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.

  16. 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

    Aim. To investigate the direct relationship between tumour hypoxia and lactate dehydrogenase (Ldh) levels in serum and tumour in two different pre-clinical murine models. Materials and methods. Experiments were performed in CDF1 or C3H/Km mice implanted with a C3H mammary carcinoma and SCCVII...... 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...

  17. Inhibition by N'-nitrosonornicotine of the catalytic activity of glutamate dehydrogenase in alpha-ketoglutarate amination.

    Science.gov (United States)

    Mao, You-An; Zhong, Ke-Jun; Wei, Wan-Zhi; Wei, Xin-Liang; Lu, Hong-Bing

    2005-02-01

    The effect of N'-nitrosonornicotine (NNN), one of the tobacco-specific nitrosamines, on the catalytic activity of glutamate dehydrogenase (GLDH) in the alpha-ketoglutarate amination, using reduced nicotinamide adenine dinucleotide as coenzyme, was studied by a chronoamperometric method. The maximum reaction rate of the enzyme-catalyzed reaction and the Michaelis-Menten constant, or the apparent Michaelis-Menten constant, were determined in the absence and presence of NNN. NNN remarkably inhibited the bio-catalysis activity of GLDH, and was a reversible competitive inhibitior with K(i), estimated as 199 micromol l(-1) at 25 degrees C and pH 8.0.

  18. 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.

  19. Taurine chloramine is more selective than hypochlorous acid at targeting critical cysteines and inactivating creatine kinase and glyceraldehyde-3-phosphate dehydrogenase.

    Science.gov (United States)

    Peskin, Alexander V; Winterbourn, Christine C

    2006-01-01

    Hypochlorous acid (HOCl) and chloramines are produced by the neutrophil enzyme, myeloperoxidase. Both react readily with thiols, although chloramines differ from HOCl in discriminating between low molecular weight thiols on the basis of their pKa. Here, we have compared the reactivity of HOCl and taurine chloramine with thiol proteins by examining inactivation of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). With both enzymes, loss of activity paralleled thiol loss. For CK both were complete at a 1:1 taurine chloramine:thiol mole ratio. For GAPDH each chloramine oxidized two thiols. Three times more HOCl than taurine chloramine was required for inactivation, indicating that HOCl is less thiol specific. Competition studies showed that thiols of CK were 4 times more reactive with taurine chloramine than thiols of GAPDH (rate constants of 1200 and 300 M-1s-1 respectively). These compare with 205 M-1s-1 for cysteine and are consistent with their lower pKa's. Both enzymes were equally susceptible to HOCl. GSH competed directly with the enzyme thiols for taurine chloramine and protected against oxidative inactivation. At lower GSH concentrations, mixed disulfides were formed. We propose that chloramines should preferentially attack proteins with low pKa thiols and this could be important in regulatory processes.

  20. 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.

  1. 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

    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......AIMS: The stomach is involved in first-pass metabolism of alcohol in humans. As conflicting data were published regarding the influence of age and gender on the activity of alcohol dehydrogenase (ADH) in human gastric mucosa, the present study aimed at the investigation of these and other...... 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...

  2. Stereospecific synthesis of (R)-2-hydroxy carboxylic acids using recombinant E. coli BL21 overexpressing YiaE from Escherichia coli K12 and glucose dehydrogenase from Bacillus subtilis.

    Science.gov (United States)

    Yun, Hyungdon; Choi, Hyeon-Lok; Fadnavis, Nitin W; Kim, Byung-Gee

    2005-01-01

    The yiaE gene from Escherichia coli K12 was functionally expressed in E. coli BL21 using an IPTG inducible pET expression system (2.1 U/mg), and YiaE was purified to a specific activity of 18 U/mg. The purified enzyme catalyzes reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (R)-2-hydoxy carboxylic acids using NADPH. For practical applications, the problem of NADPH recycle was effectively solved by using recombinant E. coli overexpressing YiaE and glucose dehydrogenase from Bacillus subtilis in the same cell. The recombinant E. coli was used to prepare (R)-phenyllactic acid and (R)-2-hydroxy-4-phenylbutanoic acid from the corresponding 2-oxo carboxylic acids (98% ee) while the alpha-carbonyl group of 2,4-dioxo-4-phenylbutyric acid was reduced regio- and stereospecifically to give (R)-2-hydroxy-4-oxo-4-phenylbutyric acid (97% ee) in quantitative yields. The cells could be recycled for 3 days at room temperature in 100 mM phosphate buffer (pH 7.0) without loss of activity, which reduced to 70% after 1 week.

  3. 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...

  4. Structural basis for regulation of stability and activity in glyceraldehyde-3-phosphate dehydrogenases. Differential scanning calorimetry and molecular dynamics.

    Science.gov (United States)

    Makshakova, Olga N; Semenyuk, Pavel I; Kuravsky, Mikhail L; Ermakova, Elena A; Zuev, Yuriy F; Muronetz, Vladimir I

    2015-05-01

    Tissue specific isoforms of human glyceraldehyde-3-phosphate dehydrogenase, somatic (GAPD) and sperm-specific (GAPDS), have been reported to display different levels of both stability and catalytic activity. Here we apply MD simulations to investigate molecular basis of this phenomenon. The protein is a tetramer where each subunit consists of two domains - catalytic and NAD-binding one. We demonstrated key residues responsible for intersubunit and interdomain interactions. Effect of several residues was studied by point mutations. Overall we considered three mutations (Glu96Gln, Glu244Gln and Asp311Asn) disrupting GAPDS-specific salt bridges. Comparison of calculated interaction energies with calorimetric enthalpies confirmed that intersubunit interactions were responsible for enhanced thermostability of GAPDS whereas interdomain interactions had indirect influence on intersubunit contacts. Mutation Asp311Asn was around 10Å far from the active center and corresponded to the closest natural substitution in the isoenzymes. MD simulations revealed that this residue had slight interaction with catalytic residues but influenced the hydrogen bond net and dynamics in active site. These effects can be responsible for a strong influence of this residue on catalytic activity. Overall, our results provide new insight into glyceraldehyde-3-phosphate dehydrogenase structure-function relationships and can be used for the engineering of mutant proteins with modified properties and for development of new inhibitors with indirect influence on the catalytic site. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. 15 Hypoxyprostaglandin dehydrogenase. A review

    DEFF Research Database (Denmark)

    Hansen, Harald S.

    1976-01-01

    A review is given on the enzyme 15 hydroxyprostaglandin dehydrogenase. The determination, activity, distribution, purification, properties and physiological aspects are discussed. 128 references.......A review is given on the enzyme 15 hydroxyprostaglandin dehydrogenase. The determination, activity, distribution, purification, properties and physiological aspects are discussed. 128 references....

  6. 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.

  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 substrat

  8. Is autism a disorder of fatty acid metabolism? Possible dysfunction of mitochondrial beta-oxidation by long chain acyl-CoA dehydrogenase.

    Science.gov (United States)

    Clark-Taylor, Tonya; Clark-Taylor, Benjamin E

    2004-01-01

    Long chain acyl-CoA dehydrogenase (LCAD) has recently been shown to be the mitochondrial enzyme responsible for the beta-oxidation of branched chain and unsaturated fatty acids [Biochim. Biophys. Acta 1393 (1998) 35; Biochim. Biophys. Acta 1485 (2000) 121]. Whilst disorders of short, medium and very long chain acyl dehydrogenases are known, there is no known disorder of LCAD deficiency in humans. Experimental LCAD deficiency in mice shows an acyl-carnitine profile with prominent elevations of unsaturated fatty acid metabolites C14:1 and C14:2 [Hum. Mol. Genet. 10 (2001) 2069]. A child with autism whose acyl-carnitine profile also shows these abnormalities is presented, and it is hypothesized that the child may have LCAD deficiency. Additional metabolic abnormalities seen in this patient include alterations of TCA energy production, ammonia detoxification, reduced synthesis of omega-3 DHA, and abnormal cholesterol metabolism. These metabolic changes are also seen as secondary abnormalities in dysfunction of fatty acid beta-oxidation, and have also been reported in autism. It is hypothesized that LCAD deficiency may be a cause of autism. Similarities between metabolic disturbances in autism, and those of disorders of fatty acid beta-oxidation are discussed.

  9. General (medium-chain) acyl-CoA dehydrogenase deficiency (non-ketotic dicarboxylic aciduria): quantitative urinary excretion pattern of 23 biologically significant organic acids in three cases.

    Science.gov (United States)

    Gregersen, N; Kølvraa, S; Rasmussen, K; Mortensen, P B; Divry, P; David, M; Hobolth, N

    1983-08-15

    Urinary analysis of the pattern of 23 organic acid metabolites derived from fatty acids in three patients with general (medium-chain) acyl-CoA dehydrogenase deficiency was performed. Although there exist quantitative differences in the excreted amounts of the different metabolites in the three patients the qualitative picture was the same. The excretion of adipic, suberic and sebacic acids was substantial, whereas that of dodecanedioic acid was within or just above control limit. The monounsaturated C6-C10-dicarboxylic acid excretion was only marginally or not increased. 5-OH-hexanoic acid and hexanoylglycine were excreted in excessive amounts, whereas 7-OH-octanoic acid, 9-OH-decanoic acid, octanoylglycine and decanoylglycine were excreted in limited amounts. The excreted amounts of 6-OH-hexanoic, 8-OH-octanoic and 10-OH-decanoic acids were not or only marginally elevated compared to controls. In one of the patients the excretion of ethylmalonic and methylsuccinic acids was enhanced, whereas the excretion of these two acids in the two other patients was comparable to that in controls. The urinary excretion of hexanoic, octanoic, decanoic and dodecanoic acids was just a little above the control limit, whereas the esterified hexanoic and octanoic acids were excreted in appreciable amounts. It is argued that the microsomal omega- and omega-1-oxidation systems are involved in the dicarboxylic and omega-1-OH-monocarboxylic acids formation at C10 and C12 level and that the C8-C6-dicarboxylic and omega-1-OH-monocarboxylic acids are formed from higher chained acids by beta-oxidation in both mitochondria and peroxisomes.

  10. The Role of Pyruvate Dehydrogenase Kinase in Diabetes and Obesity

    Directory of Open Access Journals (Sweden)

    In-Kyu Lee

    2014-06-01

    Full Text Available The pyruvate dehydrogenase complex (PDC is an emerging target for the treatment of metabolic syndrome. To maintain a steady-state concentration of adenosine triphosphate during the feed-fast cycle, cells require efficient utilization of fatty acid and glucose, which is controlled by the PDC. The PDC converts pyruvate, coenzyme A (CoA, and oxidized nicotinamide adenine dinucleotide (NAD+ into acetyl-CoA, reduced form of nicotinamide adenine dinucleotide (NADH, and carbon dioxide. The activity of the PDC is up- and down-regulated by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase, respectively. In addition, pyruvate is a key intermediate of glucose oxidation and an important precursor for the synthesis of glucose, glycerol, fatty acids, and nonessential amino acids.

  11. 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 es

  12. 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.

  13. Delta1-pyrroline-5-carboxylic acid formed by proline dehydrogenase from the Bacillus subtilis ssp. natto expressed in Escherichia coli as a precursor for 2-acetyl-1-pyrroline.

    Science.gov (United States)

    Huang, Tzou-Chi; Huang, Yi-Wen; Hung, Hui-Ju; Ho, Chi-Tang; Wu, Mei-Li

    2007-06-27

    Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.

  14. Electron transfer from NADH bound to horse liver alcohol dehydrogenase (NAD+ dependent dehydrogenase): visualisation of the activity in the enzyme crystals and adsorption of formazan derivatives by these crystals.

    Science.gov (United States)

    Pacaud-Mercier, Karine; Blaghen, Mohamed; Lee, Kang Min; Tritsch, Denis; Biellmann, Jean-François

    2007-02-01

    The crystals of holoenzyme from native and cross-linked alcohol dehydrogenase exhibit electron transfer from NADH to phenazinium methosulfate (PMS), and then to the tetrazolium salt sodium 3,3'-{1-[(phenylamino)carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzenesulfonate (XXT). The slow dissociation of the cofactor and/or the conformational change associated can now be bypassed. The reduction product, formazan, did not diffuse out of the crystals in buffer and the crystals turned colored. In the presence of dimethyl sulfoxide or dimethoxyethane, the formazan diffused out to the solution. The reaction rates were found to be, respectively, 18% and 15% of the redox reaction rate of ethanol with cinnamaldehyde, close to the activity determined for the enzyme in solution in the presence of dimethoxyethane. The use of system PMS-tetrazolium salt is a useful tool to visualize the activity of dehydrogenases and other electron transferring systems in the crystalline state. The adsorption of formazan by the alcohol dehydrogenase crystals occurs in solution.

  15. 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.

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

    Science.gov (United States)

    Naveed, Muhammad; Ahmed, Iftikhar; Khalid, Nauman; Mumtaz, Abdul Samad

    2014-01-01

    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.

  17. 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

    Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different...... consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic...... 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....

  18. Sorbitol dehydrogenase inhibitors (SDIs): a new potent, enantiomeric SDI, 4-[2-1R-hydroxy-ethyl)-pyrimidin-4-yl]piperazine-1-sulfonic acid dimethylamide.

    Science.gov (United States)

    Mylari, B L; Oates, P J; Beebe, D A; Brackett, N S; Coutcher, J B; Dina, M S; Zembrowski, W J

    2001-08-16

    We report here on our medicinal chemistry and pharmacology efforts to provide a potent sorbitol dehydrogenase inhibitor (SDI) as a tool to probe a recently disclosed hypothesis centered on the role of sorbitol dehydrogenase (SDH) in the second step of the polyol pathway, under conditions of high glucose flux. Starting from a weak literature lead, 2, and through newly developed structure-activity relationships, we have designed and executed an unambiguous synthesis of enantiomeric SDI, 6, which is at least 10x more potent than 2. Also, 6 potently inhibits SDH in streptozotocin-diabetic rat sciatic nerve. We have described an expedient synthesis of a key building template, 33, for future research in the SDI area that may facilitate the discovery of even more potent SDIs with longer duration of action in vivo.

  19. Total lactate dehydrogenase activity of tail muscle is not cold-adapted in nocturnal lizards from cool-temperate habitats.

    Science.gov (United States)

    Hare, K M; Miller, J H; Clark, A G; Daugherty, C H

    2005-12-01

    The dependence of metabolic processes on temperature constrains the behavior, physiology and ecology of many ectothermic animals. The evolution of nocturnality in lizards, especially in temperate regions, requires adaptations for activity at low temperatures when optimal body temperatures are unlikely to be obtained. We examined whether nocturnal lizards have cold-adapted lactate dehydrogenase (LDH). LDH was chosen as a representative metabolic enzyme. We measured LDH activity of tail muscle in six lizard species (n=123: three nocturnal, two diurnal and one crepuscular) between 5 and 35 degrees C and found no differences in LDH-specific activity or thermal sensitivity among the species. Similarly, the specific activity and thermal sensitivity of LDH were similar between skinks and geckos. Similar enzyme activities among nocturnal and diurnal lizards indicate that there is no selection of temperature specific LDH enzyme activity at any temperature. As many nocturnal lizards actively thermoregulate during the day, LDH may be adapted for a broad range of temperatures rather than adapted specifically for the low temperatures encountered when the animals are active. The total activity of LDH in tropical and temperate lizards is not cold-adapted. More data are required on biochemical adaptations and whole animal thermal preferences before trends can be established.

  20. 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

    2017-08-08

    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 (NH4)2HPO4 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.

  1. Cortisone induces insulin resistance in C2C12 myotubes through activation of 11beta-hydroxysteroid dehydrogenase 1 and autocrinal regulation.

    Science.gov (United States)

    Park, Seung Yeon; Bae, Ji Hyun; Cho, Young Sik

    2014-04-01

    The enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) is known to catalyse inactive glucocorticoids into active forms, and its dysregulation in adipose and muscle tissues has been implicated in the development of metabolic syndrome. To delineate the molecular mechanism by which active cortisol has an antagonizing effect against insulin, we optimized the metabolic production of cortisol and its biological functions in myotubes (C2C12). Myotubes supplemented with cortisone actively catalysed its conversion into cortisol, which in turn abolished phosphorylation of Akt in response to insulin treatment. This led to diminished uptake of insulin-induced glucose. This was corroborated by the application of 11β-HSD1 inhibitor glycyrrhetinic acid and a glucocorticoid receptor antagonist RU-486, which reversed completely the antagonizing effects of cortisol on insulin action. Therefore, development of specific inhibitors targeting 11β-HSD1 might be a promising way to improve impaired insulin-stimulated glucose uptake. Copyright © 2013 John Wiley & Sons, Ltd.

  2. Experimentally increased codon bias in the Drosophila Adh gene leads to an increase in larval, but not adult, alcohol dehydrogenase activity.

    Science.gov (United States)

    Hense, Winfried; Anderson, Nathan; Hutter, Stephan; Stephan, Wolfgang; Parsch, John; Carlini, David B

    2010-02-01

    Although most amino acids can be encoded by more than one codon, the synonymous codons are not used with equal frequency. This phenomenon is known as codon bias and appears to be a universal feature of genomes. The translational selection hypothesis posits that the use of optimal codons, which match the most abundant species of isoaccepting tRNAs, results in increased translational efficiency and accuracy. Previous work demonstrated that the experimental reduction of codon bias in the Drosophila alcohol dehydrogenase (Adh) gene led to a significant decrease in ADH protein expression. In this study we performed the converse experiment: we replaced seven suboptimal leucine codons that occur naturally in the Drosophila melanogaster Adh gene with the optimal codon. We then compared the in vivo ADH activities imparted by the wild-type and mutant alleles. The introduction of optimal leucine codons led to an increase in ADH activity in third-instar larvae. In adult flies, however, the introduction of optimal codons led to a decrease in ADH activity. There is no evidence that other selectively constrained features of the Adh gene, or its rate of transcription, were altered by the synonymous replacements. These results are consistent with translational selection for codon bias being stronger in the larval stage and suggest that there may be a selective conflict over optimal codon usage between different developmental stages.

  3. Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum.

    Science.gov (United States)

    Pérez-Pomares, F; Ferrer, J; Camacho, M; Pire, C; LLorca, F; Bonete, M J

    1999-02-01

    The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.

  4. Aldehyde dehydrogenase 2 activation in aged heart improves the autophagy by reducing the carbonyl modification on SIRT1.

    Science.gov (United States)

    Wu, Bing; Yu, Lu; Wang, Yishi; Wang, Hongtao; Li, Chen; Yin, Yue; Yang, Jingrun; Wang, Zhifa; Zheng, Qiangsun; Ma, Heng

    2016-01-19

    Cardiac aging is characterized by accumulation of damaged proteins and decline of autophagic efficiency. Here, by forestalling SIRT1 carbonylated inactivation in aged heart, we determined the benefits of activation of aldehyde dehydrogenase 2 (ALDH2) on the autophagy. In this study, the ALDH2 KO mice progressively developed age-related heart dysfunction and showed reduction in the life span, which strongly suggests that ALDH2 ablation leads to cardiac aging. What's more, aged hearts displayed a significant decrease ALDH2 activity, resulting in accumulation of 4-HNE-protein adducts and protein carbonyls, impairment in the autophagy flux, and, consequently, deteriorated cardiac function after starvation. Sustained Alda-1 (selective ALDH2 activator) treatment increased cardiac ALDH2 activity and abrogated these effects. Using SIRT1 deficient heterozygous (Sirt1+/-) mice, we found that SIRT1 was necessary for ALDH2 activation-induced autophagy. We further demonstrated that ALDH2 activation attenuated SIRT1 carbonylation and improved SIRT1 activity, thereby increasing the deacetylation of nuclear LC3 and FoxO1. Sequentially, ALDH2 enhanced SIRT1 regulates LC3-Atg7 interaction and FoxO1 increased Rab7 expression, which were both necessary and sufficient for restoring autophagy flux. These results highlight that both accumulation of proteotoxic carbonyl stress linkage with autophagy decline contribute to heart senescence. ALDH2 activation is adequate to improve the autophagy flux by reducing the carbonyl modification on SIRT1, which in turn plays an important role in maintaining cardiac health during aging.

  5. 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.

  6. 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.

  7. 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.

  8. E. coli histidine triad nucleotide binding protein 1 (ecHinT) is a catalytic regulator of D-alanine dehydrogenase (DadA) activity in vivo.

    Science.gov (United States)

    Bardaweel, Sanaa; Ghosh, Brahma; Chou, Tsui-Fen; Sadowsky, Michael J; Wagner, Carston R

    2011-01-01

    Histidine triad nucleotide binding proteins (Hints) are highly conserved members of the histidine triad (HIT) protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both gram-negative and gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli.

  9. E. coli histidine triad nucleotide binding protein 1 (ecHinT is a catalytic regulator of D-alanine dehydrogenase (DadA activity in vivo.

    Directory of Open Access Journals (Sweden)

    Sanaa Bardaweel

    Full Text Available Histidine triad nucleotide binding proteins (Hints are highly conserved members of the histidine triad (HIT protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both gram-negative and gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli. Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA (equivalent to D-amino acid oxidase in eukaryotes, a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli.

  10. Additivity of the Stabilization Effect of Single Amino Acid Substitutions in Triple Mutants of Recombinant Formate Dehydrogenase from the Soybean Glycine max.

    Science.gov (United States)

    Alekseeva, A A; Kargov, I S; Kleimenov, S Yu; Savin, S S; Tishkov, V I

    2015-01-01

    Recently, we demonstrated that the amino acid substitutions Ala267Met and Ala267Met/Ile272Val (Alekseeva et al., Biochemistry, 2012), Phe290Asp, Phe290Asn and Phe290Ser (Alekseeva et al., Prot. Eng. Des. Select, 2012) in recombinant formate dehydrogenase from soya Glycine max (SoyFDH) lead to a significant (up to 30-100 times) increase in the thermal stability of the enzyme. The substitutions Phe290Asp, Phe290Asn and Phe290Ser were introduced into double mutant SoyFDH Ala267Met/Ile272Val by site-directed mutagenesis. Combinations of three substitutions did not lead to a noticeable change in the catalytic properties of the mutant enzymes. The stability of the resultant triple mutants was studied through thermal inactivation kinetics and differential scanning calorimetry. The thermal stability of the new mutant SoyFDHs was shown to be much higher than that of their precursors. The stability of the best mutant SoyFDH Ala267Met/Ile272Val/Phe290Asp turned out to be comparable to that of the most stable wild-type formate dehydrogenases from other sources. The results obtained with both methods indicate a great synergistic contribution of individual amino acid substitutions to the common stabilization effect.

  11. 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.

  12. Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability.

    Science.gov (United States)

    Fedøy, Anita-Elin; Yang, Nannan; Martinez, Aurora; Leiros, Hanna-Kirsti S; Steen, Ida Helene

    2007-09-07

    Isocitrate dehydrogenase (IDH) has been studied extensively due to its central role in the Krebs cycle, catalyzing the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate to alpha-ketoglutarate and CO(2). Here, we present the first crystal structure of IDH from a psychrophilic bacterium, Desulfotalea psychrophila (DpIDH). The structural information is combined with a detailed biochemical characterization and a comparative study with IDHs from the mesophilic bacterium Desulfitobacterium hafniense (DhIDH), porcine (PcIDH), human cytosolic (HcIDH) and the hyperthermophilic Thermotoga maritima (TmIDH). DpIDH was found to have a higher melting temperature (T(m)=66.9 degrees C) than its mesophilic homologues and a suboptimal catalytic efficiency at low temperatures. The thermodynamic activation parameters indicated a disordered active site, as seen also for the drastic increase in K(m) for isocitrate at elevated temperatures. A methionine cluster situated at the dimeric interface between the two active sites and a cluster of destabilizing charged amino acids in a region close to the active site might explain the poor isocitrate affinity. On the other hand, DpIDH was optimized for interacting with NADP(+) and the crystal structure revealed unique interactions with the cofactor. The highly acidic surface, destabilizing charged residues, fewer ion pairs and reduced size of ionic networks in DpIDH suggest a flexible global structure. However, strategic placement of ionic interactions stabilizing the N and C termini, and additional ionic interactions in the clasp domain as well as two enlarged aromatic clusters might counteract the destabilizing interactions and promote the increased thermal stability. The structure analysis of DpIDH illustrates how psychrophilic enzymes can adjust their flexibility in dynamic regions during their catalytic cycle without compromising the global stability of the protein.

  13. JWH-018 ω-OH, a shared hydroxy metabolite of the two synthetic cannabinoids JWH-018 and AM-2201, undergoes oxidation by alcohol dehydrogenase and aldehyde dehydrogenase enzymes in vitro forming the carboxylic acid metabolite

    DEFF Research Database (Denmark)

    Holm, Niels Bjerre; Noble, Carolina; Linnet, Kristian

    2016-01-01

    +)-dependent alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. The sole end-product identified in HLC was the JWH-018 ω-COOH metabolite, while trapping tests with methoxyamine proved the presence of the aldehyde intermediate. ADH/ALDH and UDP-glucuronosyl-transferases (UGT) enzymes may therefore...

  14. 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 (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.

  15. 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.

  16. 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.

  17. 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.

  18. Loss of Mitochondrial Malate Dehydrogenase Activity Alters Seed Metabolism Impairing Seed Maturation and Post-Germination Growth in Arabidopsis.

    Science.gov (United States)

    Sew, Yun Shin; Ströher, Elke; Fenske, Ricarda; Millar, A Harvey

    2016-06-01

    Mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37) has multiple roles; the most commonly described is its catalysis of the interconversion of malate and oxaloacetate in the tricarboxylic acid cycle. The roles of mMDH in Arabidopsis (Arabidopsis thaliana) seed development and germination were investigated in mMDH1 and mMDH2 double knockout plants. A significant proportion of mmdh1mmdh2 seeds were nonviable and developed only to torpedo-shaped embryos, indicative of arrested seed embryo growth during embryogenesis. The viable mmdh1mmdh2 seeds had an impaired maturation process that led to slow germination rates as well as retarded post-germination growth, shorter root length, and decreased root biomass. During seed development, mmdh1mmdh2 showed a paler green phenotype than the wild type and exhibited deficiencies in reserve accumulation and reduced final seed biomass. The respiration rate of mmdh1mmdh2 seeds was significantly elevated throughout their maturation, consistent with the previously reported higher respiration rate in mmdh1mmdh2 leaves. Mutant seeds showed a consistently higher content of free amino acids (branched-chain amino acids, alanine, serine, glycine, proline, and threonine), differences in sugar and sugar phosphate levels, and lower content of 2-oxoglutarate. Seed-aging assays showed that quiescent mmdh1mmdh2 seeds lost viability more than 3 times faster than wild-type seeds. Together, these data show the important role of mMDH in the earliest phases of the life cycle of Arabidopsis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  19. 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...

  20. 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.

  1. 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.

  2. Properties and subunit structure of pig heart pyruvate dehydrogenase.

    Science.gov (United States)

    Hamada, M; Hiraoka, T; Koike, K; Ogasahara, K; Kanzaki, T

    1976-06-01

    Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

  3. Salivary aldehyde dehydrogenase - temporal and population variability, correlations with drinking and smoking habits and activity towards aldehydes contained in food.

    Science.gov (United States)

    Giebułtowicz, Joanna; Dziadek, Marta; Wroczyński, Piotr; Woźnicka, Katarzyna; Wojno, Barbara; Pietrzak, Monika; Wierzchowski, Jacek

    2010-01-01

    Fluorimetric method based on oxidation of the fluorogenic 6-methoxy-2-naphthaldehyde was applied to evaluate temporal and population variability of the specific activity of salivary aldehyde dehydrogenase (ALDH) and the degree of its inactivation in healthy human population. Analyzed was also its dependence on drinking and smoking habits, coffee consumption, and its sensitivity to N-acetylcysteine. Both the specific activity of salivary ALDH and the degree of its inactivation were highly variable during the day, with the highest activities recorded in the morning hours. The activities were also highly variable both intra- and interpersonally, and negatively correlated with age, and this correlation was stronger for the subgroup of volunteers declaring abstinence from alcohol and tobacco. Moderately positive correlations of salivary ALDH specific activity with alcohol consumption and tobacco smoking were also recorded (r(s) ~0.27; p=0.004 and r(s) =0.30; p=0.001, respectively). Moderate coffee consumption correlated positively with the inactivation of salivary ALDH, particularly in the subgroup of non-drinking and non-smoking volunteers. It was found that mechanical stimulation of the saliva flow increases the specific activity of salivary ALDH. The specific activity of the salivary ALDH was strongly and positively correlated with that of superoxide dismutase, and somewhat less with salivary peroxidase. The antioxidant-containing drug N-acetylcysteine increased activity of salivary ALDH presumably by preventing its inactivation in the oral cavity. Some food-related aldehydes, mainly cinnamic aldehyde and anisaldehyde, were excellent substrates of the salivary ALDH3A1 enzyme, while alkenals, particularly those with short chain, were characterized by lower affinity towards this enzyme but high catalytic constants. The protective role of salivary ALDH against aldehydes in food and those found in the cigarette smoke is discussed, as well as its participation in

  4. Tissue-specific strategies of the very-long chain acyl-CoA dehydrogenase-deficient (VLCAD-/- mouse to compensate a defective fatty acid β-oxidation.

    Directory of Open Access Journals (Sweden)

    Sara Tucci

    Full Text Available Very long-chain acyl-CoA dehydrogenase (VLCAD-deficiency is the most common long-chain fatty acid oxidation disorder presenting with heterogeneous phenotypes. Similar to many patients with VLCADD, VLCAD-deficient mice (VLCAD(-/- remain asymptomatic over a long period of time. In order to identify the involved compensatory mechanisms, wild-type and VLCAD(-/- mice were fed one year either with a normal diet or with a diet in which medium-chain triglycerides (MCT replaced long-chain triglycerides, as approved intervention in VLCADD. The expression of the mitochondrial long-chain acyl-CoA dehydrogenase (LCAD and medium-chain acyl-CoA dehydrogenase (MCAD was quantified at mRNA and protein level in heart, liver and skeletal muscle. The oxidation capacity of the different tissues was measured by LC-MS/MS using acyl-CoA substrates with a chain length of 8 to 20 carbons. Moreover, in white skeletal muscle the role of glycolysis and concomitant muscle fibre adaptation was investigated. In one year old VLCAD(-/- mice MCAD and LCAD play an important role in order to compensate deficiency of VLCAD especially in the heart and in the liver. However, the white gastrocnemius muscle develops alternative compensatory mechanism based on a different substrate selection and increased glucose oxidation. Finally, the application of an MCT diet over one year has no effects on LCAD or MCAD expression. MCT results in the VLCAD(-/- mice only in a very modest improvement of medium-chain acyl-CoA oxidation capacity restricted to cardiac tissue. In conclusion, VLCAD(-/- mice develop tissue-specific strategies to compensate deficiency of VLCAD either by induction of other mitochondrial acyl-CoA dehydrogenases or by enhancement of glucose oxidation. In the muscle, there is evidence of a muscle fibre type adaptation with a predominance of glycolytic muscle fibres. Dietary modification as represented by an MCT-diet does not improve these strategies long-term.

  5. Biological activities of substituted trichostatic acid derivatives

    Indian Academy of Sciences (India)

    Cédric Charrier; Joëlle Roche; Jean-Pierre Gesson; Philippe Bertrand

    2009-07-01

    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 derivatives. The first fluorinated derivatives of trichostatic acid are described, such as 6-fluoro trichostatin A, with antiproliferative activities in the micromolar range and with histone deacetylase inhibitory activity.

  6. ANTIMICROBIAL ACTIVITY OF LACTIC ACID BACTERIAL ISOLATES

    Directory of Open Access Journals (Sweden)

    Utkarsha S. Shivsharan

    2013-08-01

    Full Text Available Micro-organisms have tendency to produce antimicrobial substances which show biological activity against other kind of micro-organisms. This phenomenon of bacterial antagonism is observed in lactic acid bacteria with competitive advantages. The lactic acid bacteria are commonly present in many fermented products, fruits and milk products. The variety of antimicrobial substances produced by lactic acid bacteria showing good inhibition capacity include production of lactic acid, acetic acid, hydrogen peroxide, carbon dioxide, diacetyl and bacteriocin. Bacteriocins produced by lactic acid bacteria are the subject of intense research because of their antimicrobial activity against food born bacteria such as Listeria monocytogenes, staphylococcus aureus, Bacillus cereus, Clostridium botulinum and several others .Bacteriocins may be bacteriostatic or bactericidal with narrow or broad range of activity. The main of the study was to study the antimicrobial activity of such lactic acid bacterial isolates.

  7. 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.

  8. Assessment of lactate dehydrogenase, alkaline phosphatase and aspartate aminotransferase activities in cow's milk as an indicator of subclinical mastitis.

    Science.gov (United States)

    Babaei, H; Mansouri-Najand, L; Molaei, M M; Kheradmand, A; Sharifan, M

    2007-05-01

    This study examined the activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in the milk of lactating Holstein cows in association with subclinical mastitis (SCM). A total of 94 milk samples were collected from 58 lactating dairy cows representing stages of lactation from the second to the tenth week after calving. Those which were classified as positive by California mastitis test (CMT) were deemed to have subclinical mastitis. All the milk samples were skimmed by centrifugation at 10 000g at 0 degrees C and were used for enzyme activities estimations. The mean activities of LDH and ALP were higher in the milk from udders with SCM than in the milk from healthy udders (p CMT results and LDH and ALP values were seen at thresholds of > 180 IU/L and > 40 IU/L respectively (kappa values 0.65 and 0.79, respectively). However, the sensitivity of the tests for identifying SCM at these thresholds was higher for ALP (96.4%) than for LDH (68.5%). In this study, LDH and ALP tests were standardized for cow's milk and results showed that only the ALP test was reliable in the early diagnosis of subclinical mastitis.

  9. [Enzyme activity of an actinomycete producer of carotenes and macrotetrolides].

    Science.gov (United States)

    Nefelova, M V; Sverdlova, A N

    1982-01-01

    The activity of pyruvate dehydrogenase and dehydrogenases of the tricarboxylic acid cycle was assayed in the mycelium of Streptomyces chrysomallus var. Carotenoides growing under different conditions of the medium. The activity of the enzymes increased when acetic, citric and succinic acids were added at different periods of the growth. Moreover, addition of the acids increased the time of intensive functioning of the dehydrogenases whose activity abruptly decreased after 60 h of the growth under the control conditions.

  10. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sorbitol dehydrogenase test system. 862.1670... Systems § 862.1670 Sorbitol dehydrogenase test system. (a) Identification. A sorbitol dehydrogenase test system is a device intended to measure the activity of the enzyme sorbitol dehydrogenase in...

  11. 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...

  12. Comparison of Activity of Four Dehydrogenases in Ginseng from Different Origins%不同产地人参中4种脱氢酶活力比较

    Institute of Scientific and Technical Information of China (English)

    杨菲; 赵雨; 王思明; 刘美辰; 李晓华

    2012-01-01

    The aim was to provide theoretical basis for the cultivation and optimization of ginseng.Adopt neutral buffer solution to extract the enzyme solution of Radix Ginseng.The activities of malate dehydrogenase (MDH), lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) were detected by spectrophotometry, and compared.The clustering analysis was performed using the software SPSS 13.0 to system for 15 batch sample.There were obvious differences of the activities dehydrogenase of ginseng from different origin.The activities of four dehydrogenases from the same origin were basically same.In Antu County Wanbao Town, MDH, LDH and G6PDH had the highest activities, 124.58 LV(g·FW), 129.88 U/(g·FW) and 109.84 U/(g·FW) respectively.The four kinds of enzymes activity of two origins in Heilongjiang Province were generally low.The sample was divided into four categories.The activities of MDH, LDH, ADH and G6PDH could provide theoretical basis for the cultivation and optimization of ginseng.%为了给人参的培育和优选提供理论依据,采用中性缓冲液提取粗酶液,应用分光光度法对15个不同产地的人参中苹果酸脱氢酶(malate dehydrogenase,MDH)、乳酸脱氢酶(lactate dehydrogenase,LDH)、乙醇脱氢酶(alcohol dehydrogenase,ADH)、葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PDH)4种脱氢酶活力进行比较.运用SPSS 13.0软件对15批样品进行系统聚类分析.结果表明不同产地人参脱氢酶活力差别明显,同一产地4种脱氢酶活力趋势基本相同.其中安图县万宝镇的人参样品的MDH、LDH、G6PDH 3种酶活力均是最高值,分别为124.58 U/(g· FW)、129.88 U/(g·FW)、109.84U/(g·FW);黑龙江2个产地的4种酶活力普遍比较低.聚类分析的结果将样品分为4类.MDH、LDH、ADH、G6PDH的活力可以作为人参培育和优选提供理论依据.

  13. 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.

  14. Intense physical exercise increases systemic 11beta-hydroxysteroid dehydrogenase type 1 activity in healthy adult subjects.

    Science.gov (United States)

    Dovio, Andrea; Roveda, Eliana; Sciolla, Chiara; Montaruli, Angela; Raffaelli, Andrea; Saba, Alessandro; Calogiuri, Giovanna; De Francia, Silvia; Borrione, Paolo; Salvadori, Piero; Carandente, Franca; Angeli, Alberto

    2010-03-01

    Intense physical exercise activates the hypothalamic-pituitary-adrenocortical axis but little is known about changes in glucocorticoid sensitivity at the target cell level. No data are available on the acute effects of exercise on 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1 activity, which generates biologically active cortisol from inactive cortisone and is expressed also in skeletal muscle. Fifteen healthy, trained males (age mean +/- SE 28 +/- 1) were assessed on three non-consecutive days: at rest, during an endurance and strength sessions. During each session, between 1000 and 1600 hours, 6-h urine and four salivary samples were collected. Urinary total tetrahydrocortisol (THF) + alloTHF, tetrahydrocortisone (THE), cortisol (F) and cortisone (E) were measured with HPLC-tandem mass spectrometry; urinary-unconjugated F and E were measured by HPLC-UV. Salivary cortisol and interleukin (IL)-6 were measured by RIA and ELISA, respectively. Both endurance and strength exercises caused an increase in (THF + alloTHF)/THE ratio (mean +/- SE 1.90 +/- 0.07 and 1.82 +/- 0.05 vs. 1.63 +/- 0.06, P < 0.01 and P = 0.03, respectively), consistent with increased systemic 11beta-HSD type 1 activity. No relationship was found with age, BMI, VO(2max) maximal power load or perceived exertion. No significant change was apparent in F/E ratio, an index of 11beta-HSD type 2 activity. No effect of exercise on salivary cortisol and IL-6 was observed, whereas a significant effect of sampling time was found. Intense physical exercise acutely increases systemic 11beta-HSD type 1 activity in humans. Such an increase may lead to higher cortisol concentration in target tissues, notably in skeletal muscle where it could contribute to limit exercise-induced muscle inflammatory response.

  15. Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity.

    Science.gov (United States)

    Xu, Xiang; Zhao, Jingyue; Xu, Zhen; Peng, Baozhen; Huang, Qiuhua; Arnold, Eddy; Ding, Jianping

    2004-08-06

    Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, and regulation of the enzymatic activity of IDHs is crucial for their biological functions. Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site. Eukaryotic NADP-dependent IDHs (NADP-IDHs) have been shown to have diverse important biological functions; however, their regulatory mechanism remains unclear. Structural studies of human cytosolic NADP-IDH (HcIDH) in complex with NADP and in complex with NADP, isocitrate, and Ca2+ reveal three biologically relevant conformational states of the enzyme that differ substantially in the structure of the active site and in the overall structure. A structural segment at the active site that forms a conserved alpha-helix in all known NADP-IDH structures assumes a loop conformation in the open, inactive form of HcIDH; a partially unraveled alpha-helix in the semi-open, intermediate form; and an alpha-helix in the closed, active form. The side chain of Asp279 of this segment occupies the isocitrate-binding site and forms hydrogen bonds with Ser94 (the equivalent of the phosphorylation site in bacterial IDHs) in the inactive form and chelates the metal ion in the active form. The structural data led us to propose a novel self-regulatory mechanism for HcIDH that mimics the phosphorylation mechanism used by the bacterial homologs, consistent with biochemical and biological data. This mechanism might be applicable to other eukaryotic NADP-IDHs. The results also provide insights into the recognition and specificity of substrate and cofactor by eukaryotic NADP-IDHs.

  16. Effects of methoxychlor and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane on 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase-3 activities in human and rat testes.

    Science.gov (United States)

    Hu, G-X; Zhao, B; Chu, Y; Li, X-H; Akingbemi, B T; Zheng, Z-Q; Ge, R S

    2011-04-01

    Human and rat testis microsomes were used to investigate direct inhibitory activities of methoxychlor (MXC) and its metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3). The 3β-HSD and 17β-HSD3 enzymes are involved in the reactions that culminate in androgen biosynthesis in Leydig cells. The results demonstrated that MXC and HPTE inhibited human 3β-HSD activity at a concentration of 10 nm. The half maximal inhibitory concentration (IC(50) ) for MXC inhibition of 3β-HSD was 53.21 ± 15.52 μm (human) and 46.15 ± 17.94 μm (rat), and for HPTE, it was 8.29 ± 2.49 μm (human) and 13.82 ± 2.26 μm (rat). At the higher concentration of 100 μm, MXC did not affect human and rat 17β-HSD3 activity. However, the IC(50) for HPTE inhibition of 17β-HSD3 was 12.1 ± 1.9 μm (human) and 32 .0 ± 8.6 μm (rat). The mode of action of MXC and HPTE on 3β-HSD activity was non-competitive with the substrate pregnenolone, but was competitive with the cofactor NAD(+) . The mode of HPTE inhibition of 17β-HSD3 was non-competitive with the substrate androstenedione, but was competitive with the cofactor NADPH. In summary, our results showed that HPTE, which is the biologically active metabolite of MXC, has the capacity for direct inhibition of 3β-HSD and 17β-HSD3 enzyme activity. Inhibition of enzyme activity is presumably associated with suppression of steroidogenesis in gonadal tissues and has implications for testis function.

  17. Activation of carboxylic acids in asymmetric organocatalysis.

    Science.gov (United States)

    Monaco, Mattia Riccardo; Poladura, Belén; Diaz de Los Bernardos, Miriam; Leutzsch, Markus; Goddard, Richard; List, Benjamin

    2014-07-01

    Organocatalysis, catalysis using small organic molecules, has recently evolved into a general approach for asymmetric synthesis, complementing both metal catalysis and biocatalysis. Its success relies to a large extent upon the introduction of novel and generic activation modes. Remarkably though, while carboxylic acids have been used as catalyst directing groups in supramolecular transition-metal catalysis, a general and well-defined activation mode for this useful and abundant substance class is still lacking. Herein we propose the heterodimeric association of carboxylic acids with chiral phosphoric acid catalysts as a new activation principle for organocatalysis. This self-assembly increases both the acidity of the phosphoric acid catalyst and the reactivity of the carboxylic acid. To illustrate this principle, we apply our concept in a general and highly enantioselective catalytic aziridine-opening reaction with carboxylic acids as nucleophiles.

  18. Scaffold electrodes based on thioctic acid-capped gold nanoparticles coordinated Alcohol Dehydrogenase and Azure A films for high performance biosensor.

    Science.gov (United States)

    Gómez-Anquela, C; García-Mendiola, T; Abad, José M; Pita, M; Pariente, F; Lorenzo, E

    2015-12-01

    Nanometric size gold nanoparticles capped with thiotic acid are used to coordinate with the Zn (II) present in the catalytic center of Alcohol Dehydrogenase (ADH). In combination with the NADH oxidation molecular catalyst Azure A, electrografted onto carbon screen-printed electrodes, they are used as scaffold electrodes for the construction of a very efficient ethanol biosensor. The final biosensing device exhibits a highly efficient ethanol oxidation with low overpotential of -0.25 V besides a very good analytical performance with a detection limit of 0.14±0.01 μM and a stable response for more than one month. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. An amperometric D-amino acid biosensor prepared with a thermostable D-proline dehydrogenase and a carbon nanotube-ionic liquid gel.

    Science.gov (United States)

    Tani, Yuji; Itoyama, Yukiko; Nishi, Kenichi; Wada, Chikahiro; Shoda, Yoshio; Satomura, Takenori; Sakuraba, Haruhiko; Ohshima, Toshihisa; Hayashi, Yukako; Yabutani, Tomoki; Motonaka, Junko

    2009-07-01

    Carbon nanotube (CNT) gel, which is composed of a mixture of single-wall CNT, an ionic liquid, and a thermostable D-proline dehydrogenase (D-Pro DH) immobilized electrode was utilized for the determination of D-amino acids (DAAs) in food samples. When a critical comparison with CNT, Ketjen Black (KB), and carbon powder (CP) was also carried out, the CNT/D-Pro DH immobilized electrode showed the highest sensitivity and the lowest detection limit of D-proline. In addition, the CNT/D-Pro DH immobilized electrode was applied to detection of DAAs in rice wine and vinegar samples. The concentrations of DAAs in rice wine and vinegar samples were 0.0210 +/- 0.0001 and 0.55 +/- 0.05 mmol L(-1), respectively.

  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. Production of lactose-free galacto-oligosaccharide mixtures: comparison of two cellobiose dehydrogenases for the selective oxidation of lactose to lactobionic acid.

    Science.gov (United States)

    Maischberger, Thomas; Nguyen, Thu-Ha; Sukyai, Prakit; Kittl, Roman; Riva, Sergio; Ludwig, Roland; Haltrich, Dietmar

    2008-08-11

    Galacto-oligosaccharides, complex mixtures of various sugars, are produced by transgalactosylation from lactose using beta-galactosidase and are of great interest for food and feed applications because of their prebiotic properties. Most galacto-oligosaccharide preparations currently available in the market contain a significant amount of monosaccharides and lactose. The mixture of galacto-oligosaccharides (GalOS) in this study produced from lactose using recombinant beta-galactosidase from Lactobacillus reuteri contains 48% monosaccharides, 26.5% lactose and 25.5% GalOS. To remove efficiently both monosaccharides and lactose from this GalOS mixture containing significant amounts of prebiotic non-lactose disaccharides, a biocatalytic approach coupled with subsequent chromatographic steps was used. Lactose was first oxidised to lactobionic acid using fungal cellobiose dehydrogenases, and then lactobionic acid and monosaccharides were removed by ion-exchange and size-exclusion chromatography. Two different cellobiose dehydrogenases (CDH), originating from Sclerotium rolfsii and Myriococcum thermophilum, were compared with respect to their applicability for this process. CDH from S. rolfsii showed higher specificity for the substrate lactose, and only few other components of the GalOS mixture were oxidised during prolonged incubation. Since these sugars were only converted once lactose oxidation was almost complete, careful control of the CDH-catalysed reaction will significantly reduce the undesired oxidation, and hence subsequent removal, of any GalOS components. Removal of ions and monosaccharides by the chromatographic steps gave an essentially pure GalOS product, containing less than 0.3% lactose and monosaccharides, in a yield of 60.3%.

  2. 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.

  3. A novel dye-linked alcohol dehydrogenase activity present in some Gram-positive bacteria

    NARCIS (Netherlands)

    VANOPHEM, PW; Euverink, Gert-Jan; Dijkhuizen, Lubbert; DUINE, JA

    1991-01-01

    An assay was developed which allowed reproducible detection of methanol oxidation by cell free extracts of methanol-grown Amycolatopsis methanolica. The dye-linked activity was only observed when high concentrations of phosphate or sulphate salts were applied in the assay. The specific activity stro

  4. 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 de......, 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....

  5. Mutation of isocitrate dehydrogenase 1 induces glioma cell proliferation via nuclear factor-κB activation in a hypoxia-inducible factor 1-α dependent manner.

    Science.gov (United States)

    Wang, Guoliang; Sai, Ke; Gong, Fanghe; Yang, Qunying; Chen, Furong; Lin, Jian

    2014-05-01

    Recently, mutations of the isocitrate dehydrogenase (IDH) 1 gene, which specifically occur in the majority of low-grade and secondary high-grade gliomas, have drawn particular attention of neuro-oncologists. Mutations of the IDH1 gene have been proposed to have significant roles in the tumorigenesis, progression and prognosis of gliomas. However, the molecular mechanism of the role of IDH1 mutants in gliomagenesis remains to be elucidated. The present study, showed that forced expression of an IDH1 mutant, of which the 132th amino acid residue arginine is substituted by histidine (IDH1R132H), promoted cell proliferation in cultured cells, while wild-type IDH1 overexpression had no effect on cell proliferation. Consistent with previous studies, it was also observed that expression of hypoxia-inducible factor 1-α (HIF1-α) was upregulated in IDH1R132H expressing cells with the induction of vascular endothelial growth factor (VEGF) expression. However, knockdown of VEGF via small RNA interference had no significant influence on the cell proliferation induced by overexpression of IDH1R132H, implying that another signaling pathway may be involved. Next, forced expression of IDH1R132H was found to activate nuclear factor-κB (NF-κB), since the inhibitory IκB protein (IκBα) was highly phosphorylated and the NF-κB p65 subunit was translocated into the nucleus. Notably, knockdown of HIF1-α significantly blocked NF-κB activation, which was induced by the overexpression of IDH1 mutants. In addition, expression of IDH1 mutants markedly induced the NF-κB target gene expression, including cyclin D1 and E and c-myc, which were involved in the regulation of cell proliferation. In conclusion, it was demonstrated that the IDH1 mutant activated NF-κB in a HIF1-α‑dependent manner and was involved in the regulation of cell proliferation.

  6. Salicylic acid binding of mitochondrial alpha-ketoglutarate dehydrogenase E2 affects mitochondrial oxidative phosphorylation and electron transport chain components and plays a role in basal defense against tobacco mosaic virus in tomato.

    Science.gov (United States)

    Liao, Yangwenke; Tian, Miaoying; Zhang, Huan; Li, Xin; Wang, Yu; Xia, Xiaojian; Zhou, Jie; Zhou, Yanhong; Yu, Jingquan; Shi, Kai; Klessig, Daniel F

    2015-02-01

    Salicylic acid (SA) plays a critical role in plant defense against pathogen invasion. SA-induced viral defense in plants is distinct from the pathways mediating bacterial and fungal defense and involves a specific pathway mediated by mitochondria; however, the underlying mechanisms remain largely unknown. The SA-binding activity of the recombinant tomato (Solanum lycopersicum) alpha-ketoglutarate dehydrogenase (Slα-kGDH) E2 subunit of the tricarboxylic acid (TCA) cycle was characterized. The biological role of this binding in plant defenses against tobacco mosaic virus (TMV) was further investigated via Slα-kGDH E2 silencing and transient overexpression in plants. Slα-kGDH E2 was found to bind SA in two independent assays. SA treatment, as well as Slα-kGDH E2 silencing, increased resistance to TMV. SA did not further enhance TMV defense in Slα-kGDH E2-silenced tomato plants but did reduce TMV susceptibility in Nicotiana benthamiana plants transiently overexpressing Slα-kGDH E2. Furthermore, Slα-kGDH E2-silencing-induced TMV resistance was fully blocked by bongkrekic acid application and alternative oxidase 1a silencing. These results indicated that binding by Slα-kGDH E2 of SA acts upstream of and affects the mitochondrial electron transport chain, which plays an important role in basal defense against TMV. The findings of this study help to elucidate the mechanisms of SA-induced viral defense.

  7. 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.

  8. The Spatial Variability of Soil Dehydrogenase Activity: A Survey in Urban Soils

    OpenAIRE

    Ridvan Kizilkaya; Tayfun Aşkin

    2007-01-01

    Information on soil microorganisms and their activity used to determine microbiological characteristics are very important for soil quality and productivity. Studies of enzyme activities provide information on the biochemical processes occurring in soil. There is growing evidence that soil biological parameters may be potential and sensitive indicators of soil ecological conditions and soil management. Soil microbiological parameters may be evaluated statistically due to application of geosta...

  9. The effects of chemical and radioactive properties of Tl-201 on human erythrocyte glucose 6-phosphate dehydrogenase activity.

    Science.gov (United States)

    Sahin, Ali; Senturk, Murat; Ciftci, Mehmet; Varoglu, Erhan; Kufrevioglu, Omer Irfan

    2010-04-01

    The inhibitory effects of thallium-201 ((201)Tl) solution on human erythrocyte glucose 6-phosphate dehydrogenase (G6PD) activity were investigated. 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 (201)Tl solution including Tl(+), Fe(+3) and Cu(+2) metals and the in vitro effects of the radiation effect of the (201)Tl solution and non-radioactive Tl(+), Fe(+3) and Cu(+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 degrees C. (201)Tl solution and radiation exposure had inhibitory effects on the enzyme activity. IC(50) value of (201)Tl solution was 36.86 microl ([Tl(+)]: 0.0036 microM, [Cu(+2)]: 0.0116 microM, [Fe(+3)]: 0.0132 microM), of human erythrocytes G6PD. Seven human patients were also used for in vivo studies of (201)Tl solution. Furthermore, non-radioactive Tl(+), Fe(+3) and Cu(+2) were found not to have influenced the enzyme in vitro. Human erythrocyte G6PD activity was inhibited by exposure for up to 10 minutes to 0.057 mCi/kg (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 (201)Tl solution. Copyright 2010 Elsevier Inc. All rights reserved.

  10. 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 series of mutant strains of Lactococcus lactis were constructed with lactate dehydrogenase (LDH) activities ranging from below 1% to 133% of the wild-type activity level. The mutants with 59% to 133% of lactate dehydrogenase activity had growth rates similar to the wild-type and showed...... 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...

  11. In vitro evidence that phytanic acid compromises Na(+),K(+)-ATPase activity and the electron flow through the respiratory chain in brain cortex from young rats.

    Science.gov (United States)

    Busanello, Estela Natacha Brandt; Viegas, Carolina Maso; Moura, Alana Pimentel; Tonin, Anelise Miotti; Grings, Mateus; Vargas, Carmen R; Wajner, Moacir

    2010-09-17

    Phytanic acid (Phyt) tissue concentrations are increased in Refsum disease and other peroxisomal disorders characterized by neurologic damage and brain abnormalities. The present work investigated the in vitro effects of Phyt, at concentrations found in these peroxisomal disorders, on important parameters of energy metabolism in brain cortex of young rats. The parameters analyzed were CO(2) production from labeled acetate and glucose, the activities of the citric acid cycle enzymes citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase, as well as of the respiratory chain complexes I-IV, creatine kinase and Na(+),K(+)-ATPase. Our results show that Phyt did not alter citric acid cycle enzyme activities, or CO(2) production from acetate, reflecting no impairment of the functionality of the citric acid cycle. In contrast, respiratory chain activities were reduced at complexes I, II, I-III, II-III and IV. Membrane synaptical Na(+),K(+)-ATPase activity was also reduced by Phyt, with no alteration of creatine kinase activity. Considering the importance of the electron flow through the respiratory chain for brain energy metabolism (oxidative phosphorylation) and of Na(+),K(+)-ATPase activity for maintaining membrane potential necessary for neurotransmission, the data indicate that Phyt impairs brain bioenergetics at the level of energy formation, as well as neurotransmission. It is presumed that Phyt-induced impairment of these important systems may be involved at least in part in the neurological damage found in patients affected by disorders in which brain Phyt concentrations are increased.

  12. Decreased mitochondrial activities of malate dehydrogenase and fumarase in tomato lead to altered root growth and architecture via diverse mechanisms.

    Science.gov (United States)

    van der Merwe, Margaretha J; Osorio, Sonia; Moritz, Thomas; Nunes-Nesi, Adriano; Fernie, Alisdair R

    2009-02-01

    Transgenic tomato (Solanum lycopersicum) plants in which either mitochondrial malate dehydrogenase or fumarase was antisense inhibited have previously been characterized to exhibit altered photosynthetic metabolism. Here, we demonstrate that these manipulations also resulted in differences in root growth, with both transgenics being characterized by a dramatic reduction of root dry matter deposition and respiratory activity but opposite changes with respect to root area. A range of physiological, molecular, and biochemical experiments were carried out in order to determine whether changes in root morphology were due to altered metabolism within the root itself, alterations in the nature of the transformants' root exudation, consequences of alteration in the efficiency of photoassimilate delivery to the root, or a combination of these factors. Grafting experiments in which the transformants were reciprocally grafted to wild-type controls suggested that root length and area were determined by the aerial part of the plant but that biomass was not. Despite the transgenic roots displaying alteration in the expression of phytohormone-associated genes, evaluation of the levels of the hormones themselves revealed that, with the exception of gibberellins, they were largely unaltered. When taken together, these combined experiments suggest that root biomass and growth are retarded by root-specific alterations in metabolism and gibberellin contents. These data are discussed in the context of current models of root growth and biomass partitioning.

  13. 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.

  14. 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

    Aim. To investigate the direct relationship between tumour hypoxia and lactate dehydrogenase (Ldh) levels in serum and tumour in two different pre-clinical murine models. Materials and methods. Experiments were performed in CDF1 or C3H/Km mice implanted with a C3H mammary carcinoma and SCCVII...... 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......H/km mice was 10.5+/-2 U/ml and 12+/-2 U/ml, respectively. For C3H mammary carcinoma bearing mice, a positive correlation between tumour volume and tumour and serum Ldh was found. Tumour Ldh in SCCVII carcinomas also increased with increasing tumour volume, but no volume dependence of serum Ldh...

  15. Cloning, expression, and biochemical characterization of a novel NADP(+)-dependent 7α-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids.

    Science.gov (United States)

    Bakonyi, Daniel; Hummel, Werner

    2017-04-01

    A gene encoding a novel 7α-specific NADP(+)-dependent hydroxysteroid dehydrogenase from Clostridium difficile was cloned and heterologously expressed in Escherichia coli. The enzyme was purified using an N-terminal hexa-his-tag and biochemically characterized. The optimum temperature is at 60°C, but the enzyme is inactivated at this temperature with a half-life time of 5min. Contrary to other known 7α-HSDHs, for example from Clostridium sardiniense or E. coli, the enzyme from C. difficile does not display a substrate inhibition. In order to demonstrate the applicability of this enzyme, a small-scale biotransformation of the bile acid chenodeoxycholic acid (CDCA) into 7-ketolithocholic acid (7-KLCA) was carried out with simultaneous regeneration of NADP(+) using an NADPH oxidase that resulted in a complete conversion (<99%). Furthermore, by a structure-based site-directed mutagenesis, cofactor specificity of the 7α-HSDH from Clostridium difficile was altered to accept NAD(H). This mutant was biochemically characterized and compared to the wild-type.

  16. Diazepam- and chlordiazepoxide-mediated increases in erythrocyte aldehyde dehydrogenase activity and its possible implications.

    Science.gov (United States)

    Murthy, P; Guru, S C; Shetty, K T; Ray, R; Channabasavanna, S M

    1992-01-01

    Erythrocyte ALDH activity was assayed in alcoholic (n = 70) and nonalcoholic (n = 40) subjects. In general, alcoholics without any prior medications (n = 57) were found to have a decreased ALDH activity (mean +/- SD: 3.38 +/- 1.7 mU; p less than 0.001) as compared to control group (5.10 +/- 1.57 mU). However, a group of alcoholics who were detoxified with benzodiazepines (n = 13) prior to blood collection for enzyme assay were found to have higher ALDH activity (4.92 +/- 2.46 mU; p less than 0.05) as compared to alcoholics who were not detoxified. In vitro experiments demonstrated that both diazepam (DZM) and chlordiazepoxide (CDP) could activate the ALDH. The magnitude of enzyme activation by DZM and CDP appear to correlate with their relative potency of tranquilizing effect. Further, the observed ability of DZM to reverse the inhibition of ALDH mediated by disulfiram may explain the biochemical basis of the reported ability of benzodiazepines (BDZ) to reduce the intensity of disulfiram ethanol reaction (DER).

  17. The complex structures of isocitrate dehydrogenase from Clostridium thermocellum and Desulfotalea psychrophila suggest a new active site locking mechanism.

    Science.gov (United States)

    Leiros, Hanna-Kirsti S; Fedøy, Anita-Elin; Leiros, Ingar; Steen, Ida Helene

    2012-01-01

    Isocitrate dehydrogenase (IDH) catalyzes the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate into α-ketoglutarate and CO2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH (CtIDH), a native open apo CtIDH to 2.35 Å and a quaternary complex of CtIDH with NADP(+), isocitrate and Mg(2+) to 2.5 Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH (DpIDH) was also resolved to 1.93 Å. CtIDH and DpIDH showed similar global thermal stabilities with melting temperatures of 67.9 and 66.9 °C, respectively. CtIDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. CtIDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to DpIDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in DpIDH, were absent in CtIDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251' and Arg255 (CtIDH). These interactions lock the large domain to the small domain and direct NADP(+) into the correct orientation, which together are important for NADP(+) selectivity.

  18. 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.

  19. 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.

  20. 11beta-Hydroxysteroid dehydrogenase type 1-driven cortisone reactivation regulates plasminogen activator inhibitor type 1 in adipose tissue of obese women.

    Science.gov (United States)

    Ayachi, S Ei; Paulmyer-Lacroix, O; Verdier, M; Alessi, M-C; Dutour, A; Grino, M

    2006-03-01

    Plasminogen activator inhibitor type 1 (PAI-1) is the main inhibitor of the fibrinolytic system and contributes to an increased risk of atherothrombosis in insulin-resistant obese patients. In adipose tissue, we have shown that PAI-1 is synthesized mainly in the visceral stromal compartment and is positively regulated by glucocorticoids. We have demonstrated that adipose tissue expression of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1), an enzyme that catalyzes the conversion of inactive cortisone to active cortisol, is exaggerated in obese patients. We hypothesized that increased action of 11beta-HSD-1 in adipose tissue of obese subjects may contribute to PAI-1 overproduction. Using in situ hybridization, we studied the expression of the mRNAs coding for PAI-1 and 11beta-HSD-1 in the stromal compartment of visceral adipose tissue obtained from obese women. The regulation of PAI-1 secretion from in vitro incubated tissue explants was also investigated. Regression analysis showed a significant positive linear relationship between PAI-1 and 11beta-HSD-1 mRNAs expression. In vitro incubation of adipose tissue explants demonstrated that cortisone stimulated PAI-1 gene expression and secretion, and that these effects were inhibited by co-incubation with the 11beta-HSD inhibitor, glycyrrhetinic acid. Our data demonstrate that 11beta-HSD-1-driven cortisone reactivation regulates adipose PAI-1 synthesis and secretion. They suggest that the increased PAI-1 synthesis and secretion observed in obese patients can be also related, at least in part, to an increased local conversion of cortisone to cortisol. Therefore, local cortisol metabolism in adipose tissue may be involved in increasing the risk of cardiovascular disease in obese subjects.

  1. 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.

  2. EFFECT OF TRIGONELLA FOENUM GRAECUM ON LACTATE DEHYDROGENASE (LDH ACTIVITY OF BLOOD, LIVER AND PANCREAS IN NORMAL AND ALLOXAN- INDUCED DIABETIC MICE

    Directory of Open Access Journals (Sweden)

    Sekaran Sridhar et al.

    2012-02-01

    Full Text Available The effect of aqueous seeds extract of Trigonella foenum graecum Linn was studied on Lactate dehydrogenase (LDH activity of blood, liver and pancreas in normal and alloxan- induced diabetic mice. Our study showed that aqueous seeds extract, Oral administration of 50 mg/animal (0.5 ml of extract in alternative days up to 7 days (1st, 3rd, 5th & 7th day. In alloxan induced diabetic mice, there was a significant increase in LDH activity of all the three tissues. The enzyme Lactate dehydrogenase showed significant decrease in the diabetic group treated with aqueous extract of tested plant when compared with the diabetic group. It is clear from the current data in this study that ginseng aqueous extract was the most efficient of the tested plant.

  3. Structural characterization of a β-hydroxyacid dehydrogenase from Geobacter sulfurreducens and Geobacter metallireducens with succinic semialdehyde reductase activity.

    Science.gov (United States)

    Zhang, Yanfeng; Zheng, Yi; Qin, Ling; Wang, Shihua; Buchko, Garry W; Garavito, R Michael

    2014-09-01

    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 are observed to reduce succinic semialdehyde, a 4-carbon substrate instead of the typical β-HAD 3-carbon substrate, to γ-hydroxybutyric acid. To further explore the structural and functional characteristics of these two β-HADs with a less frequently observed 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 structures of both proteins are similar, composed of 14 α-helices and nine β-strands organized into two domains. Domain 1 (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 2 (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, with a buried molecule of NADP(+), at the interdomain cleft. Comparison of these Geobacter structures to a closely related β-HAD from Arabidopsis thaliana in the apo-NADP(+) and apo-substrate bound state suggests that NADP(+) binding effects a rigid body rotation between Domains 1 and 2. Bound

  4. 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

  5. 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.

  6. 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.

  7. 11β-Hydroxysteroid Dehydrogenase Activity in the Brain Does Not Contribute to Systemic Interconversion of Cortisol and Cortisone in Healthy Men

    OpenAIRE

    Kilgour, Alixe H M; Semple, Scott; Marshall, Ian; Andrews, Peter; Andrew, Ruth; Walker, Brian R.

    2015-01-01

    Context and Objective: 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) catalyses regeneration of cortisol in liver, adipose tissue, and skeletal muscle, making a substantial contribution to circulating cortisol as demonstrated in humans by combining stable isotope tracer infusion with arteriovenous sampling. In the brain, 11βHSD1 is a potential therapeutic target implicated in age-associated cognitive dysfunction. We aimed to quantify brain 11βHSD1 activity, both to assess its contribution ...

  8. Annotated compound data for modulators of detergent-solubilised or lipid-reconstituted respiratory type II NADH dehydrogenase activity obtained by compound library screening

    OpenAIRE

    Dunn, Elyse A.; Cook, Gregory M.; Adam Heikal

    2015-01-01

    The energy-generating membrane protein NADH dehydrogenase (NDH-2), a proposed antibacterial drug target (see “Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs” Weinstein et al. 2005 [1]), was screened for modulators of activity in either detergent-solublised or lipid reconstituted (proteolipsome) form. Here we present an annotated list of compounds identified in a small-scale screen against NDH-2. The dataset contains information regarding the li...

  9. 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......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......), tetradecadienoic acid, 14:2(n-6), and hexadecadienoic acid, 16:2(n-6). Palmitoyl-CoA and behenoyl-CoA dehydrogenase in fibroblasts were deficient. Muscle VLCAD activity was very low. DNA analysis revealed compound heterozygosity for two missense mutations in the VLCAD gene. The relatively mild clinical course may...

  10. The effect of ammonium ions on the activity of glutamate dehydrogenase, alanine aminotransferase and aspartate aminotransferase in Cucumis sativus L. seedlings

    Directory of Open Access Journals (Sweden)

    Genowefa Kubiak-Dobosz

    2014-02-01

    Full Text Available Changes in the activity of glutamate dehydrogenase (GDH, alanine aminotransferase (GPT and aspartate aminotransferase (GOT were studied in various organs of Cucumis sativus L. seedlings in relation to the uptake of mineral nitrogen (in form of N03- or NH4+ from the medium. Activity of GDH, GPT, and GOT was higher in young leaves and roots of cucumber seedlings if the plants developed- in an ammonium medium. No similar changes of aminotransferases activity were noted in the cotyledons. Factors affecting varying effect of ammonium ions upon GPT and GOT activity are discussed for particular organs of cucumber seedlings.

  11. 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

    2016-02-01

    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.

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

    Science.gov (United States)

    Kuivanen, Joosu; Arvas, Mikko; Richard, Peter

    2017-01-01

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

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

    Science.gov (United States)

    Kuivanen, Joosu; Arvas, Mikko; Richard, Peter

    2017-01-01

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

  14. 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

  15. Enhanced enzymatic activity of glycerol-3-phosphate dehydrogenase from the cryophilic Saccharomyces kudriavzevii.

    Science.gov (United States)

    Oliveira, Bruno M; Barrio, Eladio; Querol, Amparo; Pérez-Torrado, Roberto

    2014-01-01

    During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied the expression of glycerol biosynthetic pathway genes and we observed a higher expression of GPD1 gene in S. kudriavzevii compared to S. cerevisiae in micro-vinification conditions. We observed higher enzymatic activity of Gpd1p in S. kudriavzevii in response to osmotic and cold stress. Also, we determined that S. kudriavzevii Gpd1p enzyme presents increased catalytic properties that will contribute to increase glycerol production. Finally, we evaluated the glycerol production with S. cerevisiae, S. kudriavzevii or a recombinant Gpd1p variant in the same background and observed that the S. kudriavzevii enzyme produced increased glycerol levels at 12 or 28°C. This suggests that glycerol is increased in S. kudriavzevii mainly due to increased V max of the Gpd1p enzyme. All these differences indicate that S. kudriavzevii has changed the metabolism to promote the branch of the glycolytic pathway involved in glycerol production to adapt to low temperature environments and maintain the NAD(+)/NADH ratio in alcoholic fermentations. This knowledge is industrially relevant due to the potential use, for example, of S. cerevisiae-S. kudriavzevii hybrids in the wine industry where glycerol content is an important quality parameter.

  16. 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.

  17. Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen.

    Directory of Open Access Journals (Sweden)

    Anne Gründel

    Full Text Available The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhesins and release of cell-damaging factors, surface-displayed glycolytic enzymes may facilitate the further interaction between host and microbe. In this study, we described detection of the four subunits of pyruvate dehydrogenase complex (PDHA-D among the cytosolic and membrane-associated proteins of M. pneumoniae. Subunits of PDH were cloned, expressed and purified to produce specific polyclonal guinea pig antisera. Using colony blotting, fractionation of total proteins and immunofluorescence experiments, the surface localization of PDHA-C was demonstrated. All recombinant PDH subunits are able to bind to HeLa cells and human plasminogen. These interactions can be specifically blocked by the corresponding polyclonal antisera. In addition, an influence of ionic interactions on PDHC-binding to plasminogen as well as of lysine residues on the association of PDHA-D with plasminogen was confirmed. The PDHB subunit was shown to activate plasminogen and the PDHB-plasminogen complex induces degradation of human fibrinogen. Hence, our data indicate that the surface-associated PDH subunits might play a role in the pathogenesis of M. pneumoniae infections by interaction with human plasminogen.

  18. 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.

  19. Active site cysteine-null glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rescues nitric oxide-induced cell death.

    Science.gov (United States)

    Kubo, Takeya; Nakajima, Hidemitsu; Nakatsuji, Masatoshi; Itakura, Masanori; Kaneshige, Akihiro; Azuma, Yasu-Taka; Inui, Takashi; Takeuchi, Tadayoshi

    2016-02-29

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a homotetrameric enzyme involved in a key step of glycolysis, also has a role in mediating cell death under nitrosative stress. Our previous reports suggest that nitric oxide-induced intramolecular disulfide-bonding GAPDH aggregation, which occurs through oxidation of the active site cysteine (Cys-152), participates in a mechanism to account for nitric oxide-induced death signaling in some neurodegenerative/neuropsychiatric disorders. Here, we demonstrate a rescue strategy for nitric oxide-induced cell death accompanied by GAPDH aggregation in a mutant with a substitution of Cys-152 to alanine (C152A-GAPDH). Pre-incubation of purified wild-type GAPDH with C152A-GAPDH under exposure to nitric oxide inhibited wild-type GAPDH aggregation in a concentration-dependent manner in vitro. Several lines of structural analysis revealed that C152A-GAPDH extensively interfered with nitric oxide-induced GAPDH-amyloidogenesis. Overexpression of doxycycline-inducible C152A-GAPDH in SH-SY5Y neuroblastoma significantly rescued nitric oxide-induced death, concomitant with the decreased formation of GAPDH aggregates. Further, both co-immunoprecipitation assays and simulation models revealed a heterotetramer composed of one dimer each of wild-type GAPDH and C152A-GAPDH. These results suggest that the C152A-GAPDH mutant acts as a dominant-negative molecule against GAPDH aggregation via the formation of this GAPDH heterotetramer. This study may contribute to a new therapeutic approach utilizing C152A-GAPDH against brain damage in nitrosative stress-related disorders.

  20. Malate dehydrogenase: a model for structure, evolution, and catalysis.

    OpenAIRE

    1994-01-01

    Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid s...

  1. A novel genetically-obese rat model with elevated 11beta-hydroxysteroid dehydrogenase type 1 activity in subcutaneous adipose tissue

    OpenAIRE

    Giridharan Nappan V; Reddy Sirisha J; Kumar Chodavarapu; Prashanth Anamthathmakula; Prasad Sakamuri; Vajreswari Ayyalasomayajula

    2010-01-01

    Abstract 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the conversion of inactive glucocorticoids to active glucocorticoids and plays an important role in the development of obesity and metabolic syndrome. 11β-HSD1 activity is lower in liver and higher in omental adipose tissue of obese rodent models like obese zucker rats, Ob/Ob and db/db mice. Here, we report the 11β-HSD1 activity in liver and adipose tissue of lean and obese rats of WNIN/Ob strain, a new genetic rat model of...

  2. 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.

  3. Active-site structure of the soluble quinoprotein glucose dehydrogenase complexed with methylhydrazine : A covalent cofactor-inhibitor complex

    NARCIS (Netherlands)

    Oubrie, Arthur; Rozeboom, Henriëtte J.; Dijkstra, Bauke W.

    1999-01-01

    Soluble glucose dehydrogenase (s-GDH) from the bacterium Acinetobacter calcoaceticus is a classical quinoprotein. It requires the cofactor pyrroloquinoline quinone (PQQ) to catalyze the oxidation of glucose to gluconolactone, The precise catalytic role of PQQ in s-GDH and several other PQQ-dependent

  4. 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

  5. The cytotoxic activity of ursolic acid derivatives.

    Science.gov (United States)

    Ma, Chao-Mei; Cai, Shao-Qing; Cui, Jing-Rong; Wang, Rui-Qing; Tu, Peng-Fei; Hattori, Masao; Daneshtalab, Mohsen

    2005-06-01

    Ursolic acid and 2alpha-hydroxyursolic acid isolated from apple peels were found to show growth inhibitory activity against four tumor cell lines, HL-60, BGC, Bel-7402 and Hela. Structural modifications were performed on the C-3, C-28 and C-11 positions of ursolic acid and the cytotoxicity of the derivatives was evaluated. The SAR revealed that the triterpenes possessing two hydrogen-bond forming groups (an H-donor and a carbonyl group) at positions 3 and 28 exhibit cytotoxic activity. The configuration at C-3 was found to be important for the activity. Introduction of an amino group increased the cytotoxicity greatly. A 3beta-amino derivative was 20 times more potent than the parent ursolic acid. The 28-aminoalkyl dimer compounds showed selective cytotoxicity.

  6. Aconitase is the main functional target of aging in the citric acid cycle of kidney mitochondria from mice

    OpenAIRE

    Yarian, Connie S.; Toroser, Dikran; Sohal, Rajindar S.

    2005-01-01

    The activities of the citric acid cycle enzymes were determined in mitochondria isolated from kidneys of relatively young, middle age, and old mice. Aconitase exhibited the most significant decrease in activity with age. The activity of α-ketoglutarate dehydrogenase exhibited a modest decrease in activity, while NADP+-isocitrate dehydrogenase (NADP+-ICD) activity increased moderately with age. Activities of citrate synthase, NAD+-isocitrate dehydrogenase (NAD+-ICD), succinyl-CoA synthetase (S...

  7. 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

  8. 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.

  9. Glucose-6-phosphate dehydrogenase

    Science.gov (United States)

    ... 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 helps red ...

  10. 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 ...

  11. Diagnostic Value of Serum Lactate Dehydrogenase Isoenzyme and Amino Acid Patterns in Several Schistosomal and Non-Schistosomal Disorders as Compared to other Biochemical Parameters

    Directory of Open Access Journals (Sweden)

    Samia A. Ahmed

    1996-01-01

    Full Text Available Serum lactate dehydrogenase (LDH isoenzyme and amino acid (a. a patterns were evaluated in comparison to several other biochemical parameters for liver and renal function with the objective of clarifying the differential diagnosis of hepatic disorders and predicting the outcome of schistosomal infection in Egyptian patients. Patients examined included those with complicated hepatic disorders and others with different stages of schistosomal infestation, hepatoma or bladder cancer, in addition to a normal control group. Several biochemical parameters appeared to be useful in establishing consistent differences or similarities between the studied groups. Examples are; elevated serum AST/ AL T ratio and methionine content in chronic schistosomiasis, elevated serum urea/creatinine ratio and leucine content in all schistosomal patients and extremely high levels of N-acetyl-β-D-glucosaminidase (NAG in the urine of non-schistosomal bladder cancer patients. In addition, characteristic LDH isoenzyme profiles distinguish between the studied groups, in particular separating chronic schistosomiasis from schistosomal bladder cancer and hepatoma from other hepatic disorders.

  12. 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.

  13. 不同农药对土壤脱氢酶活性的影响%Influence of Different Pesticides on Soil Dehydrogenase Activity

    Institute of Scientific and Technical Information of China (English)

    姜虎生; 王宏燕

    2011-01-01

    Experiment studies the influence of the Bo sprinkling, ground insect killer, hypertonic imidaclo-prid, trichloro-4 pesticides on soil dehydrogenase activity and respiration. The results showed that; soil dehy-drogenase activity at low , reduced with the addition increase of hypertonic imidacloprid, triclosan, expressed as inhibition, when the concentration exceeds a certain threshold ,stabilized. The Bo sprinkling, insect killer to increase with the amount of pesticides, inhibition of dehydrogenase activity was first re-activated; adding an equal amount (0. 01ml) of pesticides in soil samples increased with incubation time was linear growth inhibition; Different pesticides on soil respiration showed an activation.%研究了博洒、地虫克星、高渗吡虫啉、三氯4种农药对土壤脱氢酶活性及呼吸作用的影响。试验结果表明:土壤脱氢酶活性在低剂量(<2 ml)时随投加高渗吡虫啉、三氯的增加而降低,表现为抑制作用,当超过一定浓度阈值后趋于稳定。而博洒、地虫克星随农药用量的增加,脱氢酶活性先抑制再激活;加入等量(0.01m1)农药的土样随培养时间增加抑制率呈线形增长;不同农药对3种土壤呼吸作用均表现为激活作用。

  14. Growth hormone (GH) substitution in GH-deficient patients inhibits 11beta-hydroxysteroid dehydrogenase type 1 messenger ribonucleic acid expression in adipose tissue.

    Science.gov (United States)

    Paulsen, Søren Kildeberg; Pedersen, Steen Bønløkke; Jørgensen, Jens Otto Lunde; Fisker, Sanne; Christiansen, Jens Sandahl; Flyvbjerg, Allan; Richelsen, Bjørn

    2006-03-01

    Local tissue activity of glucocorticoids is in part determined by the isoenzymes 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) and 11beta-HSD2, interconverting inert cortisone and active cortisol. Increased tissue activity of cortisol may play a central role in the features of GH deficiency and the metabolic syndrome. We investigated the effects of GH treatment on adipose tissue 11beta-HSD mRNA. A randomized placebo-controlled double-blind study design was used. Twenty-three GH-deficient patients (16 males and seven females) were randomized to 4 months of GH treatment (2 IU/m2) (n = 11) or placebo treatment (n = 12). Adipose tissue biopsies and blood samples were obtained before and after treatment. Biopsies were obtained from the abdominal sc depot at the level of the umbilicus and do not necessarily reflect the metabolically more important visceral adipose tissue. Gene expressions were determined by real-time RT-PCR. GH treatment decreased 11beta-HSD1 mRNA 66% [95% confidence interval (CI), 23-107%; P adipose tissue. Serum IGF-I and IGF-I mRNA increased in the GH-treated group by 187% (95% CI, 122-250%; P cortisol in adipose tissue.

  15. Investigation of the possible protective role of gallic acid on paraoxanase and arylesterase activities in livers of rats with acute alcohol intoxication.

    Science.gov (United States)

    Kartkaya, Kazim; Oğlakçi, Ayşegül; Şentürk, Hakan; Bayramoğlu, Gökhan; Canbek, Mediha; Kanbak, Güngör

    2013-04-01

    Gallic acid, a polyphenyl class natural product from gallnut and green tea, is known to be antioxidant, anti-inflammatory and radical scavenger. In this study, we aimed to investigate the possible protective effects of gallic acid on paraoxonase and arylesterase activities in liver exposed to acute alcohol intoxication. Paraoxonase and arylesterase activities in liver tissue and serum aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase levels were measured. Histological investigations were also made. In our study, we observed a significant increase of serum alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase activities, which are indicators of liver damage after acute ethanol consumption. Gallic acid therapy has significantly reduced the increase in these biomarkers, indicating a possible hepatoprotective effect of gallic acid. Ethanol consumption caused a significant decrease in liver paraoxonase activity (P Gallic acid treatment partly restored this decreased paraoxonase activity, which resulted from ethanol administration. A gallic acid dose of 100 mg/kg was observed as highest restoring effect for paraoxonase activity (P gallic acid treatment restored the loss of this activity due to ethanol exposure (P gallic acid ameliorates the liver damage caused by excessive alcohol consumption in a dose-dependent way. Our results in this study showed that gallic acid might have a protective effect against alcoholic liver disease.

  16. [Effect of reproduction of the LPP-3 cyanophage on glutamate dehydrogenase and glutamine synthetase activity in the cyanobacterium Plectonema boryanum].

    Science.gov (United States)

    Mendzhul, M I; Koltukova, N V; Lysenko, T G; Shainskaia, O A; Perepelitsa, S I

    1995-01-01

    The effect of cyanophage LPP-3 reproduction on glutamate dehydrogenase and glutamine synthetase (GS) in P boryanum cells have been studied. It was determined that the both reactions are intensified by 135% and 220%, accordingly. Isoenzymes of GS were purified from native and infected cell of cyanobacteria. Their physical-and-chemical properties are different. The cyanophage development probably causes specific modification of the cell enzymes.

  17. 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.

  18. Aqueous soluble tetrazolium/formazan MTS as an indicator of NADH- and NADPH-dependent dehydrogenase activity.

    Science.gov (United States)

    Dunigan, D D; Waters, S B; Owen, T C

    1995-10-01

    Recently a new tetrazolium was described for the use of monitoring cell viability in culture. This tetrazolium, commonly referred to as MTS [3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], has the unusual property that it can be reduced to a water-soluble formazan. beta-Nicotinamide adenine dinucleotide/reduced (NADH) and beta-nicotinamide adenine dinucleotide phosphate/reduced (NADPH) are examples of physiologically important reducing agents. In cell-free studies, MTS was reduce to the soluble formazan in the presence of NADH and NADPH, and reaction were compared to those with dithiothreitol (DTT) or 2-mercaptoethanol (2-ME). The efficiency of these reactions was enhanced 1000-fold by the presence of phenazine methosulfate. Selectivity in the electron transfer from NADPH was slightly greater than NADH, and NADPH or NADH was much greater than the thiols DTT or 2-ME. Generation of either NADH or NADPH in solution by malate dehydrogenase or isocitrate dehydrogenase, respectively, was monitored by the MTS reduction reaction. The rate of formazan formation was comparable to the formation of NADH or NADPH. This system represents a useful tool for evaluating reaction kinetics in solutions of NAD- or NADP-dependent dehydrogenase enzymes, and these reactions can be performed in typical biological buffers containing reducing agents without significant interference to the MTS/formazan system.

  19. Glucose-stimulated insulin secretion does not require activation of pyruvate dehydrogenase: impact of adenovirus-mediated overexpression of PDH kinase and PDH phosphate phosphatase in pancreatic islets.

    Science.gov (United States)

    Nicholls, Linda I; Ainscow, Edward K; Rutter, Guy A

    2002-03-01

    Glucose-stimulated increases in mitochondrial metabolism are generally thought to be important for the activation of insulin secretion. Pyruvate dehydrogenase (PDH) is a key regulatory enzyme, believed to govern the rate of pyruvate entry into the citrate cycle. We show here that elevated glucose concentrations (16 or 30 vs 3 mM) cause an increase in PDH activity in both isolated rat islets, and in a clonal beta-cell line (MIN6). However, increases in PDH activity elicited with either dichloroacetate, or by adenoviral expression of the catalytic subunit of pyruvate dehydrogenase phosphatase, were without effect on glucose-induced increases in mitochondrial pyridine nucleotide levels, or cytosolic ATP concentration, in MIN6 cells, and insulin secretion from isolated rat islets. Similarly, the above parameters were unaffected by blockade of the glucose-induced increase in PDH activity by adenovirus-mediated over-expression of PDH kinase (PDK). Thus, activation of the PDH complex plays an unexpectedly minor role in stimulating glucose metabolism and in triggering insulin release.

  20. The activity of 11β-hydroxysteroid dehydrogenase type 2 enzyme and cortisol secretion in patients with adrenal incidentalomas.

    Science.gov (United States)

    Morelli, Valentina; Polledri, Elisa; Mercadante, Rosa; Zhukouskaya, Volha; Palmieri, Serena; Beck-Peccoz, Paolo; Spada, Anna; Fustinoni, Silvia; Chiodini, Iacopo

    2016-09-01

    In adrenal incidentaloma (AI) patients, beside the cortisol secretion, a different 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity, measurable by 24-h urinary cortisol/cortisone ratio (R-UFF/UFE) (the higher R-UFF/UFE the lower HSD11B2 activity), could influence the occurrence of the subclinical hypercortisolism (SH)-related complications (hypertension, type 2 diabetes, obesity). We evaluated whether in AI patients, UFF levels are associated to UFE levels, and the HSD11B2 activity to the complications presence. In 156 AI patients (93F, age 65.2 ± 9.5 years), the following were measured: serum cortisol after 1 mg-dexamethasone test (1 mg-DST), ACTH, UFF, UFE levels, and R-UFF/UFE (by liquid chromatography-tandem mass spectrometry), the latter was also evaluated in 63 matched-controls. We diagnosed SH (n = 22) in the presence of ≥2 among ACTH 83 nmol/L. Patients showed higher UFF levels and R-UFF/UFE than controls (75.9 ± 43.1 vs 54.4 ± 22.9 nmol/24 h and 0.26 ± 0.12 vs 0.20 ± 0.07, p < 0.005, respectively) but comparable UFE levels (291 ± 91.1 vs 268 ± 61.5, p = 0.069). The R-UFF/UFE was higher in patients with high (h-UFF, n = 28, 0.41 ± 0.20) than in those with normal (n-UFF, 0.22 ± 0.10, p < 0.005) UFF levels and in patients with SH than in those without SH (0.30 ± 0.12 vs 0.25 ± 0.12, p = 0.04). UFF levels were associated with R-UFF/UFE (r = 0.849, p < 0.001) in n-UFF, but not in h-UFF patients. Among h-UFF patients, the complications prevalence was not associated with R-UFF/UFE values. In AI patients, the UFF increase is not associated with a UFE increase. The HSD11B2 activity is inversely associated with UFF levels in n-UFF patients but not in h-UFF patients, and it is not associated with the SH complications.

  1. 4-Dihydromethyltrisporate dehydrogenase, an enzyme of the sex hormone pathway in Mucor mucedo, is constitutively transcribed but its activity is differently regulated in (+) and (-) mating types.

    Science.gov (United States)

    Schimek, Christine; Petzold, Annett; Schultze, Kornelia; Wetzel, Jana; Wolschendorf, Frank; Burmester, Anke; Wöstemeyer, Johannes

    2005-09-01

    4-Dihydromethyltrisporate dehydrogenase (TDH) converts the (+) mating type sex pheromone 4-dihydromethyltrisporate into methyltrisporate. In Mucor mucedo, this conversion is required only in the (-) mating type. Expression of the TDH encoding TSP1 gene was analyzed qualitatively using reverse-transcribed PCR. TSP1 is constitutively transcribed in the (+) and in the (-) mating type, irrespective of the mating situation. By immunodetection, the translation product is also formed constitutively. In contrast to gene expression, TDH enzyme activity depends on the sexual status of the mycelium. Activity is restricted to the sexually stimulated (-) mating type. Non-stimulated (-), as well as stimulated and non-stimulated (+) mycelia exhibit no activity and do not influence activity in stimulated (-) mycelia. Time course analysis shows strongly increased enzyme activity at 80 min after stimulation. Low activity exists from the onset of stimulation, indicating that additional regulation mechanisms are involved in TDH function.

  2. 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

  3. 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.

  4. 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.

  5. 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...... enhanced in the strain deleted for lactate dehydrogenase. What is more surprising is that the enzyme had a strong negative control (C- LDH(F1)J=-1.3) on the flux to formate at the wild-type level of lactate dehydrogenase. Furthermore, we showed that L. lactis has limited excess of capacity of lactate...

  6. 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.

  7. Effect of Pd(II and Ni(II coordination compounds with 4-amino-3-mercapto-5-methyl-1,2,4-triazole on the mitochondrial dehydrogenases activity

    Directory of Open Access Journals (Sweden)

    S. I. Orysyk

    2015-02-01

    Full Text Available Pd(II and Ni(II complex compounds: [Pd(AMMT2]Cl2 (1, [Pd(AMMT4]Cl2 (2 and [Ni(AMMT2(H2O2](NO32 (3 with 4-amino-3-mercapto-5-methyl-1,2,4-triazole (AMMT have been synthesized. The spectral characteristics of 1, 2 were studied by 1H (13C NMR and UV-Vis spectroscopy. X-ray diffraction studies established that all complexes contain the AMMT molecule, which are coordinated to the central metal ion in the thione tautomeric form. At the ratio M : L = 1 : 2 ligand is coordinated in bidentate chelate manner by the nitrogen of amino- and sulfur of mercapto group (compounds 1, 3. But the molar ratio M : L = 1 : 4 leads to monodentate coordination of AMMT molecules only by sulfur of mercaptogroup (complex 2. Vacant coordination sites of the metal ion are occupied by water molecules (complex 3. The screening of complexes 1−3 and starting compounds [АММТ, K2PdCl4 (4, Ni(NO32∙6H2O (5] by their mitochondrial dehydrogenase activity have been performed by us for the first time, resulting in established that the Pd(II complexes (1, 2, Pd(II salt (4 and AMMT normalize the activity of mitochondrial dehydrogenases of cancer HeLa cells, identified by MTT-test. In contrast, the Ni(II complex (3 and Ni(II salt (5 do not stimulate the activity of mitochondrial dehydrogenases. It has been found, that all investigated compounds do not affect on the cell cycle and the level of apoptotic cells as well as do not show a toxic effect. Thus, these results indicate that AMMT and Pd(II complexes may be used as modifiers of mitochondrial respiration, which dysfunction is particularly evident in the tumor cells.

  8. Detection of activity and mass spectrometric identification of mouse liver carboxylesterase and aldehyde dehydrogenase separated by non-denaturing two-dimensional electrophoresis after extraction with detergents.

    Science.gov (United States)

    Shimazaki, Youji; Manabe, Takashi

    2005-05-20

    To examine the activities and identity of enzymes associated with organelles such as microsomes and mitochondria, proteins from mouse liver were extracted using the non-ionic detergents Nonidet P-40 (NP-40), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene isooctylphenyl ester (Triton X), n-octyl beta-D-glucoside (octyl glycoside) or anionic detergent sodium dodecylsulfate (SDS) after the removal of cytosolic proteins. The proteins extracted by detergents were separated by non-denaturing two-dimensional electrophoresis (2-DE). The activities of esterase and aldehyde dehydrogenase were retained by non-denaturing 2-DE after treatment with each non-ionic detergent, but the activities were reduced or lost when the proteins were extracted with more than 0.5% SDS. For proteomic analysis of the organelle-associated proteins in mouse liver, proteins were separated by non-denaturing 2-DE and were identified using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after the proteins were solubilized by octyl glycoside, NP-40 and 0.1% SDS. Several organelle-associated proteins such as carboxylesterase, aldehyde dehydrogenase, glucose regulated protein and HSP60 were identified. These results indicate that the activities and identity of detergent-soluble enzymes can be examined by this non-denaturing 2-DE and mass spectrometry.

  9. In vitro evidence that D-serine disturbs the citric acid cycle through inhibition of citrate synthase activity in rat cerebral cortex.

    Science.gov (United States)

    Zanatta, Angela; Schuck, Patrícia Fernanda; Viegas, Carolina Maso; Knebel, Lisiane Aurélio; Busanello, Estela Natacha Brandt; Moura, Alana Pimentel; Wajner, Moacir

    2009-11-17

    The present work investigated the in vitro effects of D-serine (D-Ser) on important parameters of energy metabolism in cerebral cortex of young rats. The parameters analyzed were CO(2) generation from glucose and acetate, glucose uptake and the activities of the respiratory chain complexes I-IV, of the citric acid cycle enzymes citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase and of creatine kinase and Na(+),K(+)-ATPase. Our results show that D-Ser significantly reduced CO(2) production from acetate, but not from glucose, reflecting an impairment of the citric acid cycle function. Furthermore, D-Ser did not affect glucose uptake. We also observed that the activity of the mitochondrial enzyme citrate synthase from mitochondrial preparations and purified citrate synthase was significantly inhibited by D-Ser, whereas the other activities of the citric acid cycle as well as the activities of complexes I-III, II-III, II and IV of the respiratory chain, creatine kinase and Na(+),K(+)-ATPase were not affected by this D-amino acid. We also found that L-serine did not affect citrate synthase activity from mitochondrial preparations and purified enzyme. The data indicate that D-Ser impairs the citric acid cycle activity via citrate synthase inhibition, therefore compromising energy metabolism production in cerebral cortex of young rats. Therefore, it is presumed that this mechanism may be involved at least in part in the neurological damage found in patients affected by disorders in which D-Ser metabolism is impaired, with altered cerebral concentrations of this D-amino acid.

  10. Often Ignored Facts about the Control of the 2-Oxoglutarate Dehydrogenase Complex

    Science.gov (United States)

    Strumilo, Slawomir

    2005-01-01

    Information about the control of the activity of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme in the citric acid cycle, is not well covered in the biochemical education literature, especially as it concerns the allosteric regulation of OGDHC by adenine nucleotide and ortophosphate. From experimental work published during the last…

  11. Often Ignored Facts about the Control of the 2-Oxoglutarate Dehydrogenase Complex

    Science.gov (United States)

    Strumilo, Slawomir

    2005-01-01

    Information about the control of the activity of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme in the citric acid cycle, is not well covered in the biochemical education literature, especially as it concerns the allosteric regulation of OGDHC by adenine nucleotide and ortophosphate. From experimental work published during the last…

  12. 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.

  13. Divergent effects of retinoic acids on the expression of ERalpha and 17beta-hydroxysteroid dehydrogenase type 2 in endometrial carcinoma cells (RL 95-2).

    Science.gov (United States)

    Li, Xiao-Hong; Li, Hui; Xiao, Zhi-Jie; Piao, Yun-Shang

    2002-02-01

    The effects of E2 are dependent on ERs and local E2 concentration in target cells. Modulation of intracellular E2 concentration involves the action of 17beta-hydroxysteroid dehydrogenase (17HSD) type 2, the enzyme converting E2 to estrone. In the present study, the influence of RAs on the growth of endometrial cancer cell line RL 95-2 as well as the expression of ERs and 17HSD type 2 have been investigated. It was found that RAs repress the growth of RL 95-2 cells, which express all subtypes of RXR and RAR, as examined by RT-PCR. Also, quantitative RT-PCR analysis showed that both ERalpha and ERbeta are present in RL 95-2 cells, and Western blot assay further revealed that ERalpha expression was decreased by all trans-RA treatment. In contrast, RAs induced 17HSD type 2 mRNA expression in a dose- and time-dependent fashion. This stimulatory effect was also detected at the level of in vivo oxidative 17HSD activity in cultured cells. On the other hand, the abundance of 17HSD type 2 mRNA was not altered by RAs in cultured normal epithelial cells isolated from human early- and late-secretory endometrium. The data indicate that RAs have an inhibitory effect on the growth of RL 95-2 cells and a cross-talk with the estrogen pathway in estrogen-responsive endometrial cancer cells.

  14. Folic acid supplementation during pregnancy induces sex-specific changes in methylation and expression of placental 11β-hydroxysteroid dehydrogenase 2 in rats.

    Directory of Open Access Journals (Sweden)

    Reyna Penailillo

    Full Text Available In the placenta, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2 limits fetal glucocorticoid exposure and its inhibition has been associated to low birth weight. Its expression, encoded by the HSD11B2 gene is regulated by DNA methylation. We hypothesized that maternal diets supplemented with folic acid (FA during pregnancy modify the expression of placental HSD11B2 through gene methylation. Wistar rats were fed with high (8 mg/kg or normal low (1mg/kg, control levels of FA during pregnancy. Concentrations of mRNA and protein in placentas were determined by qRT-PCR and Western blot respectively. Methylation in five CpG sites of the placental HSD11B2 promoter (-378 to -275 was analyzed by bacterial cloning and subsequent sequencing. In the FA-supplemented group, mRNA and protein levels of 11β-HSD2 decreased by 58% and increased by 89%, respectively, only in placentas attached to males. In controls, most CpG sites were not methylated except for the CpG2 site which was 80% methylated. CpG2 methylation level increased under the FA treatment; however, only in placentas attached to females was this increase significant (113%. This change was not related to HSD11B2 expression. Fetal weight of females from FA- supplemented mothers was 6% higher than females from control mothers. In conclusion, this is the first study reporting that FA over supplementation during pregnancy modifies the placental HSD11B2 gene expression and methylation in a sex-dependent manner, suggesting that maternal diets with high content of FA can induce early sex-specific responses, which may lead to long-term consequences for the offspring.

  15. Folic acid supplementation during pregnancy induces sex-specific changes in methylation and expression of placental 11β-hydroxysteroid dehydrogenase 2 in rats.

    Science.gov (United States)

    Penailillo, Reyna; Guajardo, Angelica; Llanos, Miguel; Hirsch, Sandra; Ronco, Ana Maria

    2015-01-01

    In the placenta, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) limits fetal glucocorticoid exposure and its inhibition has been associated to low birth weight. Its expression, encoded by the HSD11B2 gene is regulated by DNA methylation. We hypothesized that maternal diets supplemented with folic acid (FA) during pregnancy modify the expression of placental HSD11B2 through gene methylation. Wistar rats were fed with high (8 mg/kg) or normal low (1mg/kg, control) levels of FA during pregnancy. Concentrations of mRNA and protein in placentas were determined by qRT-PCR and Western blot respectively. Methylation in five CpG sites of the placental HSD11B2 promoter (-378 to -275) was analyzed by bacterial cloning and subsequent sequencing. In the FA-supplemented group, mRNA and protein levels of 11β-HSD2 decreased by 58% and increased by 89%, respectively, only in placentas attached to males. In controls, most CpG sites were not methylated except for the CpG2 site which was 80% methylated. CpG2 methylation level increased under the FA treatment; however, only in placentas attached to females was this increase significant (113%). This change was not related to HSD11B2 expression. Fetal weight of females from FA- supplemented mothers was 6% higher than females from control mothers. In conclusion, this is the first study reporting that FA over supplementation during pregnancy modifies the placental HSD11B2 gene expression and methylation in a sex-dependent manner, suggesting that maternal diets with high content of FA can induce early sex-specific responses, which may lead to long-term consequences for the offspring.

  16. Effect of noise exposure (85 dB ) on testicular adrenocortical steroidogenic key enzymes, acid and alkaline phosphatase activities of sex organs in mature albino rats

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Changes in the activities of △5-3β-hydroysteroid dehydrogenase (HSD) in testis and adrenal gland, 17β-hydroxysteroid dehydrogenase in testis, acid and alkaline phosphatase in testis, prostate and seminal vesicle were observed in noise exposed mature rats at the intensity of 85 dB for 8 h/day for 45 days. The results indicated that noise exposed group showed a significant diminution in the activities of androgenic key enzymes △5-3β and 17β-HSD, acid phosphatase in testis, prostate and seminal vesicle. There was a significant elevation in the activities of adrenal △5-3β-HSD, alkaline phosphatase in testis and other accessory sex organ in noise exposed group. Gonadosomatic, prostatosomatic and seminal vesiculo-somatic indexes were decreased significantly in noise exposed group. Therefore, it is evident that noise exposure at 85dB exerts a deleterious effect on testicular and adrenocortical activities.

  17. The influence of dietary fat on the lipogenic activity and fatty acid composition of rat white adipose tissue.

    Science.gov (United States)

    Nelson, G J; Kelley, D S; Schmidt, P C; Serrato, C M

    1987-05-01

    The in vivo fatty acid synthesis rate, selected enzyme activities and fatty acid composition of rat white adipose tissue from animals fed semisynthetic diets of differing fat type and content were studied. All animals were starved for 48 hr and then refed a fat-free (FF) diet for 48 hr. They were then divided into three groups. One group was continued on the FF diet for 48 hr. Another group was fed a diet containing 44% of calories from corn oil (CO). The final group was fed a diet containing 44% of calories from completely hydrogenated soybean oil (HSO). The animals on the FF diet had a marked increase in adipose tissue fatty acid synthesis during the 96-hr feeding period (as measured by 3H incorporation into adipose fatty acids). Addition of either CO or HSO to the diets did not significantly inhibit fatty acid synthesis in dorsal or epididymal adipose tissue. The activities of the enzymes' fatty acid synthetase, ATP-citrate lyase and glucose-6-phosphate dehydrogenase increased on the FF diet and generally were not inhibited significantly by the addition of either fat to the diets. Linoleic acid was the major polyunsaturated fatty acid (ca. 22%) in adipose tissue. Monounsaturated fatty acids (palmitoleic, oleic, cis-vaccenic) made up ca. 38% of the total adipose fatty acids, while saturated fatty acids accounted for about 32% (myristic, palmitic and stearic). White adipose tissue in mature male rats was a major depot for n-3 fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Derivatives of (phenylsulfonamido-methyl)nicotine and (phenylsulfonamido-methyl)thiazole as novel 11β-hydroxysteroid dehydrogenase type 1 inhibitors: synthesis and biological activities in vitro

    Institute of Scientific and Technical Information of China (English)

    Xu ZHANG; Yang ZHOU; Yu SHEN; Li-li DU; Jun-hua CHEN; Ying LENG; Jian-hua SHEN

    2009-01-01

    Aim: To design and synthese a novel class of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors, featuring the (phenylsul-fonamido-methyl)pyridine and (phenyisulfonamido-methyl)thiazole framework. Methods: Our initial lead 4-(phenylsulfonamido-methyl)benzamides were modified. Inhibition of human and mouse 11β-HSD1 enzy-matic activities by the new compounds was determined by a scintillation proximity assay (SPA) using microsomes containing 11β-HSD1.Results: Sixteen new compounds (6a-6h, 7a-7h) were designed, synthesized and bioassayed. In dose-response studies, several com-pounds showed strong inhibitory activities with IC_(50) values at nanomolar or low nanomolar concentrations. Structure-activity relation-ships are also discussed with respect to molecular docking results. Conclusion: This study provides two promising new templates for 11β-HSD1 inhibitors.

  19. Determination of enzyme activity by chromatography and videodensitometry. I. Microassay of amino acid transforming enzymes in human tissue homogenates.

    Science.gov (United States)

    Karsai, T; Elödi, P

    1979-01-01

    A chromatographic-videodensitometric assay was found to be appropriate for measuring the activity of glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, ornithine-2-oxoacid aminotransferase and histidine ammonia-lyase in human tissue homogenates. From the assay mixtures containing substrate(s), cofactor(s), buffer and tissue extract, five or ten microliters samples were taken at different time intervals and chromatographed on Dowex 50 X 8 type resin-coated chromatosheets. On each chromatoplate 50 nmoles of the amino acid to be measured were separately run as a reference for videodensitometric evaluation. By comparing the density of the reference amino acid to that of the individual samples the molar amount of amino acids formed or consumed in the reaction could be calculated. The present findings suggest that the chromatographic-videodensitometric microassay (CV-technique) is suitable for measuring the activity of amino acid transforming enzymes in minute amounts of tissue extracts.

  20. 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.

  1. Anti-inflammatory effects of levalbuterol-induced 11β-hydroxysteroid dehydrogenase type 1 activity in airway epithelial cells

    Directory of Open Access Journals (Sweden)

    Matthew J Randall

    2015-01-01

    Full Text Available Airway epithelial NF-kB activation is observed in asthmatic subjects and is a cause of airway inflammation in mouse models of allergic asthma. Combination therapy with inhaled short-acting b2-agonists and corticosteroids significantly improves lung function and reduces inflammation in asthmatic subjects. Corticosteroids operate through a number of mechanisms to potently inhibit NF-kB activity. Since b-agonists can induce expression of 11b-HSD1, which converts inactive 11-keto corticosteroids into active 11-hydroxy corticosteroids, thereby potentiating the effects of endogenous glucocorticoids, we examined whether this mechanism is involved in the inhibition of NF-kB activation induced by the b-agonist albuterol in airway epithelial cells. Treatment of transformed murine Club cells (MTCC with (R-albuterol (levalbuterol, but not with (S- or a mixture of (R+S- (racemic albuterol, augmented mRNA expression of 11b-HSD1. MTCC were stably transfected with luciferase (luc reporter constructs under transcriptional regulation by NF-kB (NF-kB/luc or glucocorticoid response element (GRE/luc consensus motifs. Stimulation of NF-kB/luc MTCC with lipopolysaccharide (LPS or tumor necrosis factor-α (TNFα induced luciferase activity, which was inhibited by pretreatment with (R-, but not (S- or racemic albuterol. Furthermore, pretreatment of GRE/luc MTCC with (R-albuterol augmented 11-keto corticosteroid (cortisone induced luciferase activity, which was diminished by the 11β-HSD inhibitor glycyrrhetinic acid (18β-GA. LPS- and TNFα-induced NF-kB/luc activity was diminished in MTCC cells treated with a combination of cortisone and (R-albuterol, an effect that was inhibited by 18β-GA. Finally, pretreatment of MTCC cells with the combination of cortisone and (R-albuterol diminished LPS- and TNFα-induced pro-inflammatory cytokine production. These results demonstrate that levalbuterol augments conversion of inactive 11-keto corticosteroids into the active 11

  2. 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

  3. Acyl-CoA Dehydrogenase 9 Is Required for the Biogenesis of Oxidative Phosphorylation Complex I

    NARCIS (Netherlands)

    J. Nouws; L. Nijtmans; S.M. Houten; M. Brand; M. Huynen; H. Venselaar; S. Hoefs; J. Gloerich; J. Kronick; T. Hutchin; P. Willems; R. Rodenburg; R. Wanders; L. van den Heuvel; J. Smeitink; R.O. Vogel

    2010-01-01

    Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified member of the acyl-CoA dehydrogenase family. It closely resembles very long-chain acyl-CoA dehydrogenase (VLCAD), involved in mitochondria! (3 oxidation of long-chain fatty acids. Contrary to its previously proposed involvement in fatty acid

  4. 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...

  5. Orally-effective, long-acting sorbitol dehydrogenase inhibitors: synthesis, structure-activity relationships, and in vivo evaluations of novel heterocycle-substituted piperazino-pyrimidines.

    Science.gov (United States)

    Chu-Moyer, Margaret Y; Ballinger, William E; Beebe, David A; Berger, Richard; Coutcher, James B; Day, Wesley W; Li, Jiancheng; Mylari, Banavara L; Oates, Peter J; Weekly, R Matthew

    2002-01-17

    Optimization of a previously disclosed sorbitol dehydrogenase inhibitor (SDI, II) for potency and duration of action was achieved by replacing the metabolically labile N,N-dimethylsulfamoyl group with a variety of heterocycles. Specifically, this effort led to a series of novel, in vitro potent SDIs with longer serum half-lives and acceptable in vivo activity in acutely diabetic rats (e.g., 62, 67, and 69). However, the desired in vivo potency in chronically diabetic rats, ED(90) < or = 5 mg/kg/day, was achieved only through further modification of the piperazine linker. Several members of this family, including 86, showed better than the targeted potency with ED(90) values of 1-2 mg/kg/day. Compound 86 was further profiled and found to be a selective inhibitor of sorbitol dehydrogenase, with excellent pharmacodynamic/pharmacokinetic properties, demonstrating normalization of sciatic nerve fructose in a chronically diabetic rat model for approximately 17 h, when administered orally at a single dose of 2 mg/kg/day.

  6. Effect of coumarin and xanthotoxin on mitochondrial structure, oxygen uptake, and succinate dehydrogenase activity in onion root cells.

    Science.gov (United States)

    Kupidlowska, E; Dobrzynska, K; Parys, E; Zobel, A M

    1994-10-01

    At concentrations in which they occur on the plant surface and retard mitosis, coumarin and xanthotoxin lowered uptake of oxygen (by 60 and 30%, respectively) by meristematic cells ofAllium cepa root tips. They caused changes in the structure of the mitochondrial matrix to become dense, and protrusions of mitochondrial membranes were visible parallelling their hypertrophy, indicating alteration in the structure and physiology of these organelles. Coumarin and, to a lesser extent, xanthotoxin increased succinate dehydrogenase production in mitochondria and also in the cytoplasm, indicating changes in membrane permeability. Changes in oxygen uptake and mitochondrial structure, in addition to the retardation of mitosis, may be the reason these compounds act as allelochemicals after they have been removed from the plant surface and reach the root meristem.

  7. 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:

  8. 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...

  9. Glucose-6-phosphate dehydrogenase (G6PD. Response of the human erythrocyte and another cells to the decrease in their activity.

    Directory of Open Access Journals (Sweden)

    Javier Fernando Bonilla

    2009-11-01

    Full Text Available Glucose-6-phosphate dehydrogenase is the first enzyme in the pentose phosphate pathway and the main intracellular source of reduced nicotidamineadenine nucleotidephosphate (NADPH, involved in diverse physiological processes such as antioxidant defense, (for instance in the erythrocyte endothelial growth modulation, erithropoyesis, vascularization and phagocitosis. G6PDH deficiency is the most common X-chromosome-linked enzymopathy in human beings. Although it is present in any type cell, its absolute deficiency is incompatible with life. According to WHO, 400 million people are affected by G6PD deficiency in the world but in Colombia, the severe form prevalence is about 3% to 7%. There are no data related to slight and moderate alterations, that also have clinical effects. This paper reviews some G6PD biomolecular aspects, its classification according to activity and electrophoretic mobility, as well as some main clinical aspects related to its activity alteration.

  10. Clinical implications of thymidylate synthetase, dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase activity levels in colorectal carcinoma following radical resection and administration of adjuvant 5-FU chemotherapy

    Directory of Open Access Journals (Sweden)

    Ishikawa Masashi

    2008-07-01

    Full Text Available Abstract Bckground A number of studies have investigated whether the activity levels of enzymes involved in 5-fluorouracil (5-FU metabolism are prognostic factors for survival in patients with colorectal carcinoma. Most reports have examined thymidylate synthetase (TS and dihydropyrimidine dehydrogenase (DPD in unresectable or metastatic cases, therefore it is unclear whether the activity of these enzymes is of prognostic value in colorectal cancer patients treated with radical resection and adjuvant chemotherapy with 5-FU. Methods This study examined fresh frozen specimens of colorectal carcinoma from 40 patients who had undergone curative operation and were orally administered adjuvant tegafur/uracil (UFT chemotherapy. TS, DPD and orotate phosphoribosyl transferase (OPRT activities were assayed in cancer tissue and adjacent normal tissue and their association with clinicopathological variables was investigated. In addition, the relationships between TS, DPD and OPRT activities and patient survival were examined to determine whether any of these enzymes could be useful prognostic factors. Results While there was no clear relationship between pathological findings and TS or DPD activity, OPRT activity was significantly lower in tumors with lymph node metastasis than in tumors lacking lymph node metastasis. Postoperative survival was significantly better in the groups with low TS activity and/or high OPRT activity. Conclusion TS and OPRT activity levels in tumor tissue may be important prognostic factors for survival in Dukes' B and C colorectal carcinoma with radical resection and adjuvant chemotherapy with UFT.

  11. Physiological regulation of isocitrate dehydrogenase and the role of 2-oxoglutarate in Prochlorococcus sp. strain PCC 9511.

    Science.gov (United States)

    Domínguez-Martín, María Agustina; López-Lozano, Antonio; Diez, Jesús; Gómez-Baena, Guadalupe; Rangel-Zúñiga, Oriol Alberto; García-Fernández, José Manuel

    2014-01-01

    The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.

  12. Physiological regulation of isocitrate dehydrogenase and the role of 2-oxoglutarate in Prochlorococcus sp. strain PCC 9511.

    Directory of Open Access Journals (Sweden)

    María Agustina Domínguez-Martín

    Full Text Available The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42 catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.

  13. Isolation and characterization of an inducible NAD-dependent butyraldehyde dehydrogenase from clostridium acetobutylicum

    Energy Technology Data Exchange (ETDEWEB)

    Schreiber, W.; Duerre, P. [Universitaet Ulm (Germany)

    1996-12-31

    A NAD-dependent butyraldehyde dehydrogenase (BAD) has been purified from C. acetobutylicum DSM 792 and DSM 173 1. This key enzyme of butanol production, catalyzing the conversion of butyryl-CoA to butyraldehyde, was induced shortly before the onset of butanol production and proved to be oxygen-sensitive. A one step purification procedure on reactive green 19 allowed to purify the enzyme to homogeneity. The purified protein was found to be extremely unstable and could only partially be stabilized by addition of mercaptoethanol and storage below -20{degrees}C. The enzyme subunit had a molecular mass of 39.5 kDa. In the reverse reaction (butyryl-CoA-forming) the apparent pH optimum was 9.75 and Vmax was significantly higher with butyraldehyde and propionaldehyde than with acetaldehyde. BAD could also use NADP+, but NAD+ was the preferred coenzyme for the reverse reaction. The N-terminal amino acid sequence of the C. acetobutylicurn DSM 792 protein showed high homology to glyceraldehyde-3-phosphate dehydrogenases (GAP), especially to the protein of C. pasteurianum. Genomic libraries of C. acetobutylicum DSM 792 were screened by hybridization using PCR-generated heterologous probes encoding the gap gene of C. pasteurianum. Sequence analysis of the positive clones revealed high homology, but no identity to the N-terminal amino acid sequence of the butyraldehyde dehydrogenase. Thus, BAD from C. acetobutylicum is distinctly different from other reported aldehyde dehydrogenases with butyraldehyde dehydrogenase activity.

  14. L-lactate dehydrogenase and N-acetyl-beta-D-glucosaminidase activities in bovine milk as indicators of non-specific mastitis.

    Science.gov (United States)

    Chagunda, Mizeck Gg; Larsen, Torben; Bjerring, Martin; Ingvartsen, Klaus L

    2006-11-01

    Systematic factors affecting the activities of L-lactate dehydrogenase (LDH) and N-acetyl-beta-D-glucosaminidase (NAGase) and somatic cell count (SCC), the association between the activities of LDH and NAGase and SCC with respect to udder health status, and the ability of LDH and NAGase to classify cows in udder health categories for early detection of mastitis were studied. A dataset of records from 74 Danish Holstein, 76 Danish Red and 47 Jersey cows on one research farm was used. Cows were grouped into healthy and clinically mastitic. A healthy cow was defined as having no veterinary treatment and SCCmastitis and SCC >800,000 cells/ml. Breed, month of production, and days in milk significantly influenced (Pmastitis. NAGase activity had numerically higher variation in healthy cows than in clinically mastitic cows (CV=56.2% v. CV=53.5%). The relationship between LDH activity and SCC was stronger in milk from clinically mastitic than from healthy cows (r=0.76 v. r=0.48 and r=0.67 v. r=0.44 for correlation of observed values and residuals, respectively). LDH activity had higher sensitivity than NAGase activity (73-95% v. 35-77%) while specificities were in a similar range (92-99%). Further, sensitivities for LDH activity were more robust to changes in the threshold value than those for NAGase activity. Opportunities for automated, in-line real-time mastitis detection are discussed.

  15. Anticholinesterase activity of fluorochloronitroacetic acid esters

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, Yu.Ya.; Brel, V.K. Martynov, I.V.

    1984-11-01

    Results are presented from pharmacologic and biochemical experiments leading to the conclusion that fluorochloronitroacetic acid esters have anticholinesterase activity. Since the esters caused muscular weakness in mice, experiments were performed on isolated tissue preparation. The biochemical experiments consisted of finding the biomolecular constants of irreversible inhibition of acetylcholinesterase by the esters, using acetylcholinesterase from human erythrocytes, as well as horse serum cholinesterase. The ethyl and n-propyl esters of halogen nitroacetic acid were used in all experiments. It was found that the propyl ester caused an increase in the force of individual contractions in the isolated muscle specimens, plus an inability of the muscle to retain tetanus. The substances were determined to have an anticholinesterase effect. The mechanism of cholinesterase inhibition is not yet known. It is probable that the substances acylate the serine hydroxyl of the esterase center of the cholinestersase. 7 references, 1 figure.

  16. The Enzyme Activity and Substrate Specificity of Two Major Cinnamyl Alcohol Dehydrogenases in Sorghum (Sorghum bicolor), SbCAD2 and SbCAD4.

    Science.gov (United States)

    Jun, Se-Young; Walker, Alexander M; Kim, Hoon; Ralph, John; Vermerris, Wilfred; Sattler, Scott E; Kang, ChulHee

    2017-08-01

    Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis, reducing sinapaldehyde, coniferaldehyde, and p-coumaraldehyde to their corresponding alcohols in an NADPH-dependent manner. Because of its terminal location in monolignol biosynthesis, the variation in substrate specificity and activity of CAD can result in significant changes in overall composition and amount of lignin. Our in-depth characterization of two major CAD isoforms, SbCAD2 (Brown midrib 6 [bmr6]) and SbCAD4, in lignifying tissues of sorghum (Sorghum bicolor), a strategic plant for generating renewable chemicals and fuels, indicates their similarity in both structure and activity to Arabidopsis (Arabidopsis thaliana) CAD5 and Populus tremuloides sinapyl alcohol dehydrogenase, respectively. This first crystal structure of a monocot CAD combined with enzyme kinetic data and a catalytic model supported by site-directed mutagenesis allows full comparison with dicot CADs and elucidates the potential signature sequence for their substrate specificity and activity. The L119W/G301F-SbCAD4 double mutant displayed its substrate preference in the order coniferaldehyde > p-coumaraldehyde > sinapaldehyde, with higher catalytic efficiency than that of both wild-type SbCAD4 and SbCAD2. As SbCAD4 is the only major CAD isoform in bmr6 mutants, replacing SbCAD4 with L119W/G301F-SbCAD4 in bmr6 plants could produce a phenotype that is more amenable to biomass processing. © 2017 American Society of Plant Biologists. All Rights Reserved.

  17. Characterization of two β-decarboxylating dehydrogenases from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Takahashi, Kento; Nakanishi, Fumika; Tomita, Takeo; Akiyama, Nagisa; Lassak, Kerstin; Albers, Sonja-Verena; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2016-11-01

    Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two β-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg(2+), thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.

  18. Recent developments in the antiprotozoal and anticancer activities of the 2-alkynoic fatty acids

    Science.gov (United States)

    Carballeira, Néstor M.

    2013-01-01

    The 2-alkynoic fatty acids are an interesting group of synthetic compounds that display antimycobacterial, antifungal, anticancer, and pesticidal activities but their antiprotozoal activity has received little attention until recently. In this review we have summarized our present knowledge of the biomedical potential of the 2-hexadecynoic acid (2-HDA) and 2-octadecynoic acid (2-ODA) together with several mechanistic pieces of work attesting to the fact that these compounds, and their metabolites, are good fatty acid biosynthesis inhibitors. The antiprotozoal activity of 2-HDA and 2-ODA against Leishmania donovani and Plasmodium falciparum, parasites responsible for visceral leishmaniasis and malaria, respectively, is also reviewed. The evidence obtained so far supports the fact that these fatty acids are good inhibitors of the L. donovani DNA topoisomerase IB enzyme (LdTopIB) and the potency of LdTopIB inhibition is chain length dependent. We also demonstrate the generality of the antiprotozoal activity of 2-HDA and 2-ODA against P. falciparum, and review our present knowledge of their inhibition of key P. falciparum enzymes such as PfFabZ, PfFabG, and PfFabI together with some possible modes of inhibition. Recent research by our group has also demonstrated that 2-ODA displays antineoplastic activity, specifically against the neuroblastoma SH-SY5Y cell line via lactate dehydrogenase (LDH) release, which is a cell death mechanism principally associated to necrosis. This is the first comprehensive review of the medicinal chemistry of this interesting group of acetylenic fatty acids. PMID:23727443

  19. Annotated compound data for modulators of detergent-solubilised or lipid-reconstituted respiratory type II NADH dehydrogenase activity obtained by compound library screening.

    Science.gov (United States)

    Dunn, Elyse A; Cook, Gregory M; Heikal, Adam

    2016-03-01

    The energy-generating membrane protein NADH dehydrogenase (NDH-2), a proposed antibacterial drug target (see "Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs" Weinstein et al. 2005 [1]), was screened for modulators of activity in either detergent-solublised or lipid reconstituted (proteolipsome) form. Here we present an annotated list of compounds identified in a small-scale screen against NDH-2. The dataset contains information regarding the libraries screened, the identities of hit compounds and the physicochemical properties governing solubility and permeability. The implications of these data for future antibiotic discovery are discussed in our associated report, "Comparison of lipid and detergent enzyme environments for identifying inhibitors of membrane-bound energy-transducing proteins" [2].

  20. Annotated compound data for modulators of detergent-solubilised or lipid-reconstituted respiratory type II NADH dehydrogenase activity obtained by compound library screening

    Directory of Open Access Journals (Sweden)

    Elyse A. Dunn

    2016-03-01

    Full Text Available The energy-generating membrane protein NADH dehydrogenase (NDH-2, a proposed antibacterial drug target (see “Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs” Weinstein et al. 2005 [1], was screened for modulators of activity in either detergent-solublised or lipid reconstituted (proteolipsome form. Here we present an annotated list of compounds identified in a small-scale screen against NDH-2. The dataset contains information regarding the libraries screened, the identities of hit compounds and the physicochemical properties governing solubility and permeability. The implications of these data for future antibiotic discovery are discussed in our associated report, “Comparison of lipid and detergent enzyme environments for identifying inhibitors of membrane-bound energy-transducing proteins” [2].

  1. Attenuated mitochondrial NADP+-dependent isocitrate dehydrogenase activity induces apoptosis and hypertrophy of H9c2 cardiomyocytes.

    Science.gov (United States)

    Lee, Jun Ho; Park, Jeen-Woo

    2014-04-01

    Oxidative stress, characterized by the accumulation of reactive oxygen species (ROS), is known to have numerous detrimental effects on the myocardium such as the induction of apoptotic cell death, hypertrophy, fibrosis, dysfunction, and dilatation. Over the past several years, we have shown that mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) functions as an antioxidant and anti-apoptotic protein by supplying NADPH to antioxidant systems. Here, we showed that transfection of H9c2 clonal myoblastic cells with small interfering RNA (siRNA) specific for IDPm markedly attenuated IDPm expression and substantially induced apoptosis, senescence, and hypertrophy as indicated by increased atrial natriuretic peptide (ANP) gene expression, a marker of cardiomyocyte hypertrophy, and a larger cell size. Knockdown of IDPm expression resulted in the modulation of cellular and mitochondrial redox status, mitochondrial function, and cellular oxidative damage. Taken together, our results suggest that the suppression of IDPm expression by siRNA induces apoptosis and hypertrophy of cultured cardiomyocytes through the disruption of cellular redox balance.

  2. A novel genetically-obese rat model with elevated 11beta-hydroxysteroid dehydrogenase type 1 activity in subcutaneous adipose tissue

    Directory of Open Access Journals (Sweden)

    Giridharan Nappan V

    2010-11-01

    Full Text Available Abstract 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1 catalyzes the conversion of inactive glucocorticoids to active glucocorticoids and plays an important role in the development of obesity and metabolic syndrome. 11β-HSD1 activity is lower in liver and higher in omental adipose tissue of obese rodent models like obese zucker rats, Ob/Ob and db/db mice. Here, we report the 11β-HSD1 activity in liver and adipose tissue of lean and obese rats of WNIN/Ob strain, a new genetic rat model of obesity. 11β-HSD1 activity in liver, omental and subcutaneous adipose tissues of 3 month-old male WNIN/Ob lean and obese rats was assayed. As observed in other rodent models, 11β-HSD1 activity was lower in liver and higher in omental adipose tissue. In contrast to other rodent obese models, WNIN/Ob obese rats had elevated 11β-HSD1 activity in subcutaneous adipose tissue, which is in line with the observation in human obesity. Here, we conclude that dysregulation of 11β-HSD1 in WNIN/Ob obese rat model is identical to human obesity, which makes it an excellent model for studying the effect of 11β-HSD1 inhibitors in ameliorating obesity and metabolic syndrome.

  3. A novel genetically-obese rat model with elevated 11 beta-hydroxysteroid dehydrogenase type 1 activity in subcutaneous adipose tissue.

    Science.gov (United States)

    Prasad, Sakamuri S S Vara; Prashanth, Anamthathmakula; Kumar, Chodavarapu Pavan; Reddy, Sirisha J; Giridharan, Nappan V; Vajreswari, Ayyalasomayajula

    2010-11-17

    11 β-hydroxysteroid dehydrogenase type 1 (11 β-HSD1) catalyzes the conversion of inactive glucocorticoids to active glucocorticoids and plays an important role in the development of obesity and metabolic syndrome. 11 β-HSD1 activity is lower in liver and higher in omental adipose tissue of obese rodent models like obese zucker rats, Ob/Ob and db/db mice. Here, we report the 11 β-HSD1 activity in liver and adipose tissue of lean and obese rats of WNIN/Ob strain, a new genetic rat model of obesity. 11 β-HSD1 activity in liver, omental and subcutaneous adipose tissues of 3 month-old male WNIN/Ob lean and obese rats was assayed. As observed in other rodent models, 11 β-HSD1 activity was lower in liver and higher in omental adipose tissue. In contrast to other rodent obese models, WNIN/Ob obese rats had elevated 11 β-HSD1 activity in subcutaneous adipose tissue, which is in line with the observation in human obesity. Here, we conclude that dysregulation of 11 β-HSD1 in WNIN/Ob obese rat model is identical to human obesity, which makes it an excellent model for studying the effect of 11 β-HSD1 inhibitors in ameliorating obesity and metabolic syndrome.

  4. Loss of Mitochondrial Malate Dehydrogenase Activity Alters Seed Metabolism Impairing Seed Maturation and Post-Germination Growth in Arabidopsis1[OPEN

    Science.gov (United States)

    2016-01-01

    Mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37) has multiple roles; the most commonly described is its catalysis of the interconversion of malate and oxaloacetate in the tricarboxylic acid cycle. The roles of mMDH in Arabidopsis (Arabidopsis thaliana) seed development and germination were investigated in mMDH1 and mMDH2 double knockout plants. A significant proportion of mmdh1mmdh2 seeds were nonviable and developed only to torpedo-shaped embryos, indicative of arrested seed embryo growth during embryogenesis. The viable mmdh1mmdh2 seeds had an impaired maturation process that led to slow germination rates as well as retarded post-germination growth, shorter root length, and decreased root biomass. During seed development, mmdh1mmdh2 showed a paler green phenotype than the wild type and exhibited deficiencies in reserve accumulation and reduced final seed biomass. The respiration rate of mmdh1mmdh2 seeds was significantly elevated throughout their maturation, consistent with the previously reported higher respiration rate in mmdh1mmdh2 leaves. Mutant seeds showed a consistently higher content of free amino acids (branched-chain amino acids, alanine, serine, glycine, proline, and threonine), differences in sugar and sugar phosphate levels, and lower content of 2-oxoglutarate. Seed-aging assays showed that quiescent mmdh1mmdh2 seeds lost viability more than 3 times faster than wild-type seeds. Together, these data show the important role of mMDH in the earliest phases of the life cycle of Arabidopsis. PMID:27208265

  5. The effect of water loading on the urinary ratio of cortisone to cortisol in healthy subjects and a new approach to the evaluation of the ratio as an index for in vivo human 11β-hydroxysteroid dehydrogenase 2 activity.

    Science.gov (United States)

    Yokokawa, Akitomo; Takasaka, Toru; Shibasaki, Hiromi; Kasuya, Yasuji; Kawashima, Soko; Yamada, Akira; Furuta, Takashi

    2012-10-01

    Factors that give rise to a large variation in the urinary ratio of free cortisone to cortisol (UFE/UFF) were investigated to accurately estimate 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity in humans in vivo. A water loading test was first carried out in two healthy subjects to examine the effect of water intake or urine volume on the urinary ratio of free cortisone to cortisol (UFE/UFF). The ratio was found to increase by water loading. We also examined urinary concentrations and amounts of cortisol, cortisone, creatinine, Na(+), K(+), and Cl(-), and urine volume, as possible factors affecting the urinary ratio (UFE/UFF), in 60 urine samples obtained from 15 healthy volunteers. Among these factors tested, the urinary concentration of cortisol was most highly correlated with the UFE/UFF ratio (r=-0.858), indicating that the in vivo activity of 11β-HSD2 (UFE/UFF) should fluctuate with the changes of the urinary concentration of cortisol. Based on the findings, we proposed a new estimation method of in vivo activity of 11β-HSD2 in humans, using the UFE/UFF ratio correlated with the urinary concentration of cortisol (UFE/UFF-cortisol concentration). Taking into consideration the intra-individual variabilities in the urinary concentration of cortisol, there were no significant within-day variations in 11β-HSD2 activity. The findings indicate that 11β-HSD2 activities can be accurately evaluated by simply measuring free cortisol and cortisone concentrations in spot urine samples. Furthermore, administrations of glycyrrhetinic acid in three healthy volunteers were performed to confirm the usefulness of the present assessment for the activity of 11β-HSD2. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Synthesis and structures of bis(dithiolene)tungsten(IV,VI) thiolate and selenolate complexes: approaches to the active sites of molybdenum and tungsten formate dehydrogenases.

    Science.gov (United States)

    Groysman, Stanislav; Holm, R H

    2007-05-14

    Formate dehydrogenases are molybdenum- or tungsten-containing enzymes that catalyze the oxidation of formate to carbon dioxide. Among the significant characteristics of the mononuclear active sites are coordination of two pyranopterindithiolene ligands and selenocysteinate to the metal in oxidation states IV-VI. The first detailed investigation of the synthesis and structures of bis(dithiolene)tungsten selenolate and analogous thiolate complexes of relevance to formate dehydrogenases has been undertaken. Some 17 complexes of the types [WIV(QR)(S2C2Me2)2]-, [WVIO(QR)(S2C2Me2)2]-, and [WVIS(QR)(S2C2Me2)2]- (Q = S, Se; R = tert-butyl, 1-adamantyl) and the desoxo species [WVI(SR)(OSiR'3)(S2C2Me2)2] (R' = Me, Ph) were prepared. Ten structures of representative members of these types were determined; WIV complexes are square-pyramidal and WVI complexes are six-coordinate, with geometries intermediate between octahedral and trigonal-prismatic. Selenolate complexes are less stable than similar thiolate species; decomposition products were identified as [WV2(mu2-Q)2(S2C2Me2)2]2- and [WIV,V2(mu2-Se)(S2C2Me2)4]-. The several [MoIV(QR)(S2C2Me2)2]- complexes prepared earlier and the tungsten compounds synthesized in this work form a family of molecules whose overall stereochemistry and metric features are those expected in the absence of protein structural constraints.

  7. Differential decolorization of textile dyes in mixtures and the joint effect of laccase and cellobiose dehydrogenase activities present in extracellular extracts from Funalia trogii.

    Science.gov (United States)

    Tilli, Silvia; Ciullini, Ilaria; Scozzafava, Andrea; Briganti, Fabrizio

    2011-10-10

    The largest part of the bio-decolorization investigations have been performed to date on a single dye without exploring the behavior in complex mixtures as the real dyeing baths. Therefore, mixtures of dyes belonging to azo and anthraquinonic classes, chosen among the most utilized in textile wool dyeing, were employed for comparative enzymatic decolorization studies using the extracellular extracts from the white rot fungus Funalia trogii, to understand how the concomitant presence of more than one dye could influence their degradation course and yield. Fungal extracts containing laccase activity only were capable to partially decolorize dyes mixtures from the different classes analyzed. The deconvolution of the decolorization with time allowed to monitor the degradation of the single dyes in the mixtures evidencing a time dependent differential decolorization not observed for the singles alone. Some dyes in the blend were in fact decolorized only when the most easily converted dyes were largely transformed. These experiments would allow to help the dyeing factories in the selection of the most readily degraded dyes. Since F. trogii grown on different media and activators shows diverse levels of expression of the redox enzymes laccase and cellobiose dehydrogenase (CDH), the dyes mixtures recalcitrant to decolorization by laccase activity alone, were subjected to the combined action of extracts containing laccase and CDH. The use of CDH, in support to the activity of laccase, resulted in substantial decolorization increases (>84%) for all the refractory dyes mixtures.

  8. Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme.

    Science.gov (United States)

    Wakamatsu, Taisuke; Sakuraba, Haruhiko; Kitamura, Megumi; Hakumai, Yuichi; Fukui, Kenji; Ohnishi, Kouhei; Ashiuchi, Makoto; Ohshima, Toshihisa

    2017-01-15

    l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P)(+)-dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD(+) Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD(+)/NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the β-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme.

  9. Essential histidine residue in 3-ketosteroid-Δ1-dehydrogenase

    OpenAIRE

    Matsushita, Hiroyuki; Itagaki, Eiji; 板垣, 英治

    1992-01-01

    The variation with pH of kinetic parameters was examined for 3-ketosteroid-Δ1-dehydrogenase from Nocardia corallina. The V(max)/K(m) profile for 4-androstenedione indicates that activity is lost upon protonation of a cationic acid-type group with a pK value of 7.7. The enzyme was inactivated by diethylpyrocarbonate at pH 7.4 and the inactivation was substantially prevented by androstadienedione. Analyses of reactivation with neutral hydroxylamine, pH variation, and spectral changes of the ina...

  10. 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.

  11. Stress-induced changes in glutamate dehydrogenase activity imply its role in adaptation to C and N metabolism in lupine embryos.

    Science.gov (United States)

    Lehmann, Teresa; Skrok, Albert; Dabert, Mirosława

    2010-01-01

    The modifying effect of sucrose on glutamate dehydrogenase (GDH) activity and isoenzyme pattern was investigated in isolated embryos of lupine (Lupinus luteus L.), cultured in vitro in a medium with sucrose (+S) or without sucrose (-S) and exposed to cadmium (Cd) and lead (Pb) stress. Sucrose starvation of lupine embryos led to a rapid increase in the specific activity of GDH, immunoreactive beta-polypeptide and it was accompanied by appearance of new cathodal isoforms of enzyme. This suggests that isoenzymes induced in lupine embryos by sucrose starvation combine into GDH hexamers with the predominance of beta-GDH subunits synthetized under GDH1 gene control. The addition of sucrose to the medium caused an opposite effect. Along with upregulation of catabolic activity of GDH by sucrose starvation, activity of proteolytic enzymes was also induced. These data can point to regulatory mechanism implying a sucrose dependent repression of the GDH1 gene according to the mechanism of catabolic repression. Treatment of embryos with Cd(2+) or Pb(2+) resulted in ammonium accumulation in the tissues, accompanied by an increase in anabolic activity of GDH and activity of anodal isoenzymes, in both (+S) and (-S) embryos without new de novo synthesis of alpha subunit proteins. Thus, GDH isoenzyme profiles may reflect the physiological function of GDH, which appears to be an important link of metabolic adaptation in cells, aimed at using carbon sources other than sugar during carbohydrate starvation (catabolic activity of GDH) and protecting plant tissues against ammonium accumulated because of heavy metal stress (anabolic activity of GDH).

  12. The active (ADHa) and inactive (ADHi) forms of the PQQ-alcohol dehydrogenase from Gluconacetobacter diazotrophicus differ in their respective oligomeric structures and redox state of their corresponding prosthetic groups.

    Science.gov (United States)

    Gómez-Manzo, Saúl; González-Valdez, Alejandra Abigail; Oria-Hernández, Jesús; Reyes-Vivas, Horacio; Arreguín-Espinosa, Roberto; Kroneck, Peter M H; Sosa-Torres, Martha Elena; Escamilla, Jose E

    2012-03-01

    The membrane-bound alcohol dehydrogenase of Gluconacetobacter diazotrophicus contains one pyrroloquinoline quinone moiety (PQQ), one [2Fe-2S] cluster, and four c-type cytochromes. Here, we describe a novel and inactive enzyme. ADHi, similarly to ADHa, is a heterodimer of 72- and 44-kDa subunits and contains the expected prosthetic groups. However, ADHa showed a threefold molecular mass as compared to ADHi. Noteworthy, the PQQ, the [2Fe-2S] and most of the cytochromes in purified ADHi is in the oxidized form, contrasting with ADHa where the PQQ-semiquinone is detected and the [2Fe-2S] cluster as well as the cytochromes c remained fully reduced after purification. Reduction kinetics of the ferricyanide-oxidized enzymes showed that while ADHa was brought back by ethanol to its full reduction state, in ADHi, only one-quarter of the total heme c was reduced. The dithionite-reduced ADHi was largely oxidized by ubiquinone-2, thus indicating that intramolecular electron transfer is not impaired in ADHi. The acidic pH of the medium might be deleterious for the membrane-bound ADH by causing conformational changes leading to changes in the relative orientation of heme groups and shift of corresponding redox potential to higher values. This would hamper electron transfer resulting in the low activity observed in ADHi.

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

    Science.gov (United States)

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

    2012-03-01

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

  14. The structure of retinal dehydrogenase type II at 2.7 A resolution: implications for retinal specificity.

    Science.gov (United States)

    Lamb, A L; Newcomer, M E

    1999-05-11

    Retinoic acid, a hormonally active form of vitamin A, is produced in vivo in a two step process: retinol is oxidized to retinal and retinal is oxidized to retinoic acid. Retinal dehydrogenase type II (RalDH2) catalyzes this last step in the production of retinoic acid in the early embryo, possibly producing this putative morphogen to initiate pattern formation. The enzyme is also found in the adult animal, where it is expressed in the testis, lung, and brain among other tissues. The crystal structure of retinal dehydrogenase type II cocrystallized with nicotinamide adenine dinucleotide (NAD) has been determined at 2.7 A resolution. The structure was solved by molecular replacement using the crystal structure of a mitochondrial aldehyde dehydrogenase (ALDH2) as a model. Unlike what has been described for the structures of two aldehyde dehydrogenases involved in the metabolism of acetaldehyde, the substrate access channel is not a preformed cavity into which acetaldehyde can readily diffuse. Retinal dehydrogenase appears to utilize a disordered loop in the substrate access channel to discriminate between retinaldehyde and short-chain aldehydes.

  15. The Isoenzyme 7 of Tobacco NAD(H)-Dependent Glutamate Dehydrogenase Exhibits High Deaminating and Low Aminating Activities in Vivo1[OA

    Science.gov (United States)

    Skopelitis, Damianos S.; Paranychianakis, Nikolaos V.; Kouvarakis, Antonios; Spyros, Apostolis; Stephanou, Euripides G.; Roubelakis-Angelakis, Kalliopi A.

    2007-01-01

    Following the discovery of glutamine synthetase/glutamate (Glu) synthase, the physiological roles of Glu dehydrogenase (GDH) in nitrogen metabolism in plants remain obscure and is the subject of considerable controversy. Recently, transgenics were used to overexpress the gene encoding for the β-subunit polypeptide of GDH, resulting in the GDH-isoenzyme 1 deaminating in vivo Glu. In this work, we present transgenic tobacco (Nicotiana tabacum) plants overexpressing the plant gdh gene encoding for the α-subunit polypeptide of GDH. The levels of transcript correlated well with the levels of total GDH protein, the α-subunit polypeptide, and the abundance of GDH-anionic isoenzymes. Assays of transgenic plant extracts revealed high in vitro aminating and low deaminating activities. However, gas chromatography/mass spectrometry analysis of the metabolic fate of 15NH4 or [15N]Glu revealed that GDH-isoenzyme 7 mostly deaminates Glu and also exhibits low ammonium assimilating activity. These and previous results firmly establish the direction of the reactions catalyzed by the anionic and cationic isoenzymes of GDH in vivo under normal growth conditions and reveal a paradox between the in vitro and in vivo enzyme activities. PMID:17932305

  16. Chemical composition and antioxidant activity of an acidic polysaccharide extracted from Cucurbita moschata Duchesne ex Poiret.

    Science.gov (United States)

    Yang, Xingbin; Zhao, Yan; Lv, You

    2007-06-13

    A simple and sensitive high-performance capillary electrophoresis (HPCE) method was designed for quantitative analysis of the component monosaccharides of an acidic polysaccharide extracted from pumpkin. In this method, the polysaccharide was hydrolyzed into component monosaccharides with 2.0 M trifluoroacetic acid at 100 degrees C for 6 h and then labeled with 1-phenyl-3-methyl-5-pyrazolone, and subsequently the labeled monosaccharide derivatives were separated by HPCE. As a result, glucose (21.7%) and glucuronic acid (18.9%) were identified to be the main component monosaccharides, followed by galactose (11.5%), arabinose (9.8%), xylose (4.4%), and rhamnose (2.8%). Furthermore, the pumpkin polysaccharide was also demonstrated to effectively inhibit the H2O2-caused decrease of cell viability, lactate dehydrogenase leakage, and malondialdehyde formation, and also reduced the H2O2-caused decline of superoxide dismutase activity and glutathione depletion in cultured mouse peritoneal macrophages, indicating that pumpkin polysaccharide possessed significant cytoprotective effect and antioxidative activity.

  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.

  18. Isolation and biochemical characterization of a glucose dehydrogenase from a hay infusion metagenome.

    Science.gov (United States)

    Basner, Alexander; Antranikian, Garabed

    2014-01-01

    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.

  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. Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates

    Energy Technology Data Exchange (ETDEWEB)

    Sukumar, Narayanasami [NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Building 436E, Argonne National Laboratory, Argonne, IL 60439 (United States); Dewanti, Asteriani [Department of Chemistry and Physics, Western Carolina University, Cullowhee, NC 28723 (United States); Merli, Angelo; Rossi, Gian Luigi [Department of Biochemistry and Molecular Biology, University of Parma, Parma (Italy); Mitra, Bharati [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Mathews, F. Scott, E-mail: mathews@biochem.wustl.edu [Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110 (United States); NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Building 436E, Argonne National Laboratory, Argonne, IL 60439 (United States)

    2009-06-01

    The crystal structure of the G81A mutant form of the chimera of (S)-mandelate dehydrogenase and of its complexes with two of its substrates reveal productive and non-productive modes of binding for the catalytic reaction. The structure also indicates the role of G81A in lowering the redox potential of the flavin co-factor leading to an ∼200-fold slower catalytic rate of substrate oxidation. (S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed ∼100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 Å resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by

  1. Antioxidant and antimicrobial activities of cinnamic acid derivatives.

    Science.gov (United States)

    Sova, M

    2012-07-01

    Cinnamic acid is an organic acid occurring naturally in plants that has low toxicity and a broad spectrum of biological activities. In the search for novel pharmacologically active compounds, cinnamic acid derivatives are important and promising compounds with high potential for development into drugs. Many cinnamic acid derivatives, especially those with the phenolic hydroxyl group, are well-known antioxidants and are supposed to have several health benefits due to their strong free radical scavenging properties. It is also well known that cinnamic acid has antimicrobial activity. Cinnamic acid derivatives, both isolated from plant material and synthesized, have been reported to have antibacterial, antiviral and antifungal properties. Acids, esters, amides, hydrazides and related derivatives of cinnamic acid with such activities are here reviewed.

  2. 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.

  3. 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

    -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...

  4. 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...

  5. A novel glutamate dehydrogenase from bovine brain: purification and characterization.

    Science.gov (United States)

    Lee, J; Kim, S W; Cho, S W

    1995-08-01

    A soluble form of novel glutamate dehydrogenase has been purified from bovine brain. The preparation was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and composed of six identical subunits having a subunit size of 57,500 Da. The biochemical properties of glutamate dehydrogenase such as N-terminal amino acids sequences, kinetic parameters, amino acids analysis, and optimum pH were examined in both reductive amination of alpha-ketoglutarate and oxidative deamination of glutamate. N-terminal amino acid sequences of the bovine brain enzyme showed the significant differences in the first 5 amino acids compared to other glutamate dehydrogenases from various sources. These results indicate that glutamate dehydrogenase isolated from bovine brain is a novel polypeptide.

  6. Serum lactic dehydrogenase isoenzymes and serum hydroxy butyric dehydrogenase in myocardial infarction

    Directory of Open Access Journals (Sweden)

    Kanekar D

    1979-01-01

    Full Text Available Total serum lactate dehydrogenase activity in cases of myocar-dial infarct is difficult to interpret as abnormal values can occur in diseases of liver, kidney and skeletal muscle. The estimation of its isoenzymes is of better diagnostic help because of its tissue specificity. Serum LDH isoenzymes were studied in patients o f myocardial infarction and results are quantitated by densitometry. As LDH 1 represents serum hydroxybutyric dehydrogenase when 2-oxylbutyrate is used as substrate, serum hydroxybutyric dehydro-genase was also estimated in above patients. Greater specificity in diagnosis is achieved with SHBDH because of its myocardial nature and lower incidence of false positive results.

  7. Evaluation of hepatic 11 beta-hydroxysteroid dehydrogenase activity by cortisone acetate test in young adults with diabetes mellitus type 1.

    Science.gov (United States)

    Šimůnková, K; Hampl, R; Hill, M; Kříž, L; Vrbíková, J; Kvasničková, H; Vondra, K

    2011-01-01

    Cortisone acetate test was performed in twelve young adult patients with diabetes mellitus type 1, after dexamethasone administration to suppress endogenous cortisol production. Previous screening revealed that all of the subjects had peak cortisol responses in the range from subnormal to normal, as determined by a low-dose Synacthen test. The aim was to find out whether these patients would exhibit different conversion of cortisone to cortisol by 11beta-hydroxysteroid dehydrogenase. Using multifactorial ANOVA the following significant relationships were obtained between cortisol or cortisol/cortisone ratio measured during the test and other parameters examined a) before dexamethasone suppression and b) during the test: a) Cortisol at 120(th) minute negatively correlated with daily insulin dose and positively with basal aldosterone. Cortisol/cortisone ratio at 60(th), 120(th), 180(th), and 240(th) minute negatively correlated with basal aldosterone/plasma renin activity ratio, urinary free cortisol/24 hours and positively with basal dehydroepindrosterone sulphate. b) Cortisol at 120(th) minute negatively correlated with suppressed basal serum glycemia; cortisol/cortisone ratio during the whole test negatively correlated with supressed basal ACTH. The examination of peripheral metabolism of cortisol using cortisone acetate test in patients with diabetes mellitus type 1 showed adaptive changes of 11beta-hydroxysteroid dehydrogenace activity associated with altered cortisol tissue supply.

  8. NADH:ubiquinone reductase and succinate dehydrogenase activity in the liver of rats with acetaminophen-induced toxic hepatitis on the background of alimentary protein deficiency

    Directory of Open Access Journals (Sweden)

    G. P. Kopylchuk

    2015-02-01

    Full Text Available The ratio between the redox forms of the nicotinamide coenzymes and key enzymatic activity of the I and II respiratory chain complexes in the liver cells mitochondria of rats with acetaminophen-induced hepatitis under the conditions of alimentary deprivation of protein was studied. It was estimated, that under the conditions of acute acetaminophen-induced hepatitis of rats kept on a low-protein diet during 4 weeks a significant decrease of the NADH:ubiquinone reductase and succinate dehydrogenase activity with simultaneous increase of the ratio between redox forms of the nicotinamide coenzymes (NAD+/NADН is observed compared to the same indices in the liver cells of animals with experimental hepatitis kept on the ration balanced by all nutrients. Results of research may become basic ones for the biochemical rationale for the approaches directed to the correction and elimination of the consequences­ of energy exchange in the toxic hepatitis, induced on the background of protein deficiency.

  9. Effects of methoxychlor and its metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane on 11β-hydroxysteroid dehydrogenase activities in vitro.

    Science.gov (United States)

    Guo, Jingjing; Deng, Haiyun; Li, Hongzhi; Zhu, Qiqi; Zhao, Binghai; Chen, Bingbing; Chu, Yanhui; Ge, Ren-Shan

    2013-03-27

    Methoxychlor (MXC) is primarily used as a pesticide and widely present in the environment. The objective of the present study is to investigate the direct effects of MXC and its metabolite 2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on two isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD1 and 11β-HSD2) in vitro. Human liver microsome, rat testis microsome and adult Leydig cells were used for the measurement of 11β-HSD1 activity. Human placental and rat kidney microsomes were used for 11β-HSD2 activity. The IC(50) values on human 11β-HSD1 by MXC and HPTE were 1.91±0.07 and 8.88 ± 0.08 μM, respectively. HPTE inhibited rat 11β-HSD1 with IC(50) of 9.15±0.05μM, while MXC did not inhibit the enzyme. MXC and HPTE were competitive inhibitors of 11β-HSD1. HPTE also inhibited human and rat 11β-HSD2 with IC(50) values of 55.57 ± 0.08 and 12.96 ± 0.11 μM, respectively, while MXC did not inhibit 11β-HSD2. In summary, our results showed that MXC and its metabolite HPTE inhibited both isoforms of 11β-HSD in a species- and chemical structure-dependent manner.

  10. α-(Substituted-phenoxyacetoxy)-α-heterocyclylmethylphosphonates: synthesis, herbicidal activity, inhibition on pyruvate dehydrogenase complex (PDHc), and application as postemergent herbicide against broadleaf weeds.

    Science.gov (United States)

    He, Hong-Wu; Peng, Hao; Wang, Tao; Wang, Chubei; Yuan, Jun-Lin; Chen, Ting; He, Junbo; Tan, Xiaosong

    2013-03-13

    Pyruvate dehydrogenase complex (PDHc) is the site of action of a new class of herbicides. On the basis of the previous work for O,O'-dimethyl α-(substituted-phenoxyacetoxy)alkylphosphonates (I), further synthetic modifications were made by introducing a fural and a thienyl group to structure I. A series of α-(substituted-phenoxyacetoxy)-α-heterocyclylmethylphosphonate derivatives (II) were synthesized as potential inhibitors of PDHc. The postemergent activity of the title compounds II was evaluated in greenhouse experiments. The in vitro efficacy of II against PDHc was also examined. Compounds II with fural as R(3) and 2,4-dichloro as X and Y showed significant herbicidal activity and effective inhibition against PDHc from plants. O,O'-Dimethyl α-(2,4-dichlorophenoxyacetoxy)-α-(furan-2-yl)methylphosphonate II-17 had higher inhibitory potency against PDHc from Pisum sativum than against PDHc from Oryza sativa in vitro and was most effective against broadleaf weeds at 50 and 300 ai g/ha. II-17 was safe for maize and rice even at the dose of 900-1200 ai g/ha. Field trials at different regions in China showed that II-17 (HWS) could control a broad spectrum of broad-leaved and sedge weeds at the rate of 225-375 ai g/ha for postemergent applications in maize fields. II-17 (HWS) displayed potential utility as a selective herbicide.

  11. The conserved RGxxE motif of the bacterial FAD assembly factor SdhE is required for succinate dehydrogenase flavinylation and activity.

    Science.gov (United States)

    McNeil, Matthew B; Fineran, Peter C

    2013-10-29

    Succinate dehydrogenase (SDH) is an important respiratory enzyme that plays a critical role in the generation of energy in the majority of eukaryotes, bacteria, and archaea. The activity of SDH is dependent on the covalent attachment of the redox cofactor FAD to the flavoprotein subunit SdhA. In the Gram-negative bacteria Escherichia coli and Serratia sp. ATCC 39006, the covalent attachment of FAD to SdhA is dependent on the FAD assembly factor SdhE (YgfY). Although mechanisms have been proposed, experimental evidence that elucidates the molecular details of SdhE-mediated flavinylation are scarce. In this study, truncation and alanine swap mutagenesis of SdhE identified a highly conserved RGxxE motif that was important for SdhE function. Interestingly, RGxxE site-directed variants were not impaired in terms of protein folding or interactions with SdhA. Purification and analysis of SdhA from different mutant backgrounds demonstrated that SdhE interacts with and flavinylates folded SdhA without a requirement for the assembly of the entire SDH complex. SdhA was also partially active in the absence of SdhE, suggesting that SdhA is able to attach FAD through an inefficient autocatalytic mechanism. The results presented are of widespread relevance because SdhE and SDH are required for bacterial pathogenesis and mutations in the eukaryotic homologues of SdhE and SDH are associated with cancer in humans.

  12. Malate dehydrogenase in phototrophic purple bacteria: purification, molecular weight, and quaternary structure.

    OpenAIRE

    1987-01-01

    The citric acid cycle enzyme malate dehydrogenase was purified to homogeneity from the nonsulfur purple bacteria Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodomicrobium vannielii, and Rhodocyclus purpureus. Malate dehydrogenase was purified from each species by either a single- or a two-step protocol: triazine dye affinity chromatography was the key step in purification of malate dehydrogenase in all cases. Purification of malate dehydrogenase resulted in a 130- to 240-fold increase in ...

  13. 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.

  14. 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.

  15. Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Muro-Pastor, M I; Florencio, F J

    1992-01-15

    NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps. The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L-isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2-oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E. coli enzyme.

  16. 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.

  17. 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

  18. [Effect of electric fields on the living organism. III. Activity of fructose-1,6-diphosphate aldolase and malate dehydrogenase in whole liver homogenate and in subcellular liver fractions in guinea pigs].

    Science.gov (United States)

    Kula, B; Wardas, M

    1990-01-01

    Guinea pigs were exposed to electric field of 50 Hz in different times of day. Activity of aldolase and malate dehydrogenase in whole liver homogenate as well as in nuclear, mitochondrial and supernatant liver fractions of guinea pigs was examined. A remarkable increase in enzyme activity in all studied groups was observed which may prove that a relevant electric stimulus can result in certain disorders in carbohydrate changes in liver cells.

  19. Pro-haloacetate Nanoparticles for Efficient Cancer Therapy via Pyruvate Dehydrogenase Kinase Modulation

    Science.gov (United States)

    Misra, Santosh K.; Ye, Mao; Ostadhossein, Fatemeh; Pan, Dipanjan

    2016-06-01

    Anticancer agents based on haloacetic acids are developed for inhibition of pyruvate dehydrogenase kinase (PDK), an enzyme responsible for reversing the suppression of mitochondria-dependent apoptosis. Through molecular docking studies mono- and dihaloacetates are identified as potent PDK2 binders and matched their efficiency with dichloroacetic acid. In silico screening directed their conversion to phospholipid prodrugs, which were subsequently self-assembled to pro-haloacetate nanoparticles. Following a thorough physico-chemical characterization, the functional activity of these novel agents was established in wide ranges of human cancer cell lines in vitro and in vivo in rodents. Results indicated that the newly explored PDK modulators can act as efficient agent for cancer regression. A Pyruvate dehydrogenase (PDH) assay mechanistically confirmed that these agents trigger their activity through the mitochondria-dependent apoptosis.

  20. Common catabolic enzyme patterns in a microplankton community of the Humboldt Current System off northern and central-south Chile: Malate dehydrogenase activity as an index of water-column metabolism in an oxygen minimum zone

    Science.gov (United States)

    González, R. R.; Quiñones, R. A.

    2009-07-01

    An extensive subsurface oxygen minimum zone off northern and central-south Chile, associated with the Peru-Chile undercurrent, has important effects on the metabolism of the organisms inhabiting therein. Planktonic species deal with the hypoxic and anoxic environments by relying on biochemical as well as physiological processes related to their anaerobic metabolisms. Here we characterize, for the first time, the potential enzymatic activities involved in the aerobic and anaerobic energy production pathways of microplanktonic organisms (catabolic pathways in the oxygen minimum zone. Malate dehydrogenase had the highest oxidizing activity of nicotinamide adenine dinucleotide (reduced form) in the batch of catabolic enzymatic activities assayed, including potential pyruvate oxidoreductases activity, the electron transport system, and dissimilatory nitrate reductase. Malate dehydrogenase correlated significantly with almost all the enzymes analyzed within and above the oxygen minimum zone, and also with the oxygen concentration and microplankton biomass in the water column of the Humboldt Current System, especially in the oxygen minimum zone off Iquique. These results suggest a possible specific pattern for the catabolic activity of the microplanktonic realm associated with the oxygen minimum zone spread along the Humboldt Current System off Chile. We hypothesize that malate dehydrogenase activity could be an appropriate indicator of microplankton catabolism in the oxygen minimum zone and adjacent areas.

  1. Relationship of lipogenic enzyme activities to the rate of rat liver fatty acid synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, G.; Kelley, D.; Schmidt, P.; Virk, S.; Serrato, C.

    1986-05-01

    The mechanism by which diet regulates liver lipogenesis is unclear. Here the authors report how dietary alterations effect the activities of key enzymes of fatty acid (FA) synthesis. Male Sprague-Dawley rats, 400-500 g, were fasted for 48h and then refed a fat-free, high carbohydrate (HC) diet (75% cal. from sucrose) for 0,3,9,24 and 48h, or refed a HC diet for 48h, then fed a high-fat (HF) diet (44% cal. from corn oil) for 3,9,24 and 48h. The FA synthesis rate and the activities of acetyl CoA carboxylase (AC), fatty acid synthase (FAS), ATP citrate lyase (CL), and glucose 6-phosphate dehydrogenase (G6PDH) were determined in the livers. FA synthesis was assayed with /sup 3/H/sub 2/O, enzyme activities were measured spectrophotometrically except for AC which was assayed with /sup 14/C-bicarbonate. There was no change in the activity of AC during fasting or on the HC diet. Fasting decreased the rate of FA synthesis by 25% and the activities of FAS and CL by 50%; refeeding the HC diet induced parallel changes in FA synthesis and the activities of FAS, CL, and G6PDH. After 9h on the HF diet, FA synthesis had decreased sharply, AC activity increased significantly while no changes were detected in the other activities. Subsequently FA synthesis did not change while the activities of the enzymes decreased slowly. These enzymes did not appear to regulate FA synthesis during inhibition of lipogenesis, but FAS, CL or G6PDH may be rate limiting in the induction phase. Other key factors may regulate FA synthesis during dietary alterations.

  2. Enhanced Photosynthetic Performance and Growth as a Consequence of Decreasing Mitochondrial Malate Dehydrogenase Activity in Transgenic Tomato Plants

    National Research Council Canada - National Science Library

    Adriano Nunes-Nesi; Fernando Carrari; Anna Lytovchenko; Anna M. O. Smith; Marcelo Ehlers Loureiro; R. George Ratcliffe; Lee J. Sweetlove; Alisdair R. Fernie

    2005-01-01

    ... photosynthetic activity and aerial growth under atmospheric conditions (360 ppm CO ). In comparison to wild-type plants, carbon dioxide assimilation rates and total plant dry matter were up to 11% and 19...

  3. Quantum chemical modeling of methanol oxidation mechanisms by methanol dehydrogenase enzyme: effect of substitution of calcium by barium in the active site.

    Science.gov (United States)

    Idupulapati, Nagesh B; Mainardi, Daniela S

    2010-02-04

    Previous experimental studies have shown that the activation energy for methanol oxidation by naturally occurring Ca(2+)-containing methanol dehydrogenase (MDH) enzyme is double the methanol activation energy by Ba(2+)-MDH. However, neither the reason for this difference nor the specific transition states and intermediates involved during the methanol oxidation by Ba(2+)-MDH have been clearly stated. Hence, an MDH active site model based on the well-documented X-ray crystallographic structure of Ca(2+)-MDH is selected, where the Ca(2+) is replaced by a Ba(2+) ion at the active site center, and the addition-elimination (A-E) and hydride-transfer (H-T) methanol oxidation mechanisms, already proposed in the literature for Ca(2+)-MDH, are tested for Ba(2+)-MDH at the BLYP/DNP theory level. Changes in the geometries and energy barriers for all the steps are identified, and qualitatively, similar (when compared to Ca(2+)-MDH) intermediates and transition states associated with each step of the mechanisms are found in the case of Ba(2+)-MDH. For both the A-E and H-T mechanisms, almost all the free-energy barriers associated with all of the steps are reduced in the presence of Ba(2+)-MDH, and they are kinetically feasible. The free energy barriers for methanol oxidation by Ba(2+)-MDH, particularly for the rate-limiting steps of both mechanisms, are almost half the corresponding barriers calculated for the case of Ca(2+)-MDH, which is in agreement with experimental observations.

  4. Investigation of the Amycolatopsis sp. Strain ATCC 39116 Vanillin Dehydrogenase and Its Impact on the Biotechnical Production of Vanillin

    OpenAIRE

    Fleige, Christian; Hansen, Gunda; Kroll, Jens; Steinbüchel, Alexander

    2013-01-01

    The actinomycete Amycolatopsis sp. strain ATCC 39116 is capable of synthesizing large amounts of vanillin from ferulic acid, which is a natural cell wall component of higher plants. The desired intermediate vanillin is subject to undesired catabolism caused by the metabolic activity of a hitherto unknown vanillin dehydrogenase (VDHATCC 39116). In order to prevent the oxidation of vanillin to vanillic acid and thereby to obtain higher yields and concentrations of vanillin, the responsible vani...

  5. 藏羚羊骨骼肌肌红蛋白含量及乳酸脱氢酶、苹果酸脱氢酶活力的研究%Study on the content of myoglobin and the activity of lactate dehydrogenase and malate dehydrogenase in skeletal muscle of tibetan antelope

    Institute of Scientific and Technical Information of China (English)

    马兰; 杨应忠; 格日力

    2012-01-01

    Objective: To explore the adaptive mechanism to hypoxia in skeletal muscle of tibetan antelope. Methods: Tibetan sheep which living at the same altitude(4 300 m) with tibetan antelope and low altitude( 1 800 m) sheep as control , the content of myoglobin (Mb) and lactic acid (LA), the activity of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in skeletal muscles among three animals were analyzed by spectrophotometer. Results: The content of myoglobin in skeletal muscle of tibetan antelope significantly higher than that of tibetan sheep and low altitude sheep( P < 0.05). And the content of LA in skeletal muscle of tibetan antelope significantly lower than that of tibetan sheep and low altitude sheep( P < 0.05), activity of LDH and MDH in skeletal muscle was significantly lower and higher respectively than that of tibetan sheep and low altitude sheep( P < 0.05) . There was no significant difference between tibetan sheep and low altitude sheep. Conclusion: Tibetan antelope may improve their ability to get oxygen under hypoxia by increasing the content of myoglobin in skeletal muscle, and the proportion of aerobic metabolism is high in skeletal muscle, it may be relate that with high myoglobin content in skeletal muscle, we suppose that high myoglobin content in skeletal muscle of tibetan antelope might be one of the molecular basis to adapt hypoxia.%目的:探讨藏羚羊骨骼肌对低氧环境的适应机制.方法:以生活在同海拔高度(4 300 m)的藏绵羊和低海拔绵羊(1 800m)为对照,用分光光度法测定三种动物骨骼肌中肌红蛋白(Mb)含量、乳酸(LA)含量,酶活力法测定三种动物骨骼肌中乳酸脱氢酶(LDH)和苹果酸脱氢酶(MDH)活力.结果:藏羚羊骨骼肌中Mb含量明显高于藏绵羊和低海拔绵羊(P<0.05),而藏绵羊和低海拔绵羊间无明显差异.LA含量和LDH活力明显低于藏绵羊和低海拔绵羊(P<0.05),而MDH活力及MDH/LDH比值显著高于藏绵羊和低海拔绵羊(P<0

  6. Stable Suppression of Lactate Dehydrogenase Activity during Anoxia in the Foot Muscle of Littorina littorea and the Potential Role of Acetylation as a Novel Posttranslational Regulatory Mechanism.

    Science.gov (United States)

    Shahriari, Ali; Dawson, Neal J; Bell, Ryan A V; Storey, Kenneth B

    2013-01-01

    The intertidal marine snail, Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH) is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD(+), which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 1.1.1.28) from foot muscle of aerobic control versus 24 h anoxia-exposed L. littorea. Anoxic LDH displayed a near 50% decrease in V max (pyruvate-reducing direction) as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves.

  7. Stable Suppression of Lactate Dehydrogenase Activity during Anoxia in the Foot Muscle of Littorina littorea and the Potential Role of Acetylation as a Novel Posttranslational Regulatory Mechanism

    Directory of Open Access Journals (Sweden)

    Ali Shahriari

    2013-01-01

    Full Text Available The intertidal marine snail, Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD+, which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 1.1.1.28 from foot muscle of aerobic control versus 24 h anoxia-exposed L. littorea. Anoxic LDH displayed a near 50% decrease in Vmax (pyruvate-reducing direction as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves.

  8. Modelling the cloud condensation nucleus activity of organic acids

    Directory of Open Access Journals (Sweden)

    Z. Varga

    2007-04-01

    Full Text Available In this study vapour pressure osmometry was used to determine water activity in solutions of organic acids. The surface tension of the solutions was also monitored in parallel and then Köhler curves were calculated for nine organic acids (oxalic, malonic, succinic, glutaric, adipic acid, maleic acid, malic acid, citric acid and pinonic acid. Surface tension depression is negligible for most of the organic acids in dilute (≤1 w/w% solutions. Therefore, these compounds affect the supersaturation only in the beginning phase of droplet formation but not necessarily at the critical size. An exception is cis-pinonic acid which remarkably depress surface tension also in dilute (0.1 w/w% solution and hence at the critical point. The surface tension of organic acid solutions is influenced by the solubility of the compound, the length of the carbon chain and also by the polar functional groups present in the molecule. Similarly to surface tension solubility plays an important role also in water activity: compounds with higher solubility (e.g. malonic, maleic, and glutaric acid reduce water activity significantly in the early phase of droplet formation while less soluble acids (e.g. succinic and adipic acid are saturated in small droplets and the solution starts diluting only in bigger droplets. As a consequence, compounds with lower solubility have a minor effect on water activity in the early phase of droplet formation. To deduce the total effect Köhler curves were calculated and critical supersaturations were determined for the organic acids using measured surface tension and water activity. It was found that critical supersaturation grew with growing carbon number. Oxalic acid had the lowest critical supersaturation in the size range studied and it was comparable to the activation of ammonium sulfate. The Sc values obtained in this study were compared to data from CCNC measurements. In most cases good agreement was found.

  9. Mechanistic and computational studies of the reductive half-reaction of tyrosine to phenylalanine active site variants of D-arginine dehydrogenase.

    Science.gov (United States)

    Gannavaram, Swathi; Sirin, Sarah; Sherman, Woody; Gadda, Giovanni

    2014-10-