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Sample records for dehydrogenase activity electronic

  1. 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.

  2. The response of electron transport mediated by active NADPH dehydrogenase complexes to heat stress in the cyanobacterium Synechocystis 6803

    Institute of Scientific and Technical Information of China (English)

    MA WeiMin; WEI LanZhen; WANG QuanXi

    2008-01-01

    The electron-transport machinery in photosynthetic membranes is known to be very sensitive to heat. In this study, the rate of electron transport (ETR) driven by photosystem Ⅰ (PSI) and photosystem Ⅱ (PSII) during heat stress in the wild-type Synechocystis sp. strain PCC 6803 (WT) and its ndh gene inactivation mutants △ndhB (M55) and △ndhD1/ndhD2 (D1/D2) was simultaneously assessed by using the novel Dual-PAM-100 measuring system. The rate of electron transport driven by the photosystems (ETRPSs) in the WT, M55, and D1/D2 cells incubated at 30℃ and at 55℃ for 10 min was compared. Incubation at 55℃ for 10 min significantly inhibited PSII-driven ETR (ETRPSII) in the WT, M55 and D1/D2 cells, and the extent of inhibition in both the M55 and D1/D2 cells was greater than that in the WT cells. Further, PSI-driven ETR (ETRPSI) was stimulated in both the WT and D1/D2 cells, and this rate was increased to a greater extent in the D1/D2 than in the WT cells. However, ETRPSI was considerably inhibited in the M55 cells. Analysis of the effect of heat stress on ETRPSs with regard to the alterations in the 2 active NDH-1 complexes in the WT, M55, and D1/D2 cells indicated that the active NDH-1 supercomplex and mediumcomplex are essential for alleviating the heat-induced inhibition of ETRPSII and for accelerating the heat-induced stimulation of ETRPSI, respectively. Further, it is believed that these effects are most likely brought about by the electron transport mediated by each of these 2 active NDH-1 complexes.

  3. The response of electron transport mediated by active NADPH dehydrogenase complexes to heat stress in the cyanobacterium Synechocystis 6803

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The electron-transport machinery in photosynthetic membranes is known to be very sensitive to heat. In this study, the rate of electron transport (ETR) driven by photosystem I (PSI) and photosystem II (PSII) during heat stress in the wild-type Synechocystis sp. strain PCC 6803 (WT) and its ndh gene inactiva-tion mutants △ndhB (M55) and △ndhD1/ndhD2 (D1/D2) was simultaneously assessed by using the novel Dual-PAM-100 measuring system. The rate of electron transport driven by the photosystems (ETRPSs) in the WT, M55, and D1/D2 cells incubated at 30℃ and at 55℃ for 10 min was compared. Incubation at 55 ℃ for 10 min significantly inhibited PSII-driven ETR (ETRPSII) in the WT, M55 and D1/D2 cells, and the ex-tent of inhibition in both the M55 and D1/D2 cells was greater than that in the WT cells. Further, PSI-driven ETR (ETRPSI) was stimulated in both the WT and D1/D2 cells, and this rate was increased to a greater extent in the D1/D2 than in the WT cells. However, ETRPSI was considerably inhibited in the M55 cells. Analysis of the effect of heat stress on ETRPSs with regard to the alterations in the 2 active NDH-1 complexes in the WT, M55, and D1/D2 cells indicated that the active NDH-1 supercomplex and medi-umcomplex are essential for alleviating the heat-induced inhibition of ETRPSII and for accelerating the heat-induced stimulation of ETRPSI, respectively. Further, it is believed that these effects are most likely brought about by the electron transport mediated by each of these 2 active NDH-1 complexes.

  4. INFLUENCE OF SELECTED PHARMACEUTICALS ON ACTIVATED SLUDGE DEHYDROGENASE ACTIVITY

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    Agnieszka Tomska

    2016-06-01

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

  5. 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.

  6. The reaction of choline dehydrogenase with some electron acceptors.

    Science.gov (United States)

    Barrett, M C; Dawson, A P

    1975-12-01

    1. The choline dehydrogenase (EC 1.1.99.1) WAS SOLUBILIZED FROM ACETONE-DRIED POWDERS OF RAT LIVER MITOCHONDRIA BY TREATMENT WITH Naja naja venom. 2. The kinetics of the reaction of enzyme with phenazine methosulphate and ubiquinone-2 as electron acceptors were investigated. 3. With both electron acceptors the reaction mechanism appears to involve a free, modified-enzyme intermediate. 4. With some electron acceptors the maximum velocity of the reaction is independent of the nature of the acceptor. With phenazine methosulphate and ubiquinone-2 as acceptors the Km value for choline is also independent of the nature of the acceptor molecule. 5. The mechanism of the Triton X-100-solubilized enzyme is apparently the smae as that for the snake venom solubilized enzyme.

  7. 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...

  8. 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.

  9. 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.

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

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    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

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

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    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.

  12. 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.

  13. In situ Regeneration of NADH via Lipoamide Dehydrogenase-catalyzed Electron Transfer Reaction Evidenced by Spectroelectrochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Tam, Tsz Kin; Chen, Baowei; Lei, Chenghong; Liu, Jun

    2012-08-01

    NAD/NADH is a coenzyme found in all living cells, carrying electrons from one reaction to another. We report on characterizations of in situ regeneration of NADH via lipoamide dehydrogenase (LD)-catalyzed electron transfer reaction to regenerate NADH using UV-vis spectroelectrochemistry. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of NADH regeneration were measured as 0.80 {+-} 0.15 mM and 1.91 {+-} 0.09 {micro}M s-1 in a 1-mm thin-layer spectroelectrochemical cell using gold gauze as the working electrode at the applied potential -0.75 V (vs. Ag/AgCl). The electrocatalytic reduction of the NAD system was further coupled with the enzymatic conversion of pyruvate to lactate by lactate dehydrogenase to examine the coenzymatic activity of the regenerated NADH. Although the reproducible electrocatalytic reduction of NAD into NADH is known to be difficult compared to the electrocatalytic oxidation of NADH, our spectroelectrochemical results indicate that the in situ regeneration of NADH via LD-catalyzed electron transfer reaction is fast and sustainable and can be potentially applied to many NAD/NADH-dependent enzyme systems.

  14. 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.

  15. 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.

  16. Menaquinone-dependent succinate dehydrogenase of bacteria catalyzes reversed electron transport driven by the proton potential.

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    Schirawski, J; Unden, G

    1998-10-01

    Succinate dehydrogenases from bacteria and archaea using menaquinone (MK) as an electron acceptor (succinate/menaquinone oxidoreductases) contain, or are predicted to contain, two heme-B groups in the membrane-anchoring protein(s), located close to opposite sides of the membrane. All succinate/ubiquinone oxidoreductases, however, contain only one heme-B molecule. In Bacillus subtilis and other bacteria that use MK as the respiratory quinone, the succinate oxidase activity (succinate-->O2), and the succinate/menaquinone oxidoreductase activity were specifically inhibited by uncoupler (CCCP, carbonyl cyanide m-chlorophenylhydrazone) or by agents dissipating the membrane potential (valinomycin). Other parts of the respiratory chains were not affected by the agents. Succinate oxidase or succinate/ubiquinone oxidoreductase from bacteria using ubiquinone as an acceptor were not inhibited. We propose that the endergonic electron transport from succinate (Eo' = +30 mV) to MK (Eo' approximately/= -80 mV) in succinate/menaquinone oxidoreductase includes a reversed electron transport across the cytoplasmic membrane from the inner (negative) to the outer (positive) side via the two heme-B groups. The reversed electron transport is driven by the proton or electrical potential, which provides the driving force for MK reduction.

  17. 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.

  18. pH-dependent electron transfer reaction and direct bioelectrocatalysis of the quinohemoprotein pyranose dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Kouta [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan); Matsumura, Hirotoshi; Ishida, Takuya [Department of Biomaterial Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657 (Japan); Yoshida, Makoto [Department of Environmental and Natural Resource Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509 (Japan); Igarashi, Kiyohiko; Samejima, Masahiro [Department of Biomaterial Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657 (Japan); Ohno, Hiroyuki [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan); Nakamura, Nobuhumi, E-mail: nobu1@cc.tuat.ac.jp [Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan)

    2016-08-26

    A pyranose dehydrogenase from Coprinopsis cinerea (CcPDH) is an extracellular quinohemoeprotein, which consists a b-type cytochrome domain, a pyrroloquinoline-quinone (PQQ) domain, and a family 1-type carbohydrate-binding module. The electron transfer reaction of CcPDH was studied using some electron acceptors and a carbon electrode at various pH levels. Phenazine methosulfate (PMS) reacted directly at the PQQ domain, whereas cytochrome c (cyt c) reacted via the cytochrome domain of intact CcPDH. Thus, electrons are transferred from reduced PQQ in the catalytic domain of CcPDH to heme b in the N-terminal cytochrome domain, which acts as a built-in mediator and transfers electron to a heterogenous electron transfer protein. The optimal pH values of the PMS reduction (pH 6.5) and the cyt c reduction (pH 8.5) differ. The catalytic currents for the oxidation of L-fucose were observed within a range of pH 4.5 to 11. Bioelectrocatalysis of CcPDH based on direct electron transfer demonstrated that the pH profile of the biocatalytic current was similar to the reduction activity of cyt c characters. - Highlights: • pH dependencies of activity were different for the reduction of cyt c and DCPIP. • DET-based bioelectrocatalysis of CcPDH was observed. • The similar pH-dependent profile was found with cyt c and electrode. • The present results suggested that IET reaction of CcPDH shows pH dependence.

  19. Toxicity of Nitrification Inhibitors on Dehydrogenase Activity in Soils

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    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.

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

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    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.

  1. Catalytic reaction of cytokinin dehydrogenase : preference for quinones as electron acceptors

    NARCIS (Netherlands)

    Frébortová, Jitka; Fraaije, Marco W.; Galuszka, Petr; Šebela, Marek; Peč, Pavel; Hrbáč, Jan; Novák, Ondřej; Bilyeu, Kristin D.; English, James T.; Frébort, Ivo; Sebela, M.; Pec, P.; Hrbac, J.; Frebort, [No Value

    2004-01-01

    The catalytic reaction of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) was studied in detail using the recombinant flavoenzyme from maize. Determination of the redox potential of the covalently linked flavin cofactor revealed a relatively high potential dictating the type of electron acceptor that

  2. Electron transfer between a quinohemoprotein alcohol dehydrogenase and an electrode via a redox polymer network

    NARCIS (Netherlands)

    Stigter, E.C.A.; Jong, G.A.H. de; Jongejan, J.A.; Duine, J.A.; Lugt, J.P. van der; Somers, W.A.C.

    1996-01-01

    A quinohemoprotein alcohol dehydrogenase (QH-EDH) from Comamonas testosteroni was immobilized on an electrode in a redox polymer network consisting of a polyvinylpyridine partially N-complexed with osmiumbis-(bipyridine)chloride. The enzyme effectively transfers electrons to the electrode via the

  3. Electron transfer between a quinohemoprotein alcohol dehydrogenase and an electrode via a redox polymer network

    NARCIS (Netherlands)

    Stigter, E.C.A.; Jong, G.A.H. de; Jongejan, J.A.; Duine, J.A.; Lugt, J.P. van der; Somers, W.A.C.

    1996-01-01

    A quinohemoprotein alcohol dehydrogenase (QH-EDH) from Comamonas testosteroni was immobilized on an electrode in a redox polymer network consisting of a polyvinylpyridine partially N-complexed with osmiumbis-(bipyridine)chloride. The enzyme effectively transfers electrons to the electrode via the po

  4. Electron transfer between periplasmic formate dehydrogenase and cytochromes c in Desulfovibrio desulfuricans ATCC 27774.

    Science.gov (United States)

    da Silva, Sofia Marques; Pacheco, Isabel; Pereira, Inês A Cardoso

    2012-06-01

    Desulfovibrio spp. are sulfate-reducing organisms characterized by having multiple periplasmic hydrogenases and formate dehydrogenases (FDHs). In contrast to enzymes in most bacteria, these enzymes do not reduce directly the quinone pool, but transfer electrons to soluble cytochromes c. Several studies have investigated electron transfer with hydrogenases, but comparatively less is known about FDHs. In this work we conducted experiments to assess potential electron transfer pathways resulting from formate oxidation in Desulfovibrio desulfuricans ATCC 27774. This organism can grow on sulfate and on nitrate, and contains a single soluble periplasmic FDH that includes a cytochrome c (3) like subunit (FdhABC(3)). It has also a unique cytochrome c composition, including two cytochromes c not yet isolated from other species, the split-Soret and nine-heme cytochromes, besides a tetraheme type I cytochrome c (3) (TpIc (3)). The FDH activity and cytochrome composition of cells grown with lactate or formate and nitrate or sulfate were determined, and the electron transfer between FDH and these cytochromes was investigated. We studied also the reduction of the Dsr complex and of the monoheme cytochrome c-553, previously proposed to be the physiological partner of FDH. FdhABC(3) was able to reduce the c-553, TpIc (3), and split-Soret cytochromes with a high rate. For comparison, the same experiments were performed with the [NiFe] hydrogenase from the same organism. This study shows that FdhABC(3) can directly reduce the periplasmic cytochrome c network, feeding electrons into several alternative metabolic pathways, which explains the advantage of not having an associated membrane subunit.

  5. Mechanistic studies on the dehydrogenases of methylotrophic bacteria. 2. Kinetic studies on the intramolecular electron transfer in trimethylamine and dimethylamine dehydrogenase.

    Science.gov (United States)

    Steenkamp, D J; Beinert, H

    1982-01-01

    E.p.r. spectroscopy of the trimethylamine and dimethylamine dehydrogenases of Hyphomicrobium X indicates that the substrate-reduced forms of these enzymes exist in the triplet state, which arise through interaction of a reduced [4Fe-4S] cluster and flavosemiquinone, with e.p.r. signals which differ in detail from those of the trimethylamine dehydrogenase of bacterium W3A1. Under certain conditions the intramolecular electron transfer between the flavoquinol form of 6-S-cysteinyl-FMN and the [4Fe-4S] cluster in all three dehydrogenases was much slower than the preceding reduction of the flavin to the flavoquinol form. Trimethylamine dehydrogenases from both organisms show a time-dependent broadening of the e.p.r. signals centred around g = 2 after mixing with trimethylamine. The broadening of the e.p.r. signals could be correlated with an unexpected dependence of the rate of formation of the triplet state on substrate concentration. A model which accounts in a qualitative manner for the substrate dependence of the formation of the triplet state in the trimethylamine dehydrogenase of Hyphomicrobium X is proposed. The binding of the substrate to the reduced form of the enzyme seems to result in a conformational change of the enzyme to a form in which the rate of intramolecular electron transfer is decreased. This finding may be correlated with the observation of hyperbolic substrate inhibition for both trimethylamine dehydrogenases. The results indicate the transfer of an electron to the [4Fe-4S] cluster to be an obligatory step in catalysis and suggest that the transfer of electrons from these enzymes to electron acceptors is mediated solely through the [4Fe-4S] cluster. PMID:6297456

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  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. 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.

  16. 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.

  17. 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.

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

    Science.gov (United States)

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

    2017-05-31

    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.

  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. Electron Microscopic Analysis and Biochemical Characterization of a Novel Methanol Dehydrogenase from the Thermotolerant Bacillus sp. C1

    NARCIS (Netherlands)

    Vonck, Janet; Arfman, Nico; Vries, Gert E. de; Beeumen, Jozef van; Bruggen, Ernst F.J. van; Dijkhuizen, Lubbert

    1991-01-01

    Methanol dehydrogenase from the thermotolerant Bacillus sp. C1 was studied by electron microscopy and image processing. Two main projections can be distinguished: one exhibits 5-fold symmetry and has a diameter of 15 nm, the other is rectangular with sides of 15 and 9 nm. Subsequent image processing

  1. 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.

  2. 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....

  3. 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...

  4. 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...

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  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. 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...

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. CRYSTAL-STRUCTURE OF AN ELECTRON-TRANSFER COMPLEX BETWEEN METHYLAMINE DEHYDROGENASE AND AMICYANIN

    NARCIS (Netherlands)

    CHEN, LY; DURLEY, R; POLIKS, BJ; HAMADA, K; CHEN, ZW; MATHEWS, FS; DAVIDSON, VL; SATOW, Y; HUIZINGA, E; VELLIEUX, FMD; HOL, WGJ

    1992-01-01

    The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-angstrom resolution using molecular replacement. The search model was MADH from Thiobacillus ver

  5. CRYSTAL-STRUCTURE OF AN ELECTRON-TRANSFER COMPLEX BETWEEN METHYLAMINE DEHYDROGENASE AND AMICYANIN

    NARCIS (Netherlands)

    CHEN, LY; DURLEY, R; POLIKS, BJ; HAMADA, K; CHEN, ZW; MATHEWS, FS; DAVIDSON, VL; SATOW, Y; HUIZINGA, E; VELLIEUX, FMD; HOL, WGJ

    1992-01-01

    The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-angstrom resolution using molecular replacement. The search model was MADH from Thiobacillus

  6. CRYSTAL-STRUCTURE OF AN ELECTRON-TRANSFER COMPLEX BETWEEN METHYLAMINE DEHYDROGENASE AND AMICYANIN

    NARCIS (Netherlands)

    CHEN, LY; DURLEY, R; POLIKS, BJ; HAMADA, K; CHEN, ZW; MATHEWS, FS; DAVIDSON, VL; SATOW, Y; HUIZINGA, E; VELLIEUX, FMD; HOL, WGJ

    1992-01-01

    The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-angstrom resolution using molecular replacement. The search model was MADH from Thiobacillus ver

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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...

  13. 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.

  14. 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.

  15. 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.

  16. [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.

  17. Active devices for electronics

    CERN Document Server

    Sowa, Walter A

    1971-01-01

    An introduction to semiconductors ; the p-n junction ; the junction transistor ; electrical characteristics and ratings ; basic transistor amplifiers ; the field-effect transistor ; the unijunction transistor (UJT) ; the silicon controlled rectifier (SCR) ; the DIAC and TRIAC ; light operated devices ; integrated circuits (ICs) ; other solid-state devices of interest ; electron tubes.

  18. Suppression of NDA-type alternative mitochondrial NAD(P)H dehydrogenases in arabidopsis thaliana modifies growth and metabolism, but not high light stimulation of mitochondrial electron transport.

    Science.gov (United States)

    Wallström, Sabá V; Florez-Sarasa, Igor; Araújo, Wagner L; Escobar, Matthew A; Geisler, Daniela A; Aidemark, Mari; Lager, Ida; Fernie, Alisdair R; Ribas-Carbó, Miquel; Rasmusson, Allan G

    2014-05-01

    The plant respiratory chain contains several pathways which bypass the energy-conserving electron transport complexes I, III and IV. These energy bypasses, including type II NAD(P)H dehydrogenases and the alternative oxidase (AOX), may have a role in redox stabilization and regulation, but current evidence is inconclusive. Using RNA interference, we generated Arabidopsis thaliana plants simultaneously suppressing the type II NAD(P)H dehydrogenase genes NDA1 and NDA2. Leaf mitochondria contained substantially reduced levels of both proteins. In sterile culture in the light, the transgenic lines displayed a slow growth phenotype, which was more severe when the complex I inhibitor rotenone was present. Slower growth was also observed in soil. In rosette leaves, a higher NAD(P)H/NAD(P)⁺ ratio and elevated levels of lactate relative to sugars and citric acid cycle metabolites were observed. However, photosynthetic performance was unaffected and microarray analyses indicated few transcriptional changes. A high light treatment increased AOX1a mRNA levels, in vivo AOX and cytochrome oxidase activities, and levels of citric acid cycle intermediates and hexoses in all genotypes. However, NDA-suppressing plants deviated from the wild type merely by having higher levels of several amino acids. These results suggest that NDA suppression restricts citric acid cycle reactions, inducing a shift towards increased levels of fermentation products, but do not support a direct association between photosynthesis and NDA proteins.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  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. 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.

  5. Glutaryl-coenzyme A dehydrogenase from Geobacter metallireducens - interaction with electron transferring flavoprotein and kinetic basis of unidirectional catalysis.

    Science.gov (United States)

    Estelmann, Sebastian; Boll, Matthias

    2014-11-01

    Glutaryl-CoA dehydrogenases (GDHs) are FAD containing acyl-CoA dehydrogenases that usually catalyze the dehydrogenation and decarboxylation of glutaryl-CoA to crotonyl-CoA with an electron transferring flavoprotein (ETF) acting as natural electron acceptor. In anaerobic bacteria, GDHs play an important role in the benzoyl-CoA degradation pathway of monocyclic aromatic compounds. In the present study, we identified, purified and characterized the benzoate-induced BamOP as the electron accepting ETF of GDH (BamM) from the Fe(III)-respiring Geobacter metallireducens. The BamOP heterodimer contained FAD and AMP as cofactors. In the absence of an artificial electron acceptor, at pH values above 8, the BamMOP-components catalyzed the expected glutaryl-CoA oxidation to crotonyl-CoA and CO2 ; however, at pH values below 7, the redox-neutral glutaryl-CoA conversion to butyryl-CoA and CO2 became the dominant reaction. This previously unknown, strictly ETF-dependent coupled glutaryl-CoA oxidation/crotonyl-CoA reduction activity was facilitated by an unexpected two-electron transfer between FAD(BamM) and FAD(BamOP) , as well as by the similar redox potentials of the two FAD cofactors in the substrate-bound state. The strict order of electron/proton transfer and C-C-cleavage events including transient charge-transfer complexes did not allow an energetic coupling of electron transfer and decarboxylation. This explains why it was difficult to release the glutaconyl-CoA intermediate from reduced GDH. Moreover, it provides a kinetic rational for the apparent inability of BamM to catalyze the reverse reductive crotonyl-CoA carboxylation, even under thermodynamically favourable conditions. For this reason reductive crotonyl-CoA carboxylation, a key reaction in C2-assimilation via the ethylmalonyl-CoA pathway, is accomplished by a different crotonyl-CoA carboxylase/reductase via a covalent NADPH/ene-adduct.

  6. 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).

  7. 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

  8. 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.

  9. 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.

  10. Identification of Electronic and Structural Descriptors of Adenosine Analogues Related to Inhibition of Leishmanial Glyceraldehyde-3-Phosphate Dehydrogenase

    Directory of Open Access Journals (Sweden)

    Norka B. H. Lozano

    2013-04-01

    Full Text Available Quantitative structure–activity relationship (QSAR studies were performed in order to identify molecular features responsible for the antileishmanial activity of 61 adenosine analogues acting as inhibitors of the enzyme glyceraldehyde 3-phosphate dehydrogenase of Leishmania mexicana (LmGAPDH. Density functional theory (DFT was employed to calculate quantum-chemical descriptors, while several structural descriptors were generated with Dragon 5.4. Variable selection was undertaken with the ordered predictor selection (OPS algorithm, which provided a set with the most relevant descriptors to perform PLS, PCR and MLR regressions. Reliable and predictive models were obtained, as attested by their high correlation coefficients, as well as the agreement between predicted and experimental values for an external test set. Additional validation procedures were carried out, demonstrating that robust models were developed, providing helpful tools for the optimization of the antileishmanial activity of adenosine compounds.

  11. 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.

  12. The multiplicity of dehydrogenases in the electron transport chain of plant mitochondria

    DEFF Research Database (Denmark)

    Rasmusson, Allan G; Geisler, Daniela A; Møller, Ian Max

    2008-01-01

    The electron transport chain in mitochondria of different organisms contains a mixture of common and specialised components. The specialised enzymes form branches to the universal electron path, especially at the level of ubiquinone, and allow the chain to adjust to different cellular and metabolic...

  13. 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

  14. 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).

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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...

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. A novel bio-electronic tongue using different cellobiose dehydrogenases to resolve mixtures of various sugars and interfering analytes.

    Science.gov (United States)

    Cipri, Andrea; Schulz, Christopher; Ludwig, Roland; Gorton, Lo; Del Valle, Manel

    2016-05-15

    A novel application of cellobiose dehydrogenase (CDH) as sensing element for a Bioelectronic Tongue (BioET) system has been tested. In this work CDHs from various fungi, which exhibit different substrate specificities, were used to discriminate between lactose and glucose in presence of the interfering matrix compound Ca(2+) in various mixtures. This work exploits the advantage of an electronic tongue system with practically zero pre-treatment of samples and operation at low voltages in a direct electron transfer mode. The Artificial Neural Network (ANN) used in the BioET system to interpret the voltammetric data was able to provide a correct prediction of the concentrations of the analytes considered. Correlation coefficients in the comparison of obtained vs. expected concentrations were highly significant, especially for lactose (R(2)=0.975) and Ca(2+) (R(2)=0.945). This BioET application has a high potential especially for the food and dairy industry and also, if further miniaturised in screen printed format, for its in-situ use.

  14. Role of Quinones in Electron Transfer of PQQ–Glucose Dehydrogenase Anodes—Mediation or Orientation Effect

    Energy Technology Data Exchange (ETDEWEB)

    Babanova, Sofia; Matanovic, Ivana; Chavez, Madelaine Seow; Atanassov, Plamen

    2015-06-24

    In this study, the influence of two quinones (1,2- and 1,4-benzoquinone) on the operation and mechanism of electron transfer in PQQ-dependent glucose dehydrogenase (PQQ–sGDH) anodes has been determined. Benzoquinones were experimentally explored as mediators present in the electrolyte. The electrochemical performance of the PQQ–sGDH anodes with and without the mediators was examined and for the first time molecular docking simulations were used to gain a fundamental understanding to explain the role of the mediator molecules in the design and operation of the enzymatic electrodes. It was proposed that the higher performance of the PQQ–sGDH anodes in the presence of 1,2- and 1,4-benzoquinones introduced in the solution is due to the shorter distance between these molecules and PQQ in the enzymatic molecule. It was also hypothesized that when 1,4-benzoquinone is adsorbed on a carbon support, it would play the dual role of a mediator and an orienting agent. At the same time, when 1,2-benzoquinone and ubiquinone are adsorbed on the electrode surface, the enzyme would transfer the electrons directly to the support, and these molecules would primarily play the role of an orienting agent.

  15. 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.

  16. 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.

  17. 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.

  18. Studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) of Acidaminococcus fermentans.

    Science.gov (United States)

    Chowdhury, Nilanjan Pal; Mowafy, Amr M; Demmer, Julius K; Upadhyay, Vikrant; Koelzer, Sebastian; Jayamani, Elamparithi; Kahnt, Joerg; Hornung, Marco; Demmer, Ulrike; Ermler, Ulrich; Buckel, Wolfgang

    2014-02-21

    Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.

  19. Crystal structure of quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni - Structural basis for substrate oxidation and electron transfer

    NARCIS (Netherlands)

    Oubrie, A; Rozeboom, HJ; Kalk, KH; Huizinga, EG; Dijkstra, BW; Huizinga, Eric G.; Dijkstra, Bauke W.

    2002-01-01

    Quinoprotein alcohol dehydrogenases are redox enzymes that participate in distinctive catabolic pathways that enable bacteria to grow on various alcohols as the sole source of carbon and energy. The x-ray structure of the quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni has been

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

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

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    Valentina Grazioli

    2016-02-01

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

  15. 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.

  16. 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

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

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    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.

  18. 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)

  19. 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.

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

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    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.

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

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    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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. A novel bio-electronic tongue using cellobiose dehydrogenase from different origins to resolve mixtures of various sugars and interfering analytes

    OpenAIRE

    2016-01-01

    A novel application of cellobiose dehydrogenase (CDH) as sensing element for a Bioelectronic Tongue (BioET) system has been tested. In this work CDHs from various fungi, which exhibit different substrate specificities, were used to discriminate between lactose and glucose in presence of the interfering matrix compound Ca²⁺ in various mixtures. This work exploits the advantage of an electronic tongue system with practically zero pre-treatment of samples and operation at low voltages in a direc...

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. [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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. Effects of Some Management Practices on Electron Transport System (ETS) Activity in Paddy Soil

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Electron transport system (ETS)/dehydrogenase activity in a paddy field soil was measured under a variety of incubation conditions using the reduction of 2-(p-iodophenyl-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium formazan (INTF). The results exhibited a high positive correlation between the ETS activity and the incubation temperature and soil moisture. Dehydrogenase/ETS activity displayed a negative correlation with insecticide concentrations, and the activity affected adversely as the concentration of the insecticide increased. The higher doses, 5 and 10 field rates (1 field rate = 1500 mL ha-1), of insecticide significantly inhibited ETS activity, while lower rates failed to produce any significant reducing effect. Inorganic N (as urea) of concentrations from 0 to 100 μg N g-1 soil showed a positive response to ETS activity. However, at concentrations of 200 and 400μg N g-1, the activity was reduced significantly.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. Thermodynamic basis of electron transfer in dihydroorotate dehydrogenase B from Lactococcus lactis: analysis by potentiometry, EPR spectroscopy, and ENDOR spectroscopy.

    Science.gov (United States)

    Mohsen, Al-Walid A; Rigby, Stephen E J; Jensen, Kaj Frank; Munro, Andrew W; Scrutton, Nigel S

    2004-06-01

    Dihydroorotate dehydrogenase B (DHODB) is a complex iron-sulfur flavoprotein that catalyzes the conversion of dihydroorotate to orotate and the reduction of NAD(+). The enzyme is a dimer of heterodimers containing an FMN, an FAD, and a 2Fe-2S center. UV-visible, EPR, and ENDOR spectroscopies have been used to determine the reduction potentials of the flavins and the 2Fe-2S center and to characterize radicals and their interactions. Reductive titration using dithionite indicates a five-electron capacity for DHODB. The midpoint reduction potential of the 2Fe-2S center (-212 +/- 3 mV) was determined from analysis of absorption data at 540 nm, where absorption contributions from the two flavins are small. The midpoint reduction potentials of the oxidized/semiquinone (E(1)) and semiquinone/hydroquinone (E(2)) couples for the FMN (E(1) = -301 +/- 6 mV; E(2) = -252 +/- 8 mV) and FAD (E(1) = -312 +/- 6 mV; E(2) = -297 +/- 5 mV) were determined from analysis of spectral changes at 630 nm. Corresponding values for the midpoint reduction potentials for FMN (E(1) = -298 +/- 4 mV; E(2) = -259 +/- 5 mV) in the isolated catalytic subunit (subunit D, which lacks the 2Fe-2S center and FAD) are consistent with the values determined for the FMN couples in DHODB. During reductive titration of DHODB, small amounts of the neutral blue semiquinone are observed at approximately 630 nm, consistent with the measured midpoint reduction potentials of the flavins. An ENDOR spectrum of substrate-reduced DHODB identifies hyperfine couplings to proton nuclei similar to those recorded for the blue semiquinone of free flavins in aqueous solution, thus confirming the presence of this species in DHODB. Spectral features observed during EPR spectroscopy of dithionite-reduced DHODB are consistent with the midpoint reduction potentials determined using UV-visible spectroscopy and further identify an unusual EPR signal with very small rhombic anisotropy and g values of 2.02, 1.99, and 1.96. This unusual

  7. 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.

  8. The impact of hypoxia on the activity of lactate dehydrogenase in two different pre-clinical tumour models

    DEFF Research Database (Denmark)

    Lukacova, Slavka; Sørensen, Brita; Alsner, Jan

    2008-01-01

    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...

  9. 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.

  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. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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...

  16. 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.

  17. Active learners in sustainable electronics and it

    DEFF Research Database (Denmark)

    Schultz, Ole

    This poster-presentation is about active learning in a course sustainable wireless electronics and it. Active learning understood as practical lab-exercises and a team chosen project.......This poster-presentation is about active learning in a course sustainable wireless electronics and it. Active learning understood as practical lab-exercises and a team chosen project....

  18. 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....

  19. 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.

  20. 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.

  1. 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...

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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...

  8. 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.

  9. 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....

  10. 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.

  11. 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.

  12. 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.

  13. Simultaneous immobilization of dehydrogenases on polyvinylidene difluoride resin after separation by non-denaturing two-dimensional electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Shimazaki, Youji [Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Bunkyo-cho 2-5, Matsuyama City 790-8577 (Japan)], E-mail: yoji@dpc.ehime-u.ac.jp; Kadota, Mariko [Faculty of Science, Ehime University, Matsuyama (Japan)

    2008-06-16

    We detected mouse liver malate, sorbitol and aldehyde dehydrogenases by negative staining, analysis of malate and sorbitol dehydrogenase activities using each substrate, and electron transfers including nicotinamide adenine dinucleotide (NAD) and nitroblue tetrazolium in non-denaturing two-dimensional electrophoresis (2-DE) gel. Dehydrogenases were also identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) after 2-DE separation and protein detection by negative staining. Spots of dehydrogenases separated by 2-DE were excised, and simultaneously transferred and immobilized on polyvinylidene difuoride (PVDF) resin by electrophoresis. The dehydrogenase activities remained intact after immobilization. In conclusion, resin-immobilized dehydrogenases can be simultaneously obtained after separation by non-denaturing 2-DE, detection by negative staining and transferring to resins.

  14. 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.

  15. 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.

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

    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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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...

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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...

  16. 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...

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    V. P. Bessonova

    2007-03-01

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

  5. 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

  6. 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...

  7. 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.

  8. 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.

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

    Science.gov (United States)

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

    2015-03-01

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

  10. 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...

  11. 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).

  12. 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...

  13. 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.

  14. 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.

  15. 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.

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

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    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.

  17. Characterization of a novel PQQ-dependent quinohemoprotein pyranose dehydrogenase from Coprinopsis cinerea classified into auxiliary activities family 12 in carbohydrate-active enzymes.

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    Kouta Takeda

    Full Text Available The basidiomycete Coprinopsis cinerea contains a quinohemoprotein (CcPDH named as CcSDH in our previous paper, which is a new type of pyrroloquinoline-quinone (PQQ-dependent pyranose dehydrogenase and is the first found among all eukaryotes. This enzyme has a three-domain structure consisting of an N-terminal heme b containing a cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase (CDH; EC 1.1.99.18 from the wood-rotting basidiomycete Phanerochaete chrysosporium, a C-terminal family 1-type carbohydrate-binding module, and a novel central catalytic domain containing PQQ as a cofactor. Here, we describe the biochemical and electrochemical characterization of recombinant CcPDH. UV-vis and resonance Raman spectroscopic studies clearly reveal characteristics of a 6-coordinated low-spin heme b in both the ferric and ferrous states, as well as intramolecular electron transfer from the PQQ to heme b. Moreover, the formal potential of the heme was evaluated to be 130 mV vs. NHE by cyclic voltammetry. These results indicate that the cytochrome domain of CcPDH possesses similar biophysical properties to that in CDH. A comparison of the conformations of monosaccharides as substrates and the associated catalytic efficiency (kcat/Km of CcPDH indicates that the enzyme prefers monosaccharides with equatorial C-2, C-3 hydroxyl groups and an axial C-4 hydroxyl group in the 1C4 chair conformation. Furthermore, a binding study shows a high binding affinity of CcPDH for cellulose, suggesting that CcPDH function is related to the enzymatic degradation of plant cell wall.

  18. Cyclic electron flow around photosystem I via chloroplast NAD(P)H dehydrogenase (NDH) complex performs a significant physiological role during photosynthesis and plant growth at low temperature in rice.

    Science.gov (United States)

    Yamori, Wataru; Sakata, Naoki; Suzuki, Yuji; Shikanai, Toshiharu; Makino, Amane

    2011-12-01

    The role of NAD(P)H dehydrogenase (NDH)-dependent cyclic electron flow around photosystem I in photosynthetic regulation and plant growth at several temperatures was examined in rice (Oryza sativa) that is defective in CHLORORESPIRATORY REDUCTION 6 (CRR6), which is required for accumulation of sub-complex A of the chloroplast NDH complex (crr6). NdhK was not detected by Western blot analysis in crr6 mutants, resulting in lack of a transient post-illumination increase in chlorophyll fluorescence, and confirming that crr6 mutants lack NDH activity. When plants were grown at 28 or 35°C, all examined photosynthetic parameters, including the CO(2) assimilation rate and the electron transport rate around photosystems I and II, at each growth temperature at light intensities above growth light (i.e. 800 μmol photons m(-2) sec(-1)), were similar between crr6 mutants and control plants. However, when plants were grown at 20°C, all the examined photosynthetic parameters were significantly lower in crr6 mutants than control plants, and this effect on photosynthesis caused a corresponding reduction in plant biomass. The F(v)/F(m) ratio was only slightly lower in crr6 mutants than in control plants after short-term strong light treatment at 20°C. However, after long-term acclimation to the low temperature, impairment of cyclic electron flow suppressed non-photochemical quenching and promoted reduction of the plastoquinone pool in crr6 mutants. Taken together, our experiments show that NDH-dependent cyclic electron flow plays a significant physiological role in rice during photosynthesis and plant growth at low temperature.

  19. 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

  20. Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer

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    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.

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

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    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.

  2. 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.

  3. 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 ...

  4. 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...

  5. Improved localization of glucose-6-phosphate dehydrogenase activity in cells with 5-cyano-2,3-ditolyl-tetrazolium chloride as fluorescent redox dye reveals its cell cycle-dependent regulation.

    Science.gov (United States)

    Frederiks, Wilma M; van Marle, Jan; van Oven, Carel; Comin-Anduix, Begonya; Cascante, Marta

    2006-01-01

    Since the introduction of cyano-ditolyl-tetrazolium chloride (CTC), a tetrazolium salt that gives rise to a fluorescent formazan after reduction, it has been applied to quantify activity of dehydrogenases in individual cells using flow cytometry. Confocal laser scanning microscopy (CLSM) showed that the fluorescent formazan was exclusively localized at the surface of individual cells and not at intracellular sites of enzyme activity. In the present study, the technique has been optimized to localize activity of glucose-6-phosphate dehydrogenase (G6PD) intracellularly in individual cells. Activity was demonstrated in cultured fibrosarcoma cells in different stages of the cell cycle. Cells were incubated for the detection of G6PD activity using a medium containing 6% (w/v) polyvinyl alcohol, 5 mM CTC, magnesium chloride, sodium azide, the electron carrier methoxyphenazine methosulphate, NADP, and glucose-6-phosphate. Before incubation, cells were permeabilized with 0.025% glutaraldehyde. Fluorescent formazan was localized exclusively in the cytoplasm of fibrosarcoma cells. The amount of fluorescent formazan in cells increased linearly with incubation time when measured with flow cytometry and CLSM. When combining the Hoechst staining for DNA with the CTC method for the demonstration of G6PD activity, flow cytometry showed that G6PD activity of cells in S phase and G2/M phase is 27 +/- 4% and 43 +/- 4% higher, respectively, than that of cells in G1 phase. CLSM revealed that cells in all phases of mitosis as well as during apoptosis contained considerably lower G6PD activity than cells in interphase. It is concluded that posttranslational regulation of G6PD is responsible for this cell cycle-dependent activity.

  6. Succinate dehydrogenase is the regulator of respiration in Mycobacterium tuberculosis.

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    Travis Hartman

    2014-11-01

    Full Text Available In chronic infection, Mycobacterium tuberculosis bacilli are thought to enter a metabolic program that provides sufficient energy for maintenance of the protonmotive force, but is insufficient to meet the demands of cellular growth. We sought to understand this metabolic downshift genetically by targeting succinate dehydrogenase, the enzyme which couples the growth processes controlled by the TCA cycle with the energy production resulting from the electron transport chain. M. tuberculosis contains two operons which are predicted to encode succinate dehydrogenase enzymes (sdh-1 and sdh-2; we found that deletion of Sdh1 contributes to an inability to survive long term stationary phase. Stable isotope labeling and mass spectrometry revealed that Sdh1 functions as a succinate dehydrogenase during aerobic growth, and that Sdh2 is dispensable for this catalysis, but partially overlapping activities ensure that the loss of one enzyme can incompletely compensate for loss of the other. Deletion of Sdh1 disturbs the rate of respiration via the mycobacterial electron transport chain, resulting in an increased proportion of reduced electron carrier (menaquinol which leads to increased oxygen consumption. The loss of respiratory control leads to an inability to recover from stationary phase. We propose a model in which succinate dehydrogenase is a governor of cellular respiration in the adaptation to low oxygen environments.

  7. Increased sensitivity of photosynthesis to antimycin A induced by inactivation of the chloroplast ndhB gene. Evidence for a participation of the NADH-dehydrogenase complex to cyclic electron flow around photosystem I.

    Science.gov (United States)

    Joët, T; Cournac, L; Horvath, E M; Medgyesy, P; Peltier, G

    2001-04-01

    Tobacco (Nicotiana tabacum var Petit Havana) ndhB-inactivated mutants (ndhB-) obtained by plastid transformation (E.M. Horvath, S.O. Peter, T. Joët, D. Rumeau, L. Cournac, G.V. Horvath, T.A. Kavanagh, C. Schäfer, G. Peltier, P. MedgyesyHorvath [2000] Plant Physiol 123: 1337-1350) were used to study the role of the NADH-dehydrogenase complex (NDH) during photosynthesis and particularly the involvement of this complex in cyclic electron flow around photosystem I (PSI). Photosynthetic activity was determined on leaf discs by measuring CO2 exchange and chlorophyll fluorescence quenchings during a dark-to-light transition. In the absence of treatment, both non-photochemical and photochemical fluorescence quenchings were similar in ndhB- and wild type (WT). When leaf discs were treated with 5 microM antimycin A, an inhibitor of cyclic electron flow around PSI, both quenchings were strongly affected. At steady state, maximum photosynthetic electron transport activity was inhibited by 20% in WT and by 50% in ndhB-. Under non-photorespiratory conditions (2% O2, 2,500 microL x L(-1) CO2), antimycin A had no effect on photosynthetic activity of WT, whereas a 30% inhibition was observed both on quantum yield of photosynthesis assayed by chlorophyll fluorescence and on CO2 assimilation in ndhB-. The effect of antimycin A on ndhB- could not be mimicked by myxothiazol, an inhibitor of the mitochondrial cytochrome bc1 complex, therefore showing that it is not related to an inhibition of the mitochondrial electron transport chain but rather to an inhibition of cyclic electron flow around PSI. We conclude to the existence of two different pathways of cyclic electron flow operating around PSI in higher plant chloroplasts. One of these pathways, sensitive to antimycin A, probably involves ferredoxin plastoquinone reductase, whereas the other involves the NDH complex. The absence of visible phenotype in ndhB- plants under normal conditions is explained by the complement of these two

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

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    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.

  9. INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS

    NARCIS (Netherlands)

    Bartoloni, Fernando Heering; Monteiro Leite Ciscato, Luiz Francisco; Augusto, Felipe Alberto; Baader, Wilhelm Josef

    2010-01-01

    INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS. Chemiluminescence properties of the peroxyoxalate reaction in the presence of activators bearing electron withdrawing substituents were studied, to evaluate the possible occurrence of an inverse electron tr

  10. INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS

    NARCIS (Netherlands)

    Bartoloni, Fernando Heering; Monteiro Leite Ciscato, Luiz Francisco; Augusto, Felipe Alberto; Baader, Wilhelm Josef

    2010-01-01

    INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS. Chemiluminescence properties of the peroxyoxalate reaction in the presence of activators bearing electron withdrawing substituents were studied, to evaluate the possible occurrence of an inverse electron tr

  11. INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS

    NARCIS (Netherlands)

    Bartoloni, Fernando Heering; Monteiro Leite Ciscato, Luiz Francisco; Augusto, Felipe Alberto; Baader, Wilhelm Josef

    2010-01-01

    INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS. Chemiluminescence properties of the peroxyoxalate reaction in the presence of activators bearing electron withdrawing substituents were studied, to evaluate the possible occurrence of an inverse electron

  12. 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

  13. 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.

  14. Effect of Follicular Fluid and Platelet-Activating Factor on Lactate Dehydrogenase C Expression in Human Asthenozoospermic Samples

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    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.

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

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    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.

  16. 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.

  17. 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.

  18. Differences in the response of soil dehydrogenase activity to Cd contamination are determined by the different substrates used for its determination.

    Science.gov (United States)

    Tan, Xiangping; Liu, Yanju; Yan, Kaihong; Wang, Ziquan; Lu, Guannan; He, Yike; He, Wenxiang

    2017-02-01

    Dehydrogenase activity (DHA) is an important indicator of heavy metal toxicity in contaminated soils. Different instances of DHA were determined using various substrates and which could affect the description of heavy metal toxicity. Currently, too few investigations have been done on selecting appropriate substrates. This study employed indoor simulation to determine soil DHA and its response to external cadmium (Cd) using two substrates (TTC and INT). Hormesis for DHA obtained using the TTC method (DHA-TTC) in low Cd concentration was observed which was quickly inhibited in high Cd concentration. While DHA obtained using the INT method (DHA-INT) decreased slowly when Cd concentration increased. The DHA-TTC and DHA-INT in soils at Cd concentration of 500 mg kg(-1) decreased 86% and 53%, respectively, compared to the control. The dose-response relationship of Cd to DHA can be well simulated using the logistic model (p soil Cd toxicity. Multiple stepwise regression analysis revealed that total organic matter (TOC) is the major factor influencing the toxicity of Cd to DHA-TTC, while TOC, pH and cation exchange capacity (CEC) are major factors influencing the toxicity of Cd to DHA-INT. The different responses of soil DHA-TTC and DHA-INT to Cd are due to the differences in electron transport chain characteristics between TTC and INT, as well as the influence of soil properties. Although both DHA-TTC and DHA-INT can monitor soil Cd contamination, DHA-INT is recommended as a superior bio-indicator to indicate and assess contamination of Cd in soil.

  19. 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...

  20. 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

  1. 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

  2. Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

    Directory of Open Access Journals (Sweden)

    Benedito Barraviera

    1988-10-01

    Full Text Available The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05 for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s., respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01 for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01 before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1

  3. 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.

  4. 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 ...

  5. 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 ...

  6. 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.

  7. 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。

  8. Crystal structure of quinone-dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates.

    Science.gov (United States)

    Rozeboom, Henriëtte J; Yu, Shukun; Mikkelsen, Rene; Nikolaev, Igor; Mulder, Harm J; Dijkstra, Bauke W

    2015-12-01

    The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca(2+) ion, and of the enzyme in complex with a Zn(2+) ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ-ADH displays an eight-bladed β-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca(2+) ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer. © 2015 The Protein Society.

  9. [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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Mediated Electron Transfer at Redox Active Monolayers

    Directory of Open Access Journals (Sweden)

    Michael E.G. Lyons

    2001-12-01

    Full Text Available A theoretical model describing the transport and kinetic processes involved in heterogeneous redox catalysis of solution phase reactants at electrode surfaces coated with redox active monolayers is presented. Although the analysis presented has quite general applicability, a specific focus of the paper is concerned with the idea that redox active monolayers can be used to model an ensemble of individual molecular nanoelectrodes. Three possible rate determining steps are considered: heterogeneous electron transfer between immobilized mediator and support electrode ; bimolecular chemical reaction between redox mediator and reactant species in the solution phase, and diffusional mass transport of reactant in solution. A general expression for the steady state reaction flux is derived which is valid for any degree of reversibility of both the heterogeneous electron transfer reaction involving immobilized mediator species and of the bimolecular cross exchange reaction between immobilized mediator and solution phase reactant. The influence of reactant transport in solution is also specifically considered. Simplified analytical expressions for the net reaction flux are derived for experimentally reasonable situations and a kinetic case diagram is constructed outlining the relationships between the various approximate solutions. The theory enables simple diagnostic plots to be constructed which can be used to analyse experimental data.

  18. Response of Chloroplast NAD(P)H Dehydrogenase-Mediated Cyclic Electron Flow to a Shortage or Lack in Ferredoxin-Quinone Oxidoreductase-Dependent Pathway in Rice Following Short-Term Heat Stress.

    Science.gov (United States)

    Essemine, Jemaa; Qu, Mingnan; Mi, Hualing; Zhu, Xin-Guang

    2016-01-01

    Cyclic electron flow (CEF) around photosystem I (PSI) can protect photosynthetic electron carriers under conditions of stromal over-reduction. The goal of the research reported in this paper was to investigate the responses of both PSI and photosystem II (PSII) to a short-term heat stress in two rice lines with different capacities of cyclic electron transfer, i.e., Q4149 with a high capacity (hcef) and C4023 with a low capacity (lcef). The absorbance change at 820 nm (ΔA820) was used here to assess the charge separation in the PSI reaction center (P700). The results obtained show that short-term heat stress abolishes the ferredoxin-quinone oxidoreductase (FQR)-dependent CEF in rice and accelerates the initial rate of P700 (+) re-reduction. The P700 (+) amplitude was slightly increased at a moderate heat-stress (35°C) because of a partial restriction of FQR but it was decreased following high heat-stress (42°C). Assessment of PSI and PSII activities shows that PSI is more susceptible to heat stress than PSII. Under high temperature, FQR-dependent CEF was completely removed and NDH-dependent CEF was up-regulated and strengthened to a higher extent in C4023 than in Q4149. Specifically, under normal growth temperature, hcef (Q4149) was characterized by higher FQR- and chloroplast NAD(P)H dehydrogenase (NDH)-dependent CEF rates than lcef (C4023). Following thermal stress, the activation of NDH-pathway was 130 and 10% for C4023 and Q4149, respectively. Thus, the NDH-dependent CEF may constitute the second layer of plant protection and defense against heat stress after the main route, i.e., FQR-dependent CEF, reaches its capacity. We discuss the possibility that under high heat stress, the NDH pathway serves as a safety valve to dissipate excess energy by cyclic photophosphorylation and overcome the stroma over-reduction following inhibition of CO2 assimilation and any shortage or lack in the FQR pathway. The potential role of the NDH-dependent pathway during the

  19. Variables Affecting Two Electron Transport System Assays

    OpenAIRE

    Burton, G. Allen; Lanza, Guy R.

    1986-01-01

    Several methodological variables were critical in two commonly used electron transport activity assays. The dehydrogenase assay based on triphenyl formazan production exhibited a nonlinear relationship between formazan production (dehydrogenase activity) and sediment dilution, and linear formazan production occurred for 1 h in sediment slurries. Activity decreased with increased time of sediment storage at 4°C. Extraction efficiencies of formazan from sediment varied with alcohol type; methan...

  20. 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.

  1. Decrease in nicotinamide adenine dinucleotide dehydrogenase is related to skin pigmentation.

    Science.gov (United States)

    Nakama, Mitsuo; Murakami, Yuhko; Tanaka, Hiroshi; Nakata, Satoru

    2012-03-01

    Skin pigmentation is caused by various physical and chemical factors. It might also be influenced by changes in the physiological function of skin with aging. Nicotinamide adenine dinucleotide (NADH) dehydrogenase is an enzyme related to the mitochondrial electron transport system and plays a key role in cellular energy production. It has been reported that the functional decrease in this system causes Parkinson's disease. Another study reports that the amount of NADH dehydrogenase in heart and skeletal muscle decreases with aging. A similar decrease in the skin would probably affect its physiological function. However, no reports have examined the age-related change in levels of NADH dehydrogenase in human skin. In this study, we investigated this change and its effect on skin pigmentation using cultured human epidermal keratinocytes. The mRNA expression of NDUFA1, NDUFB7, and NDUFS2, subunits of NADH dehydrogenase, and its activity were significantly decreased in late passage keratinocytes compared to early passage cells. Conversely, the mRNA expression of melanocyte-stimulating cytokines, interleukin-1 alpha and endothelin 1, was increased in late passage cells. On the other hand, the inhibition of NADH dehydrogenase upregulated the mRNA expression of melanocyte-stimulating cytokines. Moreover, the level of NDUFB7 mRNA was lower in pigmented than in nonpigmented regions of skin in vivo. These results suggest the decrease in NADH dehydrogenase with aging to be involved in skin pigmentation.

  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. 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.

  4. 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.

  5. 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:

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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].

  14. 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].

  15. Thermodynamic Basis of Electron Transfer in Dihydroorotate Dehydrogenase B from Lactococcus lactis:  Analysis by Potentiometry, EPR Spectroscopy, and ENDOR Spectroscopy

    DEFF Research Database (Denmark)

    Mohnsen, Al-Walid A.; Rigby, Stephen E. J.; Jensen, Kaj Frank

    2004-01-01

    Dihydroorotate dehydrogenase B (DHODB) is a complex iron-sulfur flavoprotein that catalyzes the conversion of dihydroorotate to orotate and the reduction of NAD+. The enzyme is a dimer of heterodimers containing an FMN, an FAD, and a 2Fe-2S center. UV-visible, EPR, and ENDOR spectroscopies have...... similar to those recorded for the blue semiquinone of free flavins in aqueous solution, thus confirming the presence of this species in DHODB. Spectral features observed during EPR spectroscopy of dithionite-reduced DHODB are consistent with the midpoint reduction potentials determined using UV-visible...

  16. 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.

  17. 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.

  18. 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.

  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. 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.

  1. 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.

  2. 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.

  3. Electron Correlation Models for Optical Activity

    DEFF Research Database (Denmark)

    Höhn, E. G.; O. E. Weigang, Jr.

    1968-01-01

    A two-system no-overlap model for rotatory strength is developed for electric-dipole forbidden as well as allowed transitions. General equations which allow for full utilization of symmetry in the chromophore and in the environment are obtained. The electron correlation terms are developed in full...

  4. 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.

  5. 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

  6. 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).

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. Structural complexities in the active layers of organic electronics.

    Science.gov (United States)

    Lee, Stephanie S; Loo, Yueh-Lin

    2010-01-01

    The field of organic electronics has progressed rapidly in recent years. However, understanding the direct structure-function relationships between the morphology in electrically active layers and the performance of devices composed of these materials has proven difficult. The morphology of active layers in organic electronics is inherently complex, with heterogeneities existing across multiple length scales, from subnanometer to micron and millimeter range. A major challenge still facing the organic electronics community is understanding how the morphology across all of the length scales in active layers collectively determines the device performance of organic electronics. In this review we highlight experiments that have contributed to the elucidation of structure-function relationships in organic electronics and also point to areas in which knowledge of such relationships is still lacking. Such knowledge will lead to the ability to select active materials on the basis of their inherent properties for the fabrication of devices with prespecified characteristics.

  13. 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

  14. 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...

  15. Mitochondrial alpha-ketoglutarate dehydrogenase complex generates reactive oxygen species.

    Science.gov (United States)

    Starkov, Anatoly A; Fiskum, Gary; Chinopoulos, Christos; Lorenzo, Beverly J; Browne, Susan E; Patel, Mulchand S; Beal, M Flint

    2004-09-08

    Mitochondria-produced reactive oxygen species (ROS) are thought to contribute to cell death caused by a multitude of pathological conditions. The molecular sites of mitochondrial ROS production are not well established but are generally thought to be located in complex I and complex III of the electron transport chain. We measured H(2)O(2) production, respiration, and NADPH reduction level in rat brain mitochondria oxidizing a variety of respiratory substrates. Under conditions of maximum respiration induced with either ADP or carbonyl cyanide p-trifluoromethoxyphenylhydrazone,alpha-ketoglutarate supported the highest rate of H(2)O(2) production. In the absence of ADP or in the presence of rotenone, H(2)O(2) production rates correlated with the reduction level of mitochondrial NADPH with various substrates, with the exception of alpha-ketoglutarate. Isolated mitochondrial alpha-ketoglutarate dehydrogenase (KGDHC) and pyruvate dehydrogenase (PDHC) complexes produced superoxide and H(2)O(2). NAD(+) inhibited ROS production by the isolated enzymes and by permeabilized mitochondria. We also measured H(2)O(2) production by brain mitochondria isolated from heterozygous knock-out mice deficient in dihydrolipoyl dehydrogenase (Dld). Although this enzyme is a part of both KGDHC and PDHC, there was greater impairment of KGDHC activity in Dld-deficient mitochondria. These mitochondria also produced significantly less H(2)O(2) than mitochondria isolated from their littermate wild-type mice. The data strongly indicate that KGDHC is a primary site of ROS production in normally functioning mitochondria.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Activation of Electron-Deficient Quinones through Hydrogen-Bond-Donor-Coupled Electron Transfer.

    Science.gov (United States)

    Turek, Amanda K; Hardee, David J; Ullman, Andrew M; Nocera, Daniel G; Jacobsen, Eric N

    2016-01-11

    Quinones are important organic oxidants in a variety of synthetic and biological contexts, and they are susceptible to activation towards electron transfer through hydrogen bonding. Whereas this effect of hydrogen bond donors (HBDs) has been observed for Lewis basic, weakly oxidizing quinones, comparable activation is not readily achieved when more reactive and synthetically useful electron-deficient quinones are used. We have successfully employed HBD-coupled electron transfer as a strategy to activate electron-deficient quinones. A systematic investigation of HBDs has led to the discovery that certain dicationic HBDs have an exceptionally large effect on the rate and thermodynamics of electron transfer. We further demonstrate that these HBDs can be used as catalysts in a quinone-mediated model synthetic transformation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. [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.

  10. Engineering of pyranose dehydrogenase for increased oxygen reactivity.

    Directory of Open Access Journals (Sweden)

    Iris Krondorfer

    Full Text Available Pyranose dehydrogenase (PDH, a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organometals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of Agaricus meleagris PDH were expressed in Saccharomyces cerevisiae. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in Pichia pastoris and characterized and revealed a five-fold increase of the oxygen reactivity.

  11. A simple and sensitive method for lactose detection based on direct electron transfer between immobilised cellobiose dehydrogenase and screen-printed carbon electrodes

    Energy Technology Data Exchange (ETDEWEB)

    Safina, Gulnara, E-mail: Gulnara.Safina@chem.gu.s [Department of Analytical Chemistry/Biochemistry, Lund University, Box 124, 221 00 Lund (Sweden); Ludwig, Roland [Department of Analytical Chemistry/Biochemistry, Lund University, Box 124, 221 00 Lund (Sweden); Research Centre Applied Biocatalysis, Petersgasse 18, 8010 Graz (Austria); Gorton, Lo, E-mail: Lo.Gorton@biochemistry.lu.s [Department of Analytical Chemistry/Biochemistry, Lund University, Box 124, 221 00 Lund (Sweden)

    2010-11-01

    A rapid and simple approach of lactose analysis is proposed based on 3rd generation amperometric biosensors employing cellobiose dehydrogenase (CDH) from Trametes villosa or Phanerochaete sordida immobilised on screen-printed carbon electrodes (SPCEs). After optimisation of the working conditions of the biosensors - pH of the carrier buffer, flow rate and applied potential - the sensors were able to detect lactose in a concentration range between 0.5-200 {mu}M and 0.5-100 {mu}M employing T. villosa and P. sordida CDH, respectively. The limit of detection is 250 nM (90 {mu}g/L) for both. Biosensors based on SPCEs modified with multiwalled carbon nanotubes showed a higher sensitivity than unmodified SPCEs. Cross-linking with glutaraldehyde or poly(ethyleneglycol)diglycidyl ether improved not only the stability but also the analytical response. The developed sensor has been successfully applied for the determination of lactose in dairy (milk with different percentages of fat, lactose-free milk and yogurt) with a good reproducibility (RSD = 1.5-2.2%). No sample preparation except a simple dilution process is needed. The biosensor is easy to make and operate, is inexpensive and reveals a high sensitivity and reliability.

  12. Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria.

    Directory of Open Access Journals (Sweden)

    Seiya Watanabe

    Full Text Available Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(PH-dependent dehydrogenases (synthases, which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti plasmid. In addition to the reverse oxidative reaction(s, the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation. We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A, and exhibited dehydrogenase (but not oxidase activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB1-C-A-B2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by "subunit-exchange". To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase.

  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. 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...

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Nitrite reductase activity of rat and human xanthine oxidase, xanthine dehydrogenase, and aldehyde oxidase: evaluation of their contribution to NO formation in vivo.

    Science.gov (United States)

    Maia, Luisa B; Pereira, Vânia; Mira, Lurdes; Moura, José J G

    2015-01-27

    Nitrite is presently considered a NO "storage form" that can be made available, through its one-electron reduction, to maintain NO formation under hypoxia/anoxia. The molybdoenzymes xanthine oxidase/dehydrogenase (XO/XD) and aldehyde oxidase (AO) are two of the most promising mammalian nitrite reductases, and in this work, we characterized NO formation by rat and human XO/XD and AO. This is the first characterization of human enzymes, and our results support the employment of rat liver enzymes as suitable models of the human counterparts. A comprehensive kinetic characterization of the effect of pH on XO and AO-catalyzed nitrite reduction showed that the enzyme's specificity constant for nitrite increase 8-fold, while the Km(NO2(-)) decrease 6-fold, when the pH decreases from 7.4 to 6.3. These results demonstrate that the ability of XO/AO to trigger NO formation would be greatly enhanced under the acidic conditions characteristic of ischemia. The dioxygen inhibition was quantified, and the Ki(O2) values found (24.3-48.8 μM) suggest that in vivo NO formation would be fine-tuned by dioxygen availability. The potential in vivo relative physiological relevance of XO/XD/AO-dependent pathways of NO formation was evaluated using HepG2 and HMEC cell lines subjected to hypoxia. NO formation by the cells was found to be pH-, nitrite-, and dioxygen-dependent, and the relative contribution of XO/XD plus AO was found to be as high as 50%. Collectively, our results supported the possibility that XO/XD and AO can contribute to NO generation under hypoxia inside a living human cell. Furthermore, the molecular mechanism of XO/AO-catalyzed nitrite reduction was revised.

  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. Resonant enhancement of relativistic electron fluxes during geomagnetically active periods

    Directory of Open Access Journals (Sweden)

    I. Roth

    Full Text Available The strong increase in the flux of relativistic electrons during the recovery phase of magnetic storms and during other active periods is investigated with the help of Hamiltonian formalism and simulations of test electrons which interact with whistler waves. The intensity of the whistler waves is enhanced significantly due to injection of 10-100 keV electrons during the substorm. Electrons which drift in the gradient and curvature of the magnetic field generate the rising tones of VLF whistler chorus. The seed population of relativistic electrons which bounce along the inhomogeneous magnetic field, interacts resonantly with the whistler waves. Whistler wave propagating obliquely to the magnetic field can interact with energetic electrons through Landau, cyclotron, and higher harmonic resonant interactions when the Doppler-shifted wave frequency equals any (positive or negative integer multiple of the local relativistic gyrofrequency. Because the gyroradius of a relativistic electron may be the order of or greater than the perpendicular wavelength, numerous cyclotron, harmonics can contribute to the resonant interaction which breaks down the adiabatic invariant. A similar process diffuses the pitch angle leading to electron precipitation. The irreversible changes in the adiabatic invariant depend on the relative phase between the wave and the electron, and successive resonant interactions result in electrons undergoing a random walk in energy and pitch angle. This resonant process may contribute to the 10-100 fold increase of the relativistic electron flux in the outer radiation belt, and constitute an interesting relation between substorm-generated waves and enhancements in fluxes of relativistic electrons during geomagnetic storms and other active periods.

    Key words. Magnetospheric physics (energetic particles · trapped; plasma waves and instabilities; storms and substorms

  3. 11 beta-hydroxysteroid dehydrogenase activity in proteinuric patients and the effect of angiotensin-II receptor blockade

    NARCIS (Netherlands)

    Kerstens, MN; Buter, H; Navis, GJ; Dullaart, RPF

    Background It has been suggested that an altered setpoint of the 11betaHSD-mediated cortisol to cortisone interconversion towards cortisol contributes to sodium retention in nephrotic syndrome patients. We studied the parameters of 11betaHSD activity in proteinuric patients, in particular its

  4. Glucose-6-phosphate dehydrogenase deficiency

    Science.gov (United States)

    ... medlineplus.gov/ency/article/000528.htm Glucose-6-phosphate dehydrogenase deficiency To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a condition in which ...

  5. 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; Dewanti, Asteriani; Merli, Angelo; Rossi, Gian Luigi; Mitra, Bharati; Mathews, F. Scott; (Cornell); (Parma); (WCU); (WSU); (WU-MED)

    2009-06-12

    (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 {approx}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 {angstrom} 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 the glycine-to-alanine mutation may account for the lowered catalytic activity of the mutant enzyme, which is consistent with the 30 mV lower flavin redox potential. Furthermore, the altered binding mode of the indolelactate substrate may account for its reduced activity compared with octanoate, as observed in the crystalline state.

  6. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5’-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris, semiaquatic (Lontra longicaudis annectens and terrestrial (Sus scrofa

    Directory of Open Access Journals (Sweden)

    Myrna eBarjau Perez-Milicua

    2015-07-01

    Full Text Available Aquatic and semiaquatic mammals have the capacity of breath hold (apnea diving. Northern elephant seals (Mirounga angustirostris have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens can hold their breath for about 30 sec. Such periods of apnea may result in reduced oxygen concentration (hypoxia and reduced blood supply (ischemia to tissues. Production of adenosine 5’-triphosphate (ATP requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa, are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal (n=11, semiaquatic (neotropical river otter (n=4 and terrestrial (domestic pig (n=11. Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT was determined by spectrophotometry, and activity of inosine 5’-monophosphate dehydrogenase (IMPDH and the concentration of hypoxanthine (HX, inosine 5’-monophosphate (IMP, adenosine 5’-monophosphate (AMP, adenosine 5’-diphosphate (ADP, ATP, guanosine 5’-diphosphate (GDP, guanosine 5’-triphosphate (GTP, and xanthosine 5’-monophosphate (XMP were determined by high-performance liquid chromatography (HPLC. The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise, aquatic and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  7. 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.

  8. A trade off between catalytic activity and protein stability determines the clinical manifestations of glucose-6-phosphate dehydrogenase (G6PD) deficiency.

    Science.gov (United States)

    Boonyuen, Usa; Chamchoy, Kamonwan; Swangsri, Thitiluck; Junkree, Thanyaphorn; Day, Nicholas P J; White, Nicholas J; Imwong, Mallika

    2017-11-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common polymorphism and enzymopathy in humans, affecting approximately 400 million people worldwide. It is responsible for various clinical manifestations, including favism, hemolytic anemia, chronic non-spherocytic hemolytic anemia, spontaneous abortion, and neonatal hyperbilirubinemia. Understanding the molecular mechanisms underlying the severity of G6PD deficiency is of great importance but that of many G6PD variants are still unknown. In this study, we report the construction, expression, purification, and biochemical characterization in terms of kinetic properties and stability of five clinical G6PD variants-G6PD Bangkok, G6PD Bangkok noi, G6PD Songklanagarind, G6PD Canton+Bangkok noi, and G6PD Union+Viangchan. G6PD Bangkok and G6PD Canton+Bangkok noi showed a complete loss of catalytic activity and moderate reduction in thermal stability when compared with the native G6PD. G6PD Bangkok noi and G6PD Union+Viangchan showed a significant reduction in catalytic efficiency, whereas G6PD Songklanagarind showed a catalytic activity comparable to the wild-type enzyme. The Union+Viangchan mutation showed a remarkable effect on the global stability of the enzyme. In addition, our results indicate that the location of mutations in G6PD variants affects their catalytic activity, stability, and structure. Hence, our results provide a molecular explanation for clinical manifestations observed in individuals with G6PD deficiency. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Effects of peroxisome proliferator-activated receptor-alpha and -gamma agonists on 11beta-hydroxysteroid dehydrogenase type 1 in subcutaneous adipose tissue in men.

    Science.gov (United States)

    Wake, Deborah J; Stimson, Roland H; Tan, Garry D; Homer, Natalie Z M; Andrew, Ruth; Karpe, Fredrik; Walker, Brian R

    2007-05-01

    In animals, peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARgamma agonists down-regulate 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) mRNA and activity in liver and adipose tissue, respectively, and PPARgamma agonists reduce ACTH secretion from corticotrope cells. Our objective was to test whether PPAR agonists alter cortisol secretion and peripheral regeneration by 11beta-HSD1 in humans and whether reduced cortisol action contributes to metabolic effects of PPARgamma agonists. Three randomized placebo-controlled crossover studies were conducted at a clinical research facility. Healthy men and patients with type 2 diabetes participated. INTERVENTIONS, OUTCOME MEASURES, AND RESULTS: In nine healthy men, 7 d of PPARalpha agonist (fenofibrate) or PPARgamma agonist (rosiglitazone) had no effect on cortisol secretion, hepatic cortisol generation after oral cortisone administration, or tracer kinetics during 9,11,12,12-[(2)H](4)-cortisol infusion, although rosiglitazone marginally reduced cortisol generation in sc adipose tissue measured by in vivo microdialysis. In 12 healthy men, 4-5 wk of rosiglitazone increased insulin sensitivity during insulin infusion but did not change 11beta-HSD1 mRNA or activity in sc adipose tissue, and insulin sensitization was unaffected by glucocorticoid blockade with a combination of metyrapone and RU38486. In 12 men with type 2 diabetes 12 wk of rosiglitazone reduced arteriovenous cortisone extraction across abdominal sc adipose tissue and reduced 11beta-HSD1 mRNA in sc adipose tissue but increased plasma cortisol concentrations. Neither PPARalpha nor PPARgamma agonists down-regulate 11beta-HSD1 or cortisol secretion acutely in humans. The early insulin-sensitizing effect of rosiglitazone is not dependent on reducing intracellular glucocorticoid concentrations. Reduced adipose 11beta-HSD1 expression and increased plasma cortisol during longer therapy with rosiglitazone probably reflect indirect effects, e

  10. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5'-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa).

    Science.gov (United States)

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E; Gallo-Reynoso, Juan P

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5'-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5'-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP, guanosine 5'-diphosphate (GDP), guanosine 5'-triphosphate (GTP), and xanthosine 5'-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  11. Quality control of the documentation process in electronic economic activities

    Directory of Open Access Journals (Sweden)

    Krutova A.S.

    2017-06-01

    Full Text Available It is proved that the main tool that will provide adequate information resources e economic activities of social and economic relations are documenting quality control processes as the basis of global information space. Directions problems as formation evaluation information resources in the process of documentation, namely development tools assess the efficiency of the system components – qualitative assessment; development of mathematical modeling tools – quantitative evaluation. A qualitative assessment of electronic documentation of economic activity through exercise performance, efficiency of communication; document management efficiency; effectiveness of flow control operations; relationship management effectiveness. The concept of quality control process documents electronically economic activity to components which include: the level of workflow; forms adequacy of information; consumer quality documents; quality attributes; type of income data; condition monitoring systems; organizational level process documentation; attributes of quality, performance quality consumer; type of management system; type of income data; condition monitoring systems. Grounded components of the control system electronic document subjects of economic activity. Detected components IT-audit management system economic activity: compliance audit; audit of internal control; detailed multilevel analysis; corporate risk assessment methodology. The stages and methods of processing electronic transactions economic activity during condition monitoring of electronic economic activity.

  12. Studies on lipoamide dehydrogenase.

    NARCIS (Netherlands)

    Benen, J.A.E.

    1992-01-01

    At the onset of the investigations described in this thesis progress was being made on the elucidation of the crystal structure of the Azotobactervinelandii lipoamide dehydrogenase. Also the gene encoding this enzyme was cloned in our laboratory. By this, a firm basis was laid to start site directed

  13. Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays

    DEFF Research Database (Denmark)

    Wiggers, Henrik; Cheleski, J; Zottis, A

    2007-01-01

    .0% for MeOH and up to 7.5% for DMSO. The results show that when GAPDH is assayed in the presence of DMSO (5%, v/v) using the ITC experiment, the enzyme exhibits approximately twofold higher activity than that of GAPDH with no cosolvent added. When MeOH (5%, v/v) is the cosolvent, the GAPDH activity...... is sixfold higher. The favorable effects of the organic solvents on the Michaelis-Menten enzyme-substrate complex formation ensure the consistency of the biological assays, structural integrity of the protein, and reproducibility over the measurement time. The reaction was also kinetically monitored......In drug discovery programs, dimethyl sulfoxide (DMSO) is a standard solvent widely used in biochemical assays. Despite the extensive use and study of enzymes in the presence of organic solvents, for some enzymes the effect of organic solvent is unknown. Macromolecular targets may be affected...

  14. [Dynamics of cardiac and skeletal muscle lactate dehydrogenase activity following a single exposure to an alternating magnetic field].

    Science.gov (United States)

    Udintsev, N A; Kanskaia, N V; Shchepetil'nikova, A I; Ordina, O M; Pichurina, R A

    1976-06-01

    A rise in LDH activity and a change of the enzyme distribution in the cytostructures of the heart and skeletal muscles of albino rats was revealed during the first 48 hours after a single twenty-four-hour action of an A. C. magnetic field (200 e, 50 cps). A displacement of the enzyma ratio in the direction of M-type was noted. Complete normalization occurred in the 3rd or 4th week only.

  15. Ionospheric Electron Density during Magnetically Active Times over Istanbul

    Science.gov (United States)

    Naz Erbaş, Bute; Kaymaz, Zerefsan; Ceren Moral, Aysegul; Emine Ceren Kalafatoglu Eyiguler, R. A..

    2016-07-01

    In this study, we analyze electron density variations over Istanbul using Dynasonde observations during the magnetically active times. In order to perform statistical analyses, we first determined magnetic storms and magnetospheric substorm intervals from October 2012 to October 2015 using Kyoto's magnetic index data. Corresponding ionospheric parameters, such as critical frequency of F2 region (foF2), maximum electron density height (hmF2), total electron density (TEC) etc. were retrieved from Dynasonde data base at Istanbul Technical University's Space Weather Laboratory. To understand the behavior of electron density during the magnetically active times, we remove the background quiet time variations first and then quantify the anomalies. In this presentation, we will report results from our preliminary analyses from the selected cases corresponding to the strong magnetic storms. Initial results show lower electron densities at noon times and higher electron densities in the late afternoon toward sunset times when compared to the electron densities of magnetically quiet times. We also compare the results with IRI and TIEGCM ionospheric models in order to understand the physical and dynamical causes of these variations. During the presentation we will also discuss the role of these changes during the magnetically active times on the GPS communications through ionosphere.

  16. 11β-Hydroxysteroid dehydrogenase activity in the brain does not contribute to systemic interconversion of cortisol and cortisone in healthy men.

    Science.gov (United States)

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

    2015-02-01

    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 to systemic cortisol/cortisone turnover and to develop a tool for measuring 11βHSD1 in dementia and following administration of 11βHSD1 inhibitors. With ethical approval and informed consent, 8 healthy men aged 38.1 years (sd 16.5) underwent an ECG-gated phase-contrast magnetic resonance scan to quantify internal jugular vein blood flow and were infused with 1,2 [(2)H]2-cortisone and 9,11,12,12 [(2)H]4-cortisol for 3 h before samples were obtained from the internal jugular vein and an arterialized hand vein. Steroids were quantified by liquid chromatography-tandem mass spectrometry. Steady state tracer enrichments were achieved and systemic indices of cortisol/cortisone interconversion were consistent with previous studies in healthy men. However, there was no measurable release or production of cortisol, 9,12,12 [(2)H]3-cortisol or cortisone into the internal jugular vein. Although cerebral 11βHSD1 reductase activity may be greater in cognitively impaired patients, in healthy men any contribution of 11βHSD1 in the brain to systemic cortisol/cortisone turnover is negligible. The influence of 11βHSD1 in the brain is likely confined to subregions, notably the hippocampus. Alternative approaches are required to quantify pharmacodynamics effects of 11βHSD1 inhibitors in the human brain.

  17. The impact of mitochondrial aldehyde dehydrogenase (ALDH2) activation by Alda-1 on the behavioral and biochemical disturbances in animal model of depression.

    Science.gov (United States)

    Stachowicz, Aneta; Głombik, Katarzyna; Olszanecki, Rafał; Basta-Kaim, Agnieszka; Suski, Maciej; Lasoń, Władysław; Korbut, Ryszard

    2016-01-01

    The etiology of depression remains still unclear. Recently, it has been proposed, that mitochondrial dysfunction may be associated with development of mood disorders, such as depression, bipolar disorder and anxiety disorders. Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda-1, a small-molecule activator of ALDH2, on depressive- and anxiety-like behaviors in an animal model of depression - the prenatally stressed rats - using behavioral, molecular and proteomic methods. Prolonged Alda-1 administration significantly increased the climbing time, tended to reduce the immobility time and increased the swimming time of the prenatally stressed rats in the forced swim test. Moreover, treatment of prenatally stressed rats with Alda-1 significantly increased number of entries into the open arms of the maze and the time spent therein, as assessed by elevated plus-maze test. Such actions were associated with reduction of plasma 4-HNE-protein content, decrease of TNF-α mRNA and increase of PGC-1α (regulator of mitochondrial biogenesis) mRNA level in the frontal cortex and hippocampus of the prenatally stressed rats as well as with normalization of peripheral immune parameters and significant changes in expression of 6 and 4 proteins related to mitochondrial functions in the frontal cortex and hippocampus, respectively. Collectively, ALDH2 activation by Alda-1 led to a significant attenuation of depressive- and anxiety-like behaviors in the prenatally stressed rats. The pattern of changes suggested mitoprotective effect of Alda-1, however the exact functional consequences of the revealed alterations require further investigation.

  18. 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.

  19. 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.

  20. Flexible electronic-paper active-matrix displays

    NARCIS (Netherlands)

    Huitema, H.E.A.; Gelinck, G.H.; Lieshout, P.J.G.; Veenendaal, E. van; Touwslager, F.J.

    2006-01-01

    A 100-um-thick 320 x 240-pixel active-matrix display integrated into a functional-device prototype is presented. The active matrix is composed of alternating layers of organic materials and gold. A six-mask photolithographic process is used. An electrophoretic electronic imaging film is laminated on

  1. Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry.

    Directory of Open Access Journals (Sweden)

    Yasuhiro Idewaki

    Full Text Available Aldehyde dehydrogenase 2 (ALDH2 detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671 was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity and drinking habits (lifetime abstainers vs. former or current drinkers was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2. The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI]: *1/*1 abstainers as the referent, 0.94 [0.76-1.16] in abstainers with *2, 1.00 [0.80-1.26] in *1/*1 drinkers, 0.71 [0.54-0.93] in drinkers with *2. Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28-6.13] in abstainers with *2, 1.89 [0.89-4.51] in *1/*1 drinkers, 2.35 [1.06-5.79] in drinkers with *2. In summary, patients with type 2 diabetes and ALDH2 *2

  2. Soft electron beams in solar active and flare region

    Energy Technology Data Exchange (ETDEWEB)

    Korneev, V.V.; Mandelshtam, S.L.; Oparin, S.N.; Urnov, A.M.; Zhitnik, I.A.

    1982-01-01

    On the basis of the experimental data obtained from the high resolution X-ray spectra for solar flares and active regions the suprathermal electron model (SEM) was proposed. This model suggests the existance of the multi-temperature structure of the solar plasma emitting Fe and Ca X-rays and the presence of additional electrons with low energies (no more than 10 keV) and small densities of about 1-5 percent relative to the thermal component.

  3. Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680.

    Science.gov (United States)

    Wang, Ao; Cao, Zheng-Yu; Wang, Peng; Liu, Ai-Min; Pan, Wei; Wang, Jie; Zhu, Guo-Ping

    2011-08-01

    A monomeric NADP-dependent isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680 (SaIDH) was heteroexpressed in Escherichia coli, and the His-tagged enzyme was further purified to homogeneity. The molecular weight of SaIDH was about 80 kDa which is typical for monomeric isocitrate dehydrogenases. Structure-based sequence alignment reveals that the deduced amino acid sequence of SaIDH shows high sequence identity with known momomeric isocitrate dehydrogenase, and the coenzyme, substrate and metal ion binding sites are completely conserved. The optimal pH and temperature of SaIDH were found to be pH 9.4 and 45°C, respectively. Heat-inactivation studies showed that heating for 20 min at 50°C caused a 50% loss in enzymatic activity. In addition, SaIDH was absolutely specific for NADP+ as electron acceptor. Apparent Km values were 4.98 μM for NADP+ and 6,620 μM for NAD+, respectively, using Mn2+ as divalent cation. The enzyme performed a 33,000-fold greater specificity (kcat/Km) for NADP+ than NAD+. Moreover, SaIDH activity was entirely dependent on the presence of Mn2+ or Mg2+, but was strongly inhibited by Ca2+ and Zn2+. Taken together, our findings implicate the recombinant SaIDH is a divalent cation-dependent monomeric isocitrate dehydrogenase which presents a remarkably high cofactor preference for NADP+.

  4. Hibernation impact on the catalytic activities of the mitochondrial D-3-hydroxybutyrate dehydrogenase in liver and brain tissues of jerboa (Jaculus orientalis

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    Hafiani Assia

    2003-09-01

    Full Text Available Abstract Background Jerboa (Jaculus orientalis is a deep hibernating rodent native to subdesert highlands. During hibernation, a high level of ketone bodies i.e. acetoacetate (AcAc and D-3-hydroxybutyrate (BOH are produced in liver, which are used in brain as energetic fuel. These compounds are bioconverted by mitochondrial D-3-hydroxybutyrate dehydrogenase (BDH E.C. 1.1.1.30. Here we report, the function and the expression of BDH in terms of catalytic activities, kinetic parameters, levels of protein and mRNA in both tissues i.e brain and liver, in relation to the hibernating process. Results We found that: 1/ In euthemic jerboa the specific activity in liver is 2.4- and 6.4- fold higher than in brain, respectively for AcAc reduction and for BOH oxidation. The same differences were found in the hibernation state. 2/ In euthermic jerboa, the Michaelis constants, KM BOH and KM NAD+ are different in liver and in brain while KM AcAc, KM NADH and the dissociation constants, KD NAD+and KD NADH are similar. 3/ During prehibernating state, as compared to euthermic state, the liver BDH activity is reduced by half, while kinetic constants are strongly increased except KD NAD+. 4/ During hibernating state, BDH activity is significantly enhanced, moreover, kinetic constants (KM and KD are strongly modified as compared to the euthermic state; i.e. KD NAD+ in liver and KM AcAc in brain decrease 5 and 3 times respectively, while KD NADH in brain strongly increases up to 5.6 fold. 5/ Both protein content and mRNA level of BDH remain unchanged during the cold adaptation process. Conclusions These results cumulatively explained and are consistent with the existence of two BDH enzymatic forms in the liver and the brain. The apoenzyme would be subjected to differential conformational folding depending on the hibernation state. This regulation could be a result of either post-translational modifications and/or a modification of the mitochondrial membrane state

  5. Pharmacokinetics of 5-fluorouracil and increased hepatic dihydropyrimidine dehydrogenase activity levels in 1,2-dimethylhydrazine-induced colorectal cancer model rats.

    Science.gov (United States)

    Kobuchi, Shinji; Ito, Yukako; Okada, Kae; Imoto, Kazuki; Takada, Kanji

    2013-09-01

    To investigate the hepatic dihydropyrimidine dehydrogenase (DPD) activity in colorectal cancer (CRC), which is critically important to create a patient-specific dosing regimen, we performed 5-FU pharmacokinetic studies in 1,2-dimethylhydrazine-induced CRC model rats (CRC rats). After rats received 5-FU intravenous (IV) bolus injections, the area under the plasma concentration-time curve (AUC) and elimination half-life (t 1/2) in CRC rats (10.02 ± 0.37 μg h mL(-1), 0.30 ± 0.02 h, respectively) were significantly lower than that in control rats (13.46 ± 1.20 μg h mL(-1), 0.52 ± 0.05 h, respectively), whereas total plasma clearance (CLtot) in CRC rats (2.01 ± 0.07 L h(-1) kg(-1)) was significantly increased compared with that in control rats (1.54 ± 0.14 L h(-1) kg(-1)). Conversely, the avoidance ratio of the hepatic first-pass effect was approximately 20 % lower than that in control rats. Of interest is that hepatic DPD activity levels and the dihydrouracil-uracil ratio (UH2/Ura ratio) in plasma, which may act as a potential biomarker to evaluate hepatic DPD activity levels, were significantly increased in CRC rats. These results suggest that the decrease of hepatic availability in CRC rats is brought about by the increase in intrinsic clearance induced by the increase in DPD activity, resulting in a decrease in AUC and t 1/2 and an increase in CLtot after 5-FU IV bolus injection. Along with a proper dosing regimen for patients with CRC, a hepatic DPD activity monitoring system, such as the determination of UH2/Ura ratio in plasma, is desirable.

  6. Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities.

    Science.gov (United States)

    Kuzu, Muslum; Aslan, Abdulselam; Ahmed, Ishtiaq; Comakli, Veysel; Demirdag, Ramazan; Uzun, Naim

    2016-04-01

    Glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) are metabolically quite important enzymes. Within this study, these two enzymes were purified for the first time from the gills of Lake Van fish. In the purifying process, ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity column chromatography techniques for glucose-6-phosphate dehydrogenase, temperature degradation and 2',5'-ADP Sepharose 4B affinity column chromatography for glutathione reductase enzyme were used. The control of the enzyme purity and determination of molecular weight were done with sodium dodecyl sulfate polyacrylamide gel electrophoresis. K(M) and V(max) values were determined with Lineweaver-Burk plot. Besides, the effects of some chalcone derivatives on the purified enzymes were analyzed. For the ones showing inhibition effect, % activity-[I] figures were drawn and IC50 values were determined. K(i) value was calculated by using Cheng-Prusoff equation.

  7. NirN Protein from Pseudomonas aeruginosa is a Novel Electron-bifurcating Dehydrogenase Catalyzing the Last Step of Heme d1 Biosynthesis*

    Science.gov (United States)

    Adamczack, Julia; Hoffmann, Martin; Papke, Ulrich; Haufschildt, Kristin; Nicke, Tristan; Bröring, Martin; Sezer, Murat; Weimar, Rebecca; Kuhlmann, Uwe; Hildebrandt, Peter; Layer, Gunhild

    2014-01-01

    Heme d1 plays an important role in denitrification as the essential cofactor of the cytochrome cd1 nitrite reductase NirS. At present, the biosynthesis of heme d1 is only partially understood. The last step of heme d1 biosynthesis requires a so far unknown enzyme that catalyzes the introduction of a double bond into one of the propionate side chains of the tetrapyrrole yielding the corresponding acrylate side chain. In this study, we show that a Pseudomonas aeruginosa PAO1 strain lacking the NirN protein does not produce heme d1. Instead, the NirS purified from this strain contains the heme d1 precursor dihydro-heme d1 lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d1 was extracted from purified NirS and characterized by UV-visible absorption spectroscopy and finally identified by high-resolution electrospray ionization mass spectrometry. Moreover, we show that purified NirN from P. aeruginosa binds the dihydro-heme d1 and catalyzes the introduction of the acrylic double bond in vitro. Strikingly, NirN uses an electron bifurcation mechanism for the two-electron oxidation reaction, during which one electron ends up on its heme c cofactor and the second electron reduces the substrate/product from the ferric to the ferrous state. On the basis of our results, we propose novel roles for the proteins NirN and NirF during the biosynthesis of heme d1. PMID:25204657

  8. Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

    Directory of Open Access Journals (Sweden)

    Mädje Katharina

    2012-01-01

    Full Text Available Abstract Background Enzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(PH. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH, presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S-1-(2-chlorophenylethanol from o-chloroacetophenone. Results As compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3 that gave an intracellular enzyme activity of 400 units/gCDW, co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/gCDW was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C brought up the FDH activity threefold to a level of 250 U/gCDW while reducing the XR activity by

  9. Electron beam induced surface activation of oxide surfaces for nanofabrication

    Energy Technology Data Exchange (ETDEWEB)

    Vollnhals, Florian; Seiler, Steffen; Walz, Marie-Madeleine; Steinrueck, Hans-Peter; Marbach, Hubertus [Lehrstuhl fuer Physikalische Chemie II and Interdisciplinary Center for Molecular Materials (ICMM), Friedrich-Alexander-Universitaet Erlangen-Nuernberg, Erlangen (Germany); Woolcot, Tom; Thornton, Geoff [London Centre for Nanotechnology and Department of Chemistry, University College London (United Kingdom)

    2012-07-01

    The controlled fabrication of structures on the nanoscale is a major challenge in science and engineering. Direct-write techniques like Electron Beam Induced Deposition (EBID) were shown to be suitable tools in this context. Recently, Electron Beam Induced Surface Activation (EBISA) has been introduced as a new focused electron beam technique. In EBISA, a surface, e.g. SiO{sub 2}, is irradiated by a focused electron beam, resulting in an activation of the exposed area. The activated area can then react and decompose precursor gases like iron pentacarbonyl, Fe(CO){sub 5}. This leads to a primary deposit, which continues to grow autocatalytically as long as Fe(CO){sub 5} is supplied, resulting in pure (> 90 % at.), crystalline iron nanostructures. We expand the use of this concept by exploring EBISA to produce metallic nanostructures on TiO{sub 2}(110) in UHV; atomistic insight into the process is obtained via Scanning Tunneling Microscopy (STM) and chemical insight via Auger Electron Spectroscopy (AES).

  10. Deficiencies of active electronic radiation protection dosimeters in pulsed fields.

    Science.gov (United States)

    Ankerhold, U; Hupe, O; Ambrosi, P

    2009-07-01

    Nowadays nearly all radiation fields used for X-ray diagnostics are pulsed. These fields are characterised by a high dose rate during the pulse and a short pulse duration in the range of a few milliseconds. The use of active electronic dosimeters has increased in the past few years, but these types of dosimeters might possibly not measure reliably in pulsed radiation fields. Not only personal dosimeters but also area dosimeters that are used mainly for dose rate measurements are concerned. These cannot be substituted by using passive dosimeter types. The characteristics of active electronic dosimeters determined in a continuous radiation field cannot be transferred to those in pulsed fields. Some provisional measurements with typical electronic dosimeters in pulsed radiation fields are presented to reveal this basic problem.

  11. Electron beam injection during active experiments. I - Electromagnetic wave emissions

    Science.gov (United States)

    Winglee, R. M.; Kellogg, P. J.

    1990-01-01

    The wave emissions produced in Echo 7 experiment by active injections of electron beams were investigated to determine the properties of the electromagnetic and electrostatic fields for both the field-aligned and cross-field injection in such experiments and to evaluate the sources of free energy and relative efficiencies for the generation of the VLF and HF emissions. It is shown that, for typical beam energies in active experiments, electromagnetic effects do not substantially change the bulk properties of the beam, spacecraft charging, and plasma particle acceleration. Through simulations, beam-generated whistlers; fundamental z-mode and harmonic x-mode radiation; and electrostatic electron-cyclotron, upper-hybrid, Langmuir, and lower-hybrid waves were identified. The characteristics of the observed wave spectra were found to be sensitive to both the ratio of the electron plasma frequency to the cyclotron frequency and the angle of injection relative to the magnetic field.

  12. Optimization of companies’ activity on development of electronic product

    Directory of Open Access Journals (Sweden)

    K.V. Nalivaychenko

    2012-02-01

    Full Text Available The article considers modern processes of informatization of activity for business companies. There are generalized the makes of world companies, which create information technologies in global information environment: the electronic information product containing an universal development algorithm for it, is most effective. It is found that companies prefer opened program codes.

  13. Platform engineering of Corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate.

    Science.gov (United States)

    Buchholz, Jens; Schwentner, Andreas; Brunnenkan, Britta; Gabris, Christina; Grimm, Simon; Gerstmeir, Robert; Takors, Ralf; Eikmanns, Bernhard J; Blombach, Bastian

    2013-09-01

    Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a YP/S of 0.24 mol per mol of glucose and a QP of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

  14. Alcohol dehydrogenase – physiological and diagnostic Importance

    Directory of Open Access Journals (Sweden)

    Magdalena Łaniewska-Dunaj

    2013-08-01

    Full Text Available Alcohol dehydrogenase (ADH is a polymorphic enzyme, existing in multiple isoenzymes divided into several classes and localized in different organs. ADH plays a significant role in the metabolism of many biologically important substances, catalyzing the oxidation or reduction of a wide spectrum of specific substrates. The best characterized function of ADH is protection against excess of ethanol and some other exogenous xenobiotics and products of lipid peroxidation. The isoenzymes of alcohol dehydrogenase also participate in the metabolism of retinol and serotonin. The total alcohol dehydrogenase activity is significantly higher in cancer tissues than in healthy organs (e.g. liver, stomach, colorectum. The changes in activity of particular ADH isoenzymes in the sera of patients with different cancers (especially of the digestive system seem to be caused by release of these isoenzymes from cancer cells, and may play a potential role as markers of this cancer. The particular isoenzymes of ADH present in the serum may indicate the cancer localization. Alcohol dehydrogenase may also be useful for diagnostics of non-cancerous liver diseases (e.g. viral hepatitis, non-alcoholic cirrhosis.

  15. An Fe-S cluster in the conserved Cys-rich region in the catalytic subunit of FAD-dependent dehydrogenase complexes.

    Science.gov (United States)

    Shiota, Masaki; Yamazaki, Tomohiko; Yoshimatsu, Keiichi; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2016-12-01

    Several bacterial flavin adenine dinucleotide (FAD)-harboring dehydrogenase complexes comprise three distinct subunits: a catalytic subunit with FAD, a cytochrome c subunit containing three hemes, and a small subunit. Owing to the cytochrome c subunit, these dehydrogenase complexes have the potential to transfer electrons directly to an electrode. Despite various electrochemical applications and engineering studies of FAD-dependent dehydrogenase complexes, the intra/inter-molecular electron transfer pathway has not yet been revealed. In this study, we focused on the conserved Cys-rich region in the catalytic subunits using the catalytic subunit of FAD dependent glucose dehydrogenase complex (FADGDH) as a model, and site-directed mutagenesis and electron paramagnetic resonance (EPR) were performed. By co-expressing a hitch-hiker protein (γ-subunit) and a catalytic subunit (α-subunit), FADGDH γα complexes were prepared, and the properties of the catalytic subunit of both wild type and mutant FADGDHs were investigated. Substitution of the conserved Cys residues with Ser resulted in the loss of dye-mediated glucose dehydrogenase activity. ICP-AEM and EPR analyses of the wild-type FADGDH catalytic subunit revealed the presence of a 3Fe-4S-type iron-sulfur cluster, whereas none of the Ser-substituted mutants showed the EPR spectrum characteristic for this cluster. The results suggested that three Cys residues in the Cys-rich region constitute an iron-sulfur cluster that may play an important role in the electron transfer from FAD (intra-molecular) to the multi-heme cytochrome c subunit (inter-molecular) electron transfer pathway. These features appear to be conserved in the other three-subunit dehydrogenases having an FAD cofactor.

  16. Genetics Home Reference: lactate dehydrogenase deficiency

    Science.gov (United States)

    ... Facebook Twitter Home Health Conditions lactate dehydrogenase deficiency lactate dehydrogenase deficiency Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Lactate dehydrogenase deficiency is a condition that affects how the ...

  17. External NAD(P)H dehydrogenases in Acanthamoeba castellanii mitochondria.

    Science.gov (United States)

    Antos-Krzeminska, Nina; Jarmuszkiewicz, Wieslawa

    2014-09-01

    The mitochondrial respiratory chain of plants and some fungi contains multiple rotenone-insensitive NAD(P)H dehydrogenases, of which at least two are located on the outer surface of the inner membrane (i.e., external NADH and external NADPH dehydrogenases). Annotated sequences of the putative alternative NAD(P)H dehydrogenases of the protozoan Acanthamoeba castellanii demonstrated similarity to plant and fungal sequences. We also studied activity of these dehydrogenases in isolated A. castellanii mitochondria. External NADPH oxidation was observed for the first time in protist mitochondria. The coupling parameters were similar for external NADH oxidation and external NADPH oxidation, indicating similar efficiencies of ATP synthesis. Both external NADH oxidation and external NADPH oxidation had an optimal pH of 6.8 independent of relevant ubiquinol-oxidizing pathways, the cytochrome pathway or a GMP-stimulated alternative oxidase. The maximal oxidizing activity with external NADH was almost double that with external NADPH. However, a lower Michaelis constant (K(M)) value for external NADPH oxidation was observed compared to that for external NADH oxidation. Stimulation by Ca(2+) was approximately 10 times higher for external NADPH oxidation, while NADH dehydrogenase(s) appeared to be slightly dependent on Ca(2+). Our results indicate that external NAD(P)H dehydrogenases similar to those in plant and fungal mitochondria function in mitochondria of A. castellanii.

  18. The Activity and Localization of 3β-hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase and Release of Androstenedione and Progesterone by Uterine Tissues During Early Pregnancy and the Estrous Cycle in Pigs

    OpenAIRE

    Wojciechowicz, Bartosz; KOTWICA, Genowefa; Kolakowska, Justyna; Franczak, Anita

    2012-01-01

    Abstract Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) responsible for progesterone and androstenedione production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3β-HSD mRNA, 2)...

  19. Artemisinin inhibits chloroplast electron transport activity: mode of action.

    Directory of Open Access Journals (Sweden)

    Adyasha Bharati

    Full Text Available Artemisinin, a secondary metabolite produced in Artemisia plant species, besides having antimalarial properties is also phytotoxic. Although, the phytotoxic activity of the compound has been long recognized, no information is available on the mechanism of action of the compound on photosynthetic activity of the plant. In this report, we have evaluated the effect of artemisinin on photoelectron transport activity of chloroplast thylakoid membrane. The inhibitory effect of the compound, under in vitro condition, was pronounced in loosely and fully coupled thylakoids; being strong in the former. The extent of inhibition was drastically reduced in the presence of uncouplers like ammonium chloride or gramicidin; a characteristic feature described for energy transfer inhibitors. The compound, on the other hand, when applied to plants (in vivo, behaved as a potent inhibitor of photosynthetic electron transport. The major site of its action was identified to be the Q(B; the secondary quinone moiety of photosystemII complex. Analysis of photoreduction kinetics of para-benzoquinone and duroquinone suggest that the inhibition leads to formation of low pool of plastoquinol, which becomes limiting for electron flow through photosystemI. Further it was ascertained that the in vivo inhibitory effect appeared as a consequence of the formation of an unidentified artemisinin-metabolite rather than by the interaction of the compound per se. The putative metabolite of artemisinin is highly reactive in instituting the inhibition of photosynthetic electron flow eventually reducing the plant growth.

  20. Hybridizability of gamma-irradiated lactic dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Saito, M.

    1976-03-01

    The hybridizabilities of the gamma-irradiated chicken heart and pig muscle lactic dehydrogenases were estimated by hybridizing the irradiated enzymes with the unirradiated pig heart lactic dehydrogenase. The disc gel electrophoretic patterns of the inter- and intraspecific hybrids showed that the LDH activity of the pig heart isozyme band increased as a function of dose. This observation was analyzed upon the binomial redistribution pattern of the recombined subunits. The result shows that the hybridizabilities of both the chicken heart and pig muscle isozymes decreased along with the loss of catalytic activity and the release from substrate inhibition. The titration of free SH groups of the irradiated chicken isozyme suggested that the unfolding of the peptide chain destroyed the specific tertiary structure needed for the binding of subunits. (auth)

  1. Study on Dehydrogenase Activity of Excised Embryos of Castanea Henryi Seeds after Cryopreservation%锥栗种子离体胚超低温保存脱氢酶活性研究

    Institute of Scientific and Technical Information of China (English)

    陈礼光; 郑郁善

    2001-01-01

    运用超低温(-196 ℃)保存手段,通过对锥栗种子离体胚超低温保存后脱氢酶活性的方差分析和Q检验法多重比较分析,对其长期保存的可行性进行研究,结果表明:含水量是影响锥栗种子离体胚超低温保存的重要因素,超低温保存应进行适度脱水.无防冻剂预处理,20%含水量,缓冻缓解方式条件下,离体胚脱氢酶活性最高.%By applying cryopreservation(-196 ℃)treatment and using the methods of variance analysis and multiple comparison of Q test of TTCH content of excised embryos after cryopreservation, dehydrogenase activity of excised embryos was analysed and long-term storage feasibility was studied. The results showed moisture content(MC) was the main factors affecting the cryopreservation of C. henryi excised embryos, and the measures desiccating a little down to a medium MC should be taken in cryopreservation. Under the conditions of no cryoprotectants pretreatment, 20% MC, mild freezing, and quick thawing, the dehydrogenase activity of excised embryos was the highest.

  2. Diversity of organotrophic bacteria, activity of dehydrogenases and urease as well as seed germination and root growth Lepidium sativum, Sorghum saccharatum and Sinapis alba under the influence of polycyclic aromatic hydrocarbons.

    Science.gov (United States)

    Lipińska, Aneta; Wyszkowska, Jadwiga; Kucharski, Jan

    2015-12-01

    Polycyclic aromatic hydrocarbons are organic compounds with highly toxic, carcinogenic, and mutagenic properties, which adversely affect the basic biological parameters of the soil, including the count of microorganisms, and the enzymatic activity. In addition to disturbances to the biological activity of the soil, PAHs may also exhibit toxic effects on plants. In view of the above, the study involved testing aimed at the determination of the effects of polycyclic aromatic hydrocarbons in a form of naphthalene, phenanthrene, anthracene and pyrene on the count, colony development (CD) index, ecophysiological (EP) diversity index of organotrophic bacteria, and the activity of soil dehydrogenases and soil urease. Moreover, an attempt was made to determine the soil's resistance based on the activity of the above-listed enzymes, and the effect of polycyclic aromatic hydrocarbons on seed germination and root growth was assessed by Lepidium sativum, Sorghum saccharatum, and Sinapis alba. In addition, the species of bacteria found in a soil subjected to strong pressure of polycyclic aromatic hydrocarbons were isolated. The experiment was performed in a laboratory on samples of loamy sand. Polycyclic aromatic hydrocarbons were introduced into the soil in an amount of 0, 1000, 2000, and 4000 mg kg(-1) of soil dry matter. Germination and growth of cress (L. sativum), white mustard (S. alba), and sweet sorghum (S. saccharatum) were determined using Phytotoxkit tests. It was found that the tested PAHs increased the average colony counts of organotrophic soil bacteria; pyrene did so to the greatest extent (2.2-fold relative to non-contaminated soil), phenanthrene to the smallest extent (1.4-fold relative to non-contaminated soil). None of the PAHs changed the value of the bacterial colony development (CD) index, while anthracene and pyrene increased the value of the eco-physiological (EP) diversity indicator. PAHs lowered the activity of the tested enzymes. The activity of

  3. 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.

  4. P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations

    NARCIS (Netherlands)

    Beyer, Nina; Kulig, Justyna K; Bartsch, Anette; Hayes, Martin A; Janssen, Dick B; Fraaije, Marco W

    2016-01-01

    To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regener

  5. Effect of ageing and ischemia on enzymatic activities linked to Krebs' cycle, electron transfer chain, glutamate and aminoacids metabolism of free and intrasynaptic mitochondria of cerebral cortex.

    Science.gov (United States)

    Villa, Roberto Federico; Gorini, Antonella; Hoyer, Siegfried

    2009-12-01

    The effect of ageing and the relationships between the catalytic properties of enzymes linked to Krebs' cycle, electron transfer chain, glutamate and aminoacid metabolism of cerebral cortex, a functional area very sensitive to both age and ischemia, were studied on mitochondria of adult and aged rats, after complete ischemia of 15 minutes duration. The maximum rate (Vmax) of the following enzyme activities: citrate synthase, malate dehydrogenase, succinate dehydrogenase for Krebs' cycle; NADH-cytochrome c reductase as total (integrated activity of Complex I-III), rotenone sensitive (Complex I) and cytochrome oxidase (Complex IV) for electron transfer chain; glutamate dehydrogenase, glutamate-oxaloacetate-and glutamate-pyruvate transaminases for glutamate metabolism were assayed in non-synaptic, perikaryal mitochondria and in two populations of intra-synaptic mitochondria, i.e., the light and heavy mitochondrial fraction. The results indicate that in normal, steady-state cerebral cortex, the value of the same enzyme activity markedly differs according (a) to the different populations of mitochondria, i.e., non-synaptic or intra-synaptic light and heavy, (b) and respect to ageing. After 15 min of complete ischemia, the enzyme activities of mitochondria located near the nucleus (perikaryal mitochondria) and in synaptic structures (intra-synaptic mitochondria) of the cerebral tissue were substantially modified by ischemia. Non-synaptic mitochondria seem to be more affected by ischemia in adult and particularly in aged animals than the intra-synaptic light and heavy mitochondria. The observed modifications in enzyme activities reflect the metabolic state of the tissue at each specific experimental condition, as shown by comparative evaluation with respect to the content of energy-linked metabolites and substrates. The derangements in enzyme activities due to ischemia is greater in aged than in adult animals and especially the non-synaptic and the intra-synaptic light

  6. 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...

  7. Promoting direct interspecies electron transfer with activated carbon

    DEFF Research Database (Denmark)

    Liu, Fanghua; Rotaru, Amelia-Elena; Shrestha, Pravin M.

    2012-01-01

    Granular activated carbon (GAC) is added to methanogenic digesters to enhance conversion of wastes to methane, but the mechanism(s) for GAC’s stimulatory effect are poorly understood. GAC has high electrical conductivity and thus it was hypothesized that one mechanism for GAC stimulation of metha......Granular activated carbon (GAC) is added to methanogenic digesters to enhance conversion of wastes to methane, but the mechanism(s) for GAC’s stimulatory effect are poorly understood. GAC has high electrical conductivity and thus it was hypothesized that one mechanism for GAC stimulation...... of methanogenesis might be to facilitate direct interspecies electron transfer (DIET) between bacteria and methanogens. Metabolism was substantially accelerated when GAC was added to co-cultures of Geobacter metallireducens and Geobacter sulfurreducens grown under conditions previously shown to require DIET. Cells...... were attached to GAC, but did not aggregate as they do when making biological electrical connections between cells. Studies with a series of gene deletion mutants eliminated the possibility that GAC promoted electron exchange via interspecies hydrogen or formate transfer and demonstrated that DIET...

  8. Marked differences in drug-induced methemoglobinemia in sheep are not due to RBC glucose-6-phosphate dehydrogenase, reduced glutathione, or methemoglobin reductase activity

    Energy Technology Data Exchange (ETDEWEB)

    Martin, D.G.; Guertler, A.T.; Lagutchik, M.S.; Woodard, C.L.; Leonard, D.A.

    1993-05-13

    Benzocaine is a commonly used topical anesthetic that is structurally similar to current candidates for cyanide prophylaxis. Benzocaine induces profound methemoglobinemia in some sheep but not others. After topical benzocaine administration certain sheep respond to form MHb (elevated MHb 16-50% after a 56-280 mg dose, a 2-10 second spray with benzocine), while other phenotypically similar sheep fail to significantly form MHb (less than a 2% increase from baseline). Deficiencies in Glucose-6-phosphate dehydrogenase (G-6-PD), reduced glutathione (GSH), and MHb reductase increase the susceptibility to methemoglobinemia in man and animals. Sheep are used as a model for G-6-PD deficiency in man, and differences in this enzyme level could cause the variable response seen in these sheep. Similarly, differences in GSH and MHb reductase could be responsible for the observed differences in MHb formation.

  9. Piper betel leaves induces wound healing activity via proliferation of fibroblasts and reducing 11β hydroxysteriod dehydrogenase-1 expression in diabetic rat.

    Science.gov (United States)

    Ghazali, Nur Amalina; Elmy, Azree; Yuen, Lee Chee; Sani, Nurul Zaidah; Das, Srijit; Suhaimi, Farihah; Yusof, Rafizul; Yusoff, Nurul Huda; Thent, Zar Chi

    Increased oxidative stress and stress enzyme 11β hydroxysteriod dehydrogenase-1 (11β HSD-1) served as the major contributing factors for delayed wound healing in diabetes mellitus (DM). Piper betel (PB) leaves are reported to possess anti-diabetic, anti-oxidant and anti-microbial properties. The objective was to investigate the effectiveness of topical application of PB leaves extract on oxidative stress and 11β hydroxysteriod dehydrogenase-1 (11β HSD-1) expression in diabetic wounds. A total 64 male Sprague-Dawley rats were randomly chosen. The experimental rats received a single intramuscular injection of streptozotocin (45 mg/kg). Four full thickness (6 mm) wounds were created on the dorsum of each rat. The animals were equally divided (n = 8) into four groups based on the days of treatment (i.e. days 3 and 7): Control (Ctrl), diabetic untreated (DM-Ctrl), diabetic treated with 1% silver nitrate cream (DM-SN) and diabetic treated with 50 mg/kg of P. betel leaves extract (DM-PB). The rats were sacrificed on day 3 and 7 of post wound creations. Following day 7 wound creation, topical application of PB extract showed significant increase in hydroxyproline content, superoxide dismutase (SOD) level and decreased malondialdehyde (MDA) level, 11β-HSD-1 enzyme expression in the diabetic wounds compared to untreated diabetic wounds. The results were supported by the observations based on histological and ultrastructural features of the wound tissue applied with PB extract. PB leaves extract improved the delayed wound healing in diabetes mellitus by decreasing the oxidative stress markers and 11β HSD-1 expression. Copyright © 2016 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Published by Elsevier B.V. All rights reserved.

  10. Modelling of electron transport and of sawtooth activity in tokamaks

    Energy Technology Data Exchange (ETDEWEB)

    Angioni, C

    2001-10-01

    Transport phenomena in tokamak plasmas strongly limit the particle and energy confinement and represent a crucial obstacle to controlled thermonuclear fusion. Within the vast framework of transport studies, three topics have been tackled in the present thesis: first, the computation of neoclassical transport coefficients for general axisymmetric equilibria and arbitrary collisionality regime; second, the analysis of the electron temperature behaviour and transport modelling of plasma discharges in the Tokamak a configuration Variable (TCV); third, the modelling and simulation of the sawtooth activity with different plasma heating conditions. The work dedicated to neoclassical theory has been undertaken in order to first analytically identify a set of equations suited for implementation in existing Fokker-Planck codes. Modifications of these codes enabled us to compute the neoclassical transport coefficients considering different realistic magnetic equilibrium configurations and covering a large range of variation of three key parameters: aspect ratio, collisionality, and effective charge number. A comparison of the numerical results with an analytical limit has permitted the identification of two expressions for the trapped particle fraction, capable of encapsulating the geometrical effects and thus enabling each transport coefficient to be fitted with a single analytical function. This has allowed us to provide simple analytical formulae for all the neoclassical transport coefficients valid for arbitrary aspect ratio and collisionality in general realistic geometry. This work is particularly useful for a correct evaluation of the neoclassical contribution in tokamak scenarios with large bootstrap cur- rent fraction, or improved confinement regimes with low anomalous transport and for the determination of the plasma current density profile, since the plasma conductivity is usually assumed neoclassical. These results have been included in the plasma transport code

  11. Isocitrate dehydrogenase mutations in gliomas.

    Science.gov (United States)

    Waitkus, Matthew S; Diplas, Bill H; Yan, Hai

    2016-01-01

    Over the last decade, extraordinary progress has been made in elucidating the underlying genetic causes of gliomas. In 2008, our understanding of glioma genetics was revolutionized when mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) were identified in the vast majority of progressive gliomas and secondary glioblastomas (GBMs). IDH enzymes normally catalyze the decarboxylation of isocitrate to generate α-ketoglutarate (αKG), but recurrent mutations at Arg(132) of IDH1 and Arg(172) of IDH2 confer a neomorphic enzyme activity that catalyzes reduction of αKG into the putative oncometabolite D-2-hydroxyglutate (D2HG). D2HG inhibits αKG-dependent dioxygenases and is thought to create a cellular state permissive to malignant transformation by altering cellular epigenetics and blocking normal differentiation processes. Herein, we discuss the relevant literature on mechanistic studies of IDH1/2 mutations in gliomas, and we review the potential impact of IDH1/2 mutations on molecular classification and glioma therapy.

  12. Two-Dimensional Halide Perovskites: Tuning Electronic Activities of Defects.

    Science.gov (United States)

    Liu, Yuanyue; Xiao, Hai; Goddard, William A

    2016-05-11

    Two-dimensional (2D) halide perovskites are emerging as promising candidates for nanoelectronics and optoelectronics. To realize their full potential, it is important to understand the role of those defects that can strongly impact material properties. In contrast to other popular 2D semiconductors (e.g., transition metal dichalcogenides MX2) for which defects typically induce harmful traps, we show that the electronic activities of defects in 2D perovskites are significantly tunable. For example, even with a fixed lattice orientation one can change the synthesis conditions to convert a line defect (edge or grain boundary) from electron acceptor to inactive site without deep gap states. We show that this difference originates from the enhanced ionic bonding in these perovskites compared with MX2. The donors tend to have high formation energies and the harmful defects are difficult to form at a low halide chemical potential. Thus, we unveil unique properties of defects in 2D perovskites and suggest practical routes to improve them.

  13. An active electron polarized scintillating GSO target for neutrino physics

    Energy Technology Data Exchange (ETDEWEB)

    Baiboussinov, B. [INFN, Sez. di Padova, Via Marzolo 8, 35131 Padova (Italy); Braggio, C., E-mail: braggio@pd.infn.it [INFN, Sez. di Padova, Via Marzolo 8, 35131 Padova (Italy); Dipartimento di Fisica, Universita di Padova, Via Marzolo 8, 35131 Padova (Italy); Cardini, A. [INFN, Sez. di Cagliari, S.P. per Sestu Km 0.700, 09042 Monserrato (Cagliari) (Italy); Carugno, G. [INFN, Sez. di Padova, Via Marzolo 8, 35131 Padova (Italy); Congiu, F. [Dipartimento di Fisica, Universita di Cagliari, S.P. per Sestu Km 0.700, 09042 Monserrato (Cagliari) (Italy); Gain, S. [St. Petersburg State Polytechnical University, 195251 St. Petersburg, Polytekhnicheskaya 29 (Russian Federation); Galeazzi, G. [INFN, Laboratori Nazionali di Legnaro, Viale dell Universita, 2 35020 Legnaro (PD) (Italy); Lai, A. [INFN, Sez. di Cagliari, S.P. per Sestu Km 0.700, 09042 Monserrato (Cagliari) (Italy); Lehman, A.; Mocci, P.; Mura, A.; Quochi, F.; Saba, M. [Dipartimento di Fisica, Universita di Cagliari, S.P. per Sestu Km 0.700, 09042 Monserrato (Cagliari) (Italy); Saitta, B. [INFN, Sez. di Cagliari, S.P. per Sestu Km 0.700, 09042 Monserrato (Cagliari) (Italy); Dipartimento di Fisica, Universita di Cagliari, S.P. per Sestu Km 0.700, 09042 Monserrato (Cagliari) (Italy); Sartori, G. [INFN, Sez. di Padova, Via Marzolo 8, 35131 Padova (Italy)

    2012-12-01

    The feasibility of an electron-polarized, active target to be used as detector in neutrino scattering experiments, suggested by several theoretical papers, has been investigated. We report on the properties of the paramagnetic crystal Gd{sub 2}SiO{sub 5} (GSO), in which 7.7% of the total number of electrons present can be polarized by lowering the temperature and applying an intense external magnetic field. The material magnetic susceptibility has been measured down to cryogenic temperatures showing that for H=5 T and T=4 K about 80% of the maximum allowed magnetization can be attained. Also the spectral and time response of the crystal have been characterized and the scintillation process has been studied using a photomultiplier to measure the response to gamma rays irradiation and cosmic rays operating the GSO crystal at 13.5 K. An avalanche photodiode (APD) readout of the scintillation signal from the GSO crystal has also been performed, since the magnetic field-independent response of this device allows it to be placed close to the crystal in the cryogenic environment.

  14. Active Interrogation Using Electronic Neutron Generators for Nuclear Safeguards Applications

    Energy Technology Data Exchange (ETDEWEB)

    David L. Chichester; Edward H. Seabury

    2008-08-01

    Active interrogation, a measurement technique which uses a radiation source to probe materials and generate unique signatures useful for characterizing those materials, is a powerful tool for assaying special nuclear material. The most commonly used technique for performing active interrogation is to use an electronic neutron generator as the probe radiation source. Exploiting the unique operating characteristics of these devices, including their monoenergetic neutron emissions and their ability to operate in pulsed modes, presents a number of options for performing prompt and delayed signature analyses using both photon and neutron sensors. A review of literature in this area shows multiple applications of the active neutron interrogation technique for performing nuclear nonproliferation measurements. Some examples include measuring the plutonium content of spent fuel, assaying plutonium residue in spent fuel hull claddings, assaying plutonium in aqueous fuel reprocessing process streams, and assaying nuclear fuel reprocessing facility waste streams to detect and quantify fissile material. This paper discusses the historical use of this technique and examines its context within the scope and challenges of next-generation nuclear fuel cycles and advanced concept nuclear fuel cycle facilities.

  15. Electronic Activation of a DNA Nanodevice Using a Multilayer Nanofilm.

    Science.gov (United States)

    Jeong, Hyejoong; Ranallo, Simona; Rossetti, Marianna; Heo, Jiwoong; Shin, Jooseok; Park, Kwangyong; Ricci, Francesco; Hong, Jinkee

    2016-10-01

    A method to control activation of a DNA nanodevice by supplying a complementary DNA (cDNA) strand from an electro-responsive nanoplatform is reported. To develop functional nanoplatform, hexalayer nanofilm is precisely designed by layer-by-layer assembly technique based on electrostatic interaction with four kinds of materials: Hydrolyzed poly(β-amino ester) can help cDNA release from the film. A cDNA is used as a key building block to activate DNA nanodevice. Reduced graphene oxides (rGOs) and the conductive polymer provide conductivity. In particular, rGOs efficiently incorporate a cDNA in the film via several interactions and act as a barrier. Depending on the types of applied electronic stimuli (reductive and oxidative potentials), a cDNA released from the electrode can quantitatively control the activation of DNA nanodevice. From this report, a new system is successfully demonstrated to precisely control DNA release on demand. By applying more advanced form of DNA-based nanodevices into multilayer system, the electro-responsive nanoplatform will expand the availability of DNA nanotechnology allowing its improved application in areas such as diagnosis, biosensing, bioimaging, and drug delivery.

  16. Kinetics of soil dehydrogenase in response to exogenous Cd toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Xiangping [College of Natural Resources and Environment, Northwest A& F University, Yangling, 712100, Shaanxi (China); Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, CAS 723 Xingke Rd., Tianhe District, Guangzhou 510650 (China); Wang, Ziquan; Lu, Guannan [College of Natural Resources and Environment, Northwest A& F University, Yangling, 712100, Shaanxi (China); He, Wenxiang, E-mail: wenxianghe@nwafu.edu.cn [College of Natural Resources and Environment, Northwest A& F University, Yangling, 712100, Shaanxi (China); Key Laboratory of Plant Nutrition and Agro-environment in Northwest China, Ministry of Agriculture, Northwest A& F University, Yangling, 712100, Shaanxi (China); Wei, Gehong [College of Life Sciences, Northwest A& F University, Yangling, 712100, Shaanxi (China); Huang, Feng; Xu, Xinlan; Shen, Weijun [Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, CAS 723 Xingke Rd., Tianhe District, Guangzhou 510650 (China)

    2017-05-05

    Highlights: • pH explained 30–45% of the dehydrogenase activity (DHA), V{sub max}, and K{sub m} variations across soils. • Different inhibition mechanism of Cd to DHA varied soil types. • Soil properties and inhibition constant affect the toxicity of Cd. • Reaction constant (k) could indicate sensitively the toxicity of Cd to DHA. - Abstract: Soil dehydrogenase plays a role in the biological oxidation of soil organic matter and can be considered a good measure of the change of microbial oxidative activity under environmental pollutions. However, the kinetic characteristic of soil dehydrogenase under heavy metal stresses has not been investigated thoroughly. In this study, we characterized the kinetic characteristic of soil dehydrogenase in 14 soil types, and investigated how kinetic parameters changed under spiked with different concentrations of cadmium (Cd). The results showed that the K{sub m} and V{sub max} values of soil dehydrogenase was among 1.4–7.3 mM and 15.9–235.2 μM h{sup −1} in uncontaminated soils, respectively. In latosolic red soil and brown soil, the inhibitory kinetic mechanism of Cd to soil dehydrogenase was anticompetitive inhibition with inhibition constants (K{sub i}) of 12 and 4.7 mM, respectively; in other soils belonged to linear mixed inhibition, the values of K{sub i} were between 0.7–4.2 mM. Soil total organic carbon and K{sub i} were the major factors affecting the toxicity of Cd to dehydrogenase activity. In addition, the velocity constant (k) was more sensitive to Cd contamination compared to V{sub max} and K{sub m}, which was established as an early indicator of gross changes in soil microbial oxidative activity caused by Cd contamination.

  17. 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.

  18. [Alcohol dehydrogenase and aldehyde dehydrogenase as tumour markers and factors intensifying carcinogenesis in colorectal cancer].

    Science.gov (United States)

    Jelski, Wojciech; Orywal, Karolina; Kedra, Bogusław; Szmitkowski, Maciej

    2008-06-01

    Numerous experiments have shown that alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are present in cells of various cancers and play role in carcinogenesis. The aim of this study was to compare the capacity for ethanol metabolism measured by ADH isoenzymes and ALDH activity, between colorectal cancer and normal colonic mucosa. We have also investigated the serum activity of these enzymes in colorectal cancer patients as potential tumour markers. The activities of ADH isoenzymes and ALDH were measured in the: cancer tissue, healthy colonic mucosa and serum of 42 patients with colorectal cancer. For the measurement of the activity of class I ADH isoenzyme and ALDH activity the fluorometric methods was employed. The total ADH activity and activity of class III and IV isoenzymes was measured by the photometric method. The activity of total alcohol dehydrogenase and class I of ADH were significantly higher in cancer cells than in healthy tissues. The other tested classes of ADH had higher activities in cancer tissue but the differences were not statistically significant. The activity of ALDH was significantly lower in the cancer cells. The activities of all tested enzymes and isoenzymes in colorectal cancer tissue were not significantly higher in drinkers than in non-drinkers. Additionally we observed statistically significant increasing activity of class I ADH isoenzymes in the sera of patients with colorectal cancer. For this reason the total ADH activity was also significantly increased. The activities of ADH III and ADH IV isoenzymes and ALDH were unchanged in the sera of patients. There were no marked differences in activities of all tested enzymes and isoenzymes between drinkers and non-drinkers (with colorectal cancer). The differences in activities of total ADH and class I ADH isoenzymes between colorectal cancer tissues and healthy mucosa might be a factor of ethanol metabolism disorders, which can intensify carcinogenesis. The increased total

  19. Waste recycling by vermicomposting: Maturity and quality assessment via dehydrogenase enzyme activity, lignin, water soluble carbon, nitrogen, phosphorous and other indicators.

    Science.gov (United States)

    Alidadi, Hossein; Hosseinzadeh, Ahmad; Najafpoor, Ali Asghar; Esmaili, Habibollah; Zanganeh, Jafar; Dolatabadi Takabi, Maryam; Piranloo, Fardin Ghasemy

    2016-11-01

    Present study aims to examine the dynamics of maturation and qualification indicators in various vermicompost treatments and selection of the best treatment along with best maturation time in this regard. In this empirical study, dynamics of chemical (pH, electrical conductivity (EC), total nitrogen (TN), phosphorous, lignin, water soluble carbon (WSC), C/N, NH4/NO3) and biological (dehydrogenase enzyme (DEH) and DEH/WSC) properties were investigated in four various treatments, including various ratios of compost produced from municipal solid waste (MSW) and carbonaceous materials (50:50, 70:30, 85:15 and 100:0) over 100 days. Results showed a significant fluctuation in EC, DEH and DEH/WSC proportions over the process. In addition, a noticeable increase was observed for the dynamics of TN, phosphorous and lignin. In contrast, the C/N, NH4/NO3 and WSC values gradually decreased during the process. Moreover, it was observed that the length of 75 days for the process is an appropriate time for maturation of all treatments. However, the first and second treatments resulted in better outcomes compared with the other types of treatments. From the point of view of quality obtained vermicompost was nitrogen enriched product in all treatments. Whereas, for the phosphorous elements this method is appropriate for the first treatment only. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Multi-organ abnormalities and mTORC1 activation in zebrafish model of multiple acyl-CoA dehydrogenase deficiency.

    Directory of Open Access Journals (Sweden)

    Seok-Hyung Kim

    2013-06-01

    Full Text Available Multiple Acyl-CoA Dehydrogenase Deficiency (MADD is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxa(vu463 that has an inactivating mutation in the etfa gene. dxa(vu463 recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxa(vu463 zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxa(vu463 zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1 with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.

  1. Rational Design of Benzylidenehydrazinyl-Substituted Thiazole Derivatives as Potent Inhibitors of Human Dihydroorotate Dehydrogenase with in Vivo Anti-arthritic Activity

    Science.gov (United States)

    Li, Shiliang; Luan, Guoqin; Ren, Xiaoli; Song, Wenlin; Xu, Liuxin; Xu, Minghao; Zhu, Junsheng; Dong, Dong; Diao, Yanyan; Liu, Xiaofeng; Zhu, Lili; Wang, Rui; Zhao, Zhenjiang; Xu, Yufang; Li, Honglin

    2015-01-01

    Human dihydroorotate dehydrogenase (hDHODH) is an attractive therapeutic target for the treatment of rheumatoid arthritis, transplant rejection and other autoimmune diseases. Based on the X-ray structure of hDHODH in complex with lead compound 7, a series of benzylidenehydrazinyl-substituted thiazole derivatives as potent inhibitors of hDHODH were designed and synthesized, of which 19 and 30 were the most potent with IC50 values in the double-digit nanomolar range. Moreover, compound 19 displayed significant anti-arthritic effects and favorable pharmacokinetic profiles in vivo. Further X-ray structure and SAR analyses revealed that the potencies of the designed inhibitors were partly attributable to additional water-mediated hydrogen bond networks formed by an unexpected buried water between hDHODH and the 2-(2-methylenehydrazinyl)thiazole scaffold. This work not only elucidates promising scaffolds targeting hDHODH for the treatment of rheumatoid arthritis, but also demonstrates that the water-mediated hydrogen bond interaction is an important factor in molecular design and optimization. PMID:26443076

  2. Reactive oxygen species (ROS) production triggered by prostaglandin D2 (PGD2) regulates lactate dehydrogenase (LDH) expression/activity in TM4 Sertoli cells.

    Science.gov (United States)

    Rossi, Soledad P; Windschüttl, Stefanie; Matzkin, María E; Rey-Ares, Verónica; Terradas, Claudio; Ponzio, Roberto; Puigdomenech, Elisa; Levalle, Oscar; Calandra, Ricardo S; Mayerhofer, Artur; Frungieri, Mónica B

    2016-10-15

    Reactive oxygen species (ROS) regulate testicular function in health and disease. We previously described a prostaglandin D2 (PGD2) system in Sertoli cells. Now, we found that PGD2 increases ROS and hydrogen peroxide (H2O2) generation in murine TM4 Sertoli cells, and also induces antioxidant enzymes expression suggesting that defense systems are triggered as an adaptive stress mechanism that guarantees cell survival. ROS and specially H2O2 may act as second messengers regulating signal transduction pathways and gene expression. We describe a stimulatory effect of PGD2 on lactate dehydrogenase (LDH) expression via DP1/DP2 receptors, which is prevented by the antioxidant N-acetyl-L-cysteine and the PI3K/Akt pathway inhibitor LY 294002. PGD2 also enhances Akt and CREB/ATF-1 phosphorylation. Our results provide evidence for a role of PGD2 in the regulation of the oxidant/antioxidant status in Sertoli cells and, more importantly, in the modulation of LDH expression which takes place through ROS generation and the Akt-CREB/ATF-1 pathway.

  3. Investigation of asymmetric alcohol dehydrogenase (ADH) reduction of acetophenone derivatives: effect of charge density.

    Science.gov (United States)

    Naik, Hemantkumar G; Yeniad, Bahar; Koning, Cor E; Heise, Andreas

    2012-07-01

    In an effort to study the effect of substituent groups of the substrate on the alcohol dehydrogenase (ADH) reductions of aryl-alkyl ketones, several derivatives of acetophenone have been evaluated against ADHs from Lactobacillus brevis (LB) and Thermoanaerobacter sp. (T). Interestingly, ketones with non-demanding (neutral) para-substituents were reduced to secondary alcohols by these enzymes in enantiomerically pure form whereas those with demanding (ionizable) substituents could not be reduced. The effect of substrate size, their solubility in the reaction medium, electron donating and withdrawing properties of the ligand and also the electronic charge density distribution on the substrate molecules have been studied and discussed in detail. From the results, it is observed that the electronic charge distribution in the substrate molecules is influencing the orientation of the substrate in the active site of the enzyme and hence the ability to reduce the substrate.

  4. 大鼠骨骼肌细胞异柠檬酸脱氢酶和琥珀酸脱氢酶活性在不同训练负荷时的变化%Effects of exercise training modes at different work intensities on activity of isocitrate dehydrogenase and succinic dehydrogenase in rat skeletal muscles

    Institute of Scientific and Technical Information of China (English)

    张敏; 陈立军; 周蔚

    2011-01-01

    背景:异柠檬酸脱氢酶和琥珀酸脱氢酶是糖有氧氧化过程中的关键酶,不同训练方式和训练时间对骨骼肌异柠檬酸脱氢酶和琥珀酸脱氢酶活性影响的报道较少.目的:探讨不同训练负荷条件对大鼠骨骼肌异柠檬酸脱氢酶和琥珀酸脱氢酶活性的影响.方法:参照BEDFORD TG 标准,建立大鼠有氧、无氧和有氧无氧交替运动跑台训练模型,正常饲养的大鼠作为对照.训练结束后,紫外分光光度计检测大鼠骨骼肌异柠檬酸脱氢酶和琥珀酸脱氢酶的活性.结果与结论:有氧运动4,6 周,大鼠骨骼肌异柠檬酸脱氢酶和琥珀酸脱氢酶活性均明显升高(P < 0.05);交替运动2 周,异柠檬酸脱氢酶和琥珀酸脱氢酶活性增加(P < 0.05),运动至4,6 周时,两种酶活性均下降(P < 0.05);而无氧运动对异柠檬酸脱氢酶和琥珀酸脱氢酶活性无影响.结果提示:长期的有氧运动和短时间的有氧无氧交替运动有利于提升骨骼肌异柠檬酸脱氢酶和琥珀酸脱氢酶的活性.%BACKGROUND: lsocitrate dehydrogenase (IDH) and succinic dehydrogenase (SDH) play key role in aerobic oxidation. However,reports about the effects of different training models and training periods on the activity of IDH and SDH in skeletal muscle have little coverage.OBJECTIVE: To observe the effects of different training on the activity of IDH and SDH in rat skeletal muscles.METHODS: According to BEDFORD TG standards, aerobic exercise, anaerobic exercise and aerobic and anaerobic cross-training motion models were established using treadmill running at different work intensities. Rats in control group lived in the cages quietly, and all the animals were killed immediately when the exercise training completed. Then the activity of IDH and SDH were determined by ultraviolet spectrophotometer.RESULTS AND CONCLUSION: The activity of IDH and SDH were obviously increased at 4 and 6 weeks after treadmill training (P< 0.05). To the cross

  5. Expression pattern, ethanol-metabolizing activities, and cellular localization of alcohol and aldehyde dehydrogenases in human large bowel: association of the functional polymorphisms of ADH and ALDH genes with hemorrhoids and colorectal cancer.

    Science.gov (United States)

    Chiang, Chien-Ping; Jao, Shu-Wen; Lee, Shiao-Pieng; Chen, Pei-Chi; Chung, Chia-Chi; Lee, Shou-Lun; Nieh, Shin; Yin, Shih-Jiun

    2012-02-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for metabolism of ethanol. Functional polymorphisms of ADH1B, ADH1C, and ALDH2 genes occur among racial populations. The goal of this study was to systematically determine the functional expressions and cellular localization of ADHs and ALDHs in human rectal mucosa, the lesions of adenocarcinoma and hemorrhoid, and the genetic association of allelic variations of ADH and ALDH with large bowel disorders. Twenty-one surgical specimens of rectal adenocarcinoma and the adjacent normal mucosa, including 16 paired tissues of rectal tumor, normal mucosae of rectum and sigmoid colon from the same individuals, and 18 surgical mixed hemorrhoid specimens and leukocyte DNA samples from 103 colorectal cancer patients, 67 hemorrhoid patients, and 545 control subjects recruited in previous study, were investigated. The isozyme/allozyme expression patterns of ADH and ALDH were identified by isoelectric focusing and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting using the corresponding purified class-specific antibodies; the cellular activity and protein localizations were detected by immunohistochemistry and histochemistry, respectively. Genotypes of ADH1B, ADH1C, and ALDH2 were determined by polymerase chain reaction-restriction fragment length polymorphisms. At 33mM ethanol, pH 7.5, the activity of ADH1C*1/1 phenotypes exhibited 87% higher than that of the ADH1C*1/*2 phenotypes in normal rectal mucosa. The activity of ALDH2-active phenotypes of rectal mucosa was 33% greater than ALDH2-inactive phenotypes at 200μM acetaldehyde. The protein contents in normal rectal mucosa were in the following order: ADH1>ALDH2>ADH3≈ALDH1A1, whereas those of ADH2, ADH4, and ALDH3A1 were fairly low. Both activity and content of ADH1 were significantly decreased in rectal tumors, whereas the ALDH activity remained

  6. 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.

  7. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    NARCIS (Netherlands)

    Resch, V.A.; Jin, J.; Chen, B.S.; Hanefeld, U.

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a

  8. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    NARCIS (Netherlands)

    Resch, V.A.; Jin, J.; Chen, B.S.; Hanefeld, U.

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a s

  9. Application of NAD-dependent polyol dehydrogenases for enzymatic mannitol/sorbitol production with coenzyme regeneration.

    Science.gov (United States)

    Parmentier, S; Arnaut, F; Soetaert, W; Vandamme, E J

    2003-01-01

    D-Mannitol and D-sorbitol were produced enzymatically from D-fructose using NAD-dependent polyol dehydrogenases. For the production of D-mannitol the Leuconostoc mesenteroides mannitol dehydrogenase could be used. Gluconobacter oxydans cell extract contained however both mannitol and sorbitol dehydrogenase. When this cell extract was used, the reduction of D-fructose resulted in a mixture of D-sorbitol and D-mannitol. To determine the optimal bioconversion conditions the polyol dehydrogenases were characterized towards pH- and temperature-optimum and -stability. As a compromise between enzyme activity and stability, the bioconversion reactions were performed at pH 6.5 and 25 degrees C. Since the polyol dehydrogenases are NADH-dependent, an efficient coenzyme regeneration was needed. Regeneration of NADH was accomplished by formate dehydrogenase-mediated oxidation of formate into CO2.

  10. Oxidation of Exogenous Lactate by Lactate Dehydrogenase C in the Midpiece of Rat Epididymal Sperm is Essential for Motility and Oxidative Activity

    Directory of Open Access Journals (Sweden)

    Hideaki Yamashiro

    2009-01-01

    Full Text Available Problem statement: To identify the metabolic reaction-glycolysis or oxidative phosphorylation that is mainly involved in the production of energy required for rat sperm mobilization. Approach: Epididymal sperm were collected from Wistar rats and extended in lactate-containing or lactate-free raffinose-modified Krebs-Ringer Bicarbonate solution (mKRB-egg yolk medium supplemented with 0, 1, 2, or 3 mM 2-Deoxy-D-Glucose (2 DG and 1, 2, or 3 mM sodium Oxamate (OX. Sperm motility, straight-line velocity (VSL and oxygen consumption were evaluated. Further, immunofluorescent localization of Lactate Dehydrogenase C (LDH-C in sperm was also performed. Results: Low concentrations of 2DG (1 and 2 mM did not significantly affect motility, VSL and oxygen consumption of sperm extended in the lactate-containing medium. While sperm motility and oxygen consumption were significantly inhibited by even 1mM 2DG in sperm extended in lactate-free medium. Sperm motility significantly inhibited in the case of sperm extended in lactate-containing and free-medium with 1 mM OX. We also found that sperm motility was not maintained in the absence of lactate throughout the 3 h incubation period. Immunofluorescence study revealed that mainly LDH-C was may be localized in the intramitochondrial region of the sperm. Conclusion: These results suggest that exogenous lactate enhances lactate oxidation by LDH-C, thereby promoting mitochondrial oxidative reactions in the midpiece and maintaining the mobilization of rat epididymal sperm.

  11. 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

  12. 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

    2014-10-21

    The flavin-mediated enzymatic oxidation of a CN bond in amino acids can occur through hydride transfer, carbanion, or polar nucleophilic mechanisms. Previous results with D-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) using multiple deuterium kinetic isotope effects (KIEs) and computational studies established preferred binding of the substrate protonated on the α-amino group, with cleavages of the NH and CH bonds occurring in asynchronous fashion, consistent with the three possible mechanisms. The hydroxyl groups of Y53 and Y249 are ≤4 Å from the imino and carboxylate groups of the reaction product iminoarginine, suggesting participation in binding and catalysis. In this study, we have investigated the reductive half-reactions of the Y53F and Y249F variants of PaDADH using substrate and solvent deuterium KIEs, solvent viscosity and pH effects, and quantum mechanical/molecular mechanical computational approaches to gain insights into the catalytic roles of the tyrosines and evaluate whether their mutations affect the transition state for substrate oxidation. Both Y53F and Y249F enzymes oxidized D-arginine with steady-state kinetic parameters similar to those of the wild-type enzyme. Rate constants for flavin reduction (k(red)) with D-leucine, a slow substrate amenable to rapid kinetics, were 3-fold smaller than the wild-type value with similar pKa values for an unprotonated group of ∼10.0. Similar pKa values were observed for (app)Kd in the variant and wild-type enzymes. However, cleavage of the substrate NH and CH bonds in the enzyme variants occurred in synchronous fashion, as suggested by multiple deuterium KIEs on k(red). These data can be reconciled with a hydride transfer mechanism, but not with carbanion and polar nucleophilic mechanisms.

  13. Transcriptional Regulation of Pyruvate Dehydrogenase Kinase

    Directory of Open Access Journals (Sweden)

    Ji Yun Jeong

    2012-10-01

    Full Text Available The pyruvate dehydrogenase complex (PDC activity is crucial to maintains blood glucose and ATP levels, which largely depends on the phosphorylation status by pyruvate dehydrogenase kinase (PDK isoenzymes. Although it has been reported that PDC is phosphorylated and inactivated by PDK2 and PDK4 in metabolically active tissues including liver, skeletal muscle, heart, and kidney during starvation and diabetes, the precise mechanisms by which expression of PDK2 and PDK4 are transcriptionally regulated still remains unclear. Insulin represses the expression of PDK2 and PDK4 via phosphorylation of FOXO through PI3K/Akt signaling pathway. Several nuclear hormone receptors activated due to fasting or increased fat supply, including peroxisome proliferator-activated receptors, glucocorticoid receptors, estrogen-related receptors, and thyroid hormone receptors, also participate in the up-regulation of PDK2 and PDK4; however, the endogenous ligands that bind those nuclear receptors have not been identified. It has been recently suggested that growth hormone, adiponectin, epinephrine, and rosiglitazone also control the expression of PDK4 in tissue-specific manners. In this review, we discuss several factors involved in the expressional regulation of PDK2 and PDK4, and introduce current studies aimed at providing a better understanding of the molecular mechanisms that underlie the development of metabolic diseases such as diabetes.

  14. RESEARCH ON THE INFLUENCE OF H+ IONS CONCENTRATION ON THE DYNAMICS OF THE ACTIVITIES OF CERTAIN DEHYDROGENASES OF THE KREBS CYCLE IN THE MONILINIA LAXA (ADERH. & RUHL. HONEY FUNGUS PARASITIC ON PLUM TREES

    Directory of Open Access Journals (Sweden)

    Tutu Elena

    2010-09-01

    Full Text Available During the process of nutrition, thus in that of their growth, microorganisms are subject to the influences of certain environmental factors that condition the microbial activity determining either the growth and reproduction, or the inhibition of activity and the inactivation of microorganisms. A well known means of expressing the H+ ions concentration in a certain environment is the pH, an important chemical factor that is closely observed when growing ascomycetes, for any alteration of its value entails conformational alterations of their enzymes, the characteristics of the substrate, such that they can no longer interact with the active site of the enzyme or be subject to catalysis. The present study comprises the results of our research on certain oxidoreductase implied in the steps of the Krebs cycle in the Monilinia laxa (Aderh.&Ruhl. Honey, a fungus that parasites the prune. The enzymatic determinations took place at 7 and 14 days from the mycelium of the fungus cultivated in Leonian media, whose pH was adjusted to values between 2.0 and 9.0 by using NaOH 1N and HCl 0,1N solutions. We registered different values of the dehydrogenasic activity, directly correlated with the physiological condition of the fungus (given its age and with the initial pH value of the culture’s environment.

  15. 重组NADH氧化酶对乳酸脱氢酶乳酸氧化活性的影响%Effects of Recombinant NADH Oxidase on the Lactate Oxidation Activity of Lactate Dehydrogenase

    Institute of Scientific and Technical Information of China (English)

    赵蕊; 霍贵成

    2013-01-01

    [目的]考察当存在其他利用NADH途径时,发酵型乳酸脱氢酶(lactate dehydrogenase,LDH)催化乳酸氧化能力的改变.[方法]PCR扩增乳酸乳球菌(Lactococcus lactis,L.lactis)中生成H2O的NADH氧化酶基因noxE,将其连接至表达载体并在大肠杆菌中过量表达;对亲和纯化的产物进行SDS-PAGE分析、光谱扫描和活性测定,考察纯化产物是否具有生物学活性;以2,4-二硝基苯肼法测定乳酸脱氢酶的乳酸氧化活性,考察添加NoxE重组蛋白对其活性的影响.[结果]重组NoxE蛋白是种黄素蛋白,具明显的生物学活性,说明noxE表达载体构建成功;添加NoxE后,LDH的乳酸氧化活性提高了3.84倍.[结论]在NADH经呼吸链代谢掉的生理条件下,LDH催化乳酸氧化的能力会明显提高.%[ Objective] To compare the lactate oxidation activity of lactate dehydrogenase (LDH) in the presence and absence of another NADH utilization pathway. [Method] The H2O-producing NADH oxidase gene (noxE) was cloned by PCR from Lactococcws lactis genome, ligated into the expression vector and expressed in E. coli. After affinity purification, the recombinant protein was analyzed by SDS-PAGE, UV-vis absorption spectrum and determination of enzyme activity. 2,4-Dinitrophenylhydrazine was used to evaluate the effect of NoxE addition on the lactate oxidation activity of LDH. [Result]NoxE was purified as a flavin protein with significant activity. When NoxE was added, the lactate oxidation activity of LDH was increased 3.84-fold. [ Conclusion]The lactate oxidation capacity of LDH will be significantly increased under physical conditions where NADH can be consumed via respiration chain.

  16. Use of a simplified spectrophotometric method for quantitative determination of glucose-6-phosphate dehydrogenase activity in normal children from two day-care centers of the city of São Paulo

    Directory of Open Access Journals (Sweden)

    Roberto Muller

    2003-06-01

    Full Text Available Objective: To evaluate the applicability of a simplified method forquantitative determination of glucose-6-phosphate dehydrogenaseactivity in normal children; to determine the mean, standarddeviation and threshold value under which the enzyme activity isconsidered deficient. Methods: Blood samples were collected from201 children from two day-care centers in the city of São Paulo.The subjects were considered normal based on physicalexamination and laboratory tests. The enzyme activity wasdetermined in red blood cells of normal children using the “TestCombination G-6-PDH®” kit. The following statistical analyses werecarried out: the results were submitted to Student’s t test,Kolmogorov-Smirnov test, lower confidence interval (one-tailedtest and Spearman’s correlation coefficient. Results: The meanhemoglobin value for girls was slightly higher than the mean valuefor boys, but this difference was not statistically significant. Therewas no statistical difference in mean enzyme activities for Caucasianand non-Caucasian children. There was no significant correlation amongenzyme activity levels, red blood cells, hemoglobin levels,hematocrit, reticulocytes, white blood cells and age of patients.The mean enzyme activity for boys was 4.448 U/g Hb, standarddeviation = 1.380 U/g Hb. For girls, the mean enzyme activity was4.531 U/g Hb, standard deviation = 1.386 U/g Hb, and the differencewas not statistically significant. Therefore, the two populationgroups were considered as one single population, presenting amean enzyme activity of 4.490 U/g Hb, standard deviation = 1.380 U/g Hb.Since the distribution curve of enzyme activity values was normal,a lower confidence interval was determined (one-tailed test, witha cutoff point of 2.227 U/g Hb. Conclusion: The method used bySolem proved to be simple, fast, very accurate and useful to detectglucose-6-phosphate dehydrogenase activity and to identifychildren with enzyme deficiency.

  17. CONTEMPORARY ISSUES USING OF ELECTRONIC CLASSBOOKS IN LEARNING ACTIVITIES

    Directory of Open Access Journals (Sweden)

    M. G. Kuzmin

    2016-01-01

    Full Text Available The article is devoted to contemporary issues of informatization of educational process in higher education through the use of electronic classbooks. The article deals with the concept and content of the electronic textbook examples of foreign experience in the use of such training facilities. Also, there is question of didactic features of the use of electronic classbooks in the framework of students‟ personal work. Support for the full cycle of didactic electronic textbook means having built into it a pedagogical goal - to achieve acceptable results all students mastering topics - and the means to achieve this by laying individual trajectory of each student based on interactivity. Additionally, the article says that the work of the electronic textbook has all the features of distance learning, the learning process using the electronic textbook should be organized as a process of blended learning. At the same time the student himself determines the duration, tempo, and time of the work. The article also highlighted the benefits that electronic textbook gives the participants of the educational process that leads to the conclusion that the electronic textbook, being modern means of education significantly improves the quality and effectiveness of training, the motivation of students and optimizes the work of the teacher.

  18. Effectiveness of Electronic Textbooks with Embedded Activities on Student Learning

    Science.gov (United States)

    Porter, Paula L.

    2010-01-01

    Current versions of electronic textbooks mimic the format and structure of printed textbooks; however, the electronic capabilities of these new versions of textbooks offer the potential of embedding interactive features of web-based learning within the context of a textbook. This dissertation research study was conducted to determine if student…

  19. 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

  20. Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15

    Directory of Open Access Journals (Sweden)

    Sakuko Ueshima

    2010-01-01

    Full Text Available The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3 were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD+-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienylserine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO2.

  1. Histochemical activity of 5-4-isomerase-3-B hydroxy steroid dehydrogenase in the ovary of the viviparous mexican lizardSceloporus mucronatus (Reptilia:Prhynosomatidae) and interelationship with progesterone levels during pregnancy

    Institute of Scientific and Technical Information of China (English)

    Martn Martnez-torres; E Martha Prez-armendariz; M Elena Hernndez Caballero; Juana luis; guadalupe ortz-Lpez

    2012-01-01

    Objective:To relate the histological characteristics and histochemicalΔ5-4-isomerase-3 beta hydroxy steroid dehydrogenase(Δ5-43β-HSD) activity of the corpora lutea(CL) and the atresic vitellogenic follicles(AVF) with progesterone(P4) plasma concentrations in three different times of gestation (early, medium and late) in the viviparous lizardSceloporus mucronatus (S. mucronatus).Methods:The histological characteristics as well as histochemical activity ofΔ5-43β-HSD of theCL andAVF and their relationship with plasmaP4 levels were studied during three different times of pregnancy of the viviparous lizardS. mucronatus.Results:Corpora lutea develops during the first third of gestation.In second third, the luteal tissue reaches maturity and starts the first regressive changes.The last third of gestation was characterized by a considerably advance in the luteolysis.Activity ofΔ5-43β-HSD was observed in he luteal cell mas.The activity of this enzyme were high during the first third and scantle activity was detected in the last third.Even though atresic vitellogenic follicles are found throughout the whole period of gestation,Δ5-43β-HSD activity is very low in relation with showed byCL and does not change significantly in the studied period of time.Another hand, we observed a direct relationship among the histological aspect of the corpus luteum,Δ5-43β-HSD activity and progesterone levels. Conclusions:These observations suggests that the corpus luteum is the most important source of ovarian progesterone(P4) during pregnancy and that the participation of theAVF in the production of this hormone is little or non-existent.

  2. Activating Knowledge Through Electronic Collaboration: Vanquishing The Knowledge Paradox

    NARCIS (Netherlands)

    S. Qureshi (Sadja); P. Keen (Peter)

    2004-01-01

    textabstractElectronic collaboration has become a driver for productivity as organizations develop linkages for the planning, sourcing and execution of goods and services. These organizations require mechanisms to harness the diverse and personalized intellectual resources that are distributed acr

  3. Analysis on the Dehydrogenase Activity in Cold-resistant Bacteria in the Processor of Low-temperature Wastewater%低温污水处理器中耐冷菌脱氢酶活性分析

    Institute of Scientific and Technical Information of China (English)

    姜明; 郭立萍; 曲艳娇; 曹振岭; 韩晓云

    2009-01-01

    [Objective] The aim was to supply technical support for solving the problem that the sewage was difficult to dispose in north China in winter. [Method] The influences of pH value, temperature and time on the dehydrogenase activity in 8 strains of cold-resistant bacteria isolated, purified and identified from low-temperature biofilm were analyzed by TTC method. [Result] The optimum pH value of the strains S1, S4, S5, S7 and S8 at both optimum temperature and 4 ℃ was 7.5 and that of the other 3 strains at the 2 temperatures were different, but they were in the range of 7.5-8.5. The optimum reaction temperature of the strains S1, S3, S4, S5 and S6 was 30 ℃ and that of the strains S2, S7 and S8 was 20 ℃. Except for the strain S3, the optimum reaction time of the other strains at 4 ℃ was longer than their optimum reaction time at optimum reaction temperature. At 4 ℃, the optimum pH values of the strains S3, S4 and S6 were 8.5, 7.5 and 8.0 resp., their optimum reaction time were 0.6, 12.0 and 0.5 h resp. and their highest dehydrogenase activities were 7.19, 6.73 and 7.70 mg(TF)/g(SS) resp. [Conclusion] The dehydrogenase activities of the strains S3, S4 and S6 were higher and they were suitable to be used in processing low-temperature sewage.%[目的]为解决我国北方冬季污水处理困难的问题提供技术支持.[方法]运用TTC法分析了pH值、温度和时间对从低温生物膜中分离纯化鉴定得到8株耐冷菌中脱氢酶活性的影响.[结果]菌株S1、S4、S5、S7和S8在最适温度和4 ℃下的最适pH值都是7.5,其他3株菌在2种温度下的最适pH值不同,但都在7.5~8.5的范围内.菌株S1、S3、S4、S5和S6的最适反应温度为30 ℃,菌株S2、S7和S8的最适反应温度为20 ℃.除菌株S3外,其余菌株在4 ℃下的最适反应时间比最适反应温度下的长.在4 ℃下,菌株S3、S4和S6的最适pH值和最适反应时间分别为8.5和0.6 h、7.5和12.0 h、8.0和0.5 h,其最

  4. 殊异韦荣菌中乳酸脱氢酶重组蛋白的表达、纯化及活性分析%The recombinant expression, purification and activity analysis of lactate dehydrogenase in Veillonella dispar

    Institute of Scientific and Technical Information of China (English)

    何钟勤; 钟丞; 高心; 薛莹; 孙晓宇; 刘晓红

    2012-01-01

    Objective To determine dependence lactate dehydrogenase activity in Veillonella dispar.Methods Transformed the recombinant plasmid pET-28a-LDH in BL21 (DE3) and Rosetta(DE3).Recombinant lactate dehydrogenase(LDH) protein was induced at different conditions.Induction condition was optimized to obtain proper yield of recombinant protein.After purification by HisTRAP FF column,the protein activity was determined with LDH reactive kit.Results Sodium dodecylsulfate-polyacrylamide gel electrophoresis showed that the best protein expression conditions were isopropylthio-β-D-galactoside (IPTG)end for 0.4 mmol/L concentration in 30 degrees to induce 8 hours,or IPTG end for 0.4 mmol/L concentration in 30 degrees to induce 6 hours.Through the analysis of BandScan 5.0,the expression in BL21 (DE3) was 9.0%,higher than 6.1% in Rosetta(DE3).After purification by HisTRAP FF column,the protein active value of 13.79.Conclusions Recombinant LDH protein could be induced by IPTG with an optimal condition.%目的 进一步测定殊异韦荣菌中依赖性乳酸脱氢酶(lactate dehydrogenase,LDH)的活性,以期为研究该酶的具体功能及作用机制奠定基础,并为龋病预防和治疗提供新的思路.方法 重组质粒转入大肠杆菌BL21(DE3)和Rosetta(DE3)中,采用不同时间及6种不同浓度的异丙基硫代-β-D-半乳糖苷(isopropyhhio-β-D-galactoside,IPTG)诱导乳酸脱氢酶蛋白表达,选择最佳条件诱导LDH蛋白表达.并经His-标记蛋白纯化柱(HisTRAP FF柱)提纯,用LDH活性试剂盒测定蛋白活性.结果 pET-28a-LDH转入BL21(DE3)和Rosetta(DE3)中十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示最适表达条件分别为IPTG终浓度0.4 mmol/L、30℃诱导8h,IPTG终浓度0.4 mmol/L、30 ℃诱导6h.经BandScan 5.0软件分析,pET-28a-LDH质粒转化BL21(DE3)表达相对含量为9.0%,高于Rosetta(DE3)的6.1%;LDH活性试剂盒测定的蛋白活性值为13.79.结论 本项研究获得了LDH蛋白的最适诱导条

  5. 76 FR 56503 - Agency Information Collection Activity (VSO Access to VHA Electronic Health Records) Under OMB...

    Science.gov (United States)

    2011-09-13

    ... AFFAIRS Agency Information Collection Activity (VSO Access to VHA Electronic Health Records) Under OMB... INFORMATION: Title: VSO Access to VHA Electronic Health Records, VA Form 10- 0400. OMB Control Number: 2900... recorded in VHA electronic health records system. An agency may not conduct or sponsor, and a person is not...

  6. 76 FR 40454 - Proposed Information Collection (VSO Access to VHA Electronic Health Records) Activity; Comment...

    Science.gov (United States)

    2011-07-08

    ... AFFAIRS Proposed Information Collection (VSO Access to VHA Electronic Health Records) Activity; Comment... attorney by veterans who have medical information recorded in VHA electronic health records system... techniques or the use of other forms of information technology. Title: VSO Access to VHA Electronic Health...

  7. Activating Knowledge Through Electronic Collaboration: Vanquishing The Knowledge Paradox

    OpenAIRE

    Qureshi, Sadja; Keen, Peter

    2004-01-01

    textabstractElectronic collaboration has become a driver for productivity as organizations develop linkages for the planning, sourcing and execution of goods and services. These organizations require mechanisms to harness the diverse and personalized intellectual resources that are distributed across the world. While electronic collaboration technologies have made it possible to harness intellectual resources across space and time, knowledge management is locked in a paradox of perception – t...

  8. 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.

  9. Purification and characterization of 3-isopropylmalate dehydrogenase from Thiobacillus thiooxidans.

    Science.gov (United States)

    Kawaguchi, H; Inagaki, K; Matsunami, H; Nakayama, Y; Tano, T; Tanaka, H

    2000-01-01

    3-Isopropylmalate dehydrogenase was purified to homogeneity from the acidophilic autotroph Thiobacillus thiooxidans. The native enzyme was a dimer of molecular weight 40,000. The apparent K(m) values for 3-isopropylmalate and NAD+ were estimated to be 0.13 mM and 8.7 mM, respectively. The optimum pH for activity was 9.0 and the optimum temperature was 65 degrees C. The properties of the enzyme were similar to those of the Thiobacillus ferrooxidans enzyme, expect for substrate specificity. T. thiooxidans 3-isopropylmalate dehydrogenase could not utilize malate as a substrate.

  10. Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1

    Directory of Open Access Journals (Sweden)

    Zhou Cong-Zhao

    2007-06-01

    Full Text Available Abstract Background As the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG. Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications. Results The crystal structure of 6-phosphogluconate dehydrogenase (6PGDH/Gnd1 from Saccharomyces cerevisiae has been determined at 2.37 Å resolution by molecular replacement. The overall structure of Gnd1 is a homodimer with three domains for each monomer, a Rossmann fold NADP+ binding domain, an all-α helical domain contributing the majority to hydrophobic interaction between the two subunits and a small C-terminal domain penetrating the other subunit. In addition, two citrate molecules occupied the 6PG binding pocket of each monomer. The intact Gnd1 had a Km of 50 ± 9 μM for 6-phosphogluconate and of 35 ± 6 μM for NADP+ at pH 7.5. But the truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lost their 6PGDH activity, despite remaining the homodimer in solution. Conclusion The overall tertiary structure of Gnd1 is similar to those of 6PGDH from other species. The substrate and coenzyme binding sites are well conserved, either from the primary sequence alignment, or from the 3D structural superposition. Enzymatic activity assays suggest a sequential mechanism of catalysis, which is in agreement with previous studies. The C-terminal domain of Gnd1 functions as a hook to further tighten the dimer, but it is not

  11. 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.

  12. A role for tungsten in the biology of Campylobacter jejuni: tungstate stimulates formate dehydrogenase activity and is transported via an ultra-high affinity ABC system distinct from the molybdate transporter.

    Science.gov (United States)

    Smart, Jonathan P; Cliff, Matthew J; Kelly, David J

    2009-11-01

    The food-borne pathogen Campylobacter jejuni possesses no known tungstoenzymes, yet encodes two ABC transporters (Cj0300-0303 and Cj1538-1540) homologous to bacterial molybdate (ModABC) uptake systems and the tungstate transporter (TupABC) of Eubacterium acidaminophilum respectively. The actual substrates and physiological role of these transporters were investigated. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry of the purified periplasmic binding proteins of each system revealed that while Cj0303 is unable to discriminate between molybdate and tungstate (K(D) values for both ligands of 4-8 nM), Cj1540 binds tungstate with a K(D) of 1.0 +/- 0.2 pM; 50 000-fold more tightly than molybdate. Induction-coupled plasma mass spectroscopy of single and double mutants showed that this large difference in affinity is reflected in a lower cellular tungsten content in a cj1540 (tupA) mutant compared with a cj0303c (modA) mutant. Surprisingly, formate dehydrogenase (FDH) activity was decreased approximately 50% in the tupA strain, and supplementation of the growth medium with tungstate significantly increased FDH activity in the wild type, while inhibiting known molybdoenzymes. Our data suggest that C. jejuni possesses a specific, ultra-high affinity tungstate transporter that supplies tungsten for incorporation into FDH. Furthermore, possession of two MoeA paralogues may explain the formation of both molybdopterin and tungstopterin in this bacterium.

  13. Dietary modulation of erythrocyte insulin receptor interaction and the regulation of adipose tissue pyruvate dehydrogenase enzyme activity in growing rats; a mechanism of action of dietary fiber in metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Ogunwole, J.O.A.

    1984-01-01

    The metabolic effects of graded cellulose (a dietary fiber) intake were studied at minimal (10%) and maximal (20%) protein levels in male weanling Sprague Dawley rats. The hypothesis was tested that the hypoglycemic effect of high fiber diets is partly mediated through increased tissue sensitivity to insulin at the cell receptor level. Erythrocyte insulin receptor interaction (IRI) and percent insulin stimulation of adipose tissue pyruvate dehydrogenase (PDH) activity (PDS) were used as indices of tissue sensitivity to insulin. IRI was determined by a standardized radioceptor assay PDS by the rate of oxidation of 1-/sup 14/C-pyruvate to /sup 14/CO/sub 2/ in epidymal fat pads and serum insulin levels by radioimmunoassay. In both protein groups, the addition of fiber in the diet resulted in a significant (P < 0.05) increase in food intake (FI) for calorie compensation. Fiber and protein intake had a significant (P < 0.01) effect on IRI and both basal (PDB) and PDS activities of PDH. At all fiber levels, specific percent /sup 125/I-insulin binding (SIB) was higher in the 20% protein groups while in the fiber-free group, a higher SIB was observed in the 10% protein group.

  14. Fungal laccase, cellobiose dehydrogenase, and chemical mediators: combined actions for the decolorization of different classes of textile dyes.

    Science.gov (United States)

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

    2008-10-01

    Dyes belonging to the mono-, di-, tri- and poly-azo as well as anthraquinonic and mono-azo Cr-complexed classes, chosen among the most utilized in textile applications, were employed for a comparative enzymatic decolorization study using the extracellular crude culture extracts from the white rot fungus Funalia (Trametes) trogii grown on different culture media and activators able to trigger different levels of expression of oxidizing enzymes: laccase and cellobiose dehydrogenase. Laccase containing extracts were capable to decolorize some dyes from all the different classes analyzed, whereas the recalcitrant dyes were subjected to the combined action of laccase and the chemical mediator HBT, or laccase plus cellobiose dehydrogenase. Correlations among the decolorization degree of the various dyes and their electronic and structural diversities were rationalized and discussed. The utilization of cellobiose dehydrogenase in support to the activity of laccase for the decolorization of azo textile dyes resulted in substantial increases in decolorization for all the refractory dyes proving to be a valid alternative to more expensive and less environmentally friendly chemical treatments of textile dyes wastes.

  15. Regulation of synthesis and activity of NAD(+)-dependent 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) by dexamethasone and phorbol ester in human erythroleukemia (HEL) cells

    Energy Technology Data Exchange (ETDEWEB)

    Xun, C.Q.; Ensor, C.M.; Tai, H.H. (Univ. of Kentucky, Lexington (USA))

    1991-06-28

    Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and (35S)methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism.

  16. Study on determination of biofilm activity in BAF by TTC-dehydrogenase assay%优化TTC-脱氢酶还原法测定陶粒负载微生物活性

    Institute of Scientific and Technical Information of China (English)

    齐鲁青; 汪晓军; 詹德明

    2012-01-01

    利用氯化三苯基四氮唑(TTC)-脱氢酶还原法定量测定了曝气生物滤池中陶粒负载生物膜的活性,并对影响生物活性测定的因素进行了分析和优化.实验表明,经优化后得出最佳培养条件是:陶粒采用原位法制样,依次加入pH为8.0的Tris-HCl缓冲液,质量分数为0.4%的TTC溶液,0.1 mol/L葡萄糖溶液,在温度40℃下培养4h,最后生成的三苯基甲臢(TriphenylFormazone,TF)用甲苯萃取可以除去有色废水的颜色影响.采用TTC-脱氢酶还原法可以快速、方便的检测曝气生物滤池中陶粒负载生物膜的生物活性,并能很好的进行定量化测定.%The biofilms activity in Biological Aerated Filters (BAF) is quantitatively determined by 2,3,5-triphenyl tetrazolium chloride (TTC) -dehydrogenase assay and some major factors influencing the determination are studied in this paper. The optimum parameters are shown as follows: Tris-HCl buffers and 8. 0 of pH, 0. 4% ( mass fraction) of TTC and 0. 1 mol/L of dextrose solution. The incubation time and temperature are 4 h and 401 , respectively. The formazan reduction product can be efficiently extracted by methylbenzene, which can reduce the disturbance of dye color. This study demonstrates that the TTC-dehydrogenase assay can be successfully applied to the determination of biofilms activity in BAF.

  17. Immunolocalization of succinate dehydrogenase in the esophagus epithelium of domesticated mammals.

    Science.gov (United States)

    Meyer, W; Kacza, J; Hornickel, I N; Schoennagel, B

    2013-05-10

    Using immunohistochemistry and transmission electron microscopy (TEM), the esophagus epithelia of seven domesticated mammals (horse, cattle, goat, pig, dog, laboratory rat, cat) of three nutrition groups (herbivorous, omnivorous, carnivorous) were studied to get first information about energy generation, as demonstrated by succinate dehydrogenase (SDH) activities. Distinct reaction intensities could be observed in all esophageal cell layers of the different species studied reflecting moderate to strong metabolic activities. The generally strong staining in the stratum basale indicated that new cells are continuously produced. The latter feature was confirmed by a thick, and in the horse generally highly active stratum spinosum. Only in the pig, reaction intensity variations occurred, obviously related to differences in physical feed quality or restricted feed allocation. The immunohistochemical results were corroborated by the presence of intact mitochondria in the esophageal cells of all species and nutrition types studied, except for the horse. Possible relationships between SDH reaction intensities and feed structure, mass or consistency are discussed.

  18. Immunolocalization of succinate dehydrogenase in the esophagus epithelium of domesticated mammals

    Directory of Open Access Journals (Sweden)

    W. Meyer

    2013-05-01

    Full Text Available Using immunohistochemistry and transmission electron microscopy (TEM, the esophagus epithelia of seven domesticated mammals (horse, cattle, goat, pig, dog, laboratory rat, cat of three nutrition groups (herbivorous, omnivorous, carnivorous were studied to get first information about energy generation, as demonstrated by succinate dehydrogenase (SDH activities. Distinct reaction intensities could be observed in all esophageal cell layers of the different species studied reflecting moderate to strong metabolic activities. The generally strong staining in the stratum basale indicated that new cells are continuously produced. The latter feature was confirmed by a thick, and in the horse generally highly active stratum spinosum. Only in the pig, reaction intensity variations occurred, obviously related to differences in physical feed quality or restricted feed allocation. The immunohistochemical results were corroborated by the presence of intact mitochondria in the esophageal cells of all species and nutrition types studied, except for the horse. Possible relationships between SDH reaction intensities and feed structure, mass or consistency are discussed.

  19. Response of Chloroplast NAD(PH Dehydrogenase-Mediated Cyclic Electron Flow to a Shortage or Lack in Ferredoxin-Quinone Oxidoreductase-Dependent Pathway in Rice Following Short-Term Heat Stress

    Directory of Open Access Journals (Sweden)

    Jemaa eEssemine

    2016-03-01

    Full Text Available Cyclic electron flow around PSI can protect photosynthetic electron carriers under conditions of stromal over-reduction. The goal of the research reported in this paper was to investigate the responses of both PSI and PSII to a short-term heat stress in two rice lines with different capacities of cyclic electron transfer, i.e. Q4149 with a high capacity (hcef and C4023 with a low capacity (lcef. The absorbance change at 820 nm (ΔA820 was used here to assess the charge separation in the photosystem I (PSI reaction center (P700. The results obtained show that short-term heat stress abolishes the FQR-dependent CEF in rice and accelerates the initial rate of P700+ re-reduction. The P700+ amplitude was slightly increased at a moderate heat-stress (35°C because of a partial restriction of FQR but it was decreased following high heat-stress (42°C. Assessment of PSI and PSII activities shows that PSI is more susceptible to heat stress than photosystem II (PSII. Under high temperature, FQR-dependent CEF was completely removed and NDH-dependent CEF was up-regulated and strengthened to a higher extent in C4023 than in Q4149. Specifically, under normal growth temperature, hcef (Q4149 was characterized by higher FQR- and NDH-dependent CEF rates than lcef (C4023. Following thermal stress, the activation of NDH-pathway was 130% and 10% for C4023 and Q4149, respectively. Thus, the NDH-dependent CEF may constitute the second layer of plant protection and defence against heat stress after the main route, i.e. FQR-dependent CEF, reaches its capacity. We discuss the possibility that under high heat stress, the NDH pathway serves as a safety valve to dissipate excess energy by cyclic photophosphorylation and overcome the stroma over-reduction following inhibition of CO2 assimilation and any shortage or lack in the FQR pathway. The potential role of the NDH-dependent pathway during the evolution of C4 photosynthesis is briefly discussed.

  20. Purification and characterization of NADP(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Peptostreptococcus productus marburg.

    Science.gov (United States)

    Wohlfarth, G; Geerligs, G; Diekert, G

    1991-01-01

    The 5,10-methylenetetrahydrofolate dehydrogenase of heterotrophically grown Peptostreptococcus productus Marburg was purified to apparent homogeneity. The purified enzyme catalyzed the reversible oxidation of methylenetetrahydrofolate with NADP+ as the electron acceptor at a specific activity of 627 U/mg of protein. The Km values for methylenetetrahydrofolate and for NADP+ were 27 and 113 microM, respectively. The enzyme, which lacked 5,10-methenyltetrahydrofolate cyclohydrolase activity, was insensitive to oxygen and was thermolabile at temperatures above 40 degrees C. The apparent molecular mass of the enzyme was estimated by gel filtration to be 66 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a single subunit of 34 kDa, accounting for a dimeric alpha 2 structure of the enzyme. Kinetic studies on the initial reaction velocities with different concentrations of both substrates in the absence and presence of NADPH as the reaction product were interpreted to indicate that the enzyme followed a sequential reaction mechanism. After gentle ultracentrifugation of crude extracts, the enzyme was recovered to greater than 95% in the soluble (supernatant) fraction. Sodium (10 microM to 10 mM) had no effect on enzymatic activity. The data were taken to indicate that the enzyme was similar to the methylenetetrahydrofolate dehydrogenases of other homoacetogenic bacteria and that the enzyme is not involved in energy conservation of P. productus. PMID:1899860

  1. P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations.

    Science.gov (United States)

    Beyer, Nina; Kulig, Justyna K; Bartsch, Anette; Hayes, Martin A; Janssen, Dick B; Fraaije, Marco W

    2017-03-01

    To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase. Phosphite-driven conversions of lauric acid at restricted NADPH concentrations confirmed multiple turnovers of the cofactor. Interestingly, both the fusion enzyme and the native P450BM3 displayed enzyme concentration dependent activity and the fused enzyme reached optimal activity at a lower enzyme concentration. This suggests that the fusion enzyme has an improved tendency to form functional oligomers.To explore the constructed phosphite-driven P450BM3 as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450BM3 driven by phosphite was found to be more efficient in terms of total turnover when compared with P450BM3 driven by NADPH. The results suggest that PTDH-P450BM3 is an attractive system for use in biocatalytic and drug metabolism studies.

  2. Isolation, characterization and evaluation of the Pichia pastoris sorbitol dehydrogenase promoter for expression of heterologous proteins.

    Science.gov (United States)

    Periyasamy, Sankar; Govindappa, Nagaraj; Sreenivas, Suma; Sastry, Kedarnath

    2013-11-01

    Sorbitol is used as a non-repressive carbon source to develop fermentation process for Mut(s) recombinant clones obtained using the AOX1 promoter in Pichia pastoris. Sorbitol dehydrogenase is an enzyme in the carbohydrate metabolism that catalyzes reduction of D-fructose into D-sorbitol in the presence of NADH. The small stretch of 211bps upstream region of sorbitol dehydrogenase coding gene has all the promoter elements like CAAT box, GC box, etc. It is able to promote protein production under repressive as well as non-repressive carbon sources. In this study, the strength of the sorbitol dehydrogenase promoter was evaluated by expression of two heterologous proteins: human serum albumin and erythrina trypsin inhibitor. Sorbitol dehydrogenase promoter allowed constitutive expression of recombinant proteins in all carbon sources that were tested to grow P. pastoris and showed activity similar to GAP promoter. The sorbitol dehydrogenase promoter was active in all the growth phases of the P. pastoris.

  3. Red Algal Bromophenols as Glucose 6-Phosphate Dehydrogenase Inhibitors

    Directory of Open Access Journals (Sweden)

    Koretaro Takahashi

    2013-10-01

    Full Text Available Five bromophenols isolated from three Rhodomelaceae algae (Laurencia nipponica, Polysiphonia morrowii, Odonthalia corymbifera showed inhibitory effects against glucose 6-phosphate dehydrogenase (G6PD. Among them, the symmetric bromophenol dimer (5 showed the highest inhibitory activity against G6PD.

  4. Purification and characterization of xylitol dehydrogenase from Fusarium oxysporum

    DEFF Research Database (Denmark)

    Panagiotou, Gianni; Kekos, D.; Macris, B.J.

    2002-01-01

    An NAD(+)-dependent xylitol dehydrogenase (XDH) from Fusarium oxysporum, a key enzyme in the conversion of xylose to ethanol, was purified to homogeneity and characterised. It was homodimeric with a subunit of M-r 48 000, and pI 3.6. It was optimally active at 45degreesC and pH 9-10. It was fully...

  5. Lactate dehydrogenase in the cyanobacterium Microcystis PCC7806

    NARCIS (Netherlands)

    Moezelaar, R.; Teixeira, de M.J.; Stal, L.J.

    1995-01-01

    The cyanobacterium Microcystis PCC7806 was found to possess an NAD-dependent lactate dehydrogenase (EC 1.1.1.27) which catalyzes the reduction of pyruvate to l-lactate. The enzyme required fructose 1,6-bisphosphate for activity and displayed positive cooperativity towards pyruvate. Lactate was not

  6. Electron self-exchange activation parameters of diethyl sulfide and tetrahydrothiophene

    Directory of Open Access Journals (Sweden)

    Martin Goez

    2013-07-01

    Full Text Available Electron transfer between the title compounds and their radical cations, which were generated by photoinduced electron transfer from the sulfides to excited 2,4,6-triphenylpyrylium cations, was investigated by time-resolved measurements of chemically induced dynamic nuclear polarization (CIDNP in acetonitrile. The strongly negative activation entropies provide evidence for an associative–dissociative electron exchange involving dimeric radical cations. Despite this mechanistic complication, the free energies of activation were found to be well reproduced by the Marcus theory of electron transfer, with the activation barrier still dominated by solvent reorganization.

  7. Promoting direct interspecies electron transfer with activated carbon

    DEFF Research Database (Denmark)

    Liu, Fanghua; Rotaru, Amelia-Elena; Shrestha, Pravin M.

    2012-01-01

    of methanogenesis might be to facilitate direct interspecies electron transfer (DIET) between bacteria and methanogens. Metabolism was substantially accelerated when GAC was added to co-cultures of Geobacter metallireducens and Geobacter sulfurreducens grown under conditions previously shown to require DIET. Cells...... were attached to GAC, but did not aggregate as they do when making biological electrical connections between cells. Studies with a series of gene deletion mutants eliminated the possibility that GAC promoted electron exchange via interspecies hydrogen or formate transfer and demonstrated that DIET...

  8. Site directed spin labelling and pulsed dipolar electron paramagnetic resonance (double electron-electron resonance) of force activation in muscle

    Energy Technology Data Exchange (ETDEWEB)

    Fajer, Piotr G [Institute of Molecular Biophysics, Department of Biological Science, National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310 (United States)

    2005-05-11

    The recent development of site specific spin labelling and advances in pulsed electron paramagnetic resonance(EPR) have established spin labelling as a viable structural biology technique. Specific protein sites or whole domains can be selectively targeted for spin labelling by cysteine mutagenesis. The secondary structure of the proteins is determined from the trends in EPR signals of labels attached to consecutive residues. Solvent accessibility or label mobility display periodicities along the labelled polypeptide chain that are characteristic of {beta}-strands (periodicity of 2 residues) or {alpha}-helices (3.6 residues). Low-resolution 3D structure of proteins is determined from the distance restraints. Two spin labels placed within 60-70 A of each other create a local dipolar field experienced by the other spin labels. The strength of this field is related to the interspin distance, {proportional_to} r{sup -3}. The dipolar field can be measured by the broadening of the EPR lines for the short distances (8-20 A) or for the longer distances (17-70 A) by the pulsed EPR methods, double electron-electron resonance(DEER) and double quantum coherence (DQC). A brief review of the methodology and its applications to the multisubunit muscle protein troponin is presented below.

  9. Anti-inflammatory effect of a selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor via the stimulation of heme oxygenase-1 in LPS-activated mice and J774.1 murine macrophages

    Directory of Open Access Journals (Sweden)

    Sung Bum Park

    2016-08-01

    Full Text Available 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1 converts inactive cortisone to the active cortisol. 11β-HSD1 may be involved in the resolution of inflammation. In the present study, we investigate the anti-inflammatory effects of 2-(3-benzoyl-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344, a selective 11β-HSD1 inhibitor, in lipopolysaccharide (LPS-activated C57BL/6J mice and macrophages. LPS increased 11β-HSD1 activity and expression in macrophages, which was inhibited by KR-66344. In addition, KR-66344 increased survival rate in LPS treated C57BL/6J mice. HO-1 mRNA expression level was increased by KR-66344, and this effect was reversed by the HO competitive inhibitor, ZnPP, in macrophages. Moreover, ZnPP reversed the suppression of ROS formation and cell death induced by KR-66344. ZnPP also suppressed animal survival rate in LPS plus KR-66344 treated C57BL/6J mice. In the spleen of LPS-treated mice, KR-66344 prevented cell death via suppression of inflammation, followed by inhibition of ROS, iNOS and COX-2 expression. Furthermore, LPS increased NFκB-p65 and MAPK phosphorylation, and these effects were abolished by pretreatment with KR-66344. Taken together, KR-66344 protects against LPS-induced animal death and spleen injury by inhibition of inflammation via induction of HO-1 and inhibition of 11β-HSD1 activity. Thus, we concluded that the selective 11β-HSD1 inhibitor may provide a novel strategy in the prevention/treatment of inflammatory disorders in patients.

  10. Expression of ghrelin and the ghrelin receptor in different stages of porcine corpus luteum development and the inhibitory effects of ghrelin on progesterone secretion, 3β-hydroxysteroid dehydrogenase (3β-honestly significant difference (HSD)) activity and protein expression.

    Science.gov (United States)

    Rak-Mardyła, A; Gregoraszczuk, E L; Karpeta, A; Duda, M

    2012-05-01

    Recent studies have suggested that ghrelin plays a direct role in controlling female reproduction. The aim of the present study was to investigate the mRNA and protein expression of ghrelin and its receptor (via real time PCR, Western blot and immunohistochemistry analysis, respectively) in porcine corpora lutea (CL) collected during early (CL1: 1-2 days after ovulation), middle (CL2: 7-10 after ovulation), and late luteal phase (CL3: 13-15 after ovulation). Ghrelin expression and concentration of both acylated and unacylated forms of ghrelin significantly increased during CL development. Immunohistochemistry analysis shown localization of ghrelin protein in the cytoplasm of large luteal cells. No changes in the expression of the ghrelin receptor were observed. Direct in vitro effects of ghrelin on progesterone (P4) secretion and 3-beta-hydroxysteroid dehydrogenase (3β-honestly significant difference (HSD)) activity, which were measured by the conversion of pregnenolone (P5) to P4, and 3β-HSD protein expression were then analyzed. To assess 3β-HSD activities, mature luteal cells were first cultured for 24 h with ghrelin at 100, 250, 500 and 1000 pg/mL with P5, or with aminoglutethimide (AMG). AMG is an inhibitor of CYP11A1-mediated hydroxylation; an addition of AMG and P5 enabled P4 production to serve as an index of 3β-HSD activity. Inhibitory effects of ghrelin on P4 secretion, 3β-HSD activity and protein expression were observed. In conclusion, the presence of ghrelin and its receptor in porcine corpora lutea and the direct inhibitory effects of ghrelin on luteal P4 secretion and 3β-HSD suggest potential auto/paracrine regulation by ghrelin in the luteal phase of ovary function.

  11. The Activity and Localization of 3β-hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase and Release of Androstenedione and Progesterone by Uterine Tissues During Early Pregnancy and the Estrous Cycle in Pigs

    Science.gov (United States)

    WOJCIECHOWICZ, Bartosz; KOTWICA, Genowefa; KOLAKOWSKA, Justyna; FRANCZAK, Anita

    2012-01-01

    Abstract Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) responsible for progesterone and androstenedione production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3β-HSD mRNA, 2) uterine 3β-HSD protein activity and 3) in vitro production of A4 and P4 by uterine slices harvested from pigs on days 10 to 11, 12 to 13 and 15 to 16 of pregnancy and the estrous cycle. The expression of 3β-HSD and the presence and activity of 3β-HSD protein were different in the endometrium and the myometrium during the examined periods of pregnancy and the estrous cycle. Production of A4 by the endometrium and myometrium was highest on days 12 to 13 of pregnancy and the estrous cycle. Endometrial secretion of P4 did not differ in the course of early pregnancy and on the respective days of the estrous cycle. The gravid myometrium was the highest source of P4 in pregnant pigs on days 12 to 13. The release of P4 by the cyclic myometrium rose during the examined days of the estrous cycle. The steroidogenic activity of the uterus, as described in this study, may support early pregnancy or the luteal phase of the estrous cycle in pigs. PMID:23095516

  12. The activity and localization of 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase and release of androstenedione and progesterone by uterine tissues during early pregnancy and the estrous cycle in pigs.

    Science.gov (United States)

    Wojciechowicz, Bartosz; Kotwica, Genowefa; Kolakowska, Justyna; Franczak, Anita

    2013-01-01

    Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-HSD) responsible for progesterone and androstenedione production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3β-HSD mRNA, 2) uterine 3β-HSD protein activity and 3) in vitro production of A(4) and P(4) by uterine slices harvested from pigs on days 10 to 11, 12 to 13 and 15 to 16 of pregnancy and the estrous cycle. The expression of 3β-HSD and the presence and activity of 3β-HSD protein were different in the endometrium and the myometrium during the examined periods of pregnancy and the estrous cycle. Production of A(4) by the endometrium and myometrium was highest on days 12 to 13 of pregnancy and the estrous cycle. Endometrial secretion of P(4) did not differ in the course of early pregnancy and on the respective days of the estrous cycle. The gravid myometrium was the highest source of P(4) in pregnant pigs on days 12 to 13. The release of P(4) by the cyclic myometrium rose during the examined days of the estrous cycle. The steroidogenic activity of the uterus, as described in this study, may support early pregnancy or the luteal phase of the estrous cycle in pigs.

  13. Vitamin A decreases pre-receptor amplification of glucocorticoids in obesity: study on the effect of vitamin A on 11beta-hydroxysteroid dehydrogenase type 1 activity in liver and visceral fat of WNIN/Ob obese rats

    Directory of Open Access Journals (Sweden)

    Ayyalasomayajula Vajreswari

    2011-06-01

    Full Text Available Abstract Background 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1 catalyzes the conversion of inactive glucocorticoids to active glucocorticoids and its inhibition ameliorates obesity and metabolic syndrome. So far, no studies have reported the effect of dietary vitamin A on 11β-HSD1 activity in visceral fat and liver under normal and obese conditions. Here, we studied the effect of chronic feeding of vitamin A-enriched diet (129 mg/kg diet on 11β-HSD1 activity in liver and visceral fat of WNIN/Ob lean and obese rats. Methods Male, 5-month-old, lean and obese rats of WNIN/Ob strain (n = 16 for each phenotype were divided into two subgroups consisting of 8 rats of each phenotype. Control groups received stock diet containing 2.6 mg vitamin A/kg diet, where as experimental groups received diet containing 129 mg vitamin A/Kg diet for 20 weeks. Food and water were provided ad libitum. At the end of the experiment, tissues were collected and 11β-HSD1 activity was assayed in liver and visceral fat. Results Vitamin A supplementation significantly decreased body weight, visceral fat mass and 11β-HSD1 activity in visceral fat of WNIN/Ob obese rats. Hepatic 11β-HSD1 activity and gene expression were significantly reduced by vitamin A supplementation in both the phenotypes. CCAAT/enhancer binding protein α (C/EBPα, the main transcription factor essential for the expression of 11β-HSD1, decreased in liver of vitamin A fed-obese rats, but not in lean rats. Liver × receptor α (LXRα, a nuclear transcription factor which is known to downregulate 11β-HSD1 gene expression was significantly increased by vitamin A supplementation in both the phenotypes. Conclusions This study suggests that chronic consumption of vitamin A-enriched diet decreases 11β-HSD1 activity in liver and visceral fat of WNIN/Ob obese rats. Decreased 11β-HSD1 activity by vitamin A may result in decreased levels of active glucocorticoids in adipose tissue and possibly

  14. Detection and characterization of estrus in dairy cattle with an electronic heatmount detector and an electronic activity tag.

    Science.gov (United States)

    At-Taras, E E; Spahr, S L

    2001-04-01

    The length and onset of estrus was studied in 71 lactating dairy cows using an electronic heatmount sensor (HeatWatch; DDx Inc., Boulder, CO, DeForest, WI) and an electronic activity tag (Heat Seeker, Boumatic, Madison, WI). Three methods were used to determine estrus: 1) the electronic heatmount system, 2) an increased activity ratio algorithm determined by the Heat Seeker, and 3) an increased activity count algorithm calculated for each estrous period. Mounting and physical activity variables were characterized, and the effects of synchrony, parity, and weather on these variables were determined with data from two different trials. Cows in trial 1 were not synchronized, while cows in trial 2 were synchronized. The results of the study were consistent as follows: mean numbers of mounts were 6.70 +/- 0.7 and 5.42 +/- 0.80 for trials 1 and 2, respectively; each mount lasted 3.20 +/- 0.19 s (trial 1) and 3.36 +/- 0.42 s (trial 2). Total mounting activity averaged 5.83 +/- 0.78 h per estrous period in trial 1 and 5.57 +/- 1.02 h in trial 2. Estrus identified by the increased activity count algorithm corresponded more closely to standing mount activity (determined by the HeatWatch System) than did the increased activity ratio algorithm. Synchrony, parity, and weather did not have a direct effect on physical activity. Hot weather decreased the duration of standing mount activity significantly, but did not affect the number or duration of individual mounts. All three methods of estrus detection employed improved the efficiency of detection over visual observation.

  15. Proteomic Analysis of Mitochondria-Enriched Fraction Isolated from the Frontal Cortex and Hippocampus of Apolipoprotein E Knockout Mice Treated with Alda-1, an Activator of Mitochondrial Aldehyde Dehydrogenase (ALDH2)

    Science.gov (United States)

    Stachowicz, Aneta; Olszanecki, Rafał; Suski, Maciej; Głombik, Katarzyna; Basta-Kaim, Agnieszka; Adamek, Dariusz; Korbut, Ryszard

    2017-01-01

    The role of different genotypes of apolipoprotein E (apoE) in the etiology of Alzheimer’s disease is widely recognized. It has been shown that altered functioning of apoE may promote 4-hydroxynonenal modification of mitochondrial proteins, which may result in mitochondrial dysfunction, aggravation of oxidative stress, and neurodegeneration. Mitochondrial aldehyde dehydrogenase (ALDH2) is an enzyme considered to perform protective function in mitochondria by the detoxification of the end products of lipid peroxidation, such as 4-hydroxynonenal and other reactive aldehydes. The goal of our study was to apply a differential proteomics approach in concert with molecular and morphological techniques to elucidate the changes in the frontal cortex and hippocampus of apolipoprotein E knockout (apoE−/−) mice upon treatment with Alda-1—a small molecular weight activator of ALDH2. Despite the lack of significant morphological changes in the brain of apoE−/− mice as compared to age-matched wild type animals, the proteomic and molecular approach revealed many changes in the expression of genes and proteins, indicating the impairment of energy metabolism, neuroplasticity, and neurogenesis in brains of apoE−/− mice. Importantly, prolonged treatment of apoE−/− mice with Alda-1 led to the beneficial changes in the expression of genes and proteins related to neuroplasticity and mitochondrial function. The pattern of alterations implies mitoprotective action of Alda-1, however, the accurate functional consequences of the revealed changes require further research. PMID:28218653

  16. Beyond usage: understanding the use of electronic journals on the basis of information activity analysis. Electronic journals, Use studies, Information activity, Scientific communication

    Directory of Open Access Journals (Sweden)

    Annaïg Mahé

    2004-01-01

    Full Text Available In this article, which reports the second part of a two-part study of the use of electronic journals by researchers in two French research institutions, we attempt to explain the integration of the use of electronic journals in the scientists' information habits, going beyond usage analysis. First, we describe how the development of electronic journals use follows a three-phase innovation process - research-development, first uses, and technical acculturation. Then, we attempt to find more significant explanatory factors, and emphasis is placed on the wider context of information activity. Three main information activity types are outlined - marginal, parallel, and integrated. Each of these types corresponds to a particular attitude towards scientific information and to different levels of electronic journal use.

  17. 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

  18. NADH dehydrogenase-like behavior of nitrogen-doped graphene and its application in NAD(+)-dependent dehydrogenase biosensing.

    Science.gov (United States)

    Gai, Pan-Pan; Zhao, Cui-E; Wang, Ying; Abdel-Halim, E S; Zhang, Jian-Rong; Zhu, Jun-Jie

    2014-12-15

    A novel electrochemical biosensing platform for nicotinamide adenine dinucleotide (NAD(+))-dependent dehydrogenase catalysis was designed using the nitrogen-doped graphene (NG), which had properties similar to NADH dehydrogenase (CoI). NG mimicked flavin mononucleotide (FMN) in CoI and efficiently catalyzed NADH oxidation. NG also acted as an electron transport "bridge" from NADH to the electrode due to its excellent conductivity. In comparison with a bare gold electrode, an 800 mV decrease in the overpotential for NADH oxidation and CoI-like behavior were observed at NG-modified electrode, which is the largest decrease in overpotential for NADH oxidation reported to date. The catalytic rate constant (k) for the CoI-like behavior of NG was estimated to be 2.3×10(5) M(-1) s(-1), which is much higher than that of other previously reported FMN analogs. The Michaelis-Menten constant (Km) of NG was 26 μM, which is comparable to the Km of CoI (10 μM). Electrodes modified with NG and NG/gold nanoparticals/formate dehydrogenase (NG/AuNPs/FDH) showed excellent analytical performance for the detection of NADH and formate. This electrode fabrication strategy could be used to create a universal biosensing platform for developing NAD(+)-dependent dehydrogenase biosensors and biofuel cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Identification and overexpression of a bifunctional aldehyde/alcohol dehydrogenase responsible for ethanol production in Thermoanaerobacter mathranii.

    Science.gov (United States)

    Yao, Shuo; Mikkelsen, Marie Just

    2010-01-01

    Thermoanaerobacter mathranii contains four genes, adhA, adhB, bdhA and adhE, predicted to code for alcohol dehydrogenases involved in ethanol metabolism. These alcohol dehydrogenases were characterized as NADP(H)-dependent primary alcohol dehydrogenase (AdhA), secondary alcohol dehydrogenase (AdhB), butanol dehydrogenase (BdhA) and NAD(H)-dependent bifunctional aldehyde/alcohol dehydrogenase (AdhE), respectively. Here we observed that AdhE is an important enzyme responsible for ethanol production in T. mathranii based on the constructed adh knockout strains. An adhE knockout strain fails to produce ethanol as a fermentation product, while other adh knockout strains showed no significant difference from the wild type. Further analysis revealed that the ΔadhE strain was defective in aldehyde dehydrogenase activity, but still maintained alcohol dehydrogenase activity. This showed that AdhE is the major aldehyde dehydrogenase in the cell and functions predominantly in the acetyl-CoA reduction to acetaldehyde in the ethanol formation pathway. Finally, AdhE was conditionally expressed from a xylose-induced promoter in a recombinant strain (BG1E1) with a concomitant deletion of a lactate dehydrogenase. Overexpressions of AdhE in strain BG1E1 with xylose as a substrate facilitate the production of ethanol at an increased yield. Copyright © 2010 S. Karger AG, Basel.

  20. Novel inhibitors of mitochondrial sn-glycerol 3-phosphate dehydrogenase.

    Directory of Open Access Journals (Sweden)

    Adam L Orr

    Full Text Available Mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH is a ubiquinone-linked enzyme in the mitochondrial inner membrane best characterized as part of the glycerol phosphate shuttle that transfers reducing equivalents from cytosolic NADH into the mitochondrial electron transport chain. Despite the widespread expression of mGPDH and the availability of mGPDH-null mice, the physiological role of this enzyme remains poorly defined in many tissues, likely because of compensatory pathways for cytosolic regeneration of NAD⁺ and mechanisms for glycerol phosphate metabolism. Here we describe a novel class of cell-permeant small-molecule inhibitors of mGPDH (iGP discovered through small-molecule screening. Structure-activity analysis identified a core benzimidazole-phenyl-succinamide structure as being essential to inhibition of mGPDH while modifications to the benzimidazole ring system modulated both potency and off-target effects. Live-cell imaging provided evidence that iGPs penetrate cellular membranes. Two compounds (iGP-1 and iGP-5 were characterized further to determine potency and selectivity and found to be mixed inhibitors with IC₅₀ and K(i values between ∼1-15 µM. These novel mGPDH inhibitors are unique tools to investigate the role of glycerol 3-phosphate metabolism in both isolated and intact systems.

  1. Novel Inhibitors of Mitochondrial sn-Glycerol 3-phosphate Dehydrogenase

    Science.gov (United States)

    Orr, Adam L.; Ashok, Deepthi; Sarantos, Melissa R.; Ng, Ryan; Shi, Tong; Gerencser, Akos A.; Hughes, Robert E.; Brand, Martin D.

    2014-01-01

    Mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a ubiquinone-linked enzyme in the mitochondrial inner membrane best characterized as part of the glycerol phosphate shuttle that transfers reducing equivalents from cytosolic NADH into the mitochondrial electron transport chain. Despite the widespread expression of mGPDH and the availability of mGPDH-null mice, the physiological role of this enzyme remains poorly defined in many tissues, likely because of compensatory pathways for cytosolic regeneration of NAD+ and mechanisms for glycerol phosphate metabolism. Here we describe a novel class of cell-permeant small-molecule inhibitors of mGPDH (iGP) discovered through small-molecule screening. Structure-activity analysis identified a core benzimidazole-phenyl-succinamide structure as being essential to inhibition of mGPDH while modifications to the benzimidazole ring system modulated both potency and off-target effects. Live-cell imaging provided evidence that iGPs penetrate cellular membranes. Two compounds (iGP-1 and iGP-5) were characterized further to determine potency and selectivity and found to be mixed inhibitors with IC50 and Ki values between ∼1–15 µM. These novel mGPDH inhibitors are unique tools to investigate the role of glycerol 3-phosphate metabolism in both isolated and intact systems. PMID:24587137

  2. Yeast cell-based analysis of human lactate dehydrogenase isoforms.

    Science.gov (United States)

    Mohamed, Lulu Ahmed; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

    2015-12-01

    Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae, with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc/LDH strains are expected to be of use for versatile analyses of human LDH. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  3. Aloe vera in active and passive regions of electronic devices towards a sustainable development

    Science.gov (United States)

    Lim, Zhe Xi; Sreenivasan, Sasidharan; Wong, Yew Hoong; Cheong, Kuan Yew

    2017-07-01

    The increasing awareness towards sustainable development of electronics has driven the search for natural bio-organic materials in place of conventional electronic materials. The concept of using natural bio-organic materials in electronics provides not only an effective solution to address global electronic waste crisis, but also a compelling template for sustainable electronics manufacturing. This paper attempts to provide an overview of using Aloe vera gel as a natural bio-organic material for various electronic applications. Important concepts such as responses of living Aloe vera plant towards electrical stimuli and demonstrations of Aloe vera films as passive and active regions of electronic devices are highlighted in chronological order. The biodegradability and biocompatibility of Aloe vera can bring the world a step closer towards the ultimate goal of sustainable development of electronic devices from "all-natural" materials.

  4. Glutamate synthase activities and protein changes in relation to nitrogen nutrition in barley: the dependence on different plastidic glucose-6P dehydrogenase isoforms.

    Science.gov (United States)

    Esposito, Sergio; Guerriero, Gea; Vona, Vincenza; Di Martino Rigano, Vittoria; Carfagna, Simona; Rigano, Carmelo

    2005-01-01

    In barley (Hordeum vulgare L. var. Nure), glutamate synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) are strictly correlated biochemical processes. NADH-GOGAT was the major root isoform, whose activity increased on a medium supplied with NH4+ or NO3-; by contrast, no noticeable variations could be observed in the leaves of plants supplied with nitrogen. In the leaves, the major isoform is Fd-GOGAT, whose activity increased under nitrogen feeding. G6PDH activity increased in the roots supplied with nitrogen; no variations were observed in the leaves. Moreover, an increase of the P2 isoform in the roots was measured, giving 13.6% G6PDH activity localized in the plastids under ammonium, and 25.2% under nitrate feeding conditions. Western blots confirmed that P2-G6PDH protein was induced in the roots by nitrogen. P1-G6PDH protein was absent in the roots and increased in the leaves by nitrogen supply to the plants. The changes measured in cytosolic G6PDH seem correlated to more general cell growth processes, and do not appear to be directly involved in glutamate synthesis. The effects of light on Fd-GOGAT is discussed, together with the possibility for P2-G6PDH to sustain nitrogen assimilation upon illumination.

  5. Positive effects of electronic patient records on three clinical activities

    DEFF Research Database (Denmark)

    Hertzum, Morten; Simonsen, Jesper

    2008-01-01

    in mental workload. The physician in charge also experienced increased clarity about the importance of and responsibilities for work tasks, and reduced mental workload during ward rounds. During nursing handovers the nurses experienced fewer missing pieces of information and fewer messages to pass on after...... Purpose: To investigate the effects of a fully functional electronic patient record (EPR) system on clinicians' work during team conferences, ward rounds, and nursing handovers. Method: In collaboration with clinicians an EPR system was configured for a stroke unit and in trial use for five days...... are not to be expected to be in operational use in Denmark until at least two years from now. The EPR system was evaluated with respect to its effects on clinicians' mental workload, overview, and need for exchanging information. Effects were measured by comparing the use of electronic records with the use of paper...

  6. Simultaneous measurements of cortisol and cortisone in urine and hair for the assessment of 11β-hydroxysteroid dehydrogenase activity among methadone maintenance treatment patients with LC-ESI-MS/MS.

    Science.gov (United States)

    Chen, Zheng; Li, Jifeng; Xu, Guanyi; Yang, Jin; Zhang, Jing; Deng, Huihua

    2014-10-15

    The activity of 11β-hydroxysteroid dehydrogenases (11β-HSD) is traditionally assessed using the ratio of cortisol to cortisone in urine or saliva. However, these biomarkers only reflect the local activity of 11β-HSD, and are easily affected by circadian variation of cortisol secretion. The shortcomings might be overcome by hair analysis. The present study aimed to develop an enhanced assay for simultaneous measurements of cortisol and cortisone in both hair and urine samples. The samples were collected from 29 patients under methadone maintenance treatment. The cortisol and cortisone were extracted either by solid phase extraction from a 20-mg milled hair sample after a 14-h incubation in 1ml of methanol, or by twice liquid-liquid extraction from a 20-fold diluted urine sample. The analyses were performed using high performance liquid chromatography tandem mass spectrometry with electrospray ionization in negative mode. Limits of detection and quantification were 0.5 and 1.25pg/mg for hair steroids and 0.2 and 0.5ng/ml for urinary steroids, respectively. The recoveries were more than 97%. The intra- and inter-day coefficients of variation were less than 10%. The ratios of cortisol to cortisone in hair and urine were both less than one, but did not correlate with each other. A possible reason for the lack of correlation was that the ratios in hair and urine might mostly reflect the activity of 11β-HSD type 2 in the eccrine sweat gland and in the kidney, respectively. Additionally, a significant correlation was observed between results obtained using external standard quantification and internal standard quantification. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5′-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa)

    Science.gov (United States)

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E.; Gallo-Reynoso, Juan P.

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5′-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5′-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5′-monophosphate (IMP), adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), ATP, guanosine 5′-diphosphate (GDP), guanosine 5′-triphosphate (GTP), and xanthosine 5′-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

  8. UV exposed electronically activated damage and photoreactivation repair

    Science.gov (United States)

    Bohr, Henrik; Malik, Bary

    2007-03-01

    An investigation of the possible physics underlying the damage caused to DNA by UV radiation and its subsequent repair via a photoreactivation mechanism is presented in this study. An electronic pathway starting from the initial damage to the final repair process is proposed. UV radiation is absorbed to create a hole-excited thymine or other pyrimidine that subsequently is responsible for the formation of the thymine dimer. The negative-ion of the cofactor riboflavin, FADH-, formed by the exposure of the photolyase protein to visible light interacts with the hole-excited electronic orbital of the thymine dimer inducing a photon-less Auger transition, which restores the two thymines to the ground state, thereby detaching the lesion and repairing the DNA. Due to energy balance, the process has to involve an electronic excited state (s). The mechanism involves the least amount of energy dissipation and is charge neutral. It also avoids radiation damage in the repair process, that is, is a radiationless process.

  9. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum.

    Science.gov (United States)

    Brown, Steven D; Guss, Adam M; Karpinets, Tatiana V; Parks, Jerry M; Smolin, Nikolai; Yang, Shihui; Land, Miriam L; Klingeman, Dawn M; Bhandiwad, Ashwini;