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Sample records for malate dehydrogenase mdh

  1. Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast, Hansenula polymorpha

    NARCIS (Netherlands)

    Klei, I.J. van der; Faber, K.N.; Keizer-Gunnink, I.; Gietl, C.; Harder, W.; Veenhuis, M.

    1993-01-01

    We have studied the fate of the watermelon (Citrullus vulgaris Schrad.) glyoxysomal enzyme, malate dehydrogenase (gMDH), after synthesis in the methylotrophic yeast, Hansenula polymorpha. The gene encoding the precursor form of gMDH (pre-gMDH) was cloned in an H. polymorpha expression vector

  2. FUNCTIONAL-ANALYSIS OF THE N-TERMINAL PREPEPTIDES OF WATERMELON MITOCHONDRIAL AND GLYOXYSOMAL MALATE-DEHYDROGENASES

    NARCIS (Netherlands)

    LEHNERER, M; KEIZERGUNNIK, [No Value; VEENHUIS, M; GIETL, C

    1994-01-01

    Mitochondrial and glyoxysomal malate dehydrogenase (mMDH; gMDH; L-malate : NAD(+) oxidoreductase; EC 1.1.1.37) of watermelon (Citrullus vulgaris) cotyledons are synthesized with N-terminal cleavable presequences which are shown to specify sorting of the two proteins. The two presequences differ in

  3. Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Se Jeong [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Gu, Dong Ryun [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Jin, Su Hyun [Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Park, Keun Ha [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Lee, Seoung Hoon, E-mail: leesh2@wku.ac.kr [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Wonkwang Institute of Biomaterials and Implant, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of)

    2016-06-17

    Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reduced following the activation of adenosine 5′ monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.

  4. Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling

    International Nuclear Information System (INIS)

    Oh, Se Jeong; Gu, Dong Ryun; Jin, Su Hyun; Park, Keun Ha; Lee, Seoung Hoon

    2016-01-01

    Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reduced following the activation of adenosine 5′ monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.

  5. Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha

    NARCIS (Netherlands)

    Gietl, Christine; Faber, Klaas Nico; Klei, Ida J. van der; Veenhuis, Marten

    1994-01-01

    We have studied the significance of the N-terminal presequence of watermelon (Citrullus vulgaris) glyoxysomal malate dehydrogenase [gMDH; (S)-malate:NAD+ oxidoreductase; EC 1.1.1.37] in microbody targeting. The yeast Hansenula polymorpha was used as heterologous host for the in vivo expression of

  6. Glutamine and ornithine alpha-ketoglutarate supplementation on malate dehydrogenases expression in hepatectomized rats.

    Science.gov (United States)

    Guimarães Filho, Artur; Cunha, Rodrigo Maranguape Silva da; Vasconcelos, Paulo Roberto Leitão de; Guimarães, Sergio Botelho

    2014-06-01

    To evaluate the relative gene expression (RGE) of cytosolic (MDH1) and mitochondrial (MDH2) malate dehydrogenases enzymes in partially hepatectomized rats after glutamine (GLN) or ornithine alpha-ketoglutarate (OKG) suplementation. One-hundred and eight male Wistar rats were randomly distributed into six groups (n=18): CCaL, GLNL and OKGL and fed calcium caseinate (CCa), GLN and OKG, 0.5 g/Kg by gavage, 30 minutes before laparotomy. CCaH, GLNH and OKGH groups were likewise fed 30 minutes before 70% partial hepatectomy. Blood and liver samples were collected three, seven and 14 days after laparotomy/hepatectomy for quantification of MDH1/MDH2 enzymes using the real-time polymerase chain reaction (PCR) methodology. Relative enzymes expression was calculated by the 2-(ΔΔC)T method using the threshold cycle (CT) value for normalization. MDH1/MDH2 RGE was not different in hepatectomized rats treated with OKG compared to rats treated with CCa. However, MDH1/MDH2 RGE was greater on days 3 (321:1/26.48:1) and 7 (2.12:1/2.48:1) while MDH2 RGE was greater on day 14 (7.79:1) in hepatectomized rats treated with GLN compared to control animals. Glutamine has beneficial effects in liver regeneration in rats by promoting an up-regulation of the MDH1 and MDH2 relative gene expression.

  7. Mutations in MDH2, Encoding a Krebs Cycle Enzyme, Cause Early-Onset Severe Encephalopathy

    NARCIS (Netherlands)

    Ait-El-Mkadem, Samira; Dayem-Quere, Manal; Gusic, Mirjana; Chaussenot, Annabelle; Bannwarth, Sylvie; François, Bérengère; Genin, Emmanuelle C; Fragaki, Konstantina; Volker-Touw, Catharina L M; Vasnier, Christelle; Serre, Valérie; van Gassen, Koen L I; Lespinasse, Françoise; Richter, Susan; Eisenhofer, Graeme; Rouzier, Cécile; Mochel, Fanny; De Saint-Martin, Anne; Abi Warde, Marie-Thérèse; de Sain-van der Velden, Monique G M; Jans, Judith J M; Amiel, Jeanne; Avsec, Ziga; Mertes, Christian; Haack, Tobias B; Strom, Tim; Meitinger, Thomas; Bonnen, Penelope E; Taylor, Robert W; Gagneur, Julien; van Hasselt, Peter M; Rötig, Agnès; Delahodde, Agnès; Prokisch, Holger; Fuchs, Sabine A; Paquis-Flucklinger, Véronique

    2016-01-01

    MDH2 encodes mitochondrial malate dehydrogenase (MDH), which is essential for the conversion of malate to oxaloacetate as part of the proper functioning of the Krebs cycle. We report bi-allelic pathogenic mutations in MDH2 in three unrelated subjects presenting with early-onset generalized

  8. Regulation of human cerebrospinal fluid malate dehydrogenase 1 in sporadic Creutzfeldt-Jakob disease patients.

    Science.gov (United States)

    Schmitz, Matthias; Llorens, Franc; Pracht, Alexander; Thom, Tobias; Correia, Ângela; Zafar, Saima; Ferrer, Isidre; Zerr, Inga

    2016-11-14

    The identification of reliable diagnostic biomarkers in differential diagnosis of neurodegenerative diseases is an ongoing topic. A previous two-dimensional proteomic study on cerebrospinal fluid (CSF) revealed an elevated level of an enzyme, mitochondrial malate dehydrogenase 1 (MDH1), in sporadic Creutzfeldt-Jakob disease (sCJD) patients. Here, we could demonstrate the expression of MDH1 in neurons as well as in the neuropil. Its levels are lower in sCJD brains than in control brains. An examination of CSF-MDH1 in sCJD patients by ELISA revealed a significant elevation of CSF-MDH1 levels in sCJD patients (independently from the PRNP codon 129 MV genotype or the prion protein scrapie (PrP Sc ) type) in comparison to controls. In combination with total tau (tau), CSF-MDH1 detection exhibited a high diagnostic accuracy for sCJD diagnosis with a sensitivity of 97.5% and a specificity of 95.6%. A correlation study of MDH1 level in CSF with other neurodegenerative marker proteins revealed a significant positive correlation between MDH1 concentration with tau, 14-3-3 and neuron specific enolase level. In conclusion, our study indicated the potential of MDH1 in combination with tau as an additional biomarker in sCJD improving diagnostic accuracy of tau markedly.

  9. Effect of Punica granatum fruit peel on glucose-6-phosphate dehydrogenase and malate dehydrogenase in amphistome Gastrothylax indicus.

    Science.gov (United States)

    Aggarwal, Rama; Bagai, Upma

    2017-03-01

    Increasing anthelmintic resistance and the impact of conventional anthelmintics on the environment, it is important to look for alternative strategies against helminth parasite in sheep. Important lipogenic enzymes like glucose-6-phosphate dehydrogenase (G-6-PDH) and malate dehydrogenase (MDH) show subcellular distribution pattern. Activity of G-6-PDH was largely restricted to cytosolic fraction while MDH was found in both cytosolic and mitochondrial fraction in Gastrothylax indicus. Following in vitro treatment with ethanolic and aqueous extracts of Punica granatum fruit peel and commercial anthelmintic, albendazole G-6-PDH activity was decreased by 19-32 %, whereas MDH was suppressed by 24-41 %, compared to the respective control. Albendazole was quite effective when compared with negative control and both the extracts. The results indicate that phytochemicals of plant may act as potential vermifuge or vermicide.

  10. Glutamine and ornithine alpha-ketoglutarate supplementation on malate dehydrogenases expression in hepatectomized rats

    OpenAIRE

    Guimarães Filho, Artur; Cunha, Rodrigo Maranguape Silva da; Vasconcelos, Paulo Roberto Leitão de; Guimarães, Sergio Botelho

    2014-01-01

    PURPOSE: To evaluate the relative gene expression (RGE) of cytosolic (MDH1) and mitochondrial (MDH2) malate dehydrogenases enzymes in partially hepatectomized rats after glutamine (GLN) or ornithine alpha-ketoglutarate (OKG) suplementation. METHODS: One-hundred and eight male Wistar rats were randomly distributed into six groups (n=18): CCaL, GLNL and OKGL and fed calcium caseinate (CCa), GLN and OKG, 0.5g/Kg by gavage, 30 minutes before laparotomy. CCaH, GLNH and OKGH groups were likewise fe...

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

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    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.

  12. 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.

  13. Immunological response and protection of mice immunized with plasmid encoding Toxoplasma gondii glycolytic enzyme malate dehydrogenase.

    Science.gov (United States)

    Hassan, I A; Wang, S; Xu, L; Yan, R; Song, X; XiangRui, L

    2014-12-01

    Toxoplasma gondii Malate dehydrogenase (TgMDH) plays an important role as part of the energy production cycle. In this investigation, immunological changes and protection efficiency of this protein delivered as a DNA vaccine have been evaluated. Mice were intramuscularly immunized with pTgMDH, followed by challenge with virulent T. gondii RH strain, 2 weeks after the booster immunization. Compared to the control groups, the results showed that pTgMDH has stimulated specific humoral response as demonstrated by significant high titers of total IgG and subclasses IgG1 and IgG2a , beside IgA and IgM, but not IgE. Analysis of cytokine profiles revealed significant increases of IFN-γ, IL-4 and IL-17, while no significant changes were detected in TGF-β1. In cell-mediated response, both T lymphocytes subpopulations CD4(+) and CD8(+) were positively recruited as significant percentages were recorded in response to immunization with TgMDH. Significant long survival rate, 17 days, has been observed in the TgMDH vaccinated group, in contrast with control groups which died within 8-9 days after challenge. These results demonstrated that TgMDH could induce significant immunological responses leading to a considerable level of protection against acute toxoplasmosis infection. © 2014 John Wiley & Sons Ltd.

  14. Action of sulphite on plant malate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Ziegler, I.

    1974-01-01

    SO/sub 3//sup 2 -/ acts on NAD- and NADP-dependent malate dehydrogenase in several ways. Firstly, SO/sub 3//sup 2 -/ favours the appearance of low MW species (65000 and 39000 daltons) in Sephadex gel chromatography. Secondly, the enzyme from which is obtained by gel chromatography with dithioerythritol plus nucleotide cofactor is changed in the presence of SO/sub 3//sup 2 -/. This is indicated by the appearance of a linear reaction (instead of curvilinear), and by the abolition of the biphasic sigmoidal kinetics on varying substrate and cofactor concentrations. Thus the inhibition of initial velocity at high substrate or cofactor concentrations is even more marked than at lower ones. Thirdly, SO/sub 3//sup 2 -/ strongly reduces the activity in substrate saturating conditions.

  15. Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Decottignies, P.; Schmitter, J.M.; Miginiac-Maslow, M.; Le Marechal, P.; Jacquot, J.P.; Gadal, P.

    1988-08-25

    The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by (14C)iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two (14C)carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, the analysis of the tryptic digest of light-activated (14C)carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15.

  16. Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase

    International Nuclear Information System (INIS)

    Decottignies, P.; Schmitter, J.M.; Miginiac-Maslow, M.; Le Marechal, P.; Jacquot, J.P.; Gadal, P.

    1988-01-01

    The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by [14C]iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two [14C]carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, the analysis of the tryptic digest of light-activated [14C]carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15

  17. Isoform expression in the multiple soluble malate dehydrogenase of Hoplias malabaricus (Erythrinidae, Characiformes

    Directory of Open Access Journals (Sweden)

    M. R. Aquino-Silva

    Full Text Available Kinetic properties and thermal stabilities of Hoplias malabaricus liver and skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to further study the possible sMDH-A* locus duplication evolved from a recent tandem duplication. Both A (A1 and A2 and B isoforms had similar optima pH (7.5-8.0. While Hoplias A isoform could not be characterized as thermostable, B could as thermolabile. A isoforms differed from B isoform in having higher Km values for oxaloacetate. The possibly duplicated A2 isoform showed higher substrate affinity than the A1. Hoplias duplicated A isoforms may influence the direction of carbon flow between glycolisis and gluconeogenesis.

  18. Isoform expression in the multiple soluble malate dehydrogenase of Hoplias malabaricus (Erythrinidae, Characiformes

    Directory of Open Access Journals (Sweden)

    Aquino-Silva M. R.

    2003-01-01

    Full Text Available Kinetic properties and thermal stabilities of Hoplias malabaricus liver and skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to further study the possible sMDH-A* locus duplication evolved from a recent tandem duplication. Both A (A1 and A2 and B isoforms had similar optima pH (7.5-8.0. While Hoplias A isoform could not be characterized as thermostable, B could as thermolabile. A isoforms differed from B isoform in having higher Km values for oxaloacetate. The possibly duplicated A2 isoform showed higher substrate affinity than the A1. Hoplias duplicated A isoforms may influence the direction of carbon flow between glycolisis and gluconeogenesis.

  19. Enzymatic urea adaptation: lactate and malate dehydrogenase in elasmobranchs

    Czech Academy of Sciences Publication Activity Database

    Lagana, G.; Bellocco, E.; Mannucci, C.; Leuzzi, U.; Tellone, E.; Kotyk, Arnošt; Galtieri, A.

    2006-01-01

    Roč. 55, č. 6 (2006), s. 675-688 ISSN 0862-8408 Institutional research plan: CEZ:AV0Z50110509 Keywords : elasmobranchs * lactate dehydrogenase * malate dehydrogenase Subject RIV: CE - Biochemistry Impact factor: 2.093, year: 2006

  20. Immobilization of malate dehydrogenase on carbon nanotubes for development of malate biosensor.

    Science.gov (United States)

    Ruhal, A; Rana, J S; Kumar, S; Kumar, A

    2012-12-22

    An amperometric malic acid biosensor was developed by immobilizing malate dehydrogenase on multi-walled carbon nanotubes (MWCNT) coated on screen printed carbon electrode. The screen printed carbon electrode is made up of three electrodes viz., carbon as working, platinum as counter and silver as reference electrode. Detection of L-malic acid concentration provides important information about the ripening and shelf life of the fruits. The NADP specific malate dehydrogenase was immobilized on carboxylated multiwalled carbon nanotubes using cross linker EDC [1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide] on screen printed carbon electrode. An amperometric current was measured by differential pulse voltammetry (DPV) which increases with increasing concentrations of malic acid at fixed concentration of NADP. Enzyme electrode was characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The detection limit of malic acid by the sensor was 60 - 120 μM and sensitivity of the sensor was 60 μM with a response time of 60s. The usual detection methods of malic acid are nonspecific, time consuming and less sensitive. However, an amperometric malic acid nanosensor is quick, specific and more sensitive for detection of malic acid in test samples.

  1. The enhancement of tolerance to salt and cold stresses by modifying the redox state and salicylic acid content via the cytosolic malate dehydrogenase gene in transgenic apple plants.

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    Wang, Qing-Jie; Sun, Hong; Dong, Qing-Long; Sun, Tian-Yu; Jin, Zhong-Xin; Hao, Yu-Jin; Yao, Yu-Xin

    2016-10-01

    In this study, we characterized the role of an apple cytosolic malate dehydrogenase gene (MdcyMDH) in the tolerance to salt and cold stresses and investigated its regulation mechanism in stress tolerance. The MdcyMDH transcript was induced by mild cold and salt treatments, and MdcyMDH-overexpressing apple plants possessed improved cold and salt tolerance compared to wild-type (WT) plants. A digital gene expression tag profiling analysis revealed that MdcyMDH overexpression largely altered some biological processes, including hormone signal transduction, photosynthesis, citrate cycle and oxidation-reduction. Further experiments verified that MdcyMDH overexpression modified the mitochondrial and chloroplast metabolisms and elevated the level of reducing power, primarily caused by increased ascorbate and glutathione, as well as the increased ratios of ascorbate/dehydroascorbate and glutathione/glutathione disulphide, under normal and especially stress conditions. Concurrently, the transgenic plants produced a high H2 O2 content, but a low O2·- production rate was observed compared to the WT plants. On the other hand, the transgenic plants accumulated more free and total salicylic acid (SA) than the WT plants under normal and stress conditions. Taken together, MdcyMDH conferred the transgenic apple plants a higher stress tolerance by producing more reductive redox states and increasing the SA level; MdcyMDH could serve as a target gene to genetically engineer salt- and cold-tolerant trees. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  2. Cellular distribution, purification and electrophoretic properties of malate dehydrogenase in Trichuris ovis and inhibition by benzimidazoles and pyrimidine derivatives.

    Science.gov (United States)

    Sanchez-Moreno, M; Ortega, J E; Valero, A

    1989-12-01

    High levels of malate dehydrogenase were found in Trichuris ovis. Two molecular forms of the enzyme, of different cellular location and electrophoretic pattern, were isolated and purified. The activity of soluble malate dehydrogenase was greater than that of mitochondrial malate dehydrogenase. Both forms also displayed different electrophoretic profiles in comparison with purified extracts from goat (Capra hircus) liver. Substrate concentration directly affected enzyme activity. Host and parasite malate dehydrogenase activity were both inhibited by a series of benzimidazoles and pyrimidine-derived compounds, some of which markedly reduced parasite enzyme activity, but not host enzyme activity. Percentage inhibition by some pyrimidine derivatives was greater than that produced by benzimidazoles.

  3. Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity.

    Science.gov (United States)

    Hecht, K; Wrba, A; Jaenicke, R

    1989-07-15

    Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.

  4. Evidence for catabolite degradation in the glucose-dependent inactivation of yeast cytoplasmic malate dehydrogenase

    International Nuclear Information System (INIS)

    Neeff, J.; Haegele, E.; Nauhaus, J.; Heer, U.; Mecke, D.

    1978-01-01

    The cytoplasmic malate dehydrogenase of Saccharomyces cerevisiae was radioactively labeled during its synthesis on a glucose-free derepression medium. After purification a sensitive radioimmunoassay for this enzyme could be developed. The assay showed that after the physiological, glucose-dependent 'catabolite inactivation' of cytoplasmic malate dehydrogenase an inactive enzyme protein is immunologically not detectable. Together with the irreversibility of this reaction in vivo this finding strongly suggests a proteolytic mechanism of enzyme inactivation. For this process the term 'catabolite degradation' is used. (orig.) [de

  5. Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yongchao [ORNL; Tschaplinski, Timothy J [ORNL; Engle, Nancy L [ORNL; Hamilton, Choo Yieng [ORNL; Rodriguez, Jr., Miguel [ORNL; Liao, James C [ORNL; Schadt, Christopher Warren [ORNL; Guss, Adam M [ORNL; Yang, Yunfeng [ORNL; Graham, David E [ORNL

    2012-01-01

    Background: The model bacterium Clostridium cellulolyticum efficiently hydrolyzes crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels. Therefore genetic engineering will likely be required to improve the ethanol yield. Random mutagenesis, plasmid transformation, and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism. Results: The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh) and L-malate dehydrogenase (Ccel_0137; mdh) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products (by molarity), corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four-times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant s TCA pathway. Conclusions: The efficient intron-based gene inactivation system produced the first gene-targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to

  6. Th2-related immune responses by the Brucella abortus cellular antigens, malate dehydrogenase, elongation factor, and arginase.

    Science.gov (United States)

    Im, Young Bin; Shim, Soojin; Park, Woo Bin; Kim, Suk; Yoo, Han Sang

    2017-09-01

    Brucellosis is an important zoonotic disease caused by Brucella species. The disease is difficult to control due to the intracellular survival of the bacterium and the lack of precise understanding of pathogenesis. Despite of continuous researches on the pathogenesis of Brucella spp. infection, there is still question on the pathogenesis, especially earlier immune response in the bacterial infection. Malate dehydrogenase (MDH), elongation factor (Tsf), and arginase (RocF), which showed serological reactivity, were purified after gene cloning, and their immune modulating activities were then analyzed in a murine model. Cytokine production profiles were investigated by stimulating RAW 264.7 cells and naïve splenocytes with the three recombinant proteins. Also, immune responses were analyzed by ELISA and an ELIspot assay after immunizing mice with the three proteins. Only TNF-α was produced in stimulated RAW 264.7 cells, whereas Th1-related cytokines, IFN-γ and IL-2, were induced in naïve splenocytes. In contrast, Th2-type immune response was more strongly induced in antigen-secreting cells in the splenocytes obtained 28 days after immunizing mice with the three proteins, as were IgM and IgG. The induction of Th2-related antibody, IgG1, was higher than the Th1-related antibody, IgG2a, in immunized mice. These results suggest that the three proteins strongly induce Th2-type immune response in vivo, even though Th1-related cytokines were produced in vitro. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

    Directory of Open Access Journals (Sweden)

    Li Yongchao

    2012-01-01

    Full Text Available Abstract Background The model bacterium Clostridium cellulolyticum efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering. Results The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh and L-malate dehydrogenase (Ccel_0137; mdh genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain, resulting in a substantial shift in fermentation toward ethanol production. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products, corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant's tricarboxylic acid pathway. Conclusions The efficient intron-based gene inactivation system produced the first non-random, targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox

  8. Structure of halophilic malate dehydrogenase in multimolar KCl solutions from neutron scattering and ultracentrifugation

    International Nuclear Information System (INIS)

    Calmettes, P.

    1987-01-01

    The structure and solvent interactions of malate dehydrogenase from Halobacterium marismortui in multimolar KCl solvents are found to be similar to those in multimolar NaCl solvents reported previously (G. Zaccai, E. Wachtel and H. Eisenberg, J. Mol. Biol. 190 (1986) 97). KCl rather than NaCl is predominant in physiological medium. At salt concentrations up to about 3.0 M, the protein (a dimer of M 87000 g/mol) can be considered to occupy an invariant volume in which it is associated with about 4100 molecules of water and about 520 molecules of salt. At very low resolution, the enzyme particle appears to have a compact protein core and protruding protein parts in interaction with the water and salt components, structural features that are not observed in non-halophilic mitochondrial malate dehydrogenase. The above conclusions were drawn from the analysis of neutron scattering and ultracentrifugation data, and the complementarity of these approaches is discussed extensively. 24 refs.; 7 figs.; 4 tabs

  9. Enhancement of malate-production and increase in sensitivity to dimethyl succinate by mutation of the VID24 gene in Saccharomyces cerevisiae.

    Science.gov (United States)

    Negoro, Hiroaki; Kotaka, Atsushi; Matsumura, Kengo; Tsutsumi, Hiroko; Hata, Yoji

    2016-06-01

    Malate in sake (a Japanese alcoholic beverage) is an important component for taste that is produced by yeasts during alcoholic fermentation. To date, many researchers have developed methods for breeding high-malate-producing yeasts; however, genes responsible for the high-acidity phenotype are not known. We determined the mutated gene involved in high malate production in yeast, isolated as a sensitive mutant to dimethyl succinate. In the comparative whole genome analysis between high-malate-producing strain and its parent strain, one of the non-synonymous substitutions was identified in the VID24 gene. The mutation of VID24 resulted in enhancement of malate-productivity and sensitivity to dimethyl succinate. The mutation appeared to lead to a deficiency in Vid24p function. Furthermore, disruption of cytoplasmic malate dehydrogenase (Mdh2p) gene in the VID24 mutant inhibited the high-malate-producing phenotype. Vid24p is known as a component of the multisubunit ubiquitin ligase and participates in the degradation of gluconeogenic enzymes such as Mdh2p. We suggest that the enhancement of malate-productivity results from an accumulation of Mdh2p due to the loss of Vid24p function. These findings propose a novel mechanism for the regulation of organic acid production in yeast cells by the component of ubiquitin ligase, Vid24p. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  10. Mutation in the peroxin-coding gene PEX22 contributing to high malate production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Negoro, Hiroaki; Sakamoto, Mitsuru; Kotaka, Atsushi; Matsumura, Kengo; Hata, Yoji

    2018-02-01

    Saccharomyces cerevisiae produces organic acids such as succinate, acetate, and malate during alcoholic fermentation. Since malate contributes to the pleasant taste of sake (a Japanese alcoholic beverage), various methods for breeding high-malate-producing yeast strains have been developed. Here, a high-malate-producing yeast strain F-701H was isolated. This mutant was sensitive to dimethyl succinate (DMS) and harbored a nonsense mutation in the peroxin gene PEX22, which was identified as the cause of high malate production by comparative genome analysis. This mutation, which appeared to cause Pex22p dysfunction, was sufficient to confer increased malate productivity and DMS sensitivity to yeast cells. Next, we investigated the mechanism by which this mutation led to high malate production in yeast cells. Peroxins, such as Pex22p, maintain peroxisomal biogenesis. Analysis of 29 PEX disruptants revealed an increased malate production by deletion of the genes encoding peroxins responsible for importing proteins (containing peroxisomal targeting signal 1, PTS1) into the peroxisomal matrix, and those responsible for the assembly of peroxins themselves in the peroxisomal membrane. A defect in peroxisomal malate dehydrogenase (Mdh3p), harboring endogenous PTS1, inhibited the high malate-producing phenotype in the PEX22 mutant. Moreover, Mdh3p, which was normally sorted to the peroxisomal matrix, was potentially mislocalized to the cytosol in the PEX22 mutant. This suggested that an increase in malate production resulted from the mislocalization of Mdh3p from the peroxisome to the cytoplasm due to the loss of peroxin-mediated transportation. Thus, the present study revealed a novel mechanism for organic acid productions in yeast during sake brewing. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. Inhibition of several enzymes by gold compounds. II. beta-Glucuronidase, acid phosphatase and L-malate dehydrogenase by sodium thiomalatoraurate (I), sodium thiosulfatoaurate (I) and thioglucosoaurate (I).

    Science.gov (United States)

    Lee, M T; Ahmed, T; Haddad, R; Friedman, M E

    1989-01-01

    Bovine liver beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32), wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) and bovine liver L-malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) were inhibited by a series of gold (I) complexes that have been used as anti-inflammatory drugs. Both sodium thiosulfatoaurate (I) (Na AuTs) and sodium thiomalatoraurate (NaAuTM) effectively inhibited all three enzymes, while thioglucosoaurate (I) (AuTG) only inhibited L-malate dehydrogenase. The equilibrium constants (K1) ranged from nearly 4000 microM for the NaAuTM-beta-glucuronidase interaction to 24 microM for the NaAuTS-beta-glucuronidase interaction. The rate of covalent bond formation (kp) ranged from 0.00032 min-1 for NaAuTM-beta-glucuronidase formation to 1.7 min-1 for AuTG-L-malate dehydrogenase formation. The equilibrium data shows that the gold (I) drugs bind by several orders lower than the gold (III) compounds, suggesting a significantly stronger interaction between the more highly charged gold ion and the enzyme. Yet the rate of covalent bond formation depends as much on the structure of the active site as upon the lability of the gold-ligand bond. It was also observed that the more effective the gold inhibition the more toxic the compound.

  12. Calcium signaling in brain mitochondria: interplay of malate aspartate NADH shuttle and calcium uniporter/mitochondrial dehydrogenase pathways.

    Science.gov (United States)

    Contreras, Laura; Satrústegui, Jorgina

    2009-03-13

    Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate alpha-ketoglutarate (alphaKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that alphaKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in alphaKG efflux through the oxoglutarate carrier and in a drop in MAS activity. Inhibition of alphaKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in lactate observed in the stimulated brain in vivo.

  13. 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 (oxygen concentration and microplanktonic biomass in the oxygen minimum zone and adjacent areas of the Humboldt Current System water column. Our results demonstrate significant potential enzymatic activity of 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.

  14. Akkumulation von L-Malat und D-Lactat in Arabidopsis thaliana und Laccase/HBT-vermittelte Delignifizierung von Spartina alterniflora und Phragmites australis

    OpenAIRE

    Heil, Alexander

    2016-01-01

    The current work contains two projects "Accumulation of L-malate and D-lactate in Arabidopsis thaliana" (A) "Laccase/HBT mediated delignification of Spartina alterniflora and Phragmites australis" (B). In project A, L-malate and D-lactate accumulated in A. thaliana plants. The accumulation of L-malate is carried out by modification of the plant metabolism with the enzymes PEPC, MDH and the tonoplast dicarboxylate transporter (TDT). Gene pepci2 (Hydrilla verticillata), mdh5 (Zea mays) and tdt ...

  15. Small-angle X-ray scattering studies on the X-ray induced aggregation of ribonnuclease, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and serum albumin. A comparison with malate synthase

    International Nuclear Information System (INIS)

    Zipper, P.; Gatterer, H.G.; Schutz, J.; Durchschlag, H.

    1980-01-01

    The X-ray induced aggregation of ribonuclease, lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and serum albumin in aqueous solution was monitored in situ by means of small-angle X-ray scattering. Measurements carried out with ribonuclease, LDH and serum albumin in the absence of dithiothreitol (DTT) and with GAPDH in the presence of 0.2mM DTT established the following series for the rates of aggregation of the proteins under these conditions: ribonuclease >LDH> >GAPDH> serum albumin. Within six hours from the beginning of irradiation (i.e. about the time required for the exposure of one complete scattering curve under the conditions of our experiments) the following increases of R tilde resulted: ribonuclease 9%, LDH 7%, GAPDH 4%, serum albumin <1%. Changes of R tilde exceeding 1% are, of course, too high to be tolerated in conventional scattering experiments. Measurements carried out with LDH and GAPDH in the presence of 2mM DTT established a strong protective effect of DTT against the X-ray induced aggregation of these enzymes. The initial increase of R tilde upon irradiation of LDH and GAPDH in the presence of 2mM DTT was found to be even lower than the increase of R tilde observed when serum albumin was irradiated in the absence of DTT. However, the observed decrease of anti x of LDH and GAPDH at the early stages of irradiation suggested the occurrence of fragmentation of the enzymes as another consequence of radiation damage. This finding is discussed in context with the results from previous scattering experiments and electrophoretic studies on malate synthase. (author)

  16. la phosphoglucoisomerase et la malate deshydrogenase

    African Journals Online (AJOL)

    AISA

    dimérique du cycle de Krebs qui catalyse la réaction suivante: Malate + NAD MDH Oxaloacétate +. NADH. Dans les cellules du maïs, les formes cytosoliques sont codées par deux loci, les formes mitochondriales par 3 loci (Newton et. Schwartz, 1980). L'acide ascorbique inhibe préférentiellement les formes du cytosol.

  17. Vitality Improvement of the Mediterranean Fruit Fly, Ceratitis capitata Wied 1- Measured by using dehydrogenase Enzyme Activities

    International Nuclear Information System (INIS)

    Salama, M.S.; Shoman, A.A.; Elbermawy, S.M.; Abul Yazid, I.

    2000-01-01

    The present study searches for the improvement vitality of the Mediterranean fruit fly, Ceratitis capitata Wied. Through the induction of a specific variance (mutation) in the genetic material. Several types of treatments that were thought to cause this mutation were used, as IGR's, temperature, formaldehyde, colchicine, alcohols, several types of larval rearing media and gamma-rays. Generally, the activities of the energy enzymes alpha-glycerophosphate dehydrogenase (alpha-GPDH) enzyme lactate dehydrogenase (LDH) enzyme and malate dehydrogenase (MDH) enzyme, when used as a direct measure for the fly vitality, increased due to treatments of the egg stage by the previously mentioned treatments specially by the usage of rice hulls in the larval rearing medium alone or followed by irradiation of the pupal stage with 90 Gy

  18. Malate and fumarate extend lifespan in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Clare B Edwards

    Full Text Available Malate, the tricarboxylic acid (TCA cycle metabolite, increased lifespan and thermotolerance in the nematode C. elegans. Malate can be synthesized from fumarate by the enzyme fumarase and further oxidized to oxaloacetate by malate dehydrogenase with the accompanying reduction of NAD. Addition of fumarate also extended lifespan, but succinate addition did not, although all three intermediates activated nuclear translocation of the cytoprotective DAF-16/FOXO transcription factor and protected from paraquat-induced oxidative stress. The glyoxylate shunt, an anabolic pathway linked to lifespan extension in C. elegans, reversibly converts isocitrate and acetyl-CoA to succinate, malate, and CoA. The increased longevity provided by malate addition did not occur in fumarase (fum-1, glyoxylate shunt (gei-7, succinate dehydrogenase flavoprotein (sdha-2, or soluble fumarate reductase F48E8.3 RNAi knockdown worms. Therefore, to increase lifespan, malate must be first converted to fumarate, then fumarate must be reduced to succinate by soluble fumarate reductase and the mitochondrial electron transport chain complex II. Reduction of fumarate to succinate is coupled with the oxidation of FADH2 to FAD. Lifespan extension induced by malate depended upon the longevity regulators DAF-16 and SIR-2.1. Malate supplementation did not extend the lifespan of long-lived eat-2 mutant worms, a model of dietary restriction. Malate and fumarate addition increased oxygen consumption, but decreased ATP levels and mitochondrial membrane potential suggesting a mild uncoupling of oxidative phosphorylation. Malate also increased NADPH, NAD, and the NAD/NADH ratio. Fumarate reduction, glyoxylate shunt activity, and mild mitochondrial uncoupling likely contribute to the lifespan extension induced by malate and fumarate by increasing the amount of oxidized NAD and FAD cofactors.

  19. Effects of L-malate on physical stamina and activities of enzymes related to the malate-aspartate shuttle in liver of mice.

    Science.gov (United States)

    Wu, J L; Wu, Q P; Huang, J M; Chen, R; Cai, M; Tan, J B

    2007-01-01

    L-malate, a tricarboxylic acid cycle (TCA) intermediate, plays an important role in transporting NADH from cytosol to mitochondria for energy production and may be involved in the beneficial effects of improving physical stamina. In the present study, we investigated the effects of L-malate on the performance of forced swimming time and blood biochemical parameters related to fatigue - blood urea nitrogen (BUN), glucose (Glc), creatine kinase (CK),total protein (TP) and lactic acid (LA). To investigate the effects of L-malate on the malate-aspartate shuttle and energy metabolism in mice, the activities of enzymes related to the malate-aspartate shuttle were measured. L-malate was orally administered to mice continuously for 30 days using a feeding atraumatic needle. The swimming time was increased by 26.1 % and 28.5 %, respectively, in the 0.210 g/kg and 0.630 g/kg L-malate-treated group compared with the control group. There were no differences in the concentrations of Glc, BUN and TP between the L-malate-treated groups and the control groups. However, the levels of CK were significantly decreased in the L-malate-treated groups. The results predict a potential benefit of L-malate for improving physical stamina and minimizing muscle damage during swimming exercise. The activities of cytosolic and mitochondrial malate dehydrogenase were significantly elevated in the L-malate-treated group compared with the control group. These enzymatic activities may be useful indicators for evaluating changes affecting the malate-aspartate shuttle and energy metabolism in the liver of mice.

  20. Rewiring the reductive tricarboxylic acid pathway and L-malate transport pathway of Aspergillus oryzae for overproduction of L-malate.

    Science.gov (United States)

    Liu, Jingjing; Xie, Zhipeng; Shin, Hyun-Dong; Li, Jianghua; Du, Guocheng; Chen, Jian; Liu, Long

    2017-07-10

    Aspergillus oryzae finds wide application in the food, feed, and wine industries, and is an excellent cell factory platform for production of organic acids. In this work, we achieved the overproduction of L-malate by rewiring the reductive tricarboxylic acid (rTCA) pathway and L-malate transport pathway of A. oryzae NRRL 3488. First, overexpression of native pyruvate carboxylase and malate dehydrogenase in the rTCA pathway improved the L-malate titer from 26.1gL -1 to 42.3gL -1 in shake flask culture. Then, the oxaloacetate anaplerotic reaction was constructed by heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase from Escherichia coli, increasing the L-malate titer to 58.5gL -1 . Next, the export of L-malate from the cytoplasm to the external medium was strengthened by overexpression of a C4-dicarboxylate transporter gene from A. oryzae and an L-malate permease gene from Schizosaccharomyces pombe, improving the L-malate titer from 58.5gL -1 to 89.5gL -1 . Lastly, guided by transcription analysis of the expression profile of key genes related to L-malate synthesis, the 6-phosphofructokinase encoded by the pfk gene was identified as a potential limiting step for L-malate synthesis. Overexpression of pfk with the strong sodM promoter increased the L-malate titer to 93.2gL -1 . The final engineered A. oryzae strain produced 165gL -1 L-malate with a productivity of 1.38gL -1 h -1 in 3-L fed-batch culture. Overall, we constructed an efficient L-malate producer by rewiring the rTCA pathway and L-malate transport pathway of A. oryzae NRRL 3488, and the engineering strategy adopted here may be useful for the construction of A. oryzae cell factories to produce other organic acids. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Bringing the excitement and motivation of research to students; Using inquiry and research-based learning in a year-long biochemistry laboratory : Part II-research-based laboratory-a semester-long research approach using malate dehydrogenase as a research model.

    Science.gov (United States)

    Knutson, Kristopher; Smith, Jennifer; Nichols, Paul; Wallert, Mark A; Provost, Joseph J

    2010-09-01

    Research-based learning in a teaching environment is an effective way to help bring the excitement and experience of independent bench research to a large number of students. The program described here is the second of a two-semester biochemistry laboratory series. Here, students are empowered to design, execute and analyze their own experiments for the entire semester. This style of laboratory replaces a variety of shorter labs in favor of an in depth research-based learning experience. The concept is to allow students to function in independent research groups. The research projects are focused on a series of wild-type and mutant clones of malate dehydrogenase. A common research theme for the laboratory helps instructors administer the course and is key to delivering a research opportunity to a large number of students. The outcome of this research-based learning laboratory results in students who are much more confident and skilled in critical areas in biochemistry and molecular biology. Students with research experience have significantly higher confidence and motivation than those students without a previous research experience. We have also found that all students performed better in advanced courses and in the workplace. Copyright © 2010 International Union of Biochemistry and Molecular Biology, Inc.

  2. Processes of malate catabolism during the anaerobic metabolism of grape berries

    International Nuclear Information System (INIS)

    Flanzy, C.; Andre, P.; Buret, M.; Chambroy, Y.; Garcia, P.

    1976-01-01

    In order to precise malate fate during the anaerobic metabolism of grape, malate- 3 - 14 C was injected into Carignan berries kept in darkness at 35 0 C under carbon dioxide atmosphere. The injection of labelled malate was effected in presence or not of non-labelled oxalate which inhibits malic enzyme (EC I.I.I.40). The analyses of the samples fixed after 3 and 7 days anaerobiosis concerned the titration of various substrates, organic acids, amino-acids and glycolysis products, and the measuring of the NADP + -malic enzyme (EC I.I.I.40) and malate dehydrogenase (EC I.I.I.40). Radioactivity is mainly observed in ethanol, amino-butyrate the non-separated group glycerate-shikimate and succinate. Malic enzyme acts in the first sequence of a process leading from malate to ethanol. Alanin synthesis seems to be stimulated in presence of oxalate. The results obtained and some hypotheses presented in the literature induce to suggest a utilization scheme for malate in the anaerobic metabolism of grape [fr

  3. The Maritime Declaration of Health (MDH) as a tool to detect maritime traffic-related health risks: analysis of MDH forms submitted to Spanish ports, October 2014 to March 2015.

    Science.gov (United States)

    López-Gigosos, Rosa M; Segura, Marina; Díez-Díaz, Rosa M; Ureña, Isabel; Urzay, David; Guillot, Patricia; Guerra-Neira, Ana; Rivera, Almudena; Pérez-Cobaleda, Ángeles; Martín, Ascensión; Nuñez-Torrón, María; Alvarez, Begoña; Faraco, Mar; Barrera, José M; Calvo, María J; Gallegos, José; Bermejo, Antonio; Aramburu, Carmen; Dávila, Miguel; Carreras, Fernando; Neipp, Rosemarie; Mariscal, Alberto

    2017-06-15

    The international maritime traffic of people and goods has often contributed to the spread of pathogens affecting public health. The Maritime Declaration of Health (MDH), according to the International Health Regulations (IHR) (2005), is a document containing data related to the state of health on board a ship during passage and on arrival at port. It is a useful tool for early detection of public health risks. The main objective of our study was to evaluate compliance with the model provided in the IHR, focusing on the format and degree of completion of MDH forms received at Spanish ports. We reviewed the content of 802 MDH forms submitted to nine Spanish ports between October 2014 and March 2015. Study results show that 22% of MDH forms presented did not comply with the recommended model and 39% were incomplete. The proportion of cargo ships with correct and complete MDH forms was lower than passenger ships; thus, the nine health questions were answered less frequently by cargo ships than passenger ships (63% vs 90%, p value < 0.001). The appropriate demand and usage of MDH forms by competent authorities should improve the quality of the document as a tool and improve risk assessment. This article is copyright of The Authors, 2017.

  4. Ion recombination characteristics of the MDH 10X5-6 ionisation chamber under continuous exposure

    International Nuclear Information System (INIS)

    Cerra, F.

    1982-01-01

    Volume recombination of the induced ionization in an X-ray ionization chamber is an important factor affecting the collection efficiency of the charge when such chambers are operated at atmospheric pressure. The volume recombination process is also dependent on the X-ray exposure rate. The theory for recombination in a cylindrical ionization chamber is shown to be in agreement with experimental measurements. For the MDH 10X5-6 cylindrical ionization chamber, the recombination loss is unimportant at exposure rates consistent with its intended usage. (U.K.)

  5. Evaluation of 90-day Repeated Dose Oral Toxicity, Glycometabolism, Learning and Memory Ability, and Related Enzyme of Chromium Malate Supplementation in Sprague-Dawley Rats.

    Science.gov (United States)

    Feng, Weiwei; Wu, Huiyu; Li, Qian; Zhou, Zhaoxiang; Chen, Yao; Zhao, Ting; Feng, Yun; Mao, Guanghua; Li, Fang; Yang, Liuqing; Wu, Xiangyang

    2015-11-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the 90-day oral toxicity of chromium malate in Sprague-Dawley rats. The present study inspected the effect of chromium malate on glycometabolism, glycometabolism-related enzymes, lipid metabolism, and learning and memory ability in metabolically healthy Sprague-Dawley rats. The results showed that all rats survived and pathological, toxic, feces, and urine changes were not observed. Chromium malate did not cause measurable damage on liver, brain, and kidney. The fasting blood glucose, serum insulin, insulin resistance index, C-peptide, hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels of normal rats in chromium malate groups had no significant change when compared with control group and chromium picolinate group under physiologically relevant conditions. The serum and organ content of Cr in chromium malate groups had no significant change compared with control group. No significant changes were found in morris water maze test and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and true choline esterase (TChE) activity. The results indicated that supplementation with chromium malate did not cause measurable toxicity and has no obvious effect on glycometabolism and related enzymes, learning and memory ability, and related enzymes and lipid metabolism of female and male rats. The results of this study suggest that chromium malate is safe for human consumption.

  6. The metabolism of malate by cultured rat brain astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    McKenna, M.C.; Tildon, J.T.; Couto, R.; Stevenson, J.H.; Caprio, F.J. (Department of Pediatrics, University of Maryland School of Medicine, Baltimore (USA))

    1990-12-01

    Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-(U-14C)malate in rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain.

  7. Global Analysis of Mannitol 2-Dehydrogenase in Lactobacillus reuteri CRL 1101 during Mannitol Production through Enzymatic, Genetic and Proteomic Approaches

    Science.gov (United States)

    Ortiz, Maria Eugenia; Bleckwedel, Juliana; Fadda, Silvina; Picariello, Gianluca; Hebert, Elvira M.; Raya, Raúl R.

    2017-01-01

    Several plants, fungi, algae, and certain bacteria produce mannitol, a polyol derived from fructose. Mannitol has multiple industrial applications in the food, pharmaceutical, and medical industries, being mainly used as a non-metabolizable sweetener in foods. Many heterofermentative lactic acid bacteria synthesize mannitol when an alternative electron acceptor such as fructose is present in the medium. In previous work, we reported the ability of Lactobacillus reuteri CRL 1101 to efficiently produce mannitol from sugarcane molasses as carbon source at constant pH of 5.0; the activity of the enzyme mannitol 2-dehydrogenase (MDH) responsible for the fructose conversion into mannitol being highest during the log cell growth phase. Here, a detailed assessment of the MDH activity and relative expression of the mdh gene during the growth of L. reuteri CRL 1101 in the presence of fructose is presented. It was observed that MDH was markedly induced by the presence of fructose. A direct correlation between the maximum MDH enzyme activity and a high level of mdh transcript expression during the log-phase of cells grown in a fructose-containing chemically defined medium was detected. Furthermore, two proteomic approaches (2DE and shotgun proteomics) applied in this study confirmed the inducible expression of MDH in L. reuteri. A global study of the effect of fructose on activity, mdh gene, and protein expressions of MDH in L. reuteri is thus for the first time presented. This work represents a deep insight into the polyol formation by a Lactobacillus strain with biotechnological potential in the nutraceutics and pharmaceutical areas. PMID:28060932

  8. Quininium Malates: Partial Chiral Discrimination via Diastereomeric ...

    African Journals Online (AJOL)

    Quinine was employed as a resolving agent for racemic malic acid. The resultant product was a quininium salt containing 75 % of the D-malate anion. Quinine was also crystallized with pure L- and D-malic acids and the structures of the resulting diastereomeric salts were elucidated. The crystal packings were analyzed in ...

  9. Type 2 Diabetic Rats on Diet Supplemented With Chromium Malate Show Improved Glycometabolism, Glycometabolism-Related Enzyme Levels and Lipid Metabolism

    Science.gov (United States)

    Feng, Weiwei; Zhao, Ting; Mao, Guanghua; Wang, Wei; Feng, Yun; Li, Fang; Zheng, Daheng; Wu, Huiyu; Jin, Dun; Yang, Liuqing; Wu, Xiangyang

    2015-01-01

    Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the effect of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism in type 2 diabetic rats. Our results showed that fasting blood glucose, serum insulin level, insulin resistance index and C-peptide level in the high dose group had a significant downward trend when compared with the model group, chromium picolinate group and chromium trichloride group. The hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, Glut4, phosphor-AMPKβ1 and Akt levels in the high dose group were significantly higher than those of the model, chromium picolinate and chromium trichloride groups. Chromium malate in a high dose group can significantly increase high density lipoprotein cholesterol level while decreasing the total cholesterol, low density lipoprotein cholesterol and triglyceride levels when compared with chromium picolinate and chromium trichloride. The serum chromium content in chromium malate and chromium picolinate group is significantly higher than that of the chromium trichloride group. The results indicated that the curative effects of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism changes are better than those of chromium picolinate and chromium trichloride. Chromium malate contributes to glucose uptake and transport in order to improved glycometabolism and glycometabolism-related enzymes. PMID:25942313

  10. Alternative oxidase pathway optimizes photosynthesis during osmotic and temperature stress by regulating cellular ROS, malate valve and antioxidative systems

    Directory of Open Access Journals (Sweden)

    DINAKAR eCHALLABATHULA

    2016-02-01

    Full Text Available The present study reveals the importance of alternative oxidase (AOX pathway in optimizing photosynthesis under osmotic and temperature stress conditions in the mesophyll protoplasts of Pisum sativum. The responses of photosynthesis and respiration were monitored at saturating light intensity of 1000 µmoles m-2 s-1 at 25 oC under a range of sorbitol concentrations from 0.4 M to 1.0M to induce hyper-osmotic stress and by varying the temperature of the thermo-jacketed pre-incubation chamber from 25 oC to 10 oC to impose sub-optimal temperature stress. Compared to controls (0.4 M sorbitol and 25 OC, the mesophyll protoplasts showed remarkable decrease in NaHCO3-dependent O2 evolution (indicator of photosynthetic carbon assimilation, under both hyper-osmotic (1.0 M sorbitol and sub-optimal temperature stress conditions (10 OC, while the decrease in rates of respiratory O2 uptake were marginal. The capacity of AOX pathway increased significantly in parallel to increase in intracellular pyruvate and reactive oxygen species (ROS levels under both hyper-osmotic stress and sub-optimal temperature stress under the background of saturating light. The ratio of redox couple (Malate/OAA related to malate valve increased in contrast to the ratio of redox couple (GSH/GSSG related to antioxidative system during hyper-osmotic stress. Nevertheless, the ratio of GSH/GSSG decreased in the presence of sub-optimal temperature, while the ratio of Malate/OAA showed no visible changes. Also, the redox ratios of pyridine nucleotides increased under hyper-osmotic (NADH/NAD and sub-optimal temperature (NADPH/NADP stresses, respectively. However, upon restriction of AOX pathway by using salicylhydroxamic acid (SHAM, the observed changes in NaHCO3 dependent O2 evolution, cellular ROS, redox ratios of Malate/OAA, NAD(PH/NAD(P and GSH/GSSG were further aggravated under stress conditions with concomitant modulations in NADP-MDH and antioxidant enzymes. Taken together, the

  11. Fitness of allozyme variants in Drosophila pseudoobscura. II. Selection at the Est-5, Odh and Mdh-2 loci

    Energy Technology Data Exchange (ETDEWEB)

    Marinkovic, D; Ayala, F J

    1975-01-01

    We have studied the effects on fitness of allelic variation at three gene loci (Est-5, Odh, and Mdh-2) coding for enzymes in Drosophila pseudoobscura. Genotype has a significant effect on fitness for all six parameters measured (female fecundity, male mating capacity, egg-to-adult survival under near-optimal and under competitive conditions, and rate of development under near-optimal and under competitive conditions). No single genotype is best for all six fitness parameters; rather, genotypes with superior performance during a certain stage of the life-cycle may have low fitness at some other stage, or in different environmental conditions. Heterozygotes are sometimes best when all fitness parameters are considered. There are significant interactions between loci. The various forms of balancing selection uncovered in our experiments may account for the polymorphisms occurring in natural populations of D. pseudoobscura at the three loci studied. (auth)

  12. 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

  13. Metabolism of excised embryos of Lupinus luteus L. VI. An electrophoretic analysis of some dehydrogenases in cultured embryos as compared with the normal seedling axes

    Directory of Open Access Journals (Sweden)

    J. Czosnowski

    2015-01-01

    Full Text Available The electrophoretic patterns (disc electrophoresis of the studied dehydrogenases: glucose-6-phosphate - (A, malate - (B, glutamate - (C, alcohol - (D and lactate dehydrogenase (E, in the axial organs of isolated Lupinus luteus embryos and seedlings cultivated over 12 days are characterized by great similarities. With time, after the third day of cultivation the patterns begin to become less deyeloped. Analyses performed during the first 10 hours of imbibition of seed parts indicate that the maximal development of isozyme patterns occurs during the third hour after which the patterns become poorer. The most uniform type of pattern. and the lowest number of isozymes was shown by glutamate dehydrogenase, the richest pattern was shown by malate dehydrogenase. No band common for a 11 the 27 experimental elements was found.

  14. Aluminum-Activated Malate Transporters Can Facilitate GABA Transport.

    Science.gov (United States)

    Ramesh, Sunita A; Kamran, Muhammad; Sullivan, Wendy; Chirkova, Larissa; Okamoto, Mamoru; Degryse, Fien; McLaughlin, Michael; Gilliham, Matthew; Tyerman, Stephen D

    2018-05-01

    Plant aluminum-activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals, leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires a specific amino acid motif in ALMTs that shares similarity with a GABA binding site in mammalian GABA A receptors. Here, we explore why TaALMT1 activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA] i ) in both wheat ( Triticum aestivum ) root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA] i because TaALMT1 facilitates GABA efflux but GABA does not complex Al 3+ TaALMT1 also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14 C-GABA uptake into TaALMT1 -expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1 F213C ) prevented both GABA influx and efflux, and resulted in no correlation between malate efflux and [GABA] i We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner to regulate each other's transport, suggestive of a role for ALMTs in communicating metabolic status. © 2018 American Society of Plant Biologists. All rights reserved.

  15. An InDel in the Promoter of Al-ACTIVATED MALATE TRANSPORTER9 Selected during Tomato Domestication Determines Fruit Malate Contents and Aluminum Tolerance[OPEN

    Science.gov (United States)

    Wang, Xin; Hu, Tixu; Zhang, Fengxia; Wang, Bing; Li, Changxin; Yang, Tianxia; Li, Hanxia; Lu, Yongen; Ye, Zhibiao

    2017-01-01

    Deciphering the mechanism of malate accumulation in plants would contribute to a greater understanding of plant chemistry, which has implications for improving flavor quality in crop species and enhancing human health benefits. However, the regulation of malate metabolism is poorly understood in crops such as tomato (Solanum lycopersicum). Here, we integrated a metabolite-based genome-wide association study with linkage mapping and gene functional studies to characterize the genetics of malate accumulation in a global collection of tomato accessions with broad genetic diversity. We report that TFM6 (tomato fruit malate 6), which corresponds to Al-ACTIVATED MALATE TRANSPORTER9 (Sl-ALMT9 in tomato), is the major quantitative trait locus responsible for variation in fruit malate accumulation among tomato genotypes. A 3-bp indel in the promoter region of Sl-ALMT9 was linked to high fruit malate content. Further analysis indicated that this indel disrupts a W-box binding site in the Sl-ALMT9 promoter, which prevents binding of the WRKY transcription repressor Sl-WRKY42, thereby alleviating the repression of Sl-ALMT9 expression and promoting high fruit malate accumulation. Evolutionary analysis revealed that this highly expressed Sl-ALMT9 allele was selected for during tomato domestication. Furthermore, vacuole membrane-localized Sl-ALMT9 increases in abundance following Al treatment, thereby elevating malate transport and enhancing Al resistance. PMID:28814642

  16. The Arabidopsis vacuolar malate channel is a member of the ALMT family.

    Science.gov (United States)

    Kovermann, Peter; Meyer, Stefan; Hörtensteiner, Stefan; Picco, Cristiana; Scholz-Starke, Joachim; Ravera, Silvia; Lee, Youngsook; Martinoia, Enrico

    2007-12-01

    In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9 (A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.

  17. An InDel in the promoter of Al-activated malate transporter 9 selected during tomato domestication determines fruit malate content and aluminum tolerance

    Science.gov (United States)

    Deciphering the mechanism of malate accumulation in plants would contribute to a greater understanding of plant chemistry, which has implications for improving flavor quality in crop species and enhancing human health benefits. However, the regulation of malate metabolism is poorly understood in cro...

  18. Induction of long noncoding RNA MALAT1 in hypoxic mice

    Directory of Open Access Journals (Sweden)

    Lelli A

    2015-10-01

    Full Text Available Aurelia Lelli,1,2,* Karen A Nolan,1,2,* Sara Santambrogio,1,2 Ana Filipa Gonçalves,1,2 Miriam J Schönenberger,1,2 Anna Guinot,1,2 Ian J Frew,1,2 Hugo H Marti,3 David Hoogewijs,1,2,4 Roland H Wenger1,2 1Institute of Physiology and Zurich Center for Human Physiology (ZIHP, University of Zurich, Zurich, Switzerland; 2National Center of Competence in Research "Kidney.CH", Zurich, Switzerland; 3Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany; 4Institute of Physiology, University of Duisburg-Essen, Essen, Germany *These authors contributed equally to this work Abstract: Long thought to be “junk DNA”, in recent years it has become clear that a substantial fraction of intergenic genomic DNA is actually transcribed, forming long noncoding RNA (lncRNA. Like mRNA, lncRNA can also be spliced, capped, and polyadenylated, affecting a multitude of biological processes. While the molecular mechanisms underlying the function of lncRNAs have just begun to be elucidated, the conditional regulation of lncRNAs remains largely unexplored. In genome-wide studies our group and others recently found hypoxic transcriptional induction of a subset of lncRNAs, whereof nuclear-enriched abundant/autosomal transcript 1 (NEAT1 and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1 appear to be the lncRNAs most ubiquitously and most strongly induced by hypoxia in cultured cells. Hypoxia-inducible factor (HIF-2 rather than HIF-1 seems to be the preferred transcriptional activator of these lncRNAs. For the first time, we also found strong induction primarily of MALAT1 in organs of mice exposed to inspiratory hypoxia. Most abundant hypoxic levels of MALAT1 lncRNA were found in kidney and testis. In situ hybridization revealed that the hypoxic induction in the kidney was confined to proximal rather than distal tubular epithelial cells. Direct oxygen-dependent regulation of MALAT1 lncRNA was confirmed using isolated primary

  19. The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier.

    Science.gov (United States)

    Emmerlich, Vera; Linka, Nicole; Reinhold, Thomas; Hurth, Marco A; Traub, Michaela; Martinoia, Enrico; Neuhaus, H Ekkehard

    2003-09-16

    Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in the past to identify the vacuolar malate transporter; here, we describe the identification of the vacuolar malate transporter [A. thaliana tonoplast dicarboxylate transporter (AttDT)]. This transporter exhibits highest sequence similarity to the human sodium/dicarboxylate cotransporter. Independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] Arabidopsis mutants exhibit substantially reduced levels of leaf malate, but respire exogenously applied [14C]malate faster than the WT. An AttDT-GFP fusion protein was localized to vacuole. Vacuoles isolated from Arabidopsis WT leaves exhibited carbonylcyanide m-chlorophenylhydrazone and citrate inhibitable malate transport, which was not stimulated by sodium. Vacuoles isolated from mutant plants import [14C]-malate at strongly reduced rates, confirming that this protein is the vacuolar malate transporter.

  20. Purification of methanol dehydrogenase from mouth methylotrophic bacteria of tropical region

    Directory of Open Access Journals (Sweden)

    Waturangi, D.

    2011-01-01

    Full Text Available Aims: Purification of methanol dehydrogenase (MDH from methylotrophic bacteria was conducted to obtain pure enzyme for further research and industrial applications due to the enzyme’s unique activity that catalyzes oxidation of methanol as an important carbon source in methylotrophic bacteria.Methodology and Results: The enzyme was screened from methylotrophic bacteria isolated from human mouth. Purification of this enzyme was conducted using ammonium sulphate precipitation followed by cation exchange chromatography. Two types of media were used to produce the enzymes: luria broth and standard mineral salts media (MSM. MSM produced MDH with higher specific activity than LB. Specific activity was also increased along with the purification steps. Application of ammonium sulphate increased the purity of enzyme and was more effective for the enzyme produced in LB. Using sepharose increased the enzyme activity 10 -57 folds.Conclusion, significant and impact of this study: With this, ammonium sulphate precipitation coupled with single cation exchange chromatographic system has been proved to provide sufficient purified of methanol dehydrogenase from methylotrophic bacteria origin of human mouth with high specific activity for further application.

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

    International Nuclear Information System (INIS)

    Fukuda, Jun; Tsujimura, Seiya; Kano, Kenji

    2008-01-01

    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 μL 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 (ΔG o ') is negative; (2) α-ketoglutarate oxidation, which involves an electrochemically active coenzyme A (CoA); and (3) malate oxidation, which is thermodynamically unfavorable because of a large positive ΔG 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 α-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 ΔG o ' value negative

  2. 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.

  3. Enhanced citrate production through gene insertion in Aspergillus niger

    DEFF Research Database (Denmark)

    Jongh, Wian de; Nielsen, Jens

    2007-01-01

    The effect of inserting genes involved in the reductive branch of the tricarboxylic acid (TCA) cycle on citrate production by Aspergillus niger was evaluated. Several different genes were inserted individually and in combination, i.e. malate dehydrogenase (mdh2) from Saccharomyces cerevisiae, two...

  4. 'Dangshansuli' pear leaf

    African Journals Online (AJOL)

    ajl yemi

    2011-12-19

    Dec 19, 2011 ... metabolism of these two organic acids, including citrate synthase (CS), cytoplast aconitase ... malate dehydrogenase (MDH) (Miller et al., 1998), and ... a pH of 6.28, a soil organic matter content of 0.54% (w·w-1), a total.

  5. Development of a competitive PCR assay for the quantification of ...

    African Journals Online (AJOL)

    The E. coli malate dehydrogenase Mdh house-keeping gene was modified and used as an internal control and competitor DNA for the c-PCR. E. coli cell concentration equivalents ranging from 20 to 2 x 104 cells ml-1 could be quantified with the c-PCR. Fifty-three water samples from various sources were tested with the ...

  6. Two NAD-linked redox shuttles maintain the peroxisomal redox balance in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Al-Saryi, Nadal A.; Al-Hejjaj, Murtakab Y.; van Roermund, Carlo W. T.; Hulmes, Georgia E.; Ekal, Lakhan; Payton, Chantell; Wanders, Ronald J. A.; Hettema, Ewald H.

    2017-01-01

    In Saccharomyces cerevisiae, peroxisomes are the sole site of fatty acid beta-oxidation. During this process, NAD(+) is reduced to NADH. When cells are grown on oleate medium, peroxisomal NADH is reoxidised to NAD(+) by malate dehydrogenase (Mdh3p) and reduction equivalents are transferred to the

  7. Hazard Assessment of High-Nitrogen Explosive Compounds: A Novel In Vitro Multi-Cellular Approach

    Science.gov (United States)

    2016-03-21

    MDH1 ................................................................................ Malate dehydrogenase 1 MENA ...metabolites (2- methoxy-5-nitroaniline ( MENA ) and 2,4-diaminoanisole (DAAN)) toward various microbial targets. The resulting LC50s ranged from 8-11 mg/L...A. fischeri) to 77 mg/L (aerobic heterotrophs). Reduction of DNAN to MENA and DAAN resulted in partial detoxification. It also has been shown

  8. Development of antimigraine transdermal delivery systems of pizotifen malate.

    Science.gov (United States)

    Serna-Jiménez, C E; del Rio-Sancho, S; Calatayud-Pascual, M A; Balaguer-Fernández, C; Femenía-Font, A; López-Castellano, A; Merino, V

    2015-08-15

    The aim of this study was to develop and evaluate a transdermal delivery system of pizotifen malate. Pizotifen is frequently used in the preventive treatment of migraine, but is also indicated in eating disorders. In the course of the project, the effects of chemical enhancers such as ethanol, 1,8-cineole, limonene, azone and different fatty acids (decanoic, decenoic, dodecanoic, linoleic and oleic acids) were determined, first using a pizotifen solution. Steady state flux, diffusion and partition parameters were estimated by fitting the Scheuplein equation to the data obtained. Among the chemical enhancers studied, decenoic acid showed the highest enhancement activity, which seemed to be due to the length of its alkyl chain and unsaturation at the 9th carbon. The influence of iontophoresis and the involvement of electrotransport in said process was determined. The absorption profile obtained with iontophoresis was similar to that obtained with fatty acids and terpenes, though skin deposition of the drug was lower with the former. Transdermal delivery systems (TDS) of pizotifen were manufactured by including chemical enhancers, decenoic acid or oleic acid, and were subsequently characterized. When the results obtained with solutions were compared with those obtained with the TDS, a positive enhancement effect was observed with the latter with respect to the partitioning and diffusion of the drug across the skin. Our findings endorse the suitability of our TDS for delivering therapeutic amounts of pizotifen malate. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. [A case of respiratory dyskinesia due to clebopride malate].

    Science.gov (United States)

    Kawasaki, H; Yamamoto, M; Okayasu, H; Wakayama, Y

    1991-08-01

    Clebopride malate is therapeutically used for the treatment of peptic ulcer. This drug has potent antidopaminergic activity that causes acute dystonic reaction, parkinsonism and tardive dyskinesia as adverse effects. Here, we have reported an 86-year-old man who developed abnormal involuntary movement of respiratory muscles and lower limb muscles after this drug had been given for four months. This involuntary movement appeared spontaneously at resting state and disappeared during sleep. Surface EMG demonstrated a synchronous grouping discharge in m. orbicularis oris, m. sternocleidomastoideus and m. interstales which synchronized with diaphragmatic movement on cinefluorography. Involuntary movement of the lower limbs was synchronous bilaterally and had little relationship with diaphragmatic movement. This involuntary movement was irregular not only in rhythm but also in duration. According to this irregular nature, we diagnosed this involuntary movement as respiratory dyskinesia with limb dyskinesia that belongs to tardive dyskinesia. After cessation of clebopride malate limb dyskinesia disappeared rapidly and respiratory dyskinesia markedly decreased. We emphasize that respiratory dyskinesia should be differentiated from psychogenic hyperventilation as easily misdiagnosed on initial examination.

  10. Plant Formate Dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    John Markwell

    2005-01-10

    The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

  11. Modeling the vacuolar storage of malate shed lights on pre- and post-harvest fruit acidity.

    Science.gov (United States)

    Etienne, Audrey; Génard, Michel; Lobit, Philippe; Bugaud, Christophe

    2014-11-18

    Malate is one of the most important organic acids in many fruits and its concentration plays a critical role in organoleptic properties. Several studies suggest that malate accumulation in fruit cells is controlled at the level of vacuolar storage. However, the regulation of vacuolar malate storage throughout fruit development, and the origins of the phenotypic variability of the malate concentration within fruit species remain to be clarified. In the present study, we adapted the mechanistic model of vacuolar storage proposed by Lobit et al. in order to study the accumulation of malate in pre and postharvest fruits. The main adaptation concerned the variation of the free energy of ATP hydrolysis during fruit development. Banana fruit was taken as a reference because it has the particularity of having separate growth and post-harvest ripening stages, during which malate concentration undergoes substantial changes. Moreover, the concentration of malate in banana pulp varies greatly among cultivars which make possible to use the model as a tool to analyze the genotypic variability. The model was calibrated and validated using data sets from three cultivars with contrasting malate accumulation, grown under different fruit loads and potassium supplies, and harvested at different stages. The model predicted the pre and post-harvest dynamics of malate concentration with fairly good accuracy for the three cultivars (mean RRMSE = 0.25-0.42). The sensitivity of the model to parameters and input variables was analyzed. According to the model, vacuolar composition, in particular potassium and organic acid concentrations, had an important effect on malate accumulation. The model suggested that rising temperatures depressed malate accumulation. The model also helped distinguish differences in malate concentration among the three cultivars and between the pre and post-harvest stages by highlighting the probable importance of proton pump activity and particularly of the free

  12. Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems.

    Science.gov (United States)

    Abbrescia, Daniela Isabel; La Piana, Gianluigi; Lofrumento, Nicola Elio

    2012-02-15

    In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α-ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate aminotransferase. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Purification, crystallization and preliminary X-ray crystallographic analysis of a methanol dehydrogenase from the marine bacterium Methylophaga aminisulfidivorans MPT

    International Nuclear Information System (INIS)

    Choi, Jin Myung; Kim, Hee Gon; Kim, Jeong-Sun; Youn, Hyung-Seop; Eom, Soo Hyun; Yu, Sung-Lim; Kim, Si Wouk; Lee, Sung Haeng

    2011-01-01

    In order to obtain molecular insights into the methanol-oxidizing system of M. aminisulfidivorans, a native heterotetrameric α 2 β 2 methanol dehydrogenase complex was directly purified from M. aminisulfidivorans MP T grown in the presence of methanol and crystallized. Methylophaga aminisulfidivorans MP T is a marine methylotrophic bacterium that utilizes C 1 compounds such as methanol as a carbon and energy source. The released electron from oxidation flows through a methanol-oxidizing system (MOX) consisting of a series of electron-transfer proteins encoded by the mox operon. One of the key enzymes in the pathway is methanol dehydrogenase (MDH), which contains the prosthetic group pyrroloquinoline quinone (PQQ) and converts methanol to formaldehyde in the periplasm by transferring two electrons from the oxidation of one methanol molecule to the electron acceptor cytochrome c L . In order to obtain molecular insights into the oxidation mechanism, a native heterotetrameric α 2 β 2 MDH complex was directly purified from M. aminisulfidivorans MP T grown in the presence of methanol and crystallized. The crystal diffracted to 1.7 Å resolution and belonged to the monoclinic space group P2 1 (unit-cell parameters a = 63.9, b = 109.5, c = 95.6 Å, β = 100.5°). The asymmetric unit of the crystal contained one heterotetrameric complex, with a calculated Matthews coefficient of 2.24 Å 3 Da −1 and a solvent content of 45.0%

  14. Photosynthetic metabolism of malate and aspartate in Flaveria trinervia a C4 dicot

    International Nuclear Information System (INIS)

    Moore, B.A.

    1986-01-01

    C 4 species are known to vary in their apparent relative use of malate and aspartate to mediate carbon flux through the C 4 cycle. These studies investigate some of the adjustments in photosynthetic carbon metabolism that occur during a dark to light transition and during expansion of leaves of Flaveria trinervia, a C 4 dicot. Enzyme localization studies with isolated leaf mesophyll and bundle sheath protoplasts, indicated that both C 4 acids are formed in the mesophyll chloroplast, and that aspartate is metabolized to malate in the bundle sheath chloroplast prior to decaroxylation there. During photosynthetic induction, the partitioning of 14 CO 2 between malate and aspartate showed a single oscillation of increased aspartate labelling after 5 min of illumination. Turnover of [4-14C] (malate plus aspartate) was slow initially during illumination, prior to establishment of active pools of C 4 cycle metabolites

  15. [Properties of modified amperometric biosensors based on methanol dehydrogenase and Methylobacterium nodulans cells].

    Science.gov (United States)

    Kuznetsova, T A; Beschastnyĭ, A P; Alferov, S V; Trotsenko, Iu A

    2013-01-01

    The properties of amperometric biosensors based on methanol dehydrogenase (MDH), Methylobacterium nodulans cells, and the ferrocene-modified carbon paste electrode were investigated. It was shown that the addition ofhydroxyapatite (HA) to a carbon paste increased the sensitivity and operating stability of MDH biosensors. The linear range of the electrode was 0.0135-0.5 and 0.032-1.5 mM for methanol and formaldehyde, respectively. The detection limit of methanol and formaldehyde was 4.5 and 11.0 microM, respectively. The loss of activity of the electrode within 10 days of storage in the presence of 2.0 mM KCN did not exceed 12%. Cyanide (10 mM) completely inhibited the sensor responses to formaldehyde (1.0 mM), which allowed for the selective determination of methanol in the presence of formaldehyde. The biosensor based on cells exhibited lower stability and sensitivity toward methanol and formaldehyde; the sensitivity coefficients were 980 and 21 nA/mM, respectively.

  16. The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier

    OpenAIRE

    Emmerlich, Vera; Linka, Nicole; Reinhold, Thomas; Hurth, Marco A.; Traub, Michaela; Martinoia, Enrico; Neuhaus, H. Ekkehard

    2003-01-01

    Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in ...

  17. Prognostic value of long non-coding RNA MALAT1 in cancer patients.

    Science.gov (United States)

    Wu, Yihua; Lu, Wei; Xu, Jinming; Shi, Yu; Zhang, Honghe; Xia, Dajing

    2016-01-01

    Metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) was identified to be the first long non-coding RNA as a biomarker of independent prognostic value for early stage non-small cell lung cancer patient survival. In recent years, the association between upregulated tissue MALAT1 level and incidence of various cancers including bladder cancer, colorectal cancer, and renal cancer has been widely discussed. The aim of our present study was to assess the potential prognostic value of MALAT1 in various human cancers. PubMed, Embase, Ovid, and Cochrane Library databases were systematically searched, and eligible studies evaluating the prognostic value of MALAT1 in various cancers were included. Finally, 11 studies encompassing 1216 participants reporting with sufficient data were enrolled in the current meta-analysis. The pooled hazard ratio (HR) was 2.05 (95 % confidence interval (CI) 1.64-2.55, p < 0.01) for overall survival (OS) and 2.66 (95 % CI 1.86-3.80, p < 0.01) for disease-free survival (DFS). In conclusion, high tissue MALAT1 level was associated with an inferior clinical outcome in various cancers, suggesting that MALAT1 might serve as a potential prognostic biomarker for various cancers.

  18. Role of malate transporter in lipid accumulation of oleaginous fungus Mucor circinelloides.

    Science.gov (United States)

    Zhao, Lina; Cánovas-Márquez, José T; Tang, Xin; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Garre, Victoriano; Song, Yuanda; Ratledge, Colin

    2016-02-01

    Fatty acid biosynthesis in oleaginous fungi requires the supply of reducing power, NADPH, and the precursor of fatty acids, acetyl-CoA, which is generated in the cytosol being produced by ATP: citrate lyase which requires citrate to be, transported from the mitochondrion by the citrate/malate/pyruvate transporter. This transporter, which is within the mitochondrial membrane, transports cytosolic malate into the mitochondrion in exchange for mitochondrial citrate moving into the cytosol (Fig. 1). The role of malate transporter in lipid accumulation in oleaginous fungi is not fully understood, however. Therefore, the expression level of the mt gene, coding for a malate transporter, was manipulated in the oleaginous fungus Mucor circinelloides to analyze its effect on lipid accumulation. The results showed that mt overexpression increased the lipid content for about 70 % (from 13 to 22 % dry cell weight, CDW), whereas the lipid content in mt knockout mutant decreased about 27 % (from 13 to 9.5 % CDW) compared with the control strain. Furthermore, the extracellular malate concentration was decreased in the mt overexpressing strain and increased in the mt knockout strain compared with the wild-type strain. This work suggests that the malate transporter plays an important role in regulating lipid accumulation in oleaginous fungus M. circinelloides.

  19. Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle

    DEFF Research Database (Denmark)

    Rasmussen, Hans N; van Hall, Gerrit; Rasmussen, Ulla F

    2002-01-01

    The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation...... procedure were assayed for marker enzymes and lactate dehydrogenase (LDH). The mitochondrial fraction contained no LDH activity (detection limit approximately 0.05 % of the tissue activity) and the distribution of LDH activity among the fractions paralleled that of pyruvate kinase, i.e. LDH was fractionated...... as a cytoplasmic enzyme. Respiratory experiments with the mitochondrial fraction also indicated the absence of LDH. Lactate did not cause respiration, nor did it affect the respiration of pyruvate + malate. The major part of the native cytochrome c was retained in the isolated mitochondria, which, furthermore...

  20. Long noncoding RNA MALAT1 as a potential novel biomarker in digestive system cancers: a meta-analysis.

    Science.gov (United States)

    Song, Wei; Zhang, Run J; Zou, Shu B

    2016-08-01

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a newly discovered long non-coding RNA (lncRNA), has been reported to be overexpressed in various cancers. However, the clinical value of MALAT1 in digestive system cancers is unclear. This study was designed to investigate the potential value of MALAT1 as a prognostic biomarker in digestive system cancers. We searched the Medline, Embase and Cochrane Library databases. All studies that explored the correlation between lncRNA MALAT1 expression and survival in digestive system tumors were selected. A quantitative meta-analysis was performed for the correlation between lncRNA MALAT1 expression and survival in digestive system tumors. Five studies were eligible for analysis, which included 547 patients. Meta-analysis showed that high expression of MALAT1 could predict poor overall survival (OS) in digestive system cancers (pooled HR: 1.85, 95% CI: 1.41-2.43, Pdigestive system cancers.

  1. The Association between Abnormal Long Noncoding RNA MALAT-1 Expression and Cancer Lymph Node Metastasis: A Meta-Analysis

    Directory of Open Access Journals (Sweden)

    Jun Wang

    2016-01-01

    Full Text Available Previous studies have investigated that the expression levels of MALAT-1 were higher in cancerous tissues than matched histologically normal tissues. And, to some extent, overexpression of MALAT-1 was inclined to lymph node metastasis. This meta-analysis collected all relevant articles and explored the association between MALAT-1 expression levels and lymph node metastasis. We searched PubMed, EmBase, Web of Science, Cochrane Library, and OVID to address the level of MALAT-1 expression in cancer cases and noncancerous controls (accessed February 2015. And 8 studies comprising 696 multiple cancer patients were included to assess this association. The odds ratio (OR and its corresponding 95% confidence interval (CI were calculated to assess the strength of the association using Stata 12.0 version software. The results revealed there was a significant difference in the incidence of lymph node metastasis between high MALAT-1 expression group and low MALAT-1 expression group (OR = 1.94, 95% CI 1.15–3.28, P=0.013 random-effects model. Subgroup analysis indicated that MALAT-1 high expression had an unfavorable impact on lymph node metastasis in Chinese patients (OR = 1.87, 95% CI 1.01–2.46. This study demonstrated that the incidence of lymph node metastasis in patients detected with high MALAT-1 expression was higher than that in patients with low MALAT-1 expression in China.

  2. Prognostic value of long noncoding RNA MALAT1 in digestive system malignancies.

    Science.gov (United States)

    Zhai, Hui; Li, Xiao-Mei; Maimaiti, Ailifeire; Chen, Qing-Jie; Liao, Wu; Lai, Hong-Mei; Liu, Fen; Yang, Yi-Ning

    2015-01-01

    MALAT1, a newly discovered long noncoding RNA (lncRNA), has been reported to be highly expressed in many types of cancers. This meta-analysis summarizes its potential prognostic value in digestive system malignancies. A quantitative meta-analysis was performed through a systematic search in PubMed, Cochrane Library, Web of Science and Chinese National Knowledge Infrastructure (CNKI) for eligible papers on the prognostic impact of MALAT1 in digestive system malignancies from inception to Apr. 25, 2015. Pooled hazard ratios (HRs) with 95% confidence interval (95% CI) were calculated to summarize the effect. Five studies were included in the study, with a total of 527 patients. A significant association was observed between MALAT1 abundance and poor overall survival (OS) of patients with digestive system malignancies, with pooled hazard ratio (HR) of 7.68 (95% confidence interval [CI]: 4.32-13.66, Pdigestive system malignancies.

  3. Systemic Sunitinib Malate Treatment for Advanced Juxtapapillary Retinal Hemangioblastomas Associated with von Hippel-Lindau Disease.

    Science.gov (United States)

    Knickelbein, Jared E; Jacobs-El, Naima; Wong, Wai T; Wiley, Henry E; Cukras, Catherine A; Meyerle, Catherine B; Chew, Emily Y

    2017-01-01

    To describe the clinical course of advanced juxtapapillary retinal capillary hemangioblastomas (RCH) associated with von Hippel-Lindau (VHL) disease treated with systemic sunitinib malate, an agent that inhibits both anti-vascular endothelial growth factor and anti-platelet-derived growth factor signaling. Observational case review. Three patients with advanced VHL-related juxtapapillary RCH treated with systemic sunitinib malate. Patient 1 was followed routinely every 4 months while on systemic sunitinib prescribed by her oncologist for metastatic pancreatic neuroendocrine and kidney tumors. Patients 2 and 3 were part of a prospective clinical trial evaluating the use of systemic sunitinib for ocular VHL lesions during a period of 9 months. Visual acuity, size of RCH, and degree of exudation were recorded at each visit. Optical coherence tomography (OCT) and fluorescein angiography were also obtained at some visits. Visual acuity, size of RCH, and degree of exudation. Three patients with advanced VHL-associated juxtapapillary RCH were treated with systemic sunitinib malate. While none of the patients lost vision during therapy, treatment with sunitinib malate did not improve visual acuity or reduce the size of RCH. Improvements in RCH-associated retinal edema were observed in two patients. All patients experienced multiple adverse effects, including thyroid toxicity, thrombocytopenia, nausea, fatigue, jaundice, and muscle aches. Two of the three patients had to discontinue treatment prematurely and the third required dose reduction. Systemic sunitinib malate may be useful in slowing progression of ocular disease from VHL-associated RCH. However, significant systemic adverse effects limited its use in this small series, and systemic sunitinib malate may not be safe for treatment of RCH when used at the doses described in this report. Further studies are required to determine if this medication used at lower doses with different treatment strategies, other

  4. microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus

    DEFF Research Database (Denmark)

    Leucci, Eleonora; Patella, Francesca; Waage, Johannes

    2013-01-01

    -coding RNAs. Here we report that microRNA-9 (miR-9) regulates the expression of the Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT-1), one of the most abundant and conserved long non-coding RNAs. Intriguingly, we find that miR-9 targets AGO2-mediated regulation of MALAT1 in the nucleus. Our...

  5. Permeabilization and lysis of Pseudomonas pseudoalcaligenes cells by triton X-100 for efficient production of D-malate

    NARCIS (Netherlands)

    Werf, M.J. van der; Hartmans, S.; Tweel, W.J.J. van den

    1995-01-01

    Pseudomonas pseudoalcaligenes can only form d-malate from maleate after incubation of the cells with a solvent or a detergent. The effect of the detergent Triton X-100 on d-malate production was studied in more detail. The longer the cells were incubated with Triton X-100, the higher was the

  6. Fumarate to Malate Conversion in Infarcted Porcine Heart – a Pilot Study

    DEFF Research Database (Denmark)

    Søvsø Szocska Hansen, Esben; Tougaard, Rasmus Stilling; Nielsen, Per Mose

    2017-01-01

    Hyperpolarized MR may be a key tool for investigation cardiac metabolism and cardiac treatment response. [1,4- 13C2]Fumarate is an emerging and interesting candidate for measuring and visualizing cardiac injury after ischemia. In this study we showed an initial step for imaging cardiac cell death...... in a large animal model with [1,4- 13C2]malate. The [1,4- 13C2]malate signal correlated well with increased 13C-lactate signal and 13C-alanine absence. Overall, this shows increased metabolism in the infarcted area and ongoing necrosis....

  7. Metabolic engineering of Aspergillus oryzae for efficient production of l-malate directly from corn starch.

    Science.gov (United States)

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

    2017-11-20

    l-Malate, an important chemical building block, has been widely applied in the food, pharmaceutical, and bio-based materials industries. In previous work, we engineered Aspergillus oryzae by rewiring the reductive tricarboxylic acid pathway to produce l-malate from glucose. To decrease the production cost, here, we further engineered A. oryzae to efficiently produce l-malate directly from corn starch with simultaneous liquefaction-saccharification and fermentation. First, a promoter PN5 was constructed by quintuple tandem of the 97-bp fragment containing the cis-element of the glucoamylase gene promoter (PglaA), and with the promoter PN5, the transcriptional level of glaA gene increased by 25-45%. Then, by co-overexpression of glaA, a-amylase (amyB) and a-glucosidase (agdA) genes with the promoter PN5, the l-malate titer increased from 55.5g/L to 72.0g/L with 100g/L corn starch in shake flask. In addition, to reduce the concentration of byproducts succinate and fumarate, a fumarase from Saccharomyces cerevisiae, which facilitated the transformation of fumarate to l-malate, was overexpressed. As a result, the concentration of succinate and fumarate decreased from 12.6 and 1.29g/L to 7.8 and 0.59g/L, and the l-malate titer increased from 72.0g/L to 78.5g/L. Finally, we found that the addition of glucose at the initial fermentation stage facilitated the cell growth and l-malate synthesis, and the l-malate titer further increased to 82.3g/L by co-fermentation of 30g/L glucose and 70g/L corn starch, with a productivity of 1.18g/L/h and a yield of 0.82g/g total carbon sources. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. L-malate enhances the gene expression of carried proteins and antioxidant enzymes in liver of aged rats.

    Science.gov (United States)

    Zeng, X; Wu, J; Wu, Q; Zhang, J

    2015-01-01

    Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. To investigate the antioxidant mechanism of L-malate in the mitochondria, we analyzed the change in gene expression of two malate-aspartate shuttle (MAS)-related carried proteins (AGC, aspartate/glutamate carrier and OMC, oxoglutarate/malate carrier) in the inner mitochondrial membrane, and three antioxidant enzymes (CAT, SOD, and GSH-Px) in the mitochondria. The changes in gene expression of these proteins and enzymes were examined by real-time RT-PCR in the heart and liver of aged rats treated with L-malate. L-malate was orally administered in rats continuously for 30 days using a feeding atraumatic needle. We found that the gene expression of OMC and GSH-Px mRNA in the liver increased by 39 % and 38 %, respectively, in the 0.630 g/kg L-malate treatment group than that in the control group. The expression levels of SOD mRNA in the liver increased by 39 %, 56 %, and 78 % in the 0.105, 0.210, and 0.630 g/kg L-malate treatment groups, respectively. No difference were observed in the expression levels of AGC, OMC, CAT, SOD, and GSH-Px mRNAs in the heart of rats between the L-malate treatment and control groups. These results predicted that L-malate may increase the antioxidant capacity of mitochondria by enhancing the expression of mRNAs involved in the MAS and the antioxidant enzymes.

  9. Assessment of emerging biomarkers of liver injury in human subjects.

    Science.gov (United States)

    Schomaker, Shelli; Warner, Roscoe; Bock, Jeff; Johnson, Kent; Potter, David; Van Winkle, Joyce; Aubrecht, Jiri

    2013-04-01

    Hepatotoxicity remains a major challenge in drug development. Although alanine aminotransferase (ALT) remains the gold standard biomarker of liver injury, alternative biomarker strategies to better predict the potential for severe drug-induced liver injury (DILI) are essential. In this study, we evaluated the utility of glutamate dehydrogenase (GLDH), purine nucleoside phosphorylase (PNP), malate dehydrogenase (MDH), and paraxonase 1 (PON1) as indicators of liver injury in cohorts of human subjects, including healthy subjects across age and gender, subjects with a variety of liver impairments, and several cases of acetaminophen poisoning. In the healthy subjects, levels of GLDH and MDH were not affected by age or gender. Reference ranges for GLDH and MDH in healthy subjects were 1-10 and 79-176U/L, respectively. In contrast, the levels of PON1 and PNP were not consistent across cohorts of healthy subjects. Furthermore, GLDH and MDH had a strong correlation with elevated ALT levels and possessed a high predictive power for liver injury, as determined by ROC analysis. In contrast, PON1 and PNP did not detect liver injury in our study. Finally, evaluation of patients with acetaminophen-induced liver injury provided evidence that both GLDH and MDH might have utility as biomarkers of DILI in humans. This study is the first to evaluate GLDH, MDH, PON1, and PNP in a large number of human subjects and, and it provides an impetus for prospective clinical studies to fully evaluate the diagnostic value of GLDH and MDH for detection of liver injury.

  10. Utilization of L-aspartate, L-malate and fumarate by Pasteurella multocida

    Energy Technology Data Exchange (ETDEWEB)

    Hoefer, M.; Flossmann, K.D. (Akademie der Landwirtschaftswissenschaften der DDR, Jena. Inst. fuer Bakterielle Tierseuchenforschung)

    1981-01-01

    Strains of Pasteurella multocida use L-aspartate, L-malate and furmarate, respectively, as substrates for production of succinic acid which accumulates in the medium. As was established by studies with /sup 14/C- and /sup 3/H-labelled substrates, the degradation of these substances proceeds analogously via the citric acid cycle.

  11. Occurrence of the malate-aspartate shuttle in various tumor types.

    Science.gov (United States)

    Greenhouse, W V; Lehninger, A L

    1976-04-01

    The activity of the malate-aspartate shuttle for the reoxidation of cytoplasmic reduced nicotinamide adenine dinucleotide (NADH) by mitochondria was assessed in six lines of rodent ascites tumor cells (two strains of Ehrlich ascites carcinoma, Krebs II carcinoma, Novikoff hepatoma, AS-30D hepatoma, and L1210 mouse leukemia). All the tumor cells examined showed mitochondrial reoxidation of cytoplasmic NADH, as evidenced by the accumulation of pyruvate when the cells were incubated aerobically with L-lactate. Reoxidation of cytoplasmic NADH thus generated was completely inhibited by the transaminase inhibitor aminooxyacetate. The involvement of the respiratory chain in the reoxidation of cytoplasmic NADH was demonstrated by the action of cyanide, rotenone, and antimycin A, which strongly inhibited the formation of pyruvate from added L-lactate. Compounds that inhibit the carrier-mediated entry of malate into mitochondria, such as butylmalonate, benzenetricarboxylate, and iodobenzylmalonate, also inhibited the accumulation of pyruvate from added L-lactate by the tumor cells. The maximal rate of the malate-aspartate shuttle was established by addtion of arsenite to inhibit the mitochondrial oxidation of the pyruvate formed from added lactate. The capacity of the various tumor lines for the reoxidation of cytoplasmic NADH via the malate-aspartate shuttle approaches 20% of the total respiratory rate of the cells and thus appears to be sufficient to account for the mitochondrial reoxidation of that fraction of glycolytic NADH not reoxidized by pyruvate and lactate dehydrognenase in the cytoplasm.

  12. Utilization of L-aspartate, L-malate and fumarate by Pasteurella multocida

    International Nuclear Information System (INIS)

    Hoefer, M.; Flossmann, K.D.

    1981-01-01

    Strains of Pasteurella multocida use L-aspartate, L-malate and furmarate, respectively, as substrates for production of succinic acid which accumulates in the medium. As was established by studies with 14 C- and 3 H-labelled substrates, the degradation of these substances proceeds analogously via the citric acid cycle. (author)

  13. Increased activities of mitochondrial enzymes in white adipose tissue in trained rats

    DEFF Research Database (Denmark)

    Stallknecht, B; Vinten, J; Ploug, T

    1991-01-01

    of 8-12 rats were swim trained for 10 wk or served as either sedentary, sham swim-trained, or cold-stressed controls. White adipose tissue was removed, and the activities of the respiratory chain enzyme cytochrome-c oxidase (CCO) and of the enzyme malate dehydrogenase (MDH), which participates...... 0.05). In female rats the CCO activity expressed per milligram protein was increased 4.5-fold in the trained compared with the sedentary control rats (P less than 0.01). Neither cold stress nor sham swim training increased CCO or MDH activities in white adipose tissue (P greater than 0...

  14. Characterization of phosphorylated isocitrate dehydrogenase and purification of the isocitrate dehydrogenase kinase/phosphatase of Escherichia coli

    International Nuclear Information System (INIS)

    Malloy, P.J.

    1985-01-01

    NADP + -specific isocitrate dehydrogenase (IDH; EC 1.1.1.42) was shown to be phosphorylated with ( 32 P)-orthophosphate in vivo in several strains of Escherichia coli. In strain KC 13, an adenylate cyclase deficient mutant, the specific activity of IDH decreased 70% when acetate was added to stationary phase cultures grown on glucose. The enzyme was immunoprecipitated from sonic extracts and shown to contain 32 P by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The results demonstrate that unlike many eukaryotic protein kinases, the protein kinase involved in the phosphorylation of IDH in E. coli does not require cyclic adenosine monophosphate for catalysis. Similarly, the phosphorylation of IDH was demonstrated in E. coli mutants deficient in either isocitrate lyase or malate synthase. The incorporation of 32 P in IDH was demonstrated following SDS-PAGE and autoradiography of the immunoprecipitated enzyme. These results suggest that the conditions required for the phosphorylation of IDH do not depend on the functioning of the glyoxylate shunt. Following in vivo 32 P-labeling of E. coli strain F143/KL259 in the presence of acetate, 32 P-labeled IDH was isolated from sonicated extracts of the cells. The 32 P-enzyme was carboxylmethylated and digested with trypsin. A single 32 P-labeled peptide was isolated from the tryptic digest. Amino acid analysis of the purified 32 P-labeled peptide showed that the peptide contains seven amino acids, including a single phosphorylated serine residue

  15. The varied functions of aluminium-activated malate transporters–much more than aluminium resistance

    Science.gov (United States)

    Palmer, Antony J.; Baker, Alison; Muench, Stephen P.

    2016-01-01

    The ALMT (aluminium-activated malate transporter) family comprises a functionally diverse but structurally similar group of ion channels. They are found ubiquitously in plant species, expressed throughout different tissues, and located in either the plasma membrane or tonoplast. The first family member identified was TaALMT1, discovered in wheat root tips, which was found to be involved in aluminium resistance by means of malate exudation into the soil. However, since this discovery other family members have been shown to have many other functions such as roles in stomatal opening, general anionic homoeostasis, and in economically valuable traits such as fruit flavour. Recent evidence has also shown that ALMT proteins can act as key molecular actors in GABA (γ-aminobutyric acid) signalling, the first evidence that GABA can act as a signal transducer in plants. PMID:27284052

  16. The varied functions of aluminium-activated malate transporters-much more than aluminium resistance.

    Science.gov (United States)

    Palmer, Antony J; Baker, Alison; Muench, Stephen P

    2016-06-15

    The ALMT (aluminium-activated malate transporter) family comprises a functionally diverse but structurally similar group of ion channels. They are found ubiquitously in plant species, expressed throughout different tissues, and located in either the plasma membrane or tonoplast. The first family member identified was TaALMT1, discovered in wheat root tips, which was found to be involved in aluminium resistance by means of malate exudation into the soil. However, since this discovery other family members have been shown to have many other functions such as roles in stomatal opening, general anionic homoeostasis, and in economically valuable traits such as fruit flavour. Recent evidence has also shown that ALMT proteins can act as key molecular actors in GABA (γ-aminobutyric acid) signalling, the first evidence that GABA can act as a signal transducer in plants. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  17. Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications.

    Science.gov (United States)

    Holtgrefe, Simone; Gohlke, Jochen; Starmann, Julia; Druce, Samantha; Klocke, Susanne; Altmann, Bianca; Wojtera, Joanna; Lindermayr, Christian; Scheibe, Renate

    2008-06-01

    Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.

  18. Analysis of copper and uranyl malates by potentiometry, polarimetry and spectrophotometry

    Energy Technology Data Exchange (ETDEWEB)

    Khalil, I; Petit-Ramel, M M; Chapelet-Barbier, C [Lyon-1 Univ., 69 (France)

    1978-07-15

    The stability and optical constants of the copper malates (CuH/sub 2/Mal)/sup +/, (CuHMal), (CuH/sub 2/Mal/sub 2/)/sup 2 -/, (CuMal)/sup -/, (Cu/sub 2/Mal/sub 2/)/sup 2 -/, and (CuHsub(-1)Mal)/sup 2 -/ have been fitted by a pit-mapping method, which also gives their confidence limits, from potentiometric, visible spectroscopic and polarimetric measurements.

  19. Post-irradiation inactivation, protection, and repair of the sulfhydryl enzyme malate synthase

    International Nuclear Information System (INIS)

    Durchschlag, H.; Zipper, P.

    1985-01-01

    Malate synthase from baker's yeast, a trimeric sulfhydryl enzyme with one essential sulfhydryl group per subunit, was inactivated by 2 kGy X-irradiation in air-saturated aqueous solution (enzyme concentration: 0.5 mg/ml). The radiation induced changes of enzymic activity were registered at about 0,30,60 h after irradiation. To elucidate the role of OH - , O 2 , and H 2 O 2 in the X-ray inactivation of the enzyme, experiments were performed in the absence of presence of different concentrations of specific additives (formate, superoxide dismutase, catalase). These additives were added to malate synthase solutions before or after X-irradiation. Moreover, repairs of inactivated malate synthase were initiated at about 0 or 30 h after irradiation by means of the sulfhydryl agent dithiothreitol. Experiments yielded the following results: 1. Irradiation of malate synthase in the absence of additives inactivated the enzyme immediately to a residual activity Asub(r)=3% (corresponding to a D 37 =0.6 kGy), and led to further slow inactivation in the post-irradiation phase. Repairs, initiated at different times after irradiation, restored enzymic activity considerably. The repair initiated at t=0 led to Asub(r)=21%; repairs started later on resulted in somewhat lower activities. The decay of reparability, however, was found to progress more slowly than post-irradiation inactivation itself. After completion of repair the activities of repaired samples did not decrease significantly. 2. The presence of specific additives during irradiation caused significant protective effects against primary inactivation. The protection by formate was very pronounced (e.g., Asub(r)=72% and D 37 =6 kGy for 100 mM formate). The presence of catalytic amounts of superoxide dismutase and/or catalase exhibited only minor effects, depending on the presence and concentration of formate. (orig.)

  20. Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells.

    Science.gov (United States)

    Greenhouse, W V; Lehninger, A L

    1977-11-01

    Measurements of respiration, CO2 and lactate production, and changes in the levels of various key metabolites of the glycolytic sequence and tricarboxylic acid cycle were made on five lines of rodent ascites tumor cells (two strains of Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma, and L1210 cells) incubated aerobically in the presence of uniformly labeled D-[14C]glucose. From these data, as well as earlier evidence demonstrating that the reduced nicotinamide adenine dinucleotide (NADH) shuttle in these cells requires a transaminase step and is thus identified as the malate-aspartate shuttle (W.V.V. Greenhouse and A.L. Lehninger, Cancer Res., 36: 1392-1396, 1976), metabolic flux diagrams were constructed for the five cell lines. These diagrams show the relative rates of glycolysis, the tricarboxylic acid cycle, electron transport, and the malate-aspartate shuttle in these tumors. Large amounts of cytosolic NADH were oxidized by the mitochondrial respiratory chain via the NADH shuttle, comprising anywhere from about 20 to 80% of the total flow of reducing equivalents to oxygen in these tumors. Calculations of the sources of energy for adenosine triphosphate synthesis indicated that on the average about one-third of the respiratory adenosine triphosphate is generated by electron flow originating from cytosolic NADH via the malate-aspartate shuttle.

  1. Genes Encoding Aluminum-Activated Malate Transporter II and their Association with Fruit Acidity in Apple

    Directory of Open Access Journals (Sweden)

    Baiquan Ma

    2015-11-01

    Full Text Available A gene encoding aluminum-activated malate transporter (ALMT was previously reported as a candidate for the locus controlling acidity in apple ( × Borkh.. In this study, we found that apple genes can be divided into three families and the gene belongs to the family. Duplication of genes in apple is related to the polyploid origin of the apple genome. Divergence in expression has occurred between the gene and its homologs in the family and only the gene is significantly associated with malic acid content. The locus consists of two alleles, and . resides in the tonoplast and its ectopic expression in yeast was found to increase the influx of malic acid into yeast cells significantly, suggesting it may function as a vacuolar malate channel. In contrast, encodes a truncated protein because of a single nucleotide substitution of G with A in the last exon. As this truncated protein resides within the cell membrane, it is deemed to be nonfunctional as a vacuolar malate channel. The frequency of the genotype is very low in apple cultivars but is high in wild relatives, which suggests that apple domestication may be accompanied by selection for the gene. In addition, variations in the malic acid content of mature fruits were also observed between accessions with the same genotype in the locus. This suggests that the gene is not the only genetic determinant of fruit acidity in apple.

  2. Brownian dynamic study of an enzyme metabolon in the TCA cycle: Substrate kinetics and channeling.

    Science.gov (United States)

    Huang, Yu-Ming M; Huber, Gary A; Wang, Nuo; Minteer, Shelley D; McCammon, J Andrew

    2018-02-01

    Malate dehydrogenase (MDH) and citrate synthase (CS) are two pacemaking enzymes involved in the tricarboxylic acid (TCA) cycle. Oxaloacetate (OAA) molecules are the intermediate substrates that are transferred from the MDH to CS to carry out sequential catalysis. It is known that, to achieve a high flux of intermediate transport and reduce the probability of substrate leaking, a MDH-CS metabolon forms to enhance the OAA substrate channeling. In this study, we aim to understand the OAA channeling within possible MDH-CS metabolons that have different structural orientations in their complexes. Three MDH-CS metabolons from native bovine, wild-type porcine, and recombinant sources, published in recent work, were selected to calculate OAA transfer efficiency by Brownian dynamics (BD) simulations and to study, through electrostatic potential calculations, a possible role of charges that drive the substrate channeling. Our results show that an electrostatic channel is formed in the metabolons of native bovine and recombinant porcine enzymes, which guides the oppositely charged OAA molecules passing through the channel and enhances the transfer efficiency. However, the channeling probability in a suggested wild-type porcine metabolon conformation is reduced due to an extended diffusion length between the MDH and CS active sites, implying that the corresponding arrangements of MDH and CS result in the decrease of electrostatic steering between substrates and protein surface and then reduce the substrate transfer efficiency from one active site to another. © 2017 The Protein Society.

  3. Genetics Home Reference: dihydropyrimidine dehydrogenase deficiency

    Science.gov (United States)

    ... 5-fluorouracil and capecitabine. These drugs are not broken down efficiently by people with dihydropyrimidine dehydrogenase deficiency ... of this enzyme. Because fluoropyrimidine drugs are also broken down by the dihydropyrimidine dehydrogenase enzyme, deficiency of ...

  4. Altered Expression of a Malate-Permeable Anion Channel, OsALMT4, Disrupts Mineral Nutrition1[OPEN

    Science.gov (United States)

    Delhaize, Emmanuel

    2017-01-01

    Aluminum-activated malate transporters (ALMTs) form a family of anion channels in plants, but little is known about most of its members. This study examined the function of OsALMT4 from rice (Oryza sativa). We show that OsALMT4 is expressed in roots and shoots and that the OsALMT4 protein localizes to the plasma membrane. Transgenic rice lines overexpressing (OX) OsALMT4 released malate from the roots constitutively and had 2-fold higher malate concentrations in the xylem sap than nulls, indicating greater concentrations of malate in the apoplast. OX lines developed brown necrotic spots on the leaves that did not appear on nulls. These symptoms were not associated with altered concentrations of any mineral element in the leaves, although the OX lines had higher concentrations of Mn and B in their grain compared with nulls. While total leaf Mn concentrations were not different between the OX and null lines, Mn concentrations in the apoplast were greater in the OX plants. The OX lines also displayed increased expression of Mn transporters and were more sensitive to Mn toxicity than null plants. We showed that the growth of wild-type rice was unaffected by 100 µm Mn in hydroponics but, when combined with 1 mm malate, this concentration inhibited growth. We conclude that increasing OsALMT4 expression affected malate efflux and compartmentation within the tissues, which increased Mn concentrations in the apoplast of leaves and induced the toxicity symptoms. This study reveals new links between malate transport and mineral nutrition. PMID:29101278

  5. Long Non-Coding RNA MALAT1 Mediates Transforming Growth Factor Beta1-Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Shuai Yang

    Full Text Available To study the role of long non-coding RNA (lncRNA MALAT1 in transforming growth factor beta 1 (TGF-β1-induced epithelial-mesenchymal transition (EMT of retinal pigment epithelial (RPE cells.ARPE-19 cells were cultured and exposed to TGF-β1. The EMT of APRE-19 cells is confirmed by morphological change, as well as the increased expression of alpha-smooth muscle actin (αSMA and fibronectin, and the down-regulation of E-cadherin and Zona occludin-1(ZO-1 at both mRNA and protein levels. The expression of lncRNA MALAT1 in RPE cells were detected by quantitative real-time PCR. Knockdown of MALAT1 was achieved by transfecting a small interfering RNA (SiRNA. The effect of inhibition of MALAT1 on EMT, migration, proliferation, and TGFβ signalings were observed. MALAT1 expression was also detected in primary RPE cells incubated with proliferative vitreoretinopathy (PVR vitreous samples.The expression of MALAT1 is significantly increased in RPE cells incubated with TGFβ1. MALAT1 silencing attenuates TGFβ1-induced EMT, migration, and proliferation of RPE cells, at least partially through activating Smad2/3 signaling. MALAT1 is also significantly increased in primary RPE cells incubated with PVR vitreous samples.LncRNA MALAT1 is involved in TGFβ1-induced EMT of human RPE cells and provides new understandings for the pathogenesis of PVR.

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

    Directory of Open Access Journals (Sweden)

    Elena Ciornea

    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.

  7. 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.

  8. A chimeric protein of aluminum-activated malate transporter generated from wheat and Arabidopsis shows enhanced response to trivalent cations.

    Science.gov (United States)

    Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Yamamoto, Yoko

    2016-07-01

    TaALMT1 from wheat (Triticum aestivum) and AtALMT1 from Arabidopsis thaliana encode aluminum (Al)-activated malate transporters, which confer acid-soil tolerance by releasing malate from roots. Chimeric proteins from TaALMT1 and AtALMT1 (Ta::At, At::Ta) were previously analyzed in Xenopus laevis oocytes. Those studies showed that Al could activate malate efflux from the Ta::At chimera but not from At::Ta. Here, functions of TaALMT1, AtALMT1 and the chimeric protein Ta::At were compared in cultured tobacco BY-2 cells. We focused on the sensitivity and specificity of their activation by trivalent cations. The activation of malate efflux by Al was at least two-fold greater in the chimera than the native proteins. All proteins were also activated by lanthanides (erbium, ytterbium, gadolinium, and lanthanum), but the chimera again released more malate than TaALMT1 or AtALMT1. In Xenopus oocytes, Al, ytterbium, and erbium activated inward currents from the native TaALMT1 and the chimeric protein, but gadolinium only activated currents from the chimera. Lanthanum inhibited currents from both proteins. These results demonstrated that function of the chimera protein was altered compared to the native proteins and was more responsive to a range of trivalent cations when expressed in plant cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. BoALMT1, an Al-Induced Malate Transporter in Cabbage, Enhances Aluminum Tolerance in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Lei Zhang

    2018-01-01

    Full Text Available Aluminum (Al is present in approximately 50% of the arable land worldwide and is regarded as the main limiting factor of crop yield on acidic soil. Al-induced root malate efflux plays an important role in the Al tolerance of plants. Here, the aluminum induced malate transporter BoALMT1 (KF322104 was cloned from cabbage (Brassica oleracea. BoALMT1 showed higher expression in roots than in shoots. The expression of BoALMT1 was specifically induced by Al treatment, but not the trivalent cations lanthanum (La, cadmium (Cd, zinc (Zn, or copper (Cu. Subcellular localization studies were performed in onion epidermal cells and revealed that BoALMT1 was localized at the plasma membrane. Scanning Ion-selective Electrode Technique was used to analyze H+ flux. Xenopus oocytes and Arabidopsis thaliana expressing BoALMT1 excreted more H+ under Al treatment. Overexpressing BoALMT1 in transgenic Arabidopsis resulted in enhanced Al tolerance and increased malate secretion. The results suggested that BoALMT1 functions as an Al-resistant gene and encodes a malate transporter. Expressing BoALMT1 in Xenopus oocytes or A. thaliana indicated that BoALMT1 could increase malate secretion and H+ efflux to resist Al tolerance.

  10. Citrate, malate and alkali content in commonly consumed diet sodas: implications for nephrolithiasis treatment.

    Science.gov (United States)

    Eisner, Brian H; Asplin, John R; Goldfarb, David S; Ahmad, Ardalanejaz; Stoller, Marshall L

    2010-06-01

    Citrate is a known inhibitor of calcium stone formation. Dietary citrate and alkali intake may have an effect on citraturia. Increasing alkali intake also increases urine pH, which can help prevent uric acid stones. We determined citrate, malate and total alkali concentrations in commonly consumed diet sodas to help direct dietary recommendations in patients with hypocitraturic calcium or uric acid nephrolithiasis. Citrate and malate were measured in a lemonade beverage commonly used to treat hypocitraturic calcium nephrolithiasis and in 15 diet sodas. Anions were measured by ion chromatography. The pH of each beverage was measured to allow calculation of the unprotonated anion concentration using the known pK of citric and malic acid. Total alkali equivalents were calculated for each beverage. Statistical analysis was done using Pearson's correlation coefficient. Several sodas contained an amount of citrate equal to or greater than that of alkali and total alkali as a lemonade beverage commonly used to treat hypocitraturic calcium nephrolithiasis (6.30 mEq/l citrate as alkali and 6.30 as total alkali). These sodas were Diet Sunkist Orange, Diet 7Up, Sprite Zero, Diet Canada Dry Ginger Ale, Sierra Mist Free, Diet Orange Crush, Fresca and Diet Mountain Dew. Colas, including Caffeine Free Diet Coke, Coke Zero, Caffeine Free Diet Pepsi and Diet Coke with Lime, had the lowest total alkali (less than 1.0 mEq/l). There was no significant correlation between beverage pH and total alkali content. Several commonly consumed diet sodas contain moderate amounts of citrate as alkali and total alkali. This information is helpful for dietary recommendations in patients with calcium nephrolithiasis, specifically those with hypocitraturia. It may also be useful in patients with low urine pH and uric acid stones. Beverage malate content is also important since malate ingestion increases the total alkali delivered, which in turn augments citraturia and increases urine pH. Copyright

  11. Tingkat Kerentanan Aedes aegypti (Linn. terhadap Malation di Provinsi Sumatera Selatan

    Directory of Open Access Journals (Sweden)

    Lasbudi P. Ambarita

    2015-07-01

    Full Text Available AbstractDengue vector control program in Indonesia and also South Sumatera Province has been using malathion quite long enough. The extensive use of chemical in dengue vector control can lead to development of resistance. This study aims to determine the susceptibility of Aedes aegypti against malathion in 11 district of South Sumatera Province. Larva or pupae were collected with entomology survey kit and colonized until first generation (F1 that were used for bioassay. This test was conducted according to WHO adult susceptibility bioassay procedure.Twenty five blood-fed mosquitoes were exposed to insecticide impregnated paper in each of 4 WHO test kits and 1 control tube. Aedes aegypti from all study sites were still susceptible to operational dose of malathion (5%after 1 hour exposure. The estimated resistance ratio (ERR of knockdown time (KT to operasional dose of malathion is about 1,02 – 1,27 for KT50 and 0,96 – 1,24 for KT95. The susceptibility test of adult mosquitoes to diagnostic dose (0,8% of malathion showed a variety of susceptibility after 24 hours. Strain of 7 districts showed resistance, 3 districts toleran and 1 district still susceptible. The detection of resistance can actually help public health personnel to formulate appropriate steps in encountering the reduction in effectiveness of vector control efforts.Keywords : Aedes aegypti, Malathion, Susceptibility, South SumateraAbstrakProgram pengendalian vektor DBD di Indonesia termasuk di Provinsi Sumatera Selatan telah cukup lama menggunakan malation dengan konsentrasi 5%. Penggunaan satu jenis insektisida kimiawi secara ekstensif dapat memicu perkembangan resistensi. Penelitian ini bertujuan untuk menentukan status kerentanan Aedes aegypti terhadap malation dari 11 kabupaten/kota di Provinsi Sumatera Selatan. Larva atau pupa dikumpulkan menggunakan alat survei entomologi dan selanjutnyadipelihara hingga mendapatkan generasi pertama (F1 yang akan digunakan pada uji

  12. Serum long non coding RNA MALAT-1 protected by exosomes is up-regulated and promotes cell proliferation and migration in non-small cell lung cancer.

    Science.gov (United States)

    Zhang, Rui; Xia, Yuhong; Wang, Zhixin; Zheng, Jie; Chen, Yafei; Li, Xiaoli; Wang, Yu; Ming, Huaikun

    2017-08-19

    Circulating lncRNAs have been defined as a novel biomarker for non-small cell lung cancer (NSCLC), MALAT-1 was first identified lncRNA that was related to lung cancer metastasis. However, the relationship between exosomal lncRNAs and the diagnosis and prognosis of NSCLC was poorly understood. The aim of this study is to evaluate the clinical significance of serum exosomal MALAT-1 as a biomarker in the metastasis of NSCLC. In this study, we firstly isolated the exosomes from healthy subjects and NSCLC patients. Then we measured the expression levels of MALAT-1 contained in exosomes, and found that exosomal MALAT-1 was highly expressed in NSCLC patients, more importantly, the levels of exosomal MALAT-1 were positively associated with tumor stage and lymphatic metastasis. In addition, we decreased MALAT-1 expression by short hairpin RNA and conducted a series of assays including MTT, cell cycle, colony formation, wound-healing scratch and Annexin/V PI by flow cytometry in human lung cancer cell lines. These in vitro studies demonstrated that serum exosome-derived long noncoding RNA MALAT-1 promoted the tumor growth and migration, and prevented tumor cells from apoptosis in lung cancer cell lines. Taken together, this study shed a light on utilizing MALAT-1 in exosomes as a non-invasive serum-based tumor biomarker for diagnosis and prognosis of NSCLC. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. The BnALMT1 Protein That is an Aluminum-Activated Malate Transporter is Localized in the Plasma Membrane

    OpenAIRE

    Ligaba, Ayalew; Katsuhara, Maki; Sakamoto, Wataru; Matsumoto, Hideaki

    2007-01-01

    We have previously reported that Al-induces citrate and malate efflux from P-sufficient and P-deficient plants of rape (Brassica napus L.) and that P-deficiency alone could not induce this response. Further investigation showed that the transcript of two genes designated BnALMT1 and BnALMT2 is accumulated in roots by Al-treatment. Transgenic tobacco cells (Nicotiana tabacum) and Xenopus laevis oocytes expressing the BnALMT1 and BnALMT2 proteins released more malate than control cells in the p...

  14. BoALMT1, an Al-Induced Malate Transporter in Cabbage, Enhances Aluminum Tolerance in Arabidopsis thaliana

    OpenAIRE

    Zhang, Lei; Wu, Xin-Xin; Wang, Jinfang; Qi, Chuandong; Wang, Xiaoyun; Wang, Gongle; Li, Mingyue; Li, Xingsheng; Guo, Yang-Dong

    2018-01-01

    Aluminum (Al) is present in approximately 50% of the arable land worldwide and is regarded as the main limiting factor of crop yield on acidic soil. Al-induced root malate efflux plays an important role in the Al tolerance of plants. Here, the aluminum induced malate transporter BoALMT1 (KF322104) was cloned from cabbage (Brassica oleracea). BoALMT1 showed higher expression in roots than in shoots. The expression of BoALMT1 was specifically induced by Al treatment, but not the trivalent catio...

  15. Relations of enzymes inAspergillus repens grown under sodium chloride stress.

    Science.gov (United States)

    Kelavkar, U P; Chhatpar, H S

    1993-09-01

    Aspergillus repens, a salt-pan isolate, was halotolerant. When grown for 72 h (log phase) and 144 h (beginning of stationary phase) in a medium containing 2M sodium chloride, the activities of invertase, malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH), and glutamate dehydrogenase (GDH) were found to have increased. Control cultures grown in a medium devoid of 2M NaCl failed to show such changes. The activities of MDH, G6PDH, and GDH increased with rising concentrations of Na(+) (as NaCl) when added up to 100MM in vitro. At higher concentrations they decreased. Changes in kinetic constants, Km and Vmax of these enzymes, as well as their de novo synthesis, were found to be some of the responses to NaCl stress-mediated changes.

  16. Phosphorylation of glyoxysomal malate synthase from castor oil seed endosperm and cucumber cotyledon

    International Nuclear Information System (INIS)

    Yang, Y.P; Randall, D.D.

    1989-01-01

    Glyoxysomal malate synthase (MS) was purified to apparent homogeneity from 3-d germinating castor oil seed endosperm by a relatively simple procedure including two sucrose density gradient centrifugations. Antibodies raised to the caster oil seed MS crossreacted with MS from cucumber cotyledon. MS was phosphorylated in both tissues in an MgATP dependent reaction. The phosphorylation pattern was similar for both enzymes and both enzymes were inhibited by NaF, NaMo, (NH 4 )SO 4 , glyoxylate and high concentration of MgCl 2 (60 mM), but was not inhibited by NaCl and malate. Further characterization of the phosphorylation of MS from castor oil seed endosperms showed that the 5S form of MS is the form which is labelled by 32 P. The addition of exogenous alkaline phosphatase to MS not only decreased enzyme activity, but could also dephosphorylate phospho-MS. The relationship between dephosphorylation of MS and the decrease of MS activity is currently under investigation

  17. Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase.

    Science.gov (United States)

    Modig, Tobias; Lidén, Gunnar; Taherzadeh, Mohammad J

    2002-01-01

    The kinetics of furfural inhibition of the enzymes alcohol dehydrogenase (ADH; EC 1.1.1.1), aldehyde dehydrogenase (AlDH; EC 1.2.1.5) and the pyruvate dehydrogenase (PDH) complex were studied in vitro. At a concentration of less than 2 mM furfural was found to decrease the activity of both PDH and AlDH by more than 90%, whereas the ADH activity decreased by less than 20% at the same concentration. Furfural inhibition of ADH and AlDH activities could be described well by a competitive inhibition model, whereas the inhibition of PDH was best described as non-competitive. The estimated K(m) value of AlDH for furfural was found to be about 5 microM, which was lower than that for acetaldehyde (10 microM). For ADH, however, the estimated K(m) value for furfural (1.2 mM) was higher than that for acetaldehyde (0.4 mM). The inhibition of the three enzymes by 5-hydroxymethylfurfural (HMF) was also measured. The inhibition caused by HMF of ADH was very similar to that caused by furfural. However, HMF did not inhibit either AlDH or PDH as severely as furfural. The inhibition effects on the three enzymes could well explain previously reported in vivo effects caused by furfural and HMF on the overall metabolism of Saccharomyces cerevisiae, suggesting a critical role of these enzymes in the observed inhibition. PMID:11964178

  18. Are phloem-derived amino acids the origin of the elevated malate concentration in the xylem sap following mineral N starvation in soybean?

    Science.gov (United States)

    Vitor, Simone C; do Amarante, Luciano; Sodek, Ladaslav

    2018-05-16

    A substantial increase in malate in the xylem sap of soybean subjected to mineral N starvation originates mainly from aspartate, a prominent amino acid of the phloem. A substantial increase in xylem malate was found when non-nodulated soybean plants were transferred to a N-free medium. Nodulated plants growing in the absence of mineral N and, therefore, dependent on symbiotic N 2 fixation also contained elevated concentrations of malate in the xylem sap. When either nitrate or ammonium was supplied, malate concentrations in the xylem sap were low, both for nodulated and non-nodulated plants. Evidence was obtained that the elevated malate concentration of the xylem was derived from amino acids supplied by the phloem. Aspartate was a prominent component of the phloem sap amino acids and, therefore, a potential source of malate. Supplying the roots of intact plants with 13 C-aspartate revealed that malate of the xylem sap was readily labelled under N starvation. A hypothetical scheme is proposed whereby aspartate supplied by the phloem is metabolised in the roots and the products of this metabolism cycled back to the shoot. Under N starvation, aspartate metabolism is diverted from asparagine synthesis to supply N for the synthesis of other amino acids via transaminase activity. The by-product of aspartate transaminase activity, oxaloacetate, is transformed to malate and its export accounts for much of the elevated concentration of malate found in the xylem sap. This mechanism represents a new additional role for malate during mineral N starvation of soybean, beyond that of charge balance.

  19. 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.

  20. Investigations regarding the anthropic impact on the Krebs cycle dehydrogenases system on certain wood-species in mining areas, Suceava county

    Directory of Open Access Journals (Sweden)

    Marius Viorel Oniciuc

    2013-03-01

    Full Text Available The Krebs cycle, a second stage of cellular respiration that occurs in the mitochondrion of the leafcell and consist in a multistep processes plays a central role in catabolism of organic fuel molecules. The miningextraction technologies for both underground and surface, the preparation of copper ore and barite applied in Tarnia,respectively to the sulphur in Calimani Mountain and the excess of these elements in natural environment may causemalfunction of ecosystem. The dehydrogenases of Krebs cycle can give information on the type and the duration of theeffects of pollutants on the metabolic activity in leaves, to subsequent area pollution, therefore, the aim of the presentstudy has been to determine these effects on this enzymatic system activity. For this reason, the isocitrate dehydrogenase,the -ketoglutate dehydrogenase, the succinate ehydrogenase and the malate dehydrogenase activity was determined using the spectrophotometric method with triphenyl-tetrazolium and the analysis of experimental results shows the differences from one sample to another sample of closely related species specificity, but also the effect of environmentalfactors.

  1. Impaired Malate and Fumarate Accumulation Due to the Mutation of the Tonoplast Dicarboxylate Transporter Has Little Effects on Stomatal Behavior.

    Science.gov (United States)

    Medeiros, David B; Barros, Kallyne A; Barros, Jessica Aline S; Omena-Garcia, Rebeca P; Arrivault, Stéphanie; Sanglard, Lílian M V P; Detmann, Kelly C; Silva, Willian Batista; Daloso, Danilo M; DaMatta, Fábio M; Nunes-Nesi, Adriano; Fernie, Alisdair R; Araújo, Wagner L

    2017-11-01

    Malate is a central metabolite involved in a multiplicity of plant metabolic pathways, being associated with mitochondrial metabolism and playing significant roles in stomatal movements. Vacuolar malate transport has been characterized at the molecular level and is performed by at least one carrier protein and two channels in Arabidopsis ( Arabidopsis thaliana ) vacuoles. The absence of the Arabidopsis tonoplast Dicarboxylate Transporter (tDT) in the tdt knockout mutant was associated previously with an impaired accumulation of malate and fumarate in leaves. Here, we investigated the consequences of this lower accumulation on stomatal behavior and photosynthetic capacity as well as its putative metabolic impacts. Neither the stomatal conductance nor the kinetic responses to dark, light, or high CO 2 were highly affected in tdt plants. In addition, we did not observe any impact on stomatal aperture following incubation with abscisic acid, malate, or citrate. Furthermore, an effect on photosynthetic capacity was not observed in the mutant lines. However, leaf mitochondrial metabolism was affected in the tdt plants. Levels of the intermediates of the tricarboxylic acid cycle were altered, and increases in both light and dark respiration were observed. We conclude that manipulation of the tonoplastic organic acid transporter impacted mitochondrial metabolism, while the overall stomatal and photosynthetic capacity were unaffected. © 2017 American Society of Plant Biologists. All Rights Reserved.

  2. Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance.

    Science.gov (United States)

    Liu, Jiping; Magalhaes, Jurandir V; Shaff, Jon; Kochian, Leon V

    2009-02-01

    Aluminum-activated root malate and citrate exudation play an important role in plant Al tolerance. This paper characterizes AtMATE, a homolog of the recently discovered sorghum and barley Al-tolerance genes, shown here to encode an Al-activated citrate transporter in Arabidopsis. Together with the previously characterized Al-activated malate transporter, AtALMT1, this discovery allowed us to examine the relationship in the same species between members of the two gene families for which Al-tolerance genes have been identified. AtMATE is expressed primarily in roots and is induced by Al. An AtMATE T-DNA knockdown line exhibited very low AtMATE expression and Al-activated root citrate exudation was abolished. The AtALMT1 AtMATE double mutant lacked both Al-activated root malate and citrate exudation and showed greater Al sensitivity than the AtALMT1 mutant. Therefore, although AtALMT1 is a major contributor to Arabidopsis Al tolerance, AtMATE also makes a significant but smaller contribution. The expression patterns of AtALMT1 and AtMATE and the profiles of Al-activated root citrate and malate exudation are not affected by the presence or absence of the other gene. These results suggest that AtALMT1-mediated malate exudation and AtMATE-mediated citrate exudation evolved independently to confer Al tolerance in Arabidopsis. However, a link between regulation of expression of the two transporters in response to Al was identified through work on STOP1, a transcription factor that was previously shown to be necessary for AtALMT1 expression. Here we show that STOP1 is also required for AtMATE expression and Al-activated citrate exudation.

  3. The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera.

    Science.gov (United States)

    De Angeli, Alexis; Baetz, Ulrike; Francisco, Rita; Zhang, Jingbo; Chaves, Maria Manuela; Regalado, Ana

    2013-08-01

    Vitis vinifera L. represents an economically important fruit species. Grape and wine flavour is made from a complex set of compounds. The acidity of berries is a major parameter in determining grape berry quality for wine making and fruit consumption. Despite the importance of malic and tartaric acid (TA) storage and transport for grape berry acidity, no vacuolar transporter for malate or tartrate has been identified so far. Some members of the aluminium-activated malate transporter (ALMT) anion channel family from Arabidopsis thaliana have been shown to be involved in mediating malate fluxes across the tonoplast. Therefore, we hypothesised that a homologue of these channels could have a similar role in V. vinifera grape berries. We identified homologues of the Arabidopsis vacuolar anion channel AtALMT9 through a TBLASTX search on the V. vinifera genome database. We cloned the closest homologue of AtALMT9 from grape berry cDNA and designated it VvALMT9. The expression profile revealed that VvALMT9 is constitutively expressed in berry mesocarp tissue and that its transcription level increases during fruit maturation. Moreover, we found that VvALMT9 is targeted to the vacuolar membrane. Using patch-clamp analysis, we could show that, besides malate, VvALMT9 mediates tartrate currents which are higher than in its Arabidopsis homologue. In summary, in the present study we provide evidence that VvALMT9 is a vacuolar malate channel expressed in grape berries. Interestingly, in V. vinifera, a tartrate-producing plant, the permeability of the channel is apparently adjusted to TA.

  4. Closing plant stomata requires a homolog of an aluminum-activated malate transporter.

    Science.gov (United States)

    Sasaki, Takayuki; Mori, Izumi C; Furuichi, Takuya; Munemasa, Shintaro; Toyooka, Kiminori; Matsuoka, Ken; Murata, Yoshiyuki; Yamamoto, Yoko

    2010-03-01

    Plant stomata limit both carbon dioxide uptake and water loss; hence, stomatal aperture is carefully set as the environment fluctuates. Aperture area is known to be regulated in part by ion transport, but few of the transporters have been characterized. Here we report that AtALMT12 (At4g17970), a homolog of the aluminum-activated malate transporter (ALMT) of wheat, is expressed in guard cells of Arabidopsis thaliana. Loss-of-function mutations in AtALMT12 impair stomatal closure induced by ABA, calcium and darkness, but do not abolish either the rapidly activated or the slowly activated anion currents previously identified as being important for stomatal closure. Expressed in Xenopus oocytes, AtALMT12 facilitates chloride and nitrate currents, but not those of organic solutes. Therefore, we conclude that AtALMT12 is a novel class of anion transporter involved in stomatal closure.

  5. The lncRNA Malat1 Is Dispensable for Mouse Development but Its Transcription Plays a cis-Regulatory Role in the Adult

    Directory of Open Access Journals (Sweden)

    Bin Zhang

    2012-07-01

    Full Text Available Genome-wide studies have identified thousands of long noncoding RNAs (lncRNAs lacking protein-coding capacity. However, most lncRNAs are expressed at a very low level, and in most cases there is no genetic evidence to support their in vivo function. Malat1 (metastasis associated lung adenocarcinoma transcript 1 is among the most abundant and highly conserved lncRNAs, and it exhibits an uncommon 3′-end processing mechanism. In addition, its specific nuclear localization, developmental regulation, and dysregulation in cancer are suggestive of it having a critical biological function. We have characterized a Malat1 loss-of-function genetic model that indicates that Malat1 is not essential for mouse pre- and postnatal development. Furthermore, depletion of Malat1 does not affect global gene expression, splicing factor level and phosphorylation status, or alternative pre-mRNA splicing. However, among a small number of genes that were dysregulated in adult Malat1 knockout mice, many were Malat1 neighboring genes, thus indicating a potential cis-regulatory role of Malat1 gene transcription.

  6. D-malate production by permeabilized Pseudomonas pseudoalcaligenes; optimization of conversion and biocatalyst productivity.

    Science.gov (United States)

    Michielsen, M J; Frielink, C; Wijffels, R H; Tramper, J; Beeftink, H H

    2000-04-14

    For the development of a continuous process for the production of solid D-malate from a Ca-maleate suspension by permeabilized Pseudomonas pseudoalcaligenes, it is important to understand the effect of appropriate process parameters on the stability and activity of the biocatalyst. Previously, we quantified the effect of product (D-malate2 -) concentration on both the first-order biocatalyst inactivation rate and on the biocatalytic conversion rate. The effects of the remaining process parameters (ionic strength, and substrate and Ca2 + concentration) on biocatalyst activity are reported here. At (common) ionic strengths below 2 M, biocatalyst activity was unaffected. At high substrate concentrations, inhibition occurred. Ca2+ concentration did not affect biocatalyst activity. The kinetic parameters (both for conversion and inactivation) were determined as a function of temperature by fitting the complete kinetic model, featuring substrate inhibition, competitive product inhibition and first-order irreversible biocatalyst inactivation, at different temperatures simultaneously through three extended data sets of substrate concentration versus time. Temperature affected both the conversion and inactivation parameters. The final model was used to calculate the substrate and biocatalyst costs per mmol of product in a continuous system with biocatalyst replenishment and biocatalyst recycling. Despite the effect of temperature on each kinetic parameter separately, the overall effect of temperature on the costs was found to be negligible (between 293 and 308 K). Within pertinent ranges, the sum of the substrate and biocatalyst costs per mmol of product was calculated to decrease with the influent substrate concentration and the residence time. The sum of the costs showed a minimum as a function of the influent biocatalyst concentration.

  7. Heat inactivation of leaf phosphoenolpyruvate carboxylase: Protection by aspartate and malate in C4 plants.

    Science.gov (United States)

    Rathnam, C K

    1978-01-01

    The activity of phosphoenolpyruvate (PEP) carboxylase EC 4.1.1.31 in leaf extracts of Eleusine indica L. Gaertn., a C4 plant, exhibited a temperature optimum of 35-37° C with a complete loss of activity at 50° C. However, the enzyme was protected effectively from heat inactivation up to 55° C by L-aspartate. Activation energies (Ea) for the enzyme in the presence of aspartate were 2.5 times lower than that of the control enzyme. Arrhenius plots of PEP carboxylase activity (±aspartate) showed a break in the slope around 17-20° C with a 3-fold increase in the Ea below the break. The discontinuity in the slopes was abolished by treating the enzyme extracts with Triton X-100, suggesting that PEP carboxylase in C4 plants is associated with lipid and may be a membrane bound enzyme. Depending upon the species, the major C4 acid formed during photosynthesis (malate or aspartate) was found to be more protective than the minor C4 acid against the heat inactivation of their PEP carboxylase. Oxaloacetate, the reaction product, was less effective compared to malate or aspartate. Several allosteric inhibitors of PEP carboxylase were found to be moderately to highly effective in protecting the C4 enzyme while its activators showed no significant effect. PEP carboxylase from C3 species was not protected from thermal inactivation by the C4 acids. The physiological significance of these results is discussed in relation to the high temperature tolerance of C4 plants.

  8. Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux.

    Science.gov (United States)

    Ligaba, Ayalew; Maron, Lyza; Shaff, Jon; Kochian, Leon; Piñeros, Miguel

    2012-07-01

    Root efflux of organic acid anions underlies a major mechanism of plant aluminium (Al) tolerance on acid soils. This efflux is mediated by transporters of the Al-activated malate transporter (ALMT) or the multi-drug and toxin extrusion (MATE) families. ZmALMT2 was previously suggested to be involved in Al tolerance based on joint association-linkage mapping for maize Al tolerance. In the current study, we functionally characterized ZmALMT2 by heterologously expressing it in Xenopus laevis oocytes and transgenic Arabidopsis. In oocytes, ZmALMT2 mediated an Al-independent electrogenic transport product of organic and inorganic anion efflux. Ectopic overexpression of ZmALMT2 in an Al-hypersensitive Arabidopsis KO/KD line lacking the Al tolerance genes, AtALMT1 and AtMATE, resulted in Al-independent constitutive root malate efflux which partially restored the Al tolerance phenotype. The lack of correlation between ZmALMT2 expression and Al tolerance (e.g., expression not localized to the root tip, not up-regulated by Al, and higher in sensitive versus tolerance maize lines) also led us to question ZmALMT2's role in Al tolerance. The functional properties of the ZmALMT2 transporter presented here, along with the gene expression data, suggest that ZmALMT2 is not involved in maize Al tolerance but, rather, may play a role in mineral nutrient acquisition and transport. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.

  9. Low pH, Aluminum, and Phosphorus Coordinately Regulate Malate Exudation through GmALMT1 to Improve Soybean Adaptation to Acid Soils1[W][OA

    Science.gov (United States)

    Liang, Cuiyue; Piñeros, Miguel A.; Tian, Jiang; Yao, Zhufang; Sun, Lili; Liu, Jiping; Shaff, Jon; Coluccio, Alison; Kochian, Leon V.; Liao, Hong

    2013-01-01

    Low pH, aluminum (Al) toxicity, and low phosphorus (P) often coexist and are heterogeneously distributed in acid soils. To date, the underlying mechanisms of crop adaptation to these multiple factors on acid soils remain poorly understood. In this study, we found that P addition to acid soils could stimulate Al tolerance, especially for the P-efficient genotype HN89. Subsequent hydroponic studies demonstrated that solution pH, Al, and P levels coordinately altered soybean (Glycine max) root growth and malate exudation. Interestingly, HN89 released more malate under conditions mimicking acid soils (low pH, +P, and +Al), suggesting that root malate exudation might be critical for soybean adaptation to both Al toxicity and P deficiency on acid soils. GmALMT1, a soybean malate transporter gene, was cloned from the Al-treated root tips of HN89. Like root malate exudation, GmALMT1 expression was also pH dependent, being suppressed by low pH but enhanced by Al plus P addition in roots of HN89. Quantitative real-time PCR, transient expression of a GmALMT1-yellow fluorescent protein chimera in Arabidopsis protoplasts, and electrophysiological analysis of Xenopus laevis oocytes expressing GmALMT1 demonstrated that GmALMT1 encodes a root cell plasma membrane transporter that mediates malate efflux in an extracellular pH-dependent and Al-independent manner. Overexpression of GmALMT1 in transgenic Arabidopsis, as well as overexpression and knockdown of GmALMT1 in transgenic soybean hairy roots, indicated that GmALMT1-mediated root malate efflux does underlie soybean Al tolerance. Taken together, our results suggest that malate exudation is an important component of soybean adaptation to acid soils and is coordinately regulated by three factors, pH, Al, and P, through the regulation of GmALMT1 expression and GmALMT1 function. PMID:23341359

  10. Low pH, aluminum, and phosphorus coordinately regulate malate exudation through GmALMT1 to improve soybean adaptation to acid soils.

    Science.gov (United States)

    Liang, Cuiyue; Piñeros, Miguel A; Tian, Jiang; Yao, Zhufang; Sun, Lili; Liu, Jiping; Shaff, Jon; Coluccio, Alison; Kochian, Leon V; Liao, Hong

    2013-03-01

    Low pH, aluminum (Al) toxicity, and low phosphorus (P) often coexist and are heterogeneously distributed in acid soils. To date, the underlying mechanisms of crop adaptation to these multiple factors on acid soils remain poorly understood. In this study, we found that P addition to acid soils could stimulate Al tolerance, especially for the P-efficient genotype HN89. Subsequent hydroponic studies demonstrated that solution pH, Al, and P levels coordinately altered soybean (Glycine max) root growth and malate exudation. Interestingly, HN89 released more malate under conditions mimicking acid soils (low pH, +P, and +Al), suggesting that root malate exudation might be critical for soybean adaptation to both Al toxicity and P deficiency on acid soils. GmALMT1, a soybean malate transporter gene, was cloned from the Al-treated root tips of HN89. Like root malate exudation, GmALMT1 expression was also pH dependent, being suppressed by low pH but enhanced by Al plus P addition in roots of HN89. Quantitative real-time PCR, transient expression of a GmALMT1-yellow fluorescent protein chimera in Arabidopsis protoplasts, and electrophysiological analysis of Xenopus laevis oocytes expressing GmALMT1 demonstrated that GmALMT1 encodes a root cell plasma membrane transporter that mediates malate efflux in an extracellular pH-dependent and Al-independent manner. Overexpression of GmALMT1 in transgenic Arabidopsis, as well as overexpression and knockdown of GmALMT1 in transgenic soybean hairy roots, indicated that GmALMT1-mediated root malate efflux does underlie soybean Al tolerance. Taken together, our results suggest that malate exudation is an important component of soybean adaptation to acid soils and is coordinately regulated by three factors, pH, Al, and P, through the regulation of GmALMT1 expression and GmALMT1 function.

  11. Pengaruh Pengasapan (Thermal Fogging Insektisida Piretroid (Malation 95% Terhadap Nyamuk Aedes aegypti dan Culex quinquefasciatus di Pemukiman

    Directory of Open Access Journals (Sweden)

    Hasan Boesri

    2013-02-01

    Full Text Available Abstracts. The evaluation of piretroid insecticide (active ingredient Malation 95% was con­ducted in Sub district Tengarang, Semarang Segency, Central Java Province. The insecti­cide was applied using thermal fogging method for dosages of 125, 250, 375, 500 and 625 ml/ha (diluted in diesel to 10 litters. The evaluation of the efficacy was conducted against two mosquito species, Aedes aegypti (the main dengue haemorrhagic fever and Culex quinquefasciatus (the urban lymphatic fil­ariasis vector. Result of the evaluation was revealed that dosages of 500 and 625 ml/ha were effective against both tested mosquito species indoor and outdoor.Key Word: Aedes aegypti, Culex quinquefasciatus, insecticide Piretroid (Malation 95%, thermal fogging.

  12. Long Non-Coding RNA Malat-1 Is Dispensable during Pressure Overload-Induced Cardiac Remodeling and Failure in Mice.

    Directory of Open Access Journals (Sweden)

    Tim Peters

    Full Text Available Long non-coding RNAs (lncRNAs are a class of RNA molecules with diverse regulatory functions during embryonic development, normal life, and disease in higher organisms. However, research on the role of lncRNAs in cardiovascular diseases and in particular heart failure is still in its infancy. The exceptionally well conserved nuclear lncRNA Metastasis associated in lung adenocarcinoma transcript 1 (Malat-1 is a regulator of mRNA splicing and highly expressed in the heart. Malat-1 modulates hypoxia-induced vessel growth, activates ERK/MAPK signaling, and scavenges the anti-hypertrophic microRNA-133. We therefore hypothesized that Malat-1 may act as regulator of cardiac hypertrophy and failure during cardiac pressure overload induced by thoracic aortic constriction (TAC in mice.Absence of Malat-1 did not affect cardiac hypertrophy upon pressure overload: Heart weight to tibia length ratio significantly increased in WT mice (sham: 5.78±0.55, TAC 9.79±1.82 g/mm; p<0.001 but to a similar extend also in Malat-1 knockout (KO mice (sham: 6.21±1.12, TAC 8.91±1.74 g/mm; p<0.01 with no significant difference between genotypes. As expected, TAC significantly reduced left ventricular fractional shortening in WT (sham: 38.81±6.53%, TAC: 23.14±11.99%; p<0.01 but to a comparable degree also in KO mice (sham: 37.01±4.19%, TAC: 25.98±9.75%; p<0.05. Histological hallmarks of myocardial remodeling, such as cardiomyocyte hypertrophy, increased interstitial fibrosis, reduced capillary density, and immune cell infiltration, did not differ significantly between WT and KO mice after TAC. In line, the absence of Malat-1 did not significantly affect angiotensin II-induced cardiac hypertrophy, dysfunction, and overall remodeling. Above that, pressure overload by TAC significantly induced mRNA levels of the hypertrophy marker genes Nppa, Nppb and Acta1, to a similar extend in both genotypes. Alternative splicing of Ndrg2 after TAC was apparent in WT (isoform ratio

  13. Pre-ischemic mitochondrial substrate constraint by inhibition of malate-aspartate shuttle preserves mitochondrial function after ischemia-reperfusion

    DEFF Research Database (Denmark)

    Jespersen, Nichlas Riise; Yokota, Takashi; Støttrup, Nicolaj Brejnholt

    2017-01-01

    KEY POINTS: Pre-ischaemic administration of aminooxiacetate (AOA), an inhibitor of the malate-aspartate shuttle (MAS), provides cardioprotection against ischaemia-reperfusion injury. The underlying mechanism remains unknown. We examined whether transient inhibition of the MAS during ischaemia......, but not IPC, reduced the myocardial interstitial concentration of tricarboxylic acid cycle intermediates at the onset of reperfusion. The results obtained in the present study demonstrate that metabolic regulation by inhibition of the MAS at the onset of reperfusion may be beneficial for the preservation...... of mitochondrial function during late reperfusion in an IR-injured heart. ABSTRACT: Mitochondrial dysfunction plays a central role in ischaemia-reperfusion (IR) injury. Pre-ischaemic administration of aminooxyacetate (AOA), an inhibitor of the malate-aspartate shuttle (MAS), provides cardioprotection against IR...

  14. Correlation between myocardial malate/aspartate shuttle activity and EAAT1 protein expression in hyper- and hypothyroidism.

    Science.gov (United States)

    Ralphe, J Carter; Bedell, Kurt; Segar, Jeffrey L; Scholz, Thomas D

    2005-05-01

    In the heart, elevated thyroid hormone leads to upregulation of metabolic pathways associated with energy production and development of hypertrophy. The malate/aspartate shuttle, which transfers cytosolic-reducing equivalents into the cardiac mitochondria, is increased 33% in hyperthyroid rats. Within the shuttle, the aspartate-glutamate carrier is rate limiting. The excitatory amino acid transporter type 1 (EAAT1) functions as a glutamate carrier in the malate/aspartate shuttle. In this study, we hypothesize that EAAT1 is regulated by thyroid hormone. Adult rats were injected with triiodothyronine (T3) or saline over a period of 8-9 days or provided with propylthiouracil (PTU) in their drinking water for 2 mo. Steady-state mRNA levels of EAAT1 and aralar1 and citrin (both cardiac mitochondrial aspartate-glutamate transporters) were determined by Northern blot analysis and normalized to 18S rRNA. A spectrophotometric assay of maximal malate/aspartate shuttle activity was performed on isolated cardiac mitochondria from PTU-treated and control animals. Protein lysates from mitochondria were separated by SDS-PAGE and probed with a human anti-EAAT1 IgG. Compared with control, EAAT1 mRNA levels (arbitrary units) were increased nearly threefold in T3-treated (3.1 +/- 0.5 vs. 1.1 +/- 0.2; P Hyperthyroidism in rats is related to an increase in cardiac expression of EAAT1 mRNA and protein. The 49% increase in EAAT1 mitochondrial protein level shows that malate/aspartate shuttle activity increased in hyperthyroid rat cardiac mitochondria. Although hypothyroidism resulted in a decrease in EAAT1 mRNA, neither the EAAT1 protein level nor shuttle activity was affected. EAAT1 regulation by thyroid hormone may facilitate increased metabolic demands of the cardiomyocyte during hyperthyroidism and impact cardiac function in hyperthyroidism.

  15. The Membrane Topology of ALMT1, an Aluminum-Activated Malate Transport Protein in Wheat (Triticum aestivum)

    OpenAIRE

    Motoda, Hirotoshi; Sasaki, Takayuki; Kano, Yoshio; Ryan, Peter R; Delhaize, Emmanuel; Matsumoto, Hideaki; Yamamoto, Yoko

    2007-01-01

    The wheat ALMT1 gene encodes an aluminum (Al)-activated malate transport protein which confers Al-resistance. We investigated the membrane topology of this plasma-membrane localized protein with immunocytochemical techniques. Several green fluorescent protein (GFP)-fused and histidine (His)-tagged chimeras of ALMT1 were prepared based on a computer-predicted secondary structure and transiently expressed in cultured mammalian cells. Antibodies raised to polypeptide epitopes of ALMT1 were used ...

  16. The long non-coding RNA MALAT1 promotes the migration and invasion of hepatocellular carcinoma by sponging miR-204 and releasing SIRT1.

    Science.gov (United States)

    Hou, Zhouhua; Xu, Xuwen; Zhou, Ledu; Fu, Xiaoyu; Tao, Shuhui; Zhou, Jiebin; Tan, Deming; Liu, Shuiping

    2017-07-01

    Increasing evidence supports the significance of long non-coding RNA in cancer development. Several recent studies suggest the oncogenic activity of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in hepatocellular carcinoma. In this study, we explored the molecular mechanisms by which MALAT1 modulates hepatocellular carcinoma biological behaviors. We found that microRNA-204 was significantly downregulated in sh-MALAT1 HepG2 cell and 15 hepatocellular carcinoma tissues by quantitative real-time polymerase chain reaction analysis. Through bioinformatic screening, luciferase reporter assay, RNA-binding protein immunoprecipitation, and RNA pull-down assay, we identified microRNA-204 as a potential interacting partner for MALAT1. Functionally, wound-healing and transwell assays revealed that microRNA-204 significantly inhibited the migration and invasion of hepatocellular carcinoma cells. Notably, sirtuin 1 was recognized as a direct downstream target of microRNA-204 in HepG2 cells. Moreover, si-SIRT1 significantly inhibited cell invasion and migration process. These data elucidated, by sponging and competitive binding to microRNA-204, MALAT1 releases the suppression on sirtuin 1, which in turn promotes hepatocellular carcinoma migration and invasion. This study reveals a novel mechanism by which MALAT1 stimulates hepatocellular carcinoma progression and justifies targeting metastasis-associated lung adenocarcinoma transcript 1 as a potential therapy for hepatocellular carcinoma.

  17. Long non-coding RNA MALAT1 acts as a competing endogenous RNA to promote malignant melanoma growth and metastasis by sponging miR-22.

    Science.gov (United States)

    Luan, Wenkang; Li, Lubo; Shi, Yan; Bu, Xuefeng; Xia, Yun; Wang, Jinlong; Djangmah, Henry Siaw; Liu, Xiaohui; You, Yongping; Xu, Bin

    2016-09-27

    Long non-coding RNAs (lncRNAs) are involved in tumorigenesis. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), an lncRNAs, is associated with the growth and metastasis of many human tumors, but its biological roles in malignant melanoma remain unclear. In this study, the aberrant up-regulation of MALAT1 was detected in melanoma. We determined that MALAT1 promotes melanoma cells proliferation, invasion and migration by sponging miR-22. MiR-22 was decreased and acted as a tumor suppressor in melanoma, and MMP14 and Snail were the functional targets of miR-22. Furthermore, MALAT1 could modulate MMP14 and Snail by operating as a competing endogenous RNA (ceRNA) for miR-22. The effects of MALAT1 in malignant melanoma is verified using a xenograft model. This finding elucidates a new mechanism for MALAT1 in melanoma development and provides a potential target for melanoma therapeutic intervention.

  18. Decarboxylation of Malate in the Crassulacean Acid Metabolism Plant Bryophyllum (Kalanchoe) fedtschenkoi (Role of NAD-Malic Enzyme).

    Science.gov (United States)

    Cook, R. M.; Lindsay, J. G.; Wilkins, M. B.; Nimmo, H. G.

    1995-01-01

    The role of NAD-malic enzyme (NAD-ME) in the Crassulacean acid metabolism plant Bryophyllum (Kalanchoe) fedtschenkoi was investigated using preparations of intact and solubilized mitochondria from fully expanded leaves. Intact, coupled mitochondria isolated during the day or night did not differ in their ability to take up [14C]malic acid from the surrounding medium or to respire using malate or succinate as substrate. However, intact mitochondria isolated from plants during the day decarboxylated added malate to pyruvate significantly faster than mitochondria isolated from plants at night. NAD-ME activity in solubilized mitochondrial extracts showed hysteretic kinetics and was stimulated by a number of activators, including acetyl-coenzyme A, fructose-1,6-bisphosphate, and sulfate ions. In the absence of these effectors, reaction progress curves were nonlinear, with a pronounced acceleration phase. The lag period before a steady-state rate was reached in assays of mitochondrial extracts decreased during the photoperiod and increased slowly during the period of darkness. However, these changes in the kinetic properties of the enzyme could not account for the changes in the rate of decarboxylation of malate by intact mitochondria. Gel-filtration experiments showed that mitochondrial extracts contained three forms of NAD-ME with different molecular weights. The relative proportions of the three forms varied somewhat throughout the light/dark cycle, but this did not account for the changes in the kinetics behavior of the enzyme during the diurnal cycle. PMID:12228671

  19. 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 serum...

  20. Histochemical localization of cytokinin oxidase/dehydrogenase ...

    African Journals Online (AJOL)

    Jane

    2011-08-15

    dehydrogenase, Withania somnifera, CKX localization. INTRODUCTION. Cytokinin (Ck) is a plant hormone that plays a crucial role in many fundamental processes of plant development throughout the life cycle. These include ...

  1. Shikimate dehydrogenase from Pinu sylvestris L. needles

    International Nuclear Information System (INIS)

    Osipov, V.I.; Shein, I.V.

    1986-01-01

    Shikimate dehydrogenase was isolated by extraction from pine needles and partially purified by fractionation with ammonium sulfate. In conifers, in contrast to other plants, all three isoenzymes of shikimate dehydrogenase exhibit activity not only with NADP + , but also with NAD + . The values of K/sub m/ for shikimate, when NADP + and NAD + are used as cofactors, are 0.22 and 1.13 mM, respectively. The enzyme is maximally active at pH 10 with both cofactors. It is suggested that NAD-dependent shikimate dehydrogenase catalyzes the initial reaction of the alternative pathway of the conversion of shikimic acid to hydroxybenzoic acid. The peculiarities of the organization and regulation of the initial reactions of the shikimate pathway in conifers and in plants with shikimate dehydrogenase absolutely specific for NADP are discussed

  2. Calcium absorption from apple and orange juice fortified with calcium citrate malate (CCM).

    Science.gov (United States)

    Andon, M B; Peacock, M; Kanerva, R L; De Castro, J A

    1996-06-01

    Determine calcium (Ca) absorption from Ca fortified orange and apple juice. Absorbability was assessed by measuring 45Ca absorption in healthy women (mean age 57 years, n = 57/group) and whole body 47Ca retention in adult female beagle dogs (n = 6/group) and young adult male rats (n = 6/group). Women received 6.24 mmol (250 mg) Ca as calcium citrate malate fortified orange juice (CCM-OJ) or apple juice (CCM-AJ). Dogs received 3.12 mmol (125 mg) Ca as CCM-OJ or CCM-AJ. Rats were administered 0.15 mmol (6 mg) Ca as either milk, CCM-OJ, or CCM-AJ. Additional 47Ca whole body retention experiments in rats measured the effects of differences in the carbohydrate and organic acid contents of the juices on Ca absorption. Mean +/- SEM percent Ca fractional absorption was greater (p composition of Ca fortified juices, we found that the greater fructose and lower organic acid content of apple juice accounted for its greater Ca absorbability. CCM fortified versions of orange and apple juice have high Ca absorbability and are potentially important vehicles for increasing dietary Ca intake. The greater Ca absorption from CCM-AJ compared with CCM-OJ is accounted for by differences in the carbohydrate and organic acid content of the juices. These data suggest that by modifying common beverage ingredients, products with even greater Ca absorbability could be formulated.

  3. High Performance Liquid Chromatographic Analysis of Almotriptan Malate in Bulk and Tablets

    Directory of Open Access Journals (Sweden)

    Chandra Bala Sekaran

    2013-02-01

    Full Text Available Purpose: A simple RP-HPLC method has been developed and validated for the determination of almotriptan malate (ATM in bulk and tablets. Methods: Chromatographic separation of ATM was achieved by using a Thermo Scientific C18 column. A Mobile phase containing a mixture of methanol, water and acetic acid (4:8:0.1 v/v was pumped at the flow rate of 1 mL/min. Detection was performed at 227 nm. According to ICH guidelines, the method was validated. Results: The calibration curve was linear in the concentration range 5–60 μg/mL for the ATM with regression coefficient 0.9999. The method was precise with RSD <1.2%. Excellent recoveries of 99.60 - 100.80% proved the accuracy of the method. The limits of detection and quantification were found to be 0.025 and 0.075 μg/mL, respectively. Conclusion: The method was successfully applied for the quantification of ATM in tablets with acceptable accuracy and precision.

  4. NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells

    Science.gov (United States)

    Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

    2014-08-01

    Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

  5. Redox Specificity of 2-Hydroxyacid-Coupled NAD+/NADH Dehydrogenases: A Study Exploiting “Reactive” Arginine as a Reporter of Protein Electrostatics

    Science.gov (United States)

    Durani, Susheel

    2013-01-01

    With “reactive” arginine as a kinetic reporter, 2-hydroxyacid dehydrogenases are assessed in basis of their specialization as NAD+-reducing or NADH-oxidizing enzymes. Specifically, M4 and H4 lactate dehydrogenases (LDHs) and cytoplasmic and mitochondrial malate dehydrogenases (MDHs) are compared to assess if their coenzyme specificity may involve electrostatics of cationic or neutral nicotinamide structure as the basis. The enzymes from diverse eukaryote and prokaryote sources thus are assessed in “reactivity” of functionally-critical arginine as a function of salt concentration and pH. Electrostatic calculations were performed on “reactive” arginines and found good correspondence with experiment. The reductive and oxidative LDHs and MDHs are assessed in their count over ionizable residues and in placement details of the residues in their structures as proteins. The variants found to be high or low in ΔpKa of “reactive” arginine are found to be also strong or weak cations that preferentially oxidize NADH (neutral nicotinamide structure) or reduce NAD+ (cationic nicotinamide structure). The ionized groups of protein structure may thus be important to redox specificity of the enzyme on basis of electrostatic preference for the oxidized (cationic nicotinamide) or reduced (neutral nicotinamide) coenzyme. Detailed comparisons of isozymes establish that the residues contributing in their redox specificity are scrambled in structure of the reductive enzyme. PMID:24391777

  6. Redox specificity of 2-hydroxyacid-coupled NAD(+/NADH dehydrogenases: a study exploiting "reactive" arginine as a reporter of protein electrostatics.

    Directory of Open Access Journals (Sweden)

    Pooja Gupta

    Full Text Available With "reactive" arginine as a kinetic reporter, 2-hydroxyacid dehydrogenases are assessed in basis of their specialization as NAD(+-reducing or NADH-oxidizing enzymes. Specifically, M4 and H4 lactate dehydrogenases (LDHs and cytoplasmic and mitochondrial malate dehydrogenases (MDHs are compared to assess if their coenzyme specificity may involve electrostatics of cationic or neutral nicotinamide structure as the basis. The enzymes from diverse eukaryote and prokaryote sources thus are assessed in "reactivity" of functionally-critical arginine as a function of salt concentration and pH. Electrostatic calculations were performed on "reactive" arginines and found good correspondence with experiment. The reductive and oxidative LDHs and MDHs are assessed in their count over ionizable residues and in placement details of the residues in their structures as proteins. The variants found to be high or low in ΔpKa of "reactive" arginine are found to be also strong or weak cations that preferentially oxidize NADH (neutral nicotinamide structure or reduce NAD(+ (cationic nicotinamide structure. The ionized groups of protein structure may thus be important to redox specificity of the enzyme on basis of electrostatic preference for the oxidized (cationic nicotinamide or reduced (neutral nicotinamide coenzyme. Detailed comparisons of isozymes establish that the residues contributing in their redox specificity are scrambled in structure of the reductive enzyme.

  7. Phosphorylation site on yeast pyruvate dehydrogenase complex

    International Nuclear Information System (INIS)

    Uhlinger, D.J.

    1986-01-01

    The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast pyruvate dehydrogenase complex was achieved by cation-exchange high pressure liquid chromatography (HPLC). No endogenous pyruvate dehydrogenase kinase activity was detected during the purification. However, the yeast pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. Tryptic digestion of the 32 P-labeled complex yielded a single phosphopeptide which was purified to homogeniety. The tryptic digest was subjected to chromatography on a C-18 reverse phase HPLC column with a linear gradient of acetonitrile. Radioactive fractions were pooled, concentrated, and subjected to anion-exchange HPLC. The column was developed with a linear gradient of ammonium acetate. Final purification of the phosphopeptide was achieved by chromatography on a C-18 reverse phase HPLC column developed with a linear gradient of acetonitrile. The amino acid sequence of the homogeneous peptide was determined by manual modified Edman degradation

  8. Pengaruh Pengasapan (Thermal Fogging Insektisida Piretroid (Malation 95% Terhadap Nyamuk Aedes aegypti dan Culex quinquefasciatus di Pemukiman

    Directory of Open Access Journals (Sweden)

    Hasan Boesri

    2009-12-01

    Full Text Available The evaluation of piretroid insecticide (active ingredient Malation 95% was con-ducted in Sub district Tengarang, Semarang Segency, Central Java Province. The insecti-cide was applied using thermal fogging method for dosages of 125, 250, 375, 500 and 625 ml/ha (diluted in diesel to 10 litters. The evaluation of the efficacy was conducted against two mosquito species, Aedes aegypti (the main dengue haemorrhagic fever and Culex quinquefasciatus (the urban lymphatic fil-ariasis vector. Result of the evaluation was revealed that dosages of 500 and 625 ml/ha were effective against both tested mosquito species indoor and outdoor.

  9. Inducible xylitol dehydrogenases in enteric bacteria.

    OpenAIRE

    Doten, R C; Mortlock, R P

    1985-01-01

    Morganella morganii ATCC 25829, Providencia stuartii ATCC 25827, Serratia marcescens ATCC 13880, and Erwinia sp. strain 4D2P were found to induce a xylitol dehydrogenase when grown on a xylitol-containing medium. The xylitol dehydrogenases were partially purified from the four strains, and those from M. morganii ATCC 25829, P. stuartii ATCC 25827, and S. marcescens ATCC 13880 were all found to oxidize xylitol to D-xylulose. These three enzymes had KmS for xylitol of 7.1 to 16.4 mM and molecul...

  10. 2-Methylbutyryl-coenzyme A dehydrogenase deficiency

    DEFF Research Database (Denmark)

    Sass, Jörn Oliver; Ensenauer, Regina; Röschinger, Wulf

    2008-01-01

    2-Methylbutyryl-CoA dehydrogenase (MBD; coded by the ACADSB gene) catalyzes the step in isoleucine metabolism that corresponds to the isovaleryl-CoA dehydrogenase reaction in the degradation of leucine. Deficiencies of both enzymes may be detected by expanded neonatal screening with tandem...... individuals showed clinical symptoms attributable to MBD deficiency although the defect in isoleucine catabolism was demonstrated both in vivo and in vitro. Several mutations in the ACADSB gene were identified, including a novel one. MBD deficiency may be a harmless metabolic variant although significant...

  11. Genetics Home Reference: glucose-6-phosphate dehydrogenase deficiency

    Science.gov (United States)

    ... deficiency Encyclopedia: Glucose-6-phosphate dehydrogenase test Encyclopedia: Hemolytic anemia Encyclopedia: Newborn jaundice Health Topic: Anemia Health Topic: G6PD Deficiency Health Topic: Newborn Screening Genetic and Rare Diseases Information Center (1 link) Glucose-6-phosphate dehydrogenase ...

  12. Variability among inbred lines and RFLP mapping of sunflower isozymes

    Directory of Open Access Journals (Sweden)

    Carrera Alicia D.

    2002-01-01

    Full Text Available Eight isozyme systems were used in this study: acid phosphatase (ACP, alcohol dehydrogenase (ADH, esterase (EST, glutamate dehydrogenase (GDH, malate dehydrogenase (MDH, phosphoglucoisomerase (PGI, 6-phosphogluconate dehydrogenase (PGD, and phosphoglucomutase (PGM. The polymorphism of these enzyme systems was studied in 25 elite inbred lines. A total of 19 loci were identified, but only eight of them were polymorphic in the germplasm tested. The polymorphic index for the eight informative markers ranged from 0.08 to 0.57, with a mean value of 0.36. Five isozyme loci were mapped in F2:3 populations with existing RFLP data. Est-1, Gdh-2 and Pgi-2 were mapped to linkage groups 3, 14 and 9, respectively. As in previous reports, an ACP locus and a PGD locus were found to be linked, both located in linkage group 2 of the public sunflower map.

  13. Conjugated fatty acids and methane production by rumen microbes when incubated with linseed oil alone or mixed with fish oil and/or malate.

    Science.gov (United States)

    Li, Xiang Z; Gao, Qing S; Yan, Chang G; Choi, Seong H; Shin, Jong S; Song, Man K

    2015-08-01

    We hypothesized that manipulating metabolism with fish oil and malate as a hydrogen acceptor would affect the biohydrogenation process of α-linolenic acid by rumen microbes. This study was to examine the effect of fish oil and/or malate on the production of conjugated fatty acids and methane (CH4 ) by rumen microbes when incubated with linseed oil. Linseed oil (LO), LO with fish oil (LO-FO), LO with malate (LO-MA), or LO with fish oil and malate (LO-FO-MA) was added to diluted rumen fluid, respectively. The LO-MA and LO-FO-MA increased pH and propionate concentration compared to the other treatments. LO-MA and LO-FO-MA reduced CH4 production compared to LO. LO-MA and LO-FO-MA increased the contents of c9,t11-conjugated linoleic acid (CLA) and c9,t11,c15-conjugated linolenic acid (CLnA) compared to LO. The content of malate was rapidly reduced while that of lactate was reduced in LO-MA and LO-FO-MA from 3 h incubation time. The fold change of the quantity of methanogen related to total bacteria was decreased at both 3 h and 6 h incubation times in all treatments compared to the control. Overall data indicate that supplementation of combined malate and/or fish oil when incubated with linseed oil, could depress methane generation and increase production of propionate, CLA and CLnA under the conditions of the current in vitro study. © 2015 Japanese Society of Animal Science.

  14. Optimization of Adsorptive Immobilization of Alcohol Dehydrogenases

    NARCIS (Netherlands)

    Trivedi, Archana; Heinemann, Matthias; Spiess, Antje C.; Daussmann, Thomas; Büchs, Jochen

    2005-01-01

    In this work, a systematic examination of various parameters of adsorptive immobilization of alcohol dehydrogenases (ADHs) on solid support is performed and the impact of these parameters on immobilization efficiency is studied. Depending on the source of the enzymes, these parameters differently

  15. Altered Expression of the Malate-Permeable Anion Channel OsALMT4 Reduces the Growth of Rice Under Low Radiance

    OpenAIRE

    Jie Liu; Jie Liu; Muyun Xu; Gonzalo M. Estavillo; Emmanuel Delhaize; Rosemary G. White; Meixue Zhou; Peter R. Ryan

    2018-01-01

    We examined the function of OsALMT4 in rice (Oryza sativa L.) which is a member of the aluminum-activated malate transporter family. Previous studies showed that OsALMT4 localizes to the plasma membrane and that expression in transgenic rice lines results in a constitutive release of malate from the roots. Here, we show that OsALMT4 is expressed widely in roots, shoots, flowers, and grain but not guard cells. Expression was also affected by ionic and osmotic stress, light and to the hormones ...

  16. AtALMT1, which encodes a malate transporter, is identified as one of several genes critical for aluminum tolerance in Arabidopsis

    OpenAIRE

    Hoekenga, Owen A.; Maron, Lyza G.; Piñeros, Miguel A.; Cançado, Geraldo M. A.; Shaff, Jon; Kobayashi, Yuriko; Ryan, Peter R.; Dong, Bei; Delhaize, Emmanuel; Sasaki, Takayuki; Matsumoto, Hideaki; Yamamoto, Yoko; Koyama, Hiroyuki; Kochian, Leon V.

    2006-01-01

    Aluminum (Al) tolerance in Arabidopsis is a genetically complex trait, yet it is mediated by a single physiological mechanism based on Al-activated root malate efflux. We investigated a possible molecular determinant for Al tolerance involving a homolog of the wheat Al-activated malate transporter, ALMT1. This gene, named AtALMT1 (At1g08430), was the best candidate from the 14-memberAtALMT family to be involved with Al tolerance based on expression patterns and genomic location. Physiological...

  17. Systematic replacement of lysine with glutamine and alanine in Escherichia coli malate synthase G: effect on crystallization

    International Nuclear Information System (INIS)

    Anstrom, David M.; Colip, Leslie; Moshofsky, Brian; Hatcher, Eric; Remington, S. James

    2005-01-01

    Alanine and glutamine mutations were made to the same 15 lysine positions on the surface of E. coli malate synthase G and the impact on crystallization observed. The results support lysine replacement for improvement of crystallization and provide insight into site selection and type of amino-acid replacement. Two proposals recommend substitution of surface lysine residues as a means to improve the quality of protein crystals. In proposal I, substitution of lysine by alanine has been suggested to improve crystallization by reducing the entropic cost of ordering flexible side chains at crystal contacts. In proposal II, substitution of lysine by residues more commonly found in crystal contacts, such as glutamine, has been proposed to improve crystallization. 15 lysine residues on the surface of Escherichia coli malate synthase G, distributed over a variety of secondary structures, were individually mutated to both alanine and glutamine. For 28 variants, detailed studies of the effect on enzymatic activity and crystallization were conducted. This has permitted direct comparison of the relative effects of the two types of mutations. While none of the variants produced crystals suitable for X-ray structural determination, small crystals were obtained in a wide variety of conditions, in support of the general approach. Glutamine substitutions were found to be more effective than alanine in producing crystals, in support of proposal II. Secondary structure at the site of mutation does not appear to play a major role in determining the rate of success

  18. Ammonium intensifies CAM photosynthesis and counteracts drought effects by increasing malate transport and antioxidant capacity in Guzmania monostachia.

    Science.gov (United States)

    Pereira, Paula Natália; Gaspar, Marília; Smith, J Andrew C; Mercier, Helenice

    2018-04-09

    Guzmania monostachia (Bromeliaceae) is a tropical epiphyte capable of up-regulating crassulacean acid metabolism (CAM) in its photosynthetic tissues in response to changing nutrient and water availability. Previous studies have shown that under drought there is a gradient of increasing CAM expression from the basal (youngest) to the apical (oldest) portion of the leaves, and additionally that nitrogen deficiency can further increase CAM intensity in the leaf apex of this bromeliad. The present study investigated the inter-relationships between nitrogen source (nitrate and/or ammonium) and water deficit in regulating CAM expression in G. monostachia leaves. The highest CAM activity was observed under ammonium nutrition in combination with water deficit. This was associated with enhanced activity of the key enzyme phosphoenolpyruvate carboxylase, elevated rates of ATP- and PPi-dependent proton transport at the vacuolar membrane in the presence of malate, and increased transcript levels of the vacuolar malate channel-encoding gene, ALMT. Water deficit was consistently associated with higher levels of total soluble sugars, which were maximal under ammonium nutrition, as were the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase). Thus, ammonium nutrition, whilst associated with the highest degree of CAM induction in G. monostachia, also mitigates the effects of water deficit by osmotic adjustment and can limit oxidative damage in the leaves of this bromeliad under conditions that may be typical of its epiphytic habitat.

  19. Genetic variability in Oligosarcus paranensis (Teleostei: Characiformes from the São Francisco river, Ivaí river basin – Paraná State, Brazil - doi: 10.4025/actascibiolsci.v35i3.14179

    Directory of Open Access Journals (Sweden)

    Michele Rocha dos Santos

    2013-08-01

    Full Text Available The genetic variability of Oligosarcus paranensis was estimated from a population collected in São Francisco river, Prudentópolis county in Paraná State (Brazil using the electrophoresis in starch gel technique. Eleven enzymatic systems were analyzed: Aspartate aminotransaminase (AAT; E. C. 2.6.1, Alcohol dehydrogenase (ADH; E. C. 1.1.1.1, Esterase (EST; E. C. 3.1.1.1, Glucose-6-phosphate isomerase (GPI; E. C. 5.3.1.9, Glycerol-3-Phosphate dehydrogenase (G3PDH; E. C. 1.1.1, Isocitrate dehydrogenase (IDH; E. C. 1.1.1.42, L-lactate dehydrogenase (LDH; E. C. 1.1.1.27, Malate dehydrogenase (MDH; E. C. 1.1.1.37 , Malate dehydrogenase NADP (ME; E. C. 1.1.1.40, Phosphoglucomutase (PGM; E. C. 5.4.2.2 and Sorbitol dehydrogenase (SORB; E.C. 1.1.1.14. Twenty loci were identified through 15% corn starch gel electrophoresis of which nine (45% were polymorphic. The average expected heterozygosity was estimated as 0.1229 ± 0.1728, and the observed was 0.0586 ± 0.1069, indicating high genetic variability. The average value of FIS = 0.5145 indicates homozygote excess.  

  20. Morphologic, biometric, and isoenzyme characterization of Trichuris suis.

    Science.gov (United States)

    Oliveros, R; Cutillas, C; Arias, P; Guevara, D

    1998-06-01

    Trichuris suis isolates were collected from the cecum of Sus scrofa domestica (pig) and S. s. scrofa (wild boar). Morphology and biometry studies were carried out. Morphology studies showed the existence of typical caudal papillae in males of T. suis from wild boars, but no other difference was observed in the biometric parameters (total length, esophageal length, posterior-portion body length, and spicular length) of T. suis isolated from either host. Individual extracts were subjected to malate dehydrogenase (MDH), malic enzyme (ME), glucose 6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LDH), and superoxide dismutase (SOD) isoenzyme analysis following starch-gel electrophoresis, and the isoenzyme patterns were compared with those obtained from other species of trichurids. MDH, ME, G6PD, LDH, and SOD isoenzyme patterns were identical for T. suis from both hosts. MDH isoenzyme patterns were characterized by the presence of one cathodic isoenzyme. ME, G6PD, and LDH isoenzyme patterns indicated the presence of three phenotypes, whereas the SOD isoenzyme pattern showed only one phenotype characterized by the existence of two (anodic and cathodic) bands. Different LDH and SOD isoenzyme patterns observed for T. suis, T. ovis, and T. skrjabini confirm once more that isoenzyme patterns have potential as a diagnostic tool for differentiation of different species of Trichuris.

  1. Posttranscriptional silencing of the lncRNA MALAT1 by miR-217 inhibits the epithelial–mesenchymal transition via enhancer of zeste homolog 2 in the malignant transformation of HBE cells induced by cigarette smoke extract

    International Nuclear Information System (INIS)

    Lu, Lu; Luo, Fei; Liu, Yi; Liu, Xinlu; Shi, Le; Lu, Xiaolin; Liu, Qizhan

    2015-01-01

    Lung cancer is regarded as the leading cause of cancer-related deaths, and cigarette smoking is one of the strongest risk factors for the development of lung cancer. However, the mechanisms for cigarette smoke-induced lung carcinogenesis remain unclear. The present study investigated the effects of an miRNA (miR-217) on levels of an lncRNA (MALAT1) and examined the role of these factors in the epithelial–mesenchymal transition (EMT) induced by cigarette smoke extract (CSE) in human bronchial epithelial (HBE) cells. In these cells, CSE caused decreases of miR-217 levels and increases in lncRNA MALAT1 levels. Over-expression of miR-217 with a mimic attenuated the CSE-induced increase of MALAT1 levels, and reduction of miR-217 levels by an inhibitor enhanced expression of MALAT1. Moreover, the CSE-induced increase of MALAT1 expression was blocked by an miR-217 mimic, indicating that miR-217 negatively regulates MALAT1 expression. Knockdown of MALAT1 reversed CSE-induced increases of EZH2 (enhancer of zeste homolog 2) and H3K27me3 levels. In addition to the alteration from epithelial to spindle-like mesenchymal morphology, chronic exposure of HBE cells to CSE increased the levels of EZH2, H3K27me3, vimentin, and N-cadherin and decreased E-cadherin levels, effects that were reversed by MALAT1 siRNA or EZH2 siRNA. The results indicate that miR-217 regulation of EZH2/H3K27me3 via MALAT1 is involved in CSE-induced EMT and malignant transformation of HBE cells. The posttranscriptional silencing of MALAT1 by miR-217 provides a link, through EZH2, between ncRNAs and the EMT and establishes a mechanism for CSE-induced lung carcinogenesis. - Highlights: • CSE exposure decreases miR-217 levels and increases MALAT1 levels. • miR-217 negatively regulates MALAT1 expression. • MALAT1, via EZH2, is involved in the EMT of CSE-transformed HBE cells.

  2. Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G

    Science.gov (United States)

    2011-01-01

    Background Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA) comprises ~530 residues, the G isoform (MSG) is ~730 residues, and this third isoform (MSH-halophilic) is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea. Results We have solved the structure of a malate synthase H (MSH) isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a β8/α8 (TIM) barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A. Conclusions The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C-terminal domain of the H

  3. Functional, structural and phylogenetic analysis of domains underlying the Al-sensitivity of the aluminium-activated malate/anion transporter, TaALMT1

    Science.gov (United States)

    TaALMT1 (Triticum aestivum Aluminum Activated Malate Transporter) is the founding member of a novel gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small subgroup of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (...

  4. O-Alkyl Hydroxamates as Metaphors of Enzyme-Bound Enolate Intermediates in Hydroxy Acid Dehydrogenases. Inhibitors of Isopropylmalate Dehydrogenase, Isocitrate Dehydrogenase, and Tartrate Dehydrogenase(1).

    Science.gov (United States)

    Pirrung, Michael C.; Han, Hyunsoo; Chen, Jrlung

    1996-07-12

    The inhibition of Thermus thermophilus isopropylmalate dehydrogenase by O-methyl oxalohydroxamate was studied for comparison to earlier results of Schloss with the Salmonella enzyme. It is a fairly potent (1.2 &mgr;M), slow-binding, uncompetitive inhibitor against isopropylmalate and is far superior to an oxamide (25 mM K(i) competitive) that is isosteric with the ketoisocaproate product of the enzyme. This improvement in inhibition was attributed to its increased NH acidity, which presumably is due to the inductive effect of the hydroxylamine oxygen. This principle was extended to the structurally homologous enzyme isocitrate dehydrogenase from E. coli, for which the compound O-(carboxymethyl) oxalohydroxamate is a 30 nM inhibitor, uncompetitive against isocitrate. The pH dependence of its inhibition supports the idea that it is bound to the enzyme in the anionic form. Another recently discovered homologous enzyme, tartrate dehydrogenase from Pseudomonas putida, was studied with oxalylhydroxamate. It has a relatively low affinity for the enzyme, though it is superior to tartrate. On the basis of these leads, squaric hydroxamates with increased acidity compared to squaric amides directed toward two of these enzymes were prepared, and they also show increased inhibitory potency, though not approaching the nanomolar levels of the oxalylhydroxamates.

  5. Altered Expression of the Malate-Permeable Anion Channel OsALMT4 Reduces the Growth of Rice Under Low Radiance

    Directory of Open Access Journals (Sweden)

    Jie Liu

    2018-05-01

    Full Text Available We examined the function of OsALMT4 in rice (Oryza sativa L. which is a member of the aluminum-activated malate transporter family. Previous studies showed that OsALMT4 localizes to the plasma membrane and that expression in transgenic rice lines results in a constitutive release of malate from the roots. Here, we show that OsALMT4 is expressed widely in roots, shoots, flowers, and grain but not guard cells. Expression was also affected by ionic and osmotic stress, light and to the hormones ABA, IAA, and salicylic acid. Malate efflux from the transgenic plants over-expressing OsALMT4 was inhibited by niflumate and salicylic acid. Growth of transgenic lines with either increased OsALMT4 expression or reduced expression was measured in different environments. Light intensity caused significant differences in growth between the transgenic lines and controls. When day-time light was reduced from 700 to 300 μmol m-2s-1 independent transgenic lines with either increased or decreased OsALMT4 expression accumulated less biomass compared to their null controls. This response was not associated with differences in photosynthetic capacity, stomatal conductance or sugar concentrations in tissues. We propose that by disrupting malate fluxes across the plasma membrane carbon partitioning and perhaps signaling are affected which compromises growth under low light. We conclude that OsALMT4 is expressed widely in rice and facilitates malate efflux from different cell types. Altering OsALMT4 expression compromises growth in low-light environments.

  6. Combination of long noncoding RNA MALAT1 and carcinoembryonic antigen for the diagnosis of malignant pleural effusion caused by lung cancer.

    Science.gov (United States)

    Wang, Wan-Wei; Zhou, Xi-Lei; Song, Ying-Jian; Yu, Chang-Hua; Zhu, Wei-Guo; Tong, Yu-Suo

    2018-01-01

    Long noncoding RNAs (lncRNAs) are present in body fluids, but their potential as tumor biomarkers has never been investigated in malignant pleural effusion (MPE) caused by lung cancer. The aim of this study was to assess the clinical significance of lncRNAs in pleural effusion, which could potentially serve as diagnostic and predictive markers for lung cancer-associated MPE (LC-MPE). RNAs from pleural effusion were extracted in 217 cases of LC-MPE and 132 cases of benign pleural effusion (BPE). Thirty-one lung cancer-associated lncRNAs were measured using quantitative real-time polymerase chain reaction (qRT-PCR). The level of carcinoembryonic antigen (CEA) was also determined. The receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were established to evaluate the sensitivity and specificity of the identified lncRNAs and other biomarkers. The correlations between baseline pleural effusion lncRNAs expression and response to chemotherapy were also analyzed. Three lncRNAs ( MALAT1 , H19 , and CUDR ) were found to have potential as diagnostic markers in LC-MPE. The AUCs for MALAT1 , H19 , CUDR , and CEA were 0.891, 0.783, 0.824, and 0.826, respectively. Using a logistic model, the combination of MALAT1 and CEA (AUC, 0.924) provided higher sensitivity and accuracy in predicting LC-MPE than CEA (AUC, 0.826) alone. Moreover, baseline MALAT1 expression in pleural fluid was inversely correlated with chemotherapy response in patients with LC-MPE. Pleural effusion lncRNAs were effective in differentiating LC-MPE from BPE. The combination of MALAT1 and CEA was more effective for LC-MPE diagnosis.

  7. The contribution of stored malate and citrate to the substrate requirements of metabolism of ripening peach (Prunus persica L. Batsch) flesh is negligible. Implications for the occurrence of phosphoenolpyruvate carboxykinase and gluconeogenesis.

    Science.gov (United States)

    Famiani, Franco; Farinelli, Daniela; Moscatello, Stefano; Battistelli, Alberto; Leegood, Richard C; Walker, Robert P

    2016-04-01

    The first aim of this study was to determine the contribution of stored malate and citrate to the substrate requirements of metabolism in the ripening flesh of the peach (Prunus persica L. Batsch) cultivar Adriatica. In the flesh, stored malate accumulated before ripening could contribute little or nothing to the net substrate requirements of metabolism. This was because there was synthesis and not dissimilation of malate throughout ripening. Stored citrate could potentially contribute a very small amount (about 5.8%) of the substrate required by metabolism when the whole ripening period was considered, and a maximum of about 7.5% over the latter part of ripening. The second aim of this study was to investigate why phosphoenolpyruvate carboxykinase (PEPCK) an enzyme utilised in gluconeogenesis from malate and citrate is present in peach flesh. The occurrence and localisation of enzymes utilised in the metabolism of malate, citrate and amino acids were determined in peach flesh throughout its development. Phosphoenolpyruvate carboxylase (essential for the synthesis of malate and citrate) was present in the same cells and at the same time as PEPCK and NADP-malic enzyme (both utilised in the dissimilation of malate and citrate). A hypothesis is presented to explain the presence of these enzymes and to account for the likely occurrence of gluconeogenesis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  8. Polimorfismo enzimático em populações de Melipona quadrifasciata anthidioides Lepeletier (Hymenoptera, Apidae, Meliponinae Enzymatic polymorphism in Melipona quadrifasciata anthidioides Lepeletier populations (Hymenoptera, Apidae, Meliponinae

    Directory of Open Access Journals (Sweden)

    Davi S. Aidar

    2001-06-01

    Full Text Available Them aim scope of this study is to characterize the enzymatic polymorphism found in the Melipona quadrifasciata Lepeletier, 1936 populations from Ribeirão Preto, São Paulo and Espírito Santo, Brazil and its hybrids. Samples from each colony (about 52 were prepared for starch gel electrophoresis in order to investigate the genetic variation of the following enzimes: esterase (EST, isocitrate dehydrogenase (IDH, malic enzyme (ME, phosphoglucomutase (PGM, superoxide desmutase (SOD, α-glycerophosphate dehydrogenase (αPGD, malate dehydrogenase (MDH, leucine aminopeptidase (LAP, hexokinase (HK and phosphoglucose isomerase (PGI. The analysis showed that LAP and HK did not show enzymatic activity and EST showed two alleles(est-sand and est-f while all the others were shown to be monomorphic. The allele EST-S showed a frequency of 82,6%.

  9. Neonatal jaundice and glucose-6-phosphate dehydrogenase

    OpenAIRE

    Leite, Amauri Antiquera [UNESP

    2010-01-01

    A deficiência de glicose-6-fosfato desidrogenase em neonatos pode ser a responsável pela icterícia neonatal. Este comentário científico é decorrente do relato sobre o tema publicado neste fascículo e que preocupa diversos autores de outros países em relação às complicações em neonatos de hiperbilirrubinemia, existindo inclusive proposições de alguns autores em incluir o teste para identificar a deficiência de glicose-6-fosfato desidrogenase nos recém-nascidos.Glucose-6-phosphate dehydrogenase...

  10. Short-lived long non-coding RNAs as surrogate indicators for chemical exposure and LINC00152 and MALAT1 modulate their neighboring genes.

    Directory of Open Access Journals (Sweden)

    Hidenori Tani

    Full Text Available Whole transcriptome analyses have revealed a large number of novel long non-coding RNAs (lncRNAs. Although accumulating evidence demonstrates that lncRNAs play important roles in regulating gene expression, the detailed mechanisms of action of most lncRNAs remain unclear. We previously reported that a novel class of lncRNAs with a short half-life (t1/2 < 4 h in HeLa cells, termed short-lived non-coding transcripts (SLiTs, are closely associated with physiological and pathological functions. In this study, we focused on 26 SLiTs and nuclear-enriched abundant lncRNA, MALAT1(t1/2 of 7.6 h in HeLa cells in neural stem cells (NSCs derived from human induced pluripotent stem cells, and identified four SLiTs (TUG1, GAS5, FAM222-AS1, and SNHG15 that were affected by the following typical chemical stresses (oxidative stress, heavy metal stress and protein synthesis stress. We also found the expression levels of LINC00152 (t1/2 of 2.1 h in NSCs, MALAT1 (t1/2 of 1.8 h in NSCs, and their neighboring genes were elevated proportionally to the chemical doses. Moreover, we confirmed that the overexpression of LINC00152 or MALAT1 upregulated the expressions of their neighboring genes even in the absence of chemical stress. These results reveal that LINC00152 and MALAT1 modulate their neighboring genes, and thus provide a deeper understanding of the functions of lncRNAs.

  11. Aberrant KDM5B expression promotes aggressive breast cancer through MALAT1 overexpression and downregulation of hsa-miR-448

    International Nuclear Information System (INIS)

    Bamodu, Oluwaseun Adebayo; Huang, Wen-Chien; Lee, Wei-Hwa; Wu, Alexander; Wang, Liang Shun; Hsiao, Michael; Yeh, Chi-Tai; Chao, Tsu-Yi

    2016-01-01

    Triple negative breast cancers (TNBC) possess cell dedifferentiation characteristics, carry out activities connate to those of cancer stem cells (CSCs) and are associated with increased metastasis, as well as, poor clinical prognosis. The regulatory mechanism of this highly malignant phenotype is still poorly characterized. Accruing evidence support the role of non-coding RNAs (ncRNAs) as potent regulators of CSC and metastatic gene expression, with their dysregulation implicated in tumorigenesis and disease progression. In this study, we investigated TNBC metastasis, metastasis-associated genes and potential inhibitory mechanisms using bioinformatics, tissue microarray analyses, immunoblotting, polymerase chain reaction, loss and gain of gene function assays and comparative analyses of data obtained. Compared with other breast cancer types, the highly metastatic MDA-MB-231 cells concurrently exhibited increased expression levels of Lysine-specific demethylase 5B protein (KDM5B) and long non-coding RNA (lncRNA), MALAT1, suggesting their functional association. KDM5B-silencing in the TNBC cells correlated with the upregulation of hsa-miR-448 and led to suppression of MALAT1 expression with decreased migration, invasion and clonogenic capacity in vitro, as well as, poor survival in vivo. This projects MALAT1 as a mediator of KDM5B oncogenic potential and highlights the critical role of this microRNA, lncRNA and histone demethylase in cancer cell motility and metastatic colonization. Increased expression of KDM5B correlating with disease progression and poor clinical outcome in breast cancer was reversed by hsa-miR-448. Our findings demonstrate the critical role of KDM5B and its negative regulator hsa-miR-448 in TNBC metastasis and progression. Hsa-miR-448 disrupting KDM5B-MALAT1 signalling axis and associated activities in TNBC cells, projects it as a putative therapeutic factor for selective eradication of TNBC cells

  12. Cloning and expression analysis of alcohol dehydrogenase ( Adh ...

    African Journals Online (AJOL)

    Hybrid promoters are created by shuffling of DNA fragments while keeping intact regulatory regions crucial of promoter activity. Two fragments of alcohol dehydrogenase (Adh) promoter from Zea mays were selected to generate hybrid promoter. Sequence analysis of both alcohol dehydrogenase promoter fragments through ...

  13. Some Properties of Glutamate Dehydrogenase from the Marine Red ...

    African Journals Online (AJOL)

    Keywords: ammonia assimilation, glutamate dehydrogenase, GDH, Gracilaria sordida, red alga, enzyme activity. Glutamate dehydrogenases (GDH, EC ... Anabolic functions could be assimilation of ammonia released during photorespiration and synthesis of N-rich transport compounds. Western Indian Ocean Journal of ...

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

    African Journals Online (AJOL)

    A quick analysis of the sludge activity method based on triphenyltetrazolium chloride-dehydrogenase activity (TTC-DHA) was developed to change the rule and status of the biological activity of the activated sludge in tomato paste wastewater treatment. The results indicate that dehydrogenase activity (DHA) can effectively ...

  15. INFLUENCE OF SELECTED PHARMACEUTICALS ON ACTIVATED SLUDGE DEHYDROGENASE ACTIVITY

    Directory of Open Access Journals (Sweden)

    Agnieszka Tomska

    2016-06-01

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

  16. The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesin

    Directory of Open Access Journals (Sweden)

    Pereira Maristela

    2009-12-01

    Full Text Available Abstract Background The pathogenic fungus Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM. This is a pulmonary mycosis acquired by inhalation of fungal airborne propagules that can disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside the host, P. brasiliensis may use the glyoxylate cycle for intracellular survival. Results Here, we provide evidence that the malate synthase of P. brasiliensis (PbMLS is located on the fungal cell surface, and is secreted. PbMLS was overexpressed in Escherichia coli, and polyclonal antibody was obtained against this protein. By using Confocal Laser Scanning Microscopy, PbMLS was detected in the cytoplasm and in the cell wall of the mother, but mainly of budding cells of the P. brasiliensis yeast phase. PbMLSr and its respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis with in vitro cultured epithelial cells A549. Conclusion These observations indicated that cell wall-associated PbMLS could be mediating the binding of fungal cells to the host, thus contributing to the adhesion of fungus to host tissues and to the dissemination of infection, behaving as an anchorless adhesin.

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

    Directory of Open Access Journals (Sweden)

    Margit Winkler

    2013-08-01

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

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

    Science.gov (United States)

    Napora-Wijata, Kamila; Strohmeier, Gernot A; Sonavane, Manoj N; Avi, Manuela; Robins, Karen; Winkler, Margit

    2013-08-12

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

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

    OpenAIRE

    Napora-Wijata, Kamila; Strohmeier, Gernot A.; Sonavane, Manoj N.; Avi, Manuela; Robins, Karen; Winkler, Margit

    2013-01-01

    Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisia...

  20. A domain-based approach for analyzing the function of aluminum-activated malate transporters from wheat (Triticum aestivum) and Arabidopsis thaliana in Xenopus oocytes.

    Science.gov (United States)

    Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Furuichi, Takuya; Yamamoto, Yoko

    2014-12-01

    Wheat and Arabidopsis plants respond to aluminum (Al) ions by releasing malate from their root apices via Al-activated malate transporter. Malate anions bind with the toxic Al ions and contribute to the Al tolerance of these species. The genes encoding the transporters in wheat and Arabidopsis, TaALMT1 and AtALMT1, respectively, were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the two-electrode voltage clamp system. The Al-activated currents generated by malate efflux were detected for TaALMT1 but not for AtALMT1. Chimeric proteins were generated by swapping the N- and C-terminal halves of TaALMT1 and AtALMT1 (Ta::At and At::Ta). When these chimeras were characterized in oocytes, Al-activated malate efflux was detected for the Ta::At chimera but not for At::Ta, suggesting that the N-terminal half of TaALMT1 is necessary for function in oocytes. An additional chimera, Ta(48)::At, generated by swapping 17 residues from the N-terminus of AtALMT1 with the equivalent 48 residues from TaALMT1, was sufficient to support transport activity. This 48 residue region includes a helical region with a putative transmembrane domain which is absent in AtALMT1. The deletion of this domain from Ta(48)::At led to the complete loss of transport activity. Furthermore, truncations and a deletion at the C-terminal end of TaALMT1 indicated that a putative helical structure in this region was also required for transport function. This study provides insights into the structure-function relationships of Al-activated ALMT proteins by identifying specific domains on the N- and C-termini of TaALMT1 that are critical for basal transport function and Al responsiveness in oocytes. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  1. Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci.

    Science.gov (United States)

    Pavlova, Sylvia I; Jin, Ling; Gasparovich, Stephen R; Tao, Lin

    2013-07-01

    Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci.

  2. Genetics Home Reference: 3-beta-hydroxysteroid dehydrogenase deficiency

    Science.gov (United States)

    ... for This Page Lutfallah C, Wang W, Mason JI, Chang YT, Haider A, Rich B, Castro-Magana ... A, Copeland KC, Chang YT, Lutfallah C, Mason JI. Carriers for type II 3beta-hydroxysteroid dehydrogenase (HSD3B2) ...

  3. Properties of glucoside 3-dehydrogenase and its potential applications

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-29

    Dec 29, 2008 ... dehydrogenase has attracted considerable attention in recent years due to broad substrate specificity and excellent ... site-selective oxidation of the C-3 hydroxyl group. .... single peptide with a molecular mass of 67 kDa in.

  4. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Science.gov (United States)

    2010-04-01

    ... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver... marrow) leukemia. (b) Classification. Class I (general controls). The device is exempt from the premarket...

  5. Modeling of NAD+ analogues in horse liver alcohol dehydrogenase

    NARCIS (Netherlands)

    Beijer, N.A.; Buck, H.M.; Sluyterman, L.A.A.E.; Meijer, E.M.

    1990-01-01

    So far, the interactions of nicotinamide adenine dinucleotide (NAD+) derivatives with dehydrogenases are not very well understood. This hampers the introduction of NAD+ analogues with improved characteristics concerning industrial application. We have developed an AMBER molecular mechanics model in

  6. An improved method for the assay of platelet pyruvate dehydrogenase

    International Nuclear Information System (INIS)

    Schofield, P.J.; Griffiths, L.R.; Rogers, S.H.

    1980-01-01

    An improved method for the assay of human platelet pyruvate dehydrogenase is described. By generating the substrate [1- 14 C]pyruvate in situ from [1- 14 C]lactate plus L-lactate dehydrogenase, the rate of spontaneous decarboxylation is dramatically reduced, allowing far greater sensitivity in the assay of low activities of pyruvate dehydrogenase. In addition, no special precautions are required for the storage and use of [1- 14 C]lactate, in contrast to those for [1- 14 C]pyruvate. These factors allow a 5-10-fold increase in sensitivity compared with current methods. The pyruvate dehydrogenase activity of normal subjects as determined by the [1- 14 C]lactate system was 215+-55 pmol min -1 mg -1 protein (n=18). The advantages of this assay system are discussed. (Auth.)

  7. Genetics Home Reference: 17-beta hydroxysteroid dehydrogenase 3 deficiency

    Science.gov (United States)

    ... 000 newborns. It is more common in the Arab population of Gaza, where it affects 1 in ... fetus, resulting in the abnormalities in the external sex organs that occur in 17-beta hydroxysteroid dehydrogenase ...

  8. Rapid synthesis of triazine inhibitors of inosine monophosphate dehydrogenase.

    Science.gov (United States)

    Pitts, William J; Guo, Junqing; Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Watterson, Scott H; Bednarz, Mark S; Chen, Bang Chi; Barrish, Joel C; Bassolino, Donna; Cheney, Daniel; Fleener, Catherine A; Rouleau, Katherine A; Hollenbaugh, Diane L; Iwanowicz, Edwin J

    2002-08-19

    A series of novel triazine-based small molecule inhibitors (IV) of inosine monophosphate dehydrogenase was prepared. The synthesis and the structure-activity relationships (SAR) derived from in vitro studies are described.

  9. Novel amide-based inhibitors of inosine 5'-monophosphate dehydrogenase.

    Science.gov (United States)

    Watterson, Scott H; Liu, Chunjian; Dhar, T G Murali; Gu, Henry H; Pitts, William J; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L; Iwanowicz, Edwin J

    2002-10-21

    A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.

  10. Eucalypt NADP-Dependent Isocitrate Dehydrogenase1

    Science.gov (United States)

    Boiffin, Vincent; Hodges, Michael; Gálvez, Susana; Balestrini, Raffaella; Bonfante, Paola; Gadal, Pierre; Martin, Francis

    1998-01-01

    NADP-dependent isocitrate dehydrogenase (NADP-ICDH) activity is increased in roots of Eucalyptus globulus subsp. bicostata ex Maiden Kirkp. during colonization by the ectomycorrhizal fungus Pisolithus tinctorius Coker and Couch. To investigate the regulation of the enzyme expression, a cDNA (EgIcdh) encoding the NADP-ICDH was isolated from a cDNA library of E. globulus-P. tinctorius ectomycorrhizae. The putative polypeptide sequence of EgIcdh showed a high amino acid similarity with plant NADP-ICDHs. Because the deduced EgICDH protein lacks an amino-terminal targeting sequence and shows highest similarity to plant cytosolic ICDHs, it probably represents a cytoplasmic isoform. RNA analysis showed that the steady-state level of EgIcdh transcripts was enhanced nearly 2-fold in ectomycorrhizal roots compared with nonmycorrhizal roots. Increased accumulation of NADP-ICDH transcripts occurred as early as 2 d after contact and likely led to the observed increased enzyme activity. Indirect immunofluorescence microscopy indicated that NADP-ICDH was preferentially accumulated in the epidermis and stele parenchyma of nonmycorrhizal and ectomycorrhizal lateral roots. The putative role of cytosolic NADP-ICDH in ectomycorrhizae is discussed. PMID:9662536

  11. Glucose-6-phosphate dehydrogenase deficiency in Singapore.

    Science.gov (United States)

    Quak, S H; Saha, N; Tay, J S

    1996-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) in man is an X-linked enzyme. The deficiency of this enzyme is one of the most common inherited metabolic disorders in man. In Singapore, three clinical syndromes associated with G6PD deficiency had been described: severe haemolysis in neonates with kernicterus, haemoglobinuria and "viral hepatitis"-like syndrome. The human G6PD monomer consists of 515 amino acids. Only the tetrameric or dimeric forms composed of a single type subunit are catylitically active. The complete amino acid sequence of G6PD had been elucidated in man and various other animals. The region of high homology among the enzymes of various animals is presumably functionally active. Among the Chinese in Singapore, three common molecular variants had been identified: Canton (nt 1376 G --> T), Kaiping (nt 1388 G --> A) and Mediterranean (nt 563 C --> T) in frequencies of 24%, 21% and 10% respectively. In addition, two common mutants (Gaozhou, nt 95 A --> G and Chinese 5, nt 1024 C --> T) have been detected in Singapore Chinese in low frequencies. In Malays, 6 different deficient variants are known in Singapore (3 new, 1 Mahidol, 1 Indonesian and 1 Mediterranean).

  12. Human liver aldehyde dehydrogenase: coenzyme binding

    International Nuclear Information System (INIS)

    Kosley, L.L.; Pietruszko, R.

    1987-01-01

    The binding of [U- 14 C] NAD to mitochondrial (E2) and cytoplasmin(E1) aldehyde dehydrogenase was measured by gel filtration and sedimentation techniques. The binding data for NAD and (E1) yielded linear Scatchard plots giving a dissociation constant of 25 (+/- 8) uM and the stoichiometry of 2 mol of NAD bound per mol of E1. The binding data for NAD and (E2) gave nonlinear Scatchard plots. The binding of NADH to E2 was measured via fluorescence enhancement; this could not be done with E1 because there was no signal. The dissociation constant for E2 by this technique was 0.7 (+/- 0.4) uM and stoichiometry of 1.0 was obtained. The binding of [U- 14 C] NADH to (E1) and (E2) was also measured by the sedimentation technique. The binding data for (E1) and NADH gave linear Scatchard plots giving a dissociation constant of 13 (+/- 6) uM and the stoichiometry of 2.0. The binding data for NADH to (E2) gave nonlinear Scatchard plots. With (E1), the dissociation constants for both NAD and NADH are similar to those determined kinetically, but the stoichiometry is only half of that found by stopped flow technique. With (E2) the dissociation constant by fluorometric procedure was 2 orders of magnitude less than that from catalytic reaction

  13. Radioimmunoassay of lactate dehydrogenase, H forms

    International Nuclear Information System (INIS)

    Malvano, R.; Massaglia, A.; Zannino, M.; Palmucci, F.; Cali, V.; Zucchelli, G.C.; Consiglio Nazionale delle Ricerche, Pisa

    1979-01-01

    Antisera to H 4 -lactate dehydrogenase (LDH) were elicited in rabbits, against both human (h) and porcine (p) isoenzymes. 125 I-labelled H 4 -LDH was prepared by electrolytic iodination. A simple and fast procedure (1-h incubation for clinical assays) was set up by using polyethylene glycol for the bound-free separation. The results obtained in the antiserum characterization indicated that the heterologous homotetramer, M 4 was completely discriminated in the porcine system, while a weak cross-reaction with human antisera resulted. In both cases, for the hybrid forms, a cross-reactivity level related to the stoichiometric contents of the H-subunit in the tetramers was observed. The H 4 -LDH from other species was found to be much more effectively distinguished in the procine than in the human system. The assay for human LDH was further validated in terms of analytical suitability and clinical response. For healthy subjects the mean concentration was 0.46 +- 0.19 μg/ml (mean +- SD). Patients with acute myocardial infarction had levels ranging from 1.2 to 5.9 μg/ml. (orig.) [de

  14. Glucose 6 phosphate dehydrogenase deficiency in adults

    International Nuclear Information System (INIS)

    Khan, M.

    2004-01-01

    Objective: To determine the frequency of glucose-6-phosphate dehydrogenase (G6PD) deficiency in adults presented with anemia. Subjects and Methods: Eighteen months admission data was reviewed for G6PD deficiency as a cause of anemia. Anemia was defined by world health organization (WHO) criteria as haemoglobin less than 11.3 gm%. G6PD activity was measured by Sigma dye decolorisation method. All patients were screened for complications of hemolysis and its possible cause. Patients with more than 13 years of age were included in the study. Results: Out of 3600 patients admitted, 1440 were found anaemic and 49 as G6PD deficient. So the frequency of G6PD deficiency in anaemic patients was 3.4% and the overall frequency is 1.36%. G6PD deficiency among males and females was three and six percent respectively. Antimalarials and antibiotics containing sulphonamide group were the most common precipitating factors for hemolysis. Anemia and jaundice were the most common presentations while malaria was the most common associated disease. Acute renal failure was the most severe complication occurring in five patients with two deaths. Conclusion: G6PD deficiency is a fairly common cause of anemia with medicine as common precipitating factor for hemolysis. Such complications can be avoided with early recognition of the disease and avoiding indiscriminate use of medicine. (author)

  15. Glucose 6-phosphate dehydrogenase variants in Japan.

    Science.gov (United States)

    Miwa, S

    1980-01-01

    Fifty-four cases of glucose 6-phosphate dehydrogenase (G6PD) deficiency have so far been reported in Japan. Among them, 21 G6PD variants have been characterized. Nineteen out of the 21 variants were characterized in our laboratory and G6PD Heian and "Kyoto" by others. G6PD Tokyo, Tokushima, Ogikubo, Kurume, Fukushima, Yokohama, Yamaguchi, Wakayama, Akita, Heian and "Kyoto" were classified as Class 1, because all these cases showed chronic hemolytic anemia and severe enzyme deficiency. All these variants showed thermal instability. G6PD Mediterranean-like, Ogori, Gifu and Fukuoka were classified as Class 2, whereas G6PD Hofu, B(-) Chinese, Ube, Konan, Kamiube and Kiwa belonged to Class 3. All the 6 Class 3 variants were found as the results of the screening tests. The incidence of the deficiency in Japanese seems to be 0.1-0.5% but that of the cases which may slow drug-induced hemolysis would be much less. G6PD Ube and Konan appear to be relatively common in Japan.

  16. Pyruvate dehydrogenase complex and lactate dehydrogenase as targets for therapy of acute liver failure.

    Science.gov (United States)

    Ferriero, Rosa; Nusco, Edoardo; De Cegli, Rossella; Carissimo, Annamaria; Manco, Giuseppe; Brunetti-Pierri, Nicola

    2018-03-23

    Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate in the nucleus to regulate histone acetylation and gene expression. Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-Ab, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by Gene Ontology Enrichment Analysis. Efficacy of histone acetyltransferase inhibitor garcinol and LDH inhibitor galloflavin at reducing liver damage was evaluated in mice with induced hepatotoxicity. Levels and activities of PDHC and LDH were increased in cytoplasmatic and nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-coA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to response to damage. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. PDHC and LDH translocate to the nucleus and are targets for therapy of acute liver failure. Acute liver failure is a rapidly progressive and life-threatening deterioration of liver function resulting in high mortality and

  17. Toxicity of Nitrification Inhibitors on Dehydrogenase Activity in Soils

    OpenAIRE

    Ferisman Tindaon; Gero Benckiser; Johannes C. G. Ottow

    2011-01-01

    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...

  18. Carbon isotope composition of intermediates of the starch-malate sequence and level of the crassulacean acid metabolism in leaves of Kalanchoe blossfeldiana Tom Thumb.

    Science.gov (United States)

    Deleens, E; Garnier-Dardart, J; Queiroz, O

    1979-09-01

    Isotype analyses were performed on biochemical fractions isolated from leaves of Kalanchoe blossfeldiana Tom Thumb. during aging under long days or short days. Irrespective of the age or photoperiodic conditions, the intermediates of the starch-malate sequence (starch, phosphorylated compounds and organic acids) have a level of (13)C higher than that of soluble sugars, cellulose and hemicellulose. In short days, the activity of the crassulacean acid metabolism pathway is predominant as compared to that of C3 pathway: leaves accumulate organic acids, rich in (13)C. In long days, the activity of the crassulacean acid metabolism pathway increases as the leaves age, remaining, however, relatively low as compared to that of C3 pathway: leaves accumulate soluble sugars, poor in (13)C. After photoperiodic change (long days→short days), isotopic modifications of starch and organic acids suggest evidence for a lag phase in the establishment of the crassulacean acid metabolism pathway specific to short days. The relative proportions of carbon from a C3-origin (RuBPC acitivity as strong discriminating step, isotope discrimination in vivo=20‰) or C4-origin (PEPC activity as weak discriminating step, isotope discrimination in vivo=4‰) present in the biochemical fractions were calculated from their δ(13)C values. Under long days, 30 to 70% versus 80 to 100% under short days, of the carbon of the intermediates linked to the starch-malate sequence, or CAM pathway (starch, phosphorylated compounds and organic acids), have a C4-origin. Products connected to the C3 pathway (free sugars, cellulose, hemicellulose) have 0 to 50% of their carbon, arising from reuptake of the C4 from malate, under long days versus 30 to 70% under short days.

  19. ald of Mycobacterium tuberculosis Encodes both the Alanine Dehydrogenase and the Putative Glycine Dehydrogenase

    Science.gov (United States)

    Giffin, Michelle M.; Modesti, Lucia; Raab, Ronald W.; Wayne, Lawrence G.

    2012-01-01

    The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown. PMID:22210765

  20. Acute citrulline malate supplementation improves upper- and lower-body submaximal weightlifting exercise performance in resistance-trained females.

    Science.gov (United States)

    Glenn, Jordan M; Gray, Michelle; Wethington, Lauren N; Stone, Matthew S; Stewart, Rodger W; Moyen, Nicole E

    2017-03-01

    Citrulline malate (CM) is a nonessential amino acid that increases exercise performance in males. However, based on physiological differences between genders, these results cannot be extrapolated to females. Therefore, the purpose of this investigation was to evaluate effects of acute CM supplementation on upper- and lower-body weightlifting performance in resistance-trained females. Fifteen females (23 ± 3 years) completed two randomized, double-blind trials consuming either CM (8 g dextrose + 8 g CM) or a placebo (8 g dextrose). One hour after supplement consumption, participants performed six sets each of upper- (i.e., bench press) and lower-body (i.e., leg press) exercises to failure at 80 % of previously established one-repetition maximum. Immediately after each set, repetitions completed, heart rate and rating of perceived exertion (RPE) were recorded. Repeated-measures analysis of variance indicated that subjects completed significantly (p = .045) more repetitions throughout upper-body exercise when consuming CM versus placebo (34.1 ± 5.7 vs. 32.9 ± 6.0, respectively). When consuming CM, similar significant (p = .03) improvements in total repetitions completed were observed for lower-body exercise (66.7 ± 30.5 vs. 55.13 ± 20.64, respectively). Overall RPE score was significantly lower (p = .02) in upper-body exercise when subjects consumed CM versus placebo (7.9 ± 0.3 and 8.6 ± 0.2, respectively). The supplement consumed exhibited no significant effects on heart rate at any time point. Acute CM supplementation in females increased upper- and lower-body resistance exercise performance and decreased RPE during upper-body exercise. These data indicate that athletes competing in sports with muscular endurance-based requirements may potentially improve performance by acutely supplementing CM.

  1. 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.

  2. Determination of malation, methidathion, and chlorpyrifos ethyl pesticides using acetylcholinesterase biosensor based on Nafion/Ag@rGO-NH_2 nanocomposites

    International Nuclear Information System (INIS)

    Guler, Muhammet; Turkoglu, Vedat; Basi, Zehra

    2017-01-01

    Herein, a facile electrochemical acetylcholinesterase (EC 3.1.1.7; AChE) biosensor based on nafion (NA) and Ag nanoparticles supported on amine functionalized reduced graphene oxide (rGO-NH_2) was developed. The Ag@rGO-NH_2 nanocomposite was characterized using Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), and X-ray diffraction (XRD). After being optimized, the biosensor exhibited excellent electrochemical response to the oxidation of thiocholine, the hydrolysis product of acetylthiocholine chloride (ATCl) catalyzed by AChE. An apparent Michealis-Menten value of 20.5 μM was obtained. Under optimized conductions, the biosensor detected malation, methidathion, and chlorpyrifos ethyl in the linear range from 0.0063 to 0.077 μg/mL, from 0.012 to 0.105 μg/mL, and from 0.021 to 0.122 μg/mL, respectively. The detection limit (LoD) was 4.5 ng/mL for malation, 9.5 ng/mL for methidathion, and 14 ng/mL for chlorpyrifos ethyl. Also, the NA/Ag@rGO-NH_2/AChE/GCE biosensor showed god sensitivity, stability and repeatability, which provides a promising tool for the detection of organophosphate pesticides.

  3. Crystallization and crystallographic analysis of the ligand-binding domain of the Pseudomonas putida chemoreceptor McpS in complex with malate and succinate

    International Nuclear Information System (INIS)

    Gavira, J. A.; Lacal, J.; Ramos, J. L.; García-Ruiz, J. M.; Krell, T.; Pineda-Molina, E.

    2012-01-01

    The crystallization of the ligand-binding domain of the methyl-accepting chemotaxis protein chemoreceptor McpS (McpS-LBD) is reported. Methyl-accepting chemotaxis proteins (MCPs) are transmembrane proteins that sense changes in environmental signals, generating a chemotactic response and regulating other cellular processes. MCPs are composed of two main domains: a ligand-binding domain (LBD) and a cytosolic signalling domain (CSD). Here, the crystallization of the LBD of the chemoreceptor McpS (McpS-LBD) is reported. McpS-LBD is responsible for sensing most of the TCA-cycle intermediates in the soil bacterium Pseudomonas putida KT2440. McpS-LBD was expressed, purified and crystallized in complex with two of its natural ligands (malate and succinate). Crystals were obtained by both the counter-diffusion and the hanging-drop vapour-diffusion techniques after pre-incubation of McpS-LBD with the ligands. The crystals were isomorphous and belonged to space group C2, with two molecules per asymmetric unit. Diffraction data were collected at the ESRF synchrotron X-ray source to resolutions of 1.8 and 1.9 Å for the malate and succinate complexes, respectively

  4. 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.

  5. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci

    Directory of Open Access Journals (Sweden)

    Guillermo Hugo Peralta

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

  6. Screening of Glucose-6-Phosphate Dehydrogenase Deficiency in Cord Blood

    Directory of Open Access Journals (Sweden)

    Can Acipayam

    2014-02-01

    Aim: Glucose-6-phosphate dehydrogenase deficiency is an important factor in etiology of pathologic neonatal jaundice. The aim of this study was to indicate the significance of screening glucose-6-phosphate dehydrogenase deficiency in the cord blood of neonates and the frequency of this deficiency in the etiology of neonatal hyperbilirubinemia. Material and Method: The study was performed consecutive 1015 neonates were included. Five hundred fifty six (54.8% of them were male and 459 (45.2% were female. The following parameters were recorded: Gender, birth weight, birth height, head circumference and gestational age. The glucose-6-phosphate dehydrogenase level of neonates were measured with quantitative method in cord blood. Also, hemoglobine, hematocrite, red blood cell count and blood group were measured. The following parameters were recorded in cases with jaundice: exchange transfusion, phototherapy, physiologic and pathologic jaundice, peak bilirubin day, maximum bilirubin level, total bilirubin level at the first day of jaundice, beginning time of jaundice. Results: Enzyme deficiency was detected in 133 (13.1% of neonates and 76 (57% of them were male, 57 (43% were female. Significant difference was detected in low glucose-6-phosphate dehydrogenase enzyme level with jaundice group for total bilirubin level at the first day of jaundice, maximum total bilirubin level and pathologic jaundice (p<0.05. Discussion: The ratio of glucose-6-phosphate dehydrogenase deficiency was found in Edirne in this study and this ratio was higher than other studies conducted in our country. For this reason, glucose-6-phosphate dehydrogenase enzyme level in cord blood of neonates should be measured routinely and high risk neonates should be followed up for hyperbilirubinemia and parents should be informed in our region.

  7. Reversible inactivation of CO dehydrogenase with thiol compounds

    Energy Technology Data Exchange (ETDEWEB)

    Kreß, Oliver [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany); Gnida, Manuel [Department of Chemistry, University of Paderborn, 33098 Paderborn (Germany); Pelzmann, Astrid M. [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany); Marx, Christian [Institute of Biochemistry and Biophysics, Friedrich-Schiller-University of Jena, 07745 Jena (Germany); Meyer-Klaucke, Wolfram [Department of Chemistry, University of Paderborn, 33098 Paderborn (Germany); Meyer, Ortwin, E-mail: Ortwin.Meyer@uni-bayreuth.de [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany)

    2014-05-09

    Highlights: • Rather large thiols (e.g. coenzyme A) can reach the active site of CO dehydrogenase. • CO- and H{sub 2}-oxidizing activity of CO dehydrogenase is inhibited by thiols. • Inhibition by thiols was reversed by CO or upon lowering the thiol concentration. • Thiols coordinate the Cu ion in the [CuSMo(=O)OH] active site as a third ligand. - Abstract: Carbon monoxide dehydrogenase (CO dehydrogenase) from Oligotropha carboxidovorans is a structurally characterized member of the molybdenum hydroxylase enzyme family. It catalyzes the oxidation of CO (CO + H{sub 2}O → CO{sub 2} + 2e{sup −} + 2H{sup +}) which proceeds at a unique [CuSMo(=O)OH] metal cluster. Because of changing activities of CO dehydrogenase, particularly in subcellular fractions, we speculated whether the enzyme would be subject to regulation by thiols (RSH). Here we establish inhibition of CO dehydrogenase by thiols and report the corresponding K{sub i}-values (mM): L-cysteine (5.2), D-cysteine (9.7), N-acetyl-L-cysteine (8.2), D,L-homocysteine (25.8), L-cysteine–glycine (2.0), dithiothreitol (4.1), coenzyme A (8.3), and 2-mercaptoethanol (9.3). Inhibition of the enzyme was reversed by CO or upon lowering the thiol concentration. Electron paramagnetic resonance spectroscopy (EPR) and X-ray absorption spectroscopy (XAS) of thiol-inhibited CO dehydrogenase revealed a bimetallic site in which the RSH coordinates to the Cu-ion as a third ligand ([Mo{sup VI}(=O)OH{sub (2)}SCu{sup I}(SR)S-Cys]) leaving the redox state of the Cu(I) and the Mo(VI) unchanged. Collectively, our findings establish a regulation of CO dehydrogenase activity by thiols in vitro. They also corroborate the hypothesis that CO interacts with the Cu-ion first. The result that thiol compounds much larger than CO can freely travel through the substrate channel leading to the bimetallic cluster challenges previous concepts involving chaperone function and is of importance for an understanding how the sulfuration step in

  8. Abnormalities in the tricarboxylic acid (TCA) cycle in the brains of schizophrenia patients.

    Science.gov (United States)

    Bubber, P; Hartounian, V; Gibson, G E; Blass, J P

    2011-03-01

    Images of brain metabolism and measurements of activities of components of the electron transport chain support earlier studies that suggest that brain glucose oxidation is inherently abnormal in a significant proportion of persons with schizophrenia. Therefore, we measured the activities of enzymes of the tricarboxylic (TCA) cycle in dorsolateral-prefrontal-cortex from schizophrenia patients (N=13) and non-psychiatric disease controls (N=13): the pyruvate dehydrogenase complex (PDHC), citrate synthase (CS), aconitase, isocitrate dehydrogenase (ICDH), the alpha-ketoglutarate dehydrogenase complex (KGDHC), succinate thiokinase (STH), succinate dehydrogenase (SDH), fumarase and malate dehydrogenase (MDH). Activities of aconitase (18.4%, pTCA cycle, were lower, but SDH (18.3%, pTCA cycle and cognitive function, age or choline acetyl transferase activity, except for aconitase activity which decreased slightly with age (r=0.55, p=003). The increased activities of dehydrogenases in the second half of the TCA cycle may reflect a compensatory response to reduced activities of enzymes in the first half. Such alterations in the components of TCA cycle are adequate to alter the rate of brain metabolism. These results are consistent with the imaging studies of hypometabolism in schizophrenia. They suggest that deficiencies in mitochondrial enzymes can be associated with mental disease that takes the form of schizophrenia. Copyright © 2010 Elsevier B.V. and ECNP. All rights reserved.

  9. [Discovery of the target genes inhibited by formic acid in Candida shehatae].

    Science.gov (United States)

    Cai, Peng; Xiong, Xujie; Xu, Yong; Yong, Qiang; Zhu, Junjun; Shiyuan, Yu

    2014-01-04

    At transcriptional level, the inhibitory effects of formic acid was investigated on Candida shehatae, a model yeast strain capable of fermenting xylose to ethanol. Thereby, the target genes were regulated by formic acid and the transcript profiles were discovered. On the basis of the transcriptome data of C. shehatae metabolizing glucose and xylose, the genes responsible for ethanol fermentation were chosen as candidates by the combined method of yeast metabolic pathway analysis and manual gene BLAST search. These candidates were then quantitatively detected by RQ-PCR technique to find the regulating genes under gradient doses of formic acid. By quantitative analysis of 42 candidate genes, we finally identified 10 and 5 genes as markedly down-regulated and up-regulated targets by formic acid, respectively. With regard to gene transcripts regulated by formic acid in C. shehatae, the markedly down-regulated genes ranking declines as follows: xylitol dehydrogenase (XYL2), acetyl-CoA synthetase (ACS), ribose-5-phosphate isomerase (RKI), transaldolase (TAL), phosphogluconate dehydrogenase (GND1), transketolase (TKL), glucose-6-phosphate dehydrogenase (ZWF1), xylose reductase (XYL1), pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC); and a declining rank for up-regulated gens as follows: fructose-bisphosphate aldolase (ALD), glucokinase (GLK), malate dehydrogenase (MDH), 6-phosphofructokinase (PFK) and alcohol dehydrogenase (ADH).

  10. Alcohol consumption, alcohol dehydrogenase 3 polymorphism, and colorectal adenomas

    NARCIS (Netherlands)

    Tiemersma, E.W.; Wark, P.A.; Ocké, M.C.; Bunschoten, A.; Otten, M.H.; Kok, F.J.; Kampman, E.

    2003-01-01

    Alcohol is a probable risk factor with regard to colorectal neoplasm and is metabolized to the carcinogen acetaldehyde by the genetically polymorphic alcohol dehydrogenase 3 (ADH3) enzyme. We evaluated whether the association between alcohol and colorectal adenomas is modified by ADH3 polymorphism.

  11. Glucose-6-phosphate dehydrogenase deficiency; the single most ...

    African Journals Online (AJOL)

    Introduction: Glucose- 6-phosphate dehydrogenase deficiency is the most common enzymatic disorder of the red cell and an important risk factor for neonatal jaundice. Methodology: The aim of the study was to determine the incidence of G-6-PD deficiency among jaundiced neonates, and describe the associated morbidity ...

  12. [Genetic variations in alcohol dehydrogenase, drinking habits and alcoholism

    DEFF Research Database (Denmark)

    Tolstrup, J.S.; Rasmussen, S.; Tybjaerg-Hansen, A.

    2008-01-01

    Alcohol is degraded primarily by alcohol dehydrogenase (ADH), and genetic variation that affects the rate of alcohol degradation is found in ADH1B and ADH1C. By genotyping 9,080 white men and women from the general population, we found that men and women with ADH1B slow versus fast alcohol degrad...

  13. Efficiency of superoxide anions in the inactivation of selected dehydrogenases

    International Nuclear Information System (INIS)

    Rodacka, Aleksandra; Serafin, Eligiusz; Puchala, Mieczyslaw

    2010-01-01

    The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as · OH and ONOO - . In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH). We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion. Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.

  14. Efficiency of superoxide anions in the inactivation of selected dehydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Rodacka, Aleksandra, E-mail: olakow@biol.uni.lodz.p [Department of Molecular Biophysics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland); Serafin, Eligiusz, E-mail: serafin@biol.uni.lodz.p [Laboratory of Computer and Analytical Techniques, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland); Puchala, Mieczyslaw, E-mail: puchala@biol.uni.lodz.p [Department of Molecular Biophysics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland)

    2010-09-15

    The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as {sup {center_dot}}OH and ONOO{sup -}. In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH). We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion. Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.

  15. Expanding the clinical spectrum of 3-phosphoglycerate dehydrogenase deficiency

    NARCIS (Netherlands)

    Tabatabaie, L; Klomp, L W J; Rubio-Gozalbo, M E; Spaapen, L J M; Haagen, A A M; Dorland, L; de Koning, T J

    UNLABELLED: 3-Phosphoglycerate dehydrogenase (3-PGDH) deficiency is considered to be a rare cause of congenital microcephaly, infantile onset of intractable seizures and severe psychomotor retardation. Here, we report for the first time a very mild form of genetically confirmed 3-PGDH deficiency in

  16. Nicotinoprotein methanol dehydrogenase enzymes in Gram-positive methylotrophic bacteria

    NARCIS (Netherlands)

    Hektor, Harm J.; Kloosterman, Harm; Dijkhuizen, Lubbert

    2000-01-01

    A novel type of alcohol dehydrogenase enzyme has been characterized from Gram-positive methylotrophic (Bacillus methanolicus, the actinomycetes Amycolatopsis methanolica and Mycobacterium gastri) and non-methylotrophic bacteria (Rhodococcus strains). Its in vivo role is in oxidation of methanol and

  17. Identification of glucose 6 phosphate dehydrogenase mutations by ...

    African Journals Online (AJOL)

    Identification of glucose 6 phosphate dehydrogenase mutations by single strand conformation polymorphism and gene sequencing analysis. ... Subject: Six DNA samples from Turkish males confirmed to have G-6-PD deficiency where available for the study. Results: One subject was found to have an abnormal mobility shift ...

  18. Medium-chain acyl-CoA dehydrogenase deficiency

    DEFF Research Database (Denmark)

    Waddell, Leigh; Wiley, Veronica; Carpenter, Kevin

    2006-01-01

    The fatty acid oxidation disorder most commonly identified by tandem mass spectrometry newborn screening is the potentially fatal medium-chain acyl-CoA dehydrogenase deficiency (MCAD). In clinically presenting cases, 80% are homozygous for the common mutation, c.985A > G and 18% heterozygous. We ...

  19. New enzymatic assay, parasite lactate dehydrogenase in diagnosis ...

    African Journals Online (AJOL)

    Background: The unique ability of plasmodial lactate dehydrogenase p(LDH) to utilise 3-acetyl pyridine dinucleotide (APAD) in lieu of NAD as a coenzyme in the conversion of pyruvate to lactate, led to the development of a biochemical assay for the detection of plasmodial parasitaemia. Researchers have reported that ...

  20. Alcoholism and alcohol drinking habits predicted from alcohol dehydrogenase genes

    DEFF Research Database (Denmark)

    Tolstrup, J.S.; Nordestgaard, Børge; Rasmussen, S.

    2008-01-01

    Alcohol is degraded primarily by alcohol dehydrogenase (ADH) wherein genetic variation that affects the rate of alcohol degradation is found in ADH1B and ADH1C. It is biologically plausible that these variations may be associated with alcohol drinking habits and alcoholism. By genotyping 9080 whi...

  1. Cloning and expression of chicken 20-hydroxysteroid dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Bryndová, Jana; Klusoňová, Petra; Kučka, Marek; Vagnerová, Karla; Mikšík, Ivan; Pácha, Jiří

    2006-01-01

    Roč. 37, č. 3 (2006), s. 453-462 ISSN 0952-5041 R&D Projects: GA AV ČR(CZ) IAA6011201 Grant - others:GA UK(CZ) 216/2004 Institutional research plan: CEZ:AV0Z50110509 Keywords : 20-hydroxysteroid dehydrogenase * SDR family Subject RIV: CE - Biochemistry Impact factor: 2.988, year: 2006

  2. Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design

    NARCIS (Netherlands)

    Machielsen, M.P.; Looger, L.L.; Raedts, J.G.J.; Dijkhuizen, S.; Hummel, W.; Henneman, H.G.; Daussmann, T.; Oost, van der J.

    2009-01-01

    The R-specific alcohol dehydrogenase from Lactobacillus brevis (Lb-ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for

  3. 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...

  4. Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein ...

    African Journals Online (AJOL)

    Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein Expression Defines the Proliferative Nature of Cervical Cancer Stem Cells. ... of cervical cancer stem cells and also to validate them in initial and advanced stages of cervical cancer. Keywords: Cervical cancer, ALDH1, BALB/c-nu/nu, HeLa cells, RKIP, Sox2 ...

  5. Assay of partially purified glutamate dehydrogenase isolated from ...

    African Journals Online (AJOL)

    Glutamate dehydrogenase (E C 1.4.1.1) isolated from the seeds of asparagus beans was partially purified to a factor of 22 by dialysis after fractional precipitation with solid ammonium sulphate at 40 and 60% saturation. A specific activity of 11.78μmol min-1 mg-1 protein was calculated for the partially purified enzyme when ...

  6. Crystallization behaviour of glyceraldehyde dehydrogenase from Thermoplasma acidophilum

    Czech Academy of Sciences Publication Activity Database

    Lermark, L.; Degtjarik, Oksana; Steffler, F.; Sieber, V.; Kutá-Smatanová, Ivana

    2015-01-01

    Roč. 71, č. 12 (2015), s. 1475-1480 ISSN 2053-230X Institutional support: RVO:67179843 Keywords : TaAlDH * Thermoplasma acidophilum * bioproduction * cell-free enzyme cascade * glyceraldehyde dehydrogenase Subject RIV: CE - Biochemistry Impact factor: 0.647, year: 2015

  7. Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Nisler, Jaroslav; Kopečný, D.; Končitíková, R.; Zatloukal, Marek; Bazgier, Václav; Berka, K.; Zalabák, D.; Briozzo, P.; Strnad, Miroslav; Spíchal, Lukáš

    2016-01-01

    Roč. 92, 1-2 (2016), s. 235-248 ISSN 0167-4412 R&D Projects: GA MŠk(CZ) LO1204; GA ČR GA15-22322S Institutional support: RVO:61389030 Keywords : Cytokinin oxidase/dehydrogenase * Crystal structure * Molecular docking Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.356, year: 2016

  8. Phosphorylation of formate dehydrogenase in potato tuber mitochondria

    DEFF Research Database (Denmark)

    Bykova, N.V.; Stensballe, A.; Egsgaard, H.

    2003-01-01

    Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1alpha...

  9. Characterization of the L-lactate dehydrogenase from Aggregatibacter actinomycetemcomitans.

    Directory of Open Access Journals (Sweden)

    Stacie A Brown

    Full Text Available Aggregatibacter actinomycetemcomitans is a Gram-negative opportunistic pathogen and the proposed causative agent of localized aggressive periodontitis. A. actinomycetemcomitans is found exclusively in the mammalian oral cavity in the space between the gums and the teeth known as the gingival crevice. Many bacterial species reside in this environment where competition for carbon is high. A. actinomycetemcomitans utilizes a unique carbon resource partitioning system whereby the presence of L-lactate inhibits uptake of glucose, thus allowing preferential catabolism of L-lactate. Although the mechanism for this process is not fully elucidated, we previously demonstrated that high levels of intracellular pyruvate are critical for L-lactate preference. As the first step in L-lactate catabolism is conversion of L-lactate to pyruvate by lactate dehydrogenase, we proposed a model in which the A. actinomycetemcomitans L-lactate dehydrogenase, unlike homologous enzymes, is not feedback inhibited by pyruvate. This lack of feedback inhibition allows intracellular pyruvate to rise to levels sufficient to inhibit glucose uptake in other bacteria. In the present study, the A. actinomycetemcomitans L-lactate dehydrogenase was purified and shown to convert L-lactate, but not D-lactate, to pyruvate with a K(m of approximately 150 microM. Inhibition studies reveal that pyruvate is a poor inhibitor of L-lactate dehydrogenase activity, providing mechanistic insight into L-lactate preference in A. actinomycetemcomitans.

  10. Natural history of succinic semialdehyde dehydrogenase deficiency through adulthood

    NARCIS (Netherlands)

    Lapalme-Remis, S.; Lewis, E.C.; De Meulemeester, C.; Chakraborty, P.; Gibson, K.M.; Torres, C.; Guberman, A.; Salomons, G.; Jakobs, C.; Ali-Ridha, A.; Parviz, M.; Pearl, P.L.

    2015-01-01

    Objective: The natural history of succinic semialdehyde dehydrogenase (SSADH) deficiency in adulthood is unknown; we elucidate the clinical manifestations of the disease later in life. Methods: A 63-year-old man with long-standing intellectual disability was diagnosed with SSADH deficiency following

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

    African Journals Online (AJOL)

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

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

    African Journals Online (AJOL)

    ... in thyroid function are common endocrine disorders affecting 5-10% of individuals over ... Key words: Hyperthyroidism, hypothyroidism, lactate dehydrogenase, serum creatine kinase ... individuals depends on age, race, lean body mass and physical activity. ... measured by radioimmunoassay on AXSYM System (Abbott.

  13. Novel guanidine-based inhibitors of inosine monophosphate dehydrogenase.

    Science.gov (United States)

    Iwanowicz, Edwin J; Watterson, Scott H; Liu, Chunjian; Gu, Henry H; Mitt, Toomas; Leftheris, Katerina; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L

    2002-10-21

    A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.

  14. Prevalence of glucose-6-phosphate dehydrogenase deficiency in ...

    African Journals Online (AJOL)

    Background: Glucose-6-phosphate dehydrogenase (G6PD) is a house keeping enzyme which catalyzes the first step in the hexose monophosphate pathway of glucose metabolism. G6PD deficiency is the commonest hemolytic X-linked genetic disease, which affects approximately 400 million people worldwide.

  15. Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency in patients ...

    African Journals Online (AJOL)

    This is a study of Glucose-6-phosphate dehydrogenase(G6PD) deficiency in sickle cell anaemia patients attending the haematology clinic of the Jos University Teaching Hospital (JUTH), Jos- Nigeria. The prevalence of G6PD deficiency among the 130 sickle cell anaemia patients studied was found to be 18.5%. G6PD ...

  16. Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells

    NARCIS (Netherlands)

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

    1983-01-01

    With the aid of thin films of polyacrylamide gel containing purified glucose-6-phosphate dehydrogenase subjected to cytochemical procedures for the enzyme using tetranitro blue tetrazolium, arbitrary units of integrated absorbance obtained with a Barr & Stroud GN5 cytophotometer were converted into

  17. Synthesis, growth, structural and optical studies of a new organic three dimensional framework: 4-(aminocarbonyl)pyridine 4-(aminocarbonyl)pyridinium hydrogen L-malate

    Energy Technology Data Exchange (ETDEWEB)

    Vijayalakshmi, A. [Department of Chemistry, RMK. Engineering College, Kavaraipettai 601206 (India); Vidyavathy, B., E-mail: vidyavathybalraj@gmail.com [Department of Chemistry,Velammal Engineering College, Chennai 600066 (India); Peramaiyan, G. [Institute of Physics, Academia Sinica, Taipei 11529, Taiwan (China); Vinitha, G. [Department of Physics, VIT University, Chennai 600127 (India)

    2017-02-15

    4-(aminocarbonyl)pyridine 4-(aminocarbonyl)pyridinium hydrogen L-malate [(4ACP)(4ACP).(LM)] a new organic nonlinear optical (NLO) crystal was grown by the slow evaporation method. Single crystal X-ray diffraction analysis revealed that the [(4ACP)(4ACP).(LM)] crystal belongs to monoclinic crystal system, space group P2{sub 1}/n, with a three dimensional network. Thermogravimetry (TG) and differential thermal (DT) analyses showed that [(4ACP)(4ACP).(LM)] is thermally stable up to 165 °C. The optical transmittance window and the lower cut-off wavelength of [(4ACP)(4ACP).(LM)] were found out by UV–vis–NIR spectral study. The molecular structure of [(4ACP)(4ACP).(LM)] was further confirmed by FTIR spectral studies. The relative dielectric permittivity and dielectric loss were determined as function of frequency and temperature. The third order nonlinear optical property of [(4ACP)(4ACP).(LM)] was studied by the Z-scan technique using a 532 nm diode pumped CW Nd:YAG laser. Nonlinear refractive index, nonlinear absorption coefficient and third order nonlinear susceptibility of the grown crystal were found to be 7.38×10{sup −8} cm{sup 2}/W, 0.08×10{sup −4} cm/W and 5.36×10{sup −6} esu, respectively. The laser damage threshold value is found to be 1.75 GW/cm{sup 2} - Graphical abstract: In the crystal structure of the title complex, the asymmetric unit contains one hydrogen L-malate anion, 4-(aminocarbonyl)pyridinium cation and a neutral isonicotinamide molecule. It is stabilized by intermolecular N-H…O, C-H…O and O-H…O hydrogen bonds which generate a three dimensional network.

  18. Response of Glycine max to drought stress in relation to growth parameters and some key enzymes of carbon and nitrogen metabolism

    Directory of Open Access Journals (Sweden)

    Maryam Nasr Esfahani

    2015-06-01

    Full Text Available Drought stress is one of the major constraints for production and yield of soybean (Glycine max. For this reason, identifying mechanisms associated with drought tolerance in soybean is very impotent for improving and increasing drought resistance by genetic engineering methods. In this study, the effect of drought on growth traits (plant height, fresh and dry weight of shoot and also fresh and dry weight of root and enzyme activities of isocitrate dehydrogenase (ICDH, phosphoenolpyruvate carboxylase (PEPC, malate dehydrogenase (MDH, glutamine synthetase (GS and nitrate reductase (NR were assessed in drought sensitive and tolerant cultivars of soybean. The results showed that growth indicators are higher in drought tolerant cultivar under water availability (control and water deficient when compared with those of drought sensitive cultivar. An increase in the activity of ICDH was observed in both the cultivars under drought stress as compared with their respective control plants but this activity was higher in tolerant cultivar. The activities of PEPC, MDH, GS and NR were significantly decreased in drought sensitive cultivar whereas the activities of these enzymes were higher in another cultivar. In general, the results of this study showed different behavior in the activities of assayed enzymes in two sets of soybean cultivars differing in drought tolerance and also decline of the activities of these enzymes in drought sensitive cultivar due to water deficit stress may be one of the possible reasons for decreased growth of the soybean plants under drought.

  19. [Enzyme kinetic analysis of Oncomelania hupensis exposed to active ingredient of Buddleja lindleyana (AIBL)].

    Science.gov (United States)

    Bang-Xing, Han; Jun, Chen

    2016-07-01

    To analyze the enzyme kinetics of active ingredient of Buddleja lindleyana (AIBL) against Oncomelania hupensis , the intermediate host of Schistosoma japonicum . O . hupensis snails were placed in 1 000 ml of 3.55 mg/L AIBL solution for 24, 48 h and 72 h, respectively, and the enzyme kinetics of alanine aminotransferase (GPT) was determined by Reitman-Frankel assay, lactate dehydrogenase (LDH) by the chemical inhibition lactic acid substrate method, alkaline phosphatase (AKP) by the disodium phenyl phosphate colorimetric method, acetylcholine esterase (AChE) and malate dehydrogenas (MDH) by ELISA, and succinate dehydrogenase (SDH) by the phenazine methyl sulfate reaction method (PMS) in the soft tissues of O. hupensis before and after AIBL treatment. Following exposure to 3.55 mg/L AIBL solution for 24 h, the GPT, LDH, and AKP activities significantly improved in the soft tissues of O. hupensis , while the SDH and MDH activities were significantly lowered in the head-foot and liver. However, AIBL treatment did not cause significant effect on AChE activity in O. hupensis . AIBL causes significant damages to O. hupensis liver and can efficiently act on anaerobic and aerobic respiration loci, which will hinder energy metabolism, and cause inadequate energy supply in cells used for normal secretion, eventually leading to O. hupensis death.

  20. Evaluation of the In Vivo and In Vitro Effects of Fructose on Respiratory Chain Complexes in Tissues of Young Rats

    Directory of Open Access Journals (Sweden)

    Ernesto António Macongonde

    2015-01-01

    Full Text Available Hereditary fructose intolerance (HFI is an autosomal-recessive disorder characterized by fructose and fructose-1-phosphate accumulation in tissues and biological fluids of patients. This disease results from a deficiency of aldolase B, which metabolizes fructose in the liver, kidney, and small intestine. We here investigated the effect of acute fructose administration on the activities of mitochondrial respiratory chain complexes, succinate dehydrogenase (SDH, and malate dehydrogenase (MDH in cerebral cortex, liver, kidney, and skeletal muscle of male 30-day-old Wistar rats. The rats received subcutaneous injection of sodium chloride (0.9%; control group or fructose solution (5 μmol/g; treated group. One hour later, the animals were euthanized and the cerebral cortex, liver, kidney, and skeletal muscle were isolated and homogenized for the investigations. Acute fructose administration increased complex I-III activity in liver. On the other hand, decreased complexes II and II-III activities in skeletal muscle and MDH in kidney were found. Interestingly, none of these parameters were affected in vitro. Our present data indicate that fructose administration elicits impairment of mitochondrial energy metabolism, which may contribute to the pathogenesis of the HFI patients.

  1. The crystal structures of the tri-functional Chloroflexus aurantiacus and bi-functional Rhodobacter sphaeroides malyl-CoA lyases and comparison with CitE-like superfamily enzymes and malate synthases.

    Science.gov (United States)

    Zarzycki, Jan; Kerfeld, Cheryl A

    2013-11-09

    Malyl-CoA lyase (MCL) is a promiscuous carbon-carbon bond lyase that catalyzes the reversible cleavage of structurally related Coenzyme A (CoA) thioesters. This enzyme plays a crucial, multifunctional role in the 3-hydroxypropionate bi-cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus. A second, phylogenetically distinct MCL from Rhodobacter sphaeroides is involved in the ethylmalonyl-CoA pathway for acetate assimilation. Both MCLs belong to the large superfamily of CitE-like enzymes, which includes the name-giving β-subunit of citrate lyase (CitE), malyl-CoA thioesterases and other enzymes of unknown physiological function. The CitE-like enzyme superfamily also bears sequence and structural resemblance to the malate synthases. All of these different enzymes share highly conserved catalytic residues, although they catalyze distinctly different reactions: C-C bond formation and cleavage, thioester hydrolysis, or both (the malate synthases). Here we report the first crystal structures of MCLs from two different phylogenetic subgroups in apo- and substrate-bound forms. Both the C. aurantiacus and the R. sphaeroides MCL contain elaborations on the canonical β8/α8 TIM barrel fold and form hexameric assemblies. Upon ligand binding, changes in the C-terminal domains of the MCLs result in closing of the active site, with the C-terminal domain of one monomer forming a lid over and contributing side chains to the active site of the adjacent monomer. The distinctive features of the two MCL subgroups were compared to known structures of other CitE-like superfamily enzymes and to malate synthases, providing insight into the structural subtleties that underlie the functional versatility of these enzymes. Although the C. aurantiacus and the R. sphaeroides MCLs have divergent primary structures (~37% identical), their tertiary and quaternary structures are very similar. It can be assumed that the C-C bond formation catalyzed by the MCLs occurs as proposed for

  2. Zinc and glutamate dehydrogenase in putative glutamatergic brain structures.

    Science.gov (United States)

    Wolf, G; Schmidt, W

    1983-01-01

    A certain topographic parallelism between the distribution of histochemically (TIMM staining) identified zinc and putative glutamatergic structures in the rat brain was demonstrated. Glutamate dehydrogenase as a zinc containing protein is in consideration to be an enzyme synthesizing transmitter glutamate. In a low concentration range externally added zinc ions (10(-9) to 10(-7) M) induced an increase in the activity of glutamate dehydrogenase (GDH) originating from rat hippocampal formation, neocortex, and cerebellum up to 142.4%. With rising molarity of Zn(II) in the incubation medium, the enzyme of hippocampal formation and cerebellum showed a biphasic course of activation. Zinc ions of a concentration higher than 10(-6) M caused a strong inhibition of GDH. The effect of Zn(II) on GDH originating from spinal ganglia and liver led only to a decrease of enzyme activity. These results are discussed in connection with a functional correlation between zinc and putatively glutamatergic system.

  3. Optic neuropathy in a patient with pyruvate dehydrogenase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Small, Juan E. [Massachusetts General Hospital and Harvard Medical School, Department of Radiology, Boston, MA (United States); Gonzalez, Guido E. [Massachusetts Eye and Ear Infirmary and Harvard Medical School, Department of Radiology, Boston, MA (United States); Clinica Alemana de Santiago, Departmento de Imagenes, Santiago (Chile); Nagao, Karina E.; Walton, David S. [Massachusetts Eye and Ear Infirmary and Harvard Medical School, Department of Ophthalmology, Boston, MA (United States); Caruso, Paul A. [Massachusetts Eye and Ear Infirmary and Harvard Medical School, Department of Radiology, Boston, MA (United States)

    2009-10-15

    Pyruvate dehydrogenase (PDH) deficiency is a genetic disorder of mitochondrial metabolism. The clinical manifestations range from severe neonatal lactic acidosis to chronic neurodegeneration. Optic neuropathy is an uncommon clinical sequela and the imaging findings of optic neuropathy in these patients have not previously been described. We present a patient with PDH deficiency with bilateral decreased vision in whom MRI demonstrated bilateral optic neuropathy and chiasmopathy. (orig.)

  4. Optic neuropathy in a patient with pyruvate dehydrogenase deficiency

    International Nuclear Information System (INIS)

    Small, Juan E.; Gonzalez, Guido E.; Nagao, Karina E.; Walton, David S.; Caruso, Paul A.

    2009-01-01

    Pyruvate dehydrogenase (PDH) deficiency is a genetic disorder of mitochondrial metabolism. The clinical manifestations range from severe neonatal lactic acidosis to chronic neurodegeneration. Optic neuropathy is an uncommon clinical sequela and the imaging findings of optic neuropathy in these patients have not previously been described. We present a patient with PDH deficiency with bilateral decreased vision in whom MRI demonstrated bilateral optic neuropathy and chiasmopathy. (orig.)

  5. Influence of thorax irradiation on lactic dehydrogenase isoenzyme activity

    International Nuclear Information System (INIS)

    Valle, C.; Munnich, A.; Pasquier, C.

    The right hemi-thorax of rats was irradiated with 1200 and 3000 rads ( 60 Co) and blood samples were taken sequentially. The five lactic dehydrogenase (LDH) isoenzymes which have proved to be useful as biochemical indicators of acute pulmonary injury in other experimental animals (dogs), were assayed, after irradiation, as a function of time and as a functon of dose. There was no significant change in LDH isoenzyme activities after lung irradiation in rats [fr

  6. Bioelectrochemical fuel cell and sensor based on quinoprotein alcohol dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Davis, G; Hill, H A.O.; Aston, W J; Higgins, I J; Turner, A P.F.

    1983-09-01

    A biofuel cell, yielding a stable and continuous low-power output, based on the enzymatic oxidation of methanol to formic acid has been designed and investigated. The homogeneous kinetics of the electrochemically-coupled enzymatic oxidation reaction were investigated and optimized. The biofuel cell also functioned as a sensitive method for the detection of primary alcohols. A method for medium-scale preparation of the enzyme alcohol dehydrogenase (alcohol: (acceptor) oxidoreductase, EC 1.1.99.8) is described. (Refs. 14).

  7. Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    White, Tommi A.; Tanner, John J., E-mail: tannerjj@missouri.edu [Departments of Chemistry and Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211 (United States)

    2005-08-01

    Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ{sup 1}-pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  8. Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

    International Nuclear Information System (INIS)

    White, Tommi A.; Tanner, John J.

    2005-01-01

    Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ 1 -pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2 1 2 1 2 1 , with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative

  9. [Effects of waterlogging on the growth and energy-metabolic enzyme activities of different tree species].

    Science.gov (United States)

    Wang, Gui-Bin; Cao, Fu-Liang; Zhang, Xiao-Yan; Zhang, Wang-Xiang

    2010-03-01

    Aimed to understand the waterlogging tolerance and adaptation mechanisms of different tree species, a simulated field experiment was conducted to study the growth and energy-metabolic enzyme activities of one-year-old seedlings of Taxodium distichum, Carya illinoensis, and Sapium sebiferum. Three treatments were installed, i. e., CK, waterlogging, and flooding, with the treatment duration being 60 days. Under waterlogging and flooding, the relative growth of test tree species was in the order of T. distichum > C. illinoensis > S. sebiferum, indicating that T. distichum had the strongest tolerance against waterlogging and flooding, while S. sebiferum had the weakest one. Also under waterlogging and flooding, the root/crown ratio of the three tree species increased significantly, suggesting that more photosynthates were allocated in roots, and the lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) activities of the tree species also had a significant increase. Among the test tree species, T. distichum had the lowest increment of LDH and ADH activities under waterlogging and flooding, but the increment could maintain at a higher level in the treatment duration, while for C. illinoensis and S. sebiferum, the increment was larger during the initial and medium period, but declined rapidly during the later period of treatment. The malate dehydrogenase (MDH), phosphohexose (HPI), and glucose-6-phosphate dehydrogenase (G6PDH) -6-phosphogluconate dehydrogenase (6PGDH) activities of the tree species under waterlogging and flooding had a significant decrease, and the decrement was the largest for T. distichum, being 35.6% for MDH, 21.0% for HPI, and 22.7% for G6PDH - 6PGDH under flooding. It was suggested that under waterlogging and flooding, the tree species with strong waterlogging tolerance had a higher ability to maintain energy-metabolic balance, and thus, its growth could be maintained at a certain level.

  10. Alcohol dehydrogenase and aldehyde dehydrogenase gene polymorphisms, alcohol intake and the risk of colorectal cancer in the European Prospective Investigation into Cancer and Nutrition study

    DEFF Research Database (Denmark)

    Ferrari, P.; McKay, J. D.; Jenab, M.

    2012-01-01

    BACKGROUND/OBJECTIVES: Heavy alcohol drinking is a risk factor of colorectal cancer (CRC), but little is known on the effect of polymorphisms in the alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) on the alcohol-related risk of CRC in Caucasian populati...

  11. Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component of human α-ketoacid dehydrogenase complexes

    International Nuclear Information System (INIS)

    Pons, G.; Raefsky-Estrin, C.; Carothers, D.J.; Pepin, R.A.; Javed, A.A.; Jesse, B.W.; Ganapathi, M.K.; Samols, D.; Patel, M.S.

    1988-01-01

    cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (>98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues

  12. Pyruvate Dehydrogenase and Pyruvate Dehydrogenase Kinase Expression in Non Small Cell Lung Cancer and Tumor-Associated Stroma

    Directory of Open Access Journals (Sweden)

    Michael I. Koukourakis

    2005-01-01

    Full Text Available Pyruvate dehydrogenase (PDH catalyzes the conversion of pyruvate to acetyl-coenzyme A, which enters into the Krebs cycle, providing adenosine triphosphate (ATP to the cell. PDH activity is under the control of pyruvate dehydrogenase kinases (PDKs. Under hypoxic conditions, conversion of pyruvate to lactate occurs, a reaction catalyzed by lactate dehydrogenase 5 (LDH5. In cancer cells, however, pyruvate is transformed to lactate occurs, regardless of the presence of oxygen (aerobic glycolysis/Warburg effect. Although hypoxic intratumoral conditions account for HIFia stabilization and induction of anaerobic metabolism, recent data suggest that high pyruvate concentrations also result in HIFia stabilization independently of hypoxia. In the present immunohistochemical study, we provide evidence that the PDH/PDK pathway is repressed in 73% of non small cell lung carcinomas, which may be a key reason for HIFia stabilization and “aerobic glycolysis.” However, about half of PDHdeficient carcinomas are not able to switch on the HIF pathway, and patients harboring these tumors have an excellent postoperative outcome. A small subgroup of clinically aggressive tumors maintains a coherent PDH and HIF/LDH5 expression. In contrast to cancer cells, fibroblasts in the tumor-supporting stroma exhibit an intense PDH but reduced PDK1 expression favoring maximum PDH activity. This means that stroma may use lactic acid produced by tumor cells, preventing the creation of an intolerable intratumoral acidic environment at the same time.

  13. High-fat diet enhanced retinal dehydrogenase activity, but suppressed retinol dehydrogenase activity in liver of rats

    Directory of Open Access Journals (Sweden)

    Mian Zhang

    2015-04-01

    Full Text Available Evidence has shown that hyperlipidemia is associated with retinoid dyshomeostasis. In liver, retinol is mainly oxidized to retinal by retinol dehydrogenases (RDHs and alcohol dehydrogenases (ADHs, further converted to retinoic acid by retinal dehydrogenases (RALDHs. The aim of this study was to investigate whether high-fat diet (HFD induced hyperlipidemia affected activity and expression of hepatic ADHs/RDHs and RALDHs in rats. Results showed that retinol levels in liver, kidney and adipose tissue of HFD rats were significantly increased, while plasma retinol and hepatic retinal levels were markedly decreased. HFD rats exhibited significantly downregulated hepatic ADHs/RDHs activity and Adh1, Rdh10 and Dhrs9 expression. Oppositely, hepatic RALDHs activity and Raldh1 expression were upregulated in HFD rats. In HepG2 cells, treatment of HFD rat serum inhibited ADHs/RDHs activity and induced RALDHs activity. Among the tested abnormally altered components in HFD rat serum, cholesterol reduced ADHs/RDHs activity and RDH10 expression, while induced RALDHs activity and RALDH1 expression in HepG2 cells. Contrary to the effect of cholesterol, cholesterol-lowering agent pravastatin upregulated ADHs/RDHs activity and RDH10 expression, while suppressed RALDHs activity and RALDH1 expression. In conclusion, hyperlipidemia oppositely altered activity and expression of hepatic ADHs/RDHs and RALDHs, which is partially due to the elevated cholesterol levels.

  14. Enhanced succinic acid production in Aspergillus saccharolyticus by heterologous expression of fumarate reductase from Trypanosoma brucei

    DEFF Research Database (Denmark)

    Yang, Lei; Lübeck, Mette; Ahring, Birgitte K.

    2015-01-01

    production medium as well as the complete medium, but the measured enzyme activities were different depending on the media. Furthermore, a soluble NADH-dependent fumarate reductase gene (frd) from Trypanosoma brucei was inserted and expressed in A. saccharolyticus. The expression of the frd gene led......Aspergillus saccharolyticus exhibits great potential as a cell factory for industrial production of dicarboxylic acids. In the analysis of the organic acid profile, A. saccharolyticus was cultivated in an acid production medium using two different pH conditions. The specific activities...... of the enzymes, pyruvate carboxylase (PYC), malate dehydrogenase (MDH), and fumarase (FUM), involved in the reductive tricarboxylic acid (rTCA) branch, were examined and compared in cells harvested from the acid production medium and a complete medium. The results showed that ambient pH had a significant impact...

  15. Flow-through 3D biofuel cell anode for NAD{sup +}-dependent enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Rincon, Rosalba A.; Lau, Carolin; Garcia, Kristen E. [Department of Chemical and Nuclear Engineering, Center for Emerging Energy Technologies, University of New Mexico, Albuquerque, NM 87131 (United States); Atanassov, Plamen, E-mail: plamen@unm.ed [Department of Chemical and Nuclear Engineering, Center for Emerging Energy Technologies, University of New Mexico, Albuquerque, NM 87131 (United States)

    2011-02-01

    NAD{sup +}-dependent enzymes require the presence of catalysts for cofactor regeneration in order to be employed in enzymatic biofuel cells. Poly-(methylene green) catalysts have proven to help the oxidation reaction of NADH allowing for the use of such enzymes in electrocatalytic oxidation reactions. In this paper we present the development of 3D anode based on NAD{sup +}-dependent malate dehydrogenase. The 3D material chosen was reticulated vitreous carbon (RVC) which was modified with poly-(MG) for NADH oxidation and it also accommodated the porous immobilization matrix for MDH consisting of MWCNTs embedded in chitosan; allowing for mass transport of the substrate to the electrode. Scanning electron microscopy was used in order to characterize the poly-(MG)-modified RVC, and electrochemical evaluation of the anode was performed.

  16. Flow-through 3D biofuel cell anode for NAD+-dependent enzymes

    International Nuclear Information System (INIS)

    Rincon, Rosalba A.; Lau, Carolin; Garcia, Kristen E.; Atanassov, Plamen

    2011-01-01

    NAD + -dependent enzymes require the presence of catalysts for cofactor regeneration in order to be employed in enzymatic biofuel cells. Poly-(methylene green) catalysts have proven to help the oxidation reaction of NADH allowing for the use of such enzymes in electrocatalytic oxidation reactions. In this paper we present the development of 3D anode based on NAD + -dependent malate dehydrogenase. The 3D material chosen was reticulated vitreous carbon (RVC) which was modified with poly-(MG) for NADH oxidation and it also accommodated the porous immobilization matrix for MDH consisting of MWCNTs embedded in chitosan; allowing for mass transport of the substrate to the electrode. Scanning electron microscopy was used in order to characterize the poly-(MG)-modified RVC, and electrochemical evaluation of the anode was performed.

  17. Characterization of human short chain dehydrogenase/reductase SDR16C family members related to retinol dehydrogenase 10.

    Science.gov (United States)

    Adams, Mark K; Lee, Seung-Ah; Belyaeva, Olga V; Wu, Lizhi; Kedishvili, Natalia Y

    2017-10-01

    All-trans-retinoic acid (RA) is a bioactive derivative of vitamin A that serves as an activating ligand for nuclear transcription factors, retinoic acid receptors. RA biosynthesis is initiated by the enzymes that oxidize retinol to retinaldehyde. It is well established that retinol dehydrogenase 10 (RDH10, SDR16C4), which belongs to the 16C family of the short chain dehydrogenase/reductase (SDR) superfamily of proteins, is the major enzyme responsible for the oxidation of retinol to retinaldehyde for RA biosynthesis during embryogenesis. However, several lines of evidence point towards the existence of additional retinol dehydrogenases that contribute to RA biosynthesis in vivo. In close proximity to RDH10 gene on human chromosome 8 are located two genes that are phylogenetically related to RDH10. The predicted protein products of these genes, retinol dehydrogenase epidermal 2 (RDHE2, SDR16C5) and retinol dehydrogenase epidermal 2-similar (RDHE2S, SDR16C6), share 59% and 56% sequence similarity with RDH10, respectively. Previously, we showed that the single ortholog of the human RDHE2 and RDHE2S in frogs, Xenopus laevis rdhe2, oxidizes retinol to retinaldehyde and is essential for frog embryonic development. In this study, we explored the potential of each of the two human proteins to contribute to RA biosynthesis. The results of this study demonstrate that human RDHE2 exhibits a relatively low but reproducible activity when expressed in either HepG2 or HEK293 cells. Expression of the native RDHE2 is downregulated in the presence of elevated levels of RA. On the other hand, the protein encoded by the human RDHE2S gene is unstable when expressed in HEK293 cells. RDHE2S protein produced in Sf9 cells is stable but has no detectable catalytic activity towards retinol. We conclude that the human RDHE2S does not contribute to RA biosynthesis, whereas the low-activity RA-sensitive human RDHE2 may have a role in adjusting the cellular levels of RA in accord with

  18. 9-Hydroxyprostaglandin dehydrogenase activity in the adult rat kidney. Regional distribution and sub-fractionation.

    Science.gov (United States)

    Asciak, C P; Domazet, Z

    1975-02-20

    1. Catabolism of prostaglandin F2alpha in the adult rat kidney takes place by the following sequence of enzymatic steps: (1) 15-hydroxyprostaglandin dehydrogenase; (2) prostaglandin delta13-reductase; and (3) 9-hydroxyprostaglandin dehydrogenase. 2. 9-Hydroxyprostaglandin dehydrogenase activity was highest in the cortex with lesser amounts in the medulla and negligible activity detected in the papilla. A similar distribution was observed for 15-hydroxyprostaglandin dehydrogenase and prostaglandin delta13-reductase. 3. Most of the 9-hydroxyprostaglandin dehydrogenase activity in the homogenate was found in the high-speed supernatant as also observed for 15-hydroxyprostaglandin dehydrogenase and prostaglandin delta13-reductase. 4. These observations indicate that the rat kidney contains an abundance of prostaglandin-catabolising enzymes which favour formation of metabolites of the E-type.

  19. Molecular structure of the pyruvate dehydrogenase complex from Escherichia coli K-12.

    Science.gov (United States)

    Vogel, O; Hoehn, B; Henning, U

    1972-06-01

    The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 x 10(6). All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This "excess" component is bound differently than are the eight dimers in the core complex.

  20. Purification of 2-oxo acid dehydrogenase multienzyme complexes from ox heart by a new method.

    OpenAIRE

    Stanley, C J; Perham, R N

    1980-01-01

    A new method is described that allows the parallel purification of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart without the need for prior isolation of mitochondria. All the assayable activity of the 2-oxo acid dehydrogenase complexes in the disrupted tissue is made soluble by the inclusion of non-ionic detergents such as Triton X-100 or Tween-80 in the buffer used for the initial extraction of the enzyme complexes. The yields of the pyruvate...

  1. Increased superoxide accumulation in pyruvate dehydrogenase complex deficient fibroblasts.

    Science.gov (United States)

    Glushakova, Lyudmyla G; Judge, Sharon; Cruz, Alex; Pourang, Deena; Mathews, Clayton E; Stacpoole, Peter W

    2011-11-01

    The pyruvate dehydrogenase complex (PDC) oxidizes pyruvate to acetyl CoA and is critically important in maintaining normal cellular energy homeostasis. Loss-of-function mutations in PDC give rise to congenital lactic acidosis and to progressive cellular energy failure. However, the subsequent biochemical consequences of PDC deficiency that may contribute to the clinical manifestations of the disorder are poorly understood. We postulated that altered flux through PDC would disrupt mitochondrial electron transport, resulting in oxidative stress. Compared to cells from 4 healthy subjects, primary cultures of skin fibroblasts from 9 patients with variable mutations in the gene encoding the alpha subunit (E1α) of pyruvate dehydrogenase (PDA1) demonstrated reduced growth and viability. Superoxide (O(2)(.-)) from the Qo site of complex III of the electron transport chain accumulated in these cells and was associated with decreased activity of manganese superoxide dismutase. The expression of uncoupling protein 2 was also decreased in patient cells, but there were no significant changes in the expression of cellular markers of protein or DNA oxidative damage. The expression of hypoxia transcription factor 1 alpha (HIF1α) also increased in PDC deficient fibroblasts. We conclude that PDC deficiency is associated with an increase in O(2)(.-) accumulation coupled to a decrease in mechanisms responsible for its removal. Increased HIF1α expression may contribute to the increase in glycolytic flux and lactate production in PDC deficiency and, by trans-activating pyruvate dehydrogenase kinase, may further suppress residual PDC activity through phosphorylation of the E1α subunit. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. 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.

  3. Two different dihydroorotate dehydrogenases from yeast Saccharomyees kluyveri

    DEFF Research Database (Denmark)

    Zameitat, E.; Knecht, Wolfgang; Piskur, Jure

    2004-01-01

    Genes for two structurally and functionally different dihydroorotate dehydrogenases (DHODHs, EC 1.3.99.11), catalyzing the fourth step of pyrimidine biosynthesis, have been previously found in yeast Saccharomyces klujveri. One is closely related to the Schizosaccharomyces pombe mitochondrial family...... for their biochemical properties and interaction with inhibitors. Benzoates as pyrimidine ring analogs were shown to he selective inhibitors of cytosolic DHODs. This unique property of Saccharomyces DHODHs could appoint DHODH as a species-specific target for novel anti-fungal therapeutics....

  4. Deracemization of Secondary Alcohols by using a Single Alcohol Dehydrogenase

    KAUST Repository

    Karume, Ibrahim

    2016-03-01

    © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. We developed a single-enzyme-mediated two-step approach for deracemization of secondary alcohols. A single mutant of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase enables the nonstereoselective oxidation of racemic alcohols to ketones, followed by a stereoselective reduction process. Varying the amounts of acetone and 2-propanol cosubstrates controls the stereoselectivities of the consecutive oxidation and reduction reactions, respectively. We used one enzyme to accomplish the deracemization of secondary alcohols with up to >99% ee and >99.5% recovery in one pot and without the need to isolate the prochiral ketone intermediate.

  5. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Woodward, J. [Oak Ridge National Lab., TN (United States)

    1996-10-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based upon the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with continuous cofactor recycle. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value commodity chemical.

  6. [Genetic variations in alcohol dehydrogenase, drinking habits and alcoholism

    DEFF Research Database (Denmark)

    Tolstrup, J.S.; Rasmussen, S.; Tybjaerg-Hansen, A.

    2008-01-01

    Alcohol is degraded primarily by alcohol dehydrogenase (ADH), and genetic variation that affects the rate of alcohol degradation is found in ADH1B and ADH1C. By genotyping 9,080 white men and women from the general population, we found that men and women with ADH1B slow versus fast alcohol...... degradation drank approximately 30% more alcohol per week and had a higher risk of everyday and heavy drinking, and of alcoholism. Individuals with ADH1C slow versus fast alcohol degradation had a higher risk of heavy drinking Udgivelsesdato: 2008/8/25...

  7. The Enzymology of 2-Hydroxyglutarate, 2-Hydroxyglutaramate and 2-Hydroxysuccinamate and Their Relationship to Oncometabolites

    Directory of Open Access Journals (Sweden)

    Vivek A. Hariharan

    2017-03-01

    Full Text Available Many enzymes make “mistakes”. Consequently, repair enzymes have evolved to correct these mistakes. For example, lactate dehydrogenase (LDH and mitochondrial malate dehydrogenase (mMDH slowly catalyze the reduction of 2-oxoglutarate (2-OG to the oncometabolite l-2-hydroxyglutarate (l-2-HG. l-2-HG dehydrogenase corrects this error by converting l-2-HG to 2-OG. LDH also catalyzes the reduction of the oxo group of 2-oxoglutaramate (2-OGM; transamination product of l-glutamine. We show here that human glutamine synthetase (GS catalyzes the amidation of the terminal carboxyl of both the l- and d- isomers of 2-HG. The reaction of 2-OGM with LDH and the reaction of l-2-HG with GS generate l-2-hydroxyglutaramate (l-2-HGM. We also show that l-2-HGM is a substrate of human ω-amidase. The product (l-2-HG can then be converted to 2-OG by l-2-HG dehydrogenase. Previous work showed that 2-oxosuccinamate (2-OSM; transamination product of l-asparagine is an excellent substrate of LDH. Finally, we also show that human ω-amidase converts the product of this reaction (i.e., l-2-hydroxysuccinamate; l-2-HSM to l-malate. Thus, ω-amidase may act together with hydroxyglutarate dehydrogenases to repair certain “mistakes” of GS and LDH. The present findings suggest that non-productive pathways for nitrogen metabolism occur in mammalian tissues in vivo. Perturbations of these pathways may contribute to symptoms associated with hydroxyglutaric acidurias and to tumor progression. Finally, methods for the synthesis of l-2-HGM and l-2-HSM are described that should be useful in determining the roles of ω-amidase/4- and 5-C compounds in photorespiration in plants.

  8. The Enzymology of 2-Hydroxyglutarate, 2-Hydroxyglutaramate and 2-Hydroxysuccinamate and Their Relationship to Oncometabolites.

    Science.gov (United States)

    Hariharan, Vivek A; Denton, Travis T; Paraszcszak, Sarah; McEvoy, Kyle; Jeitner, Thomas M; Krasnikov, Boris F; Cooper, Arthur J L

    2017-03-30

    Many enzymes make "mistakes". Consequently, repair enzymes have evolved to correct these mistakes. For example, lactate dehydrogenase (LDH) and mitochondrial malate dehydrogenase (mMDH) slowly catalyze the reduction of 2-oxoglutarate (2-OG) to the oncometabolite l-2-hydroxyglutarate (l-2-HG). l-2-HG dehydrogenase corrects this error by converting l-2-HG to 2-OG. LDH also catalyzes the reduction of the oxo group of 2-oxoglutaramate (2-OGM; transamination product of l-glutamine). We show here that human glutamine synthetase (GS) catalyzes the amidation of the terminal carboxyl of both the l- and d- isomers of 2-HG. The reaction of 2-OGM with LDH and the reaction of l-2-HG with GS generate l-2-hydroxyglutaramate (l-2-HGM). We also show that l-2-HGM is a substrate of human ω-amidase. The product (l-2-HG) can then be converted to 2-OG by l-2-HG dehydrogenase. Previous work showed that 2-oxosuccinamate (2-OSM; transamination product of l-asparagine) is an excellent substrate of LDH. Finally, we also show that human ω-amidase converts the product of this reaction (i.e., l-2-hydroxysuccinamate; l-2-HSM) to l-malate. Thus, ω-amidase may act together with hydroxyglutarate dehydrogenases to repair certain "mistakes" of GS and LDH. The present findings suggest that non-productive pathways for nitrogen metabolism occur in mammalian tissues in vivo. Perturbations of these pathways may contribute to symptoms associated with hydroxyglutaric acidurias and to tumor progression. Finally, methods for the synthesis of l-2-HGM and l-2-HSM are described that should be useful in determining the roles of ω-amidase/4- and 5-C compounds in photorespiration in plants.

  9. Increased salivary aldehyde dehydrogenase 1 in non-reticular oral lichen planus.

    Science.gov (United States)

    Mansourian, Arash; Shanbehzadeh, Najmeh; Kia, Seyed Javad; Moosavi, Mahdieh-Sadat

    2017-01-01

    Oral lichen planus is a potentially malignant disorder. One of the malignant transformation markers is cancer stem cells. One of the proposed marker for the detection of cancer stem cells's in head and neck cancer is aldehyde dehydrogenase. Recently it is shown that aldehyde dehydrogenase 1 expression in tissue samples is associated with oral lichen planus malignant transformation. This study evaluates salivary aldehyde dehydrogenase 1 in oral lichen planus. Thirty patients and 30 age and sex-matched healthy volunteers were recruited. Oral lichen planus was diagnosed based on the modified World Health Organization criteria. Subjects in the case group were divided into reticular and non-reticular forms. Unstimulated salivary samples were collected at 10-12 AM. Saliva concentrations of aldehyde dehydrogenase 1 were measured by ELISA. The differences between aldehyde dehydrogenase levels in the oral lichen planus group compared with the control group were not significant but aldehyde dehydrogenase in non-reticular oral lichen planus was significantly higher than that of the reticular form. This is a cross-sectional study, thus longitudinal studies in oral lichen planus may present similar or different results. The mechanism of malignant transformation in oral lichen planus is not defined. Previous analyses revealed that the aldehyde dehydrogenase 1 expression is significantly correlated with increased risk of transformation. This finding is consistent with our results because in the erosive and ulcerative forms of oral lichen planus, which have an increased risk of transformation, salivary aldehyde dehydrogenase 1 was overexpressed. A higher salivary aldehyde dehydrogenase level in non-reticular oral lichen planus can be a defensive mechanism against higher oxidative stress in these groups. Aldehyde dehydrogenase may be one of the malignant transformation markers in oral lichen planus. Further studies are needed for introducing aldehyde dehydrogenase as a prognostic

  10. Increased salivary aldehyde dehydrogenase 1 in non-reticular oral lichen planus*

    Science.gov (United States)

    Mansourian, Arash; Shanbehzadeh, Najmeh; Kia, Seyed Javad; Moosavi, Mahdieh-Sadat

    2017-01-01

    Background Oral lichen planus is a potentially malignant disorder. One of the malignant transformation markers is cancer stem cells. One of the proposed marker for the detection of cancer stem cells's in head and neck cancer is aldehyde dehydrogenase. Recently it is shown that aldehyde dehydrogenase 1 expression in tissue samples is associated with oral lichen planus malignant transformation. Objective This study evaluates salivary aldehyde dehydrogenase 1 in oral lichen planus. Method Thirty patients and 30 age and sex-matched healthy volunteers were recruited. Oral lichen planus was diagnosed based on the modified World Health Organization criteria. Subjects in the case group were divided into reticular and non-reticular forms. Unstimulated salivary samples were collected at 10-12 AM. Saliva concentrations of aldehyde dehydrogenase 1 were measured by ELISA. Results The differences between aldehyde dehydrogenase levels in the oral lichen planus group compared with the control group were not significant but aldehyde dehydrogenase in non-reticular oral lichen planus was significantly higher than that of the reticular form. Limitations of the study This is a cross-sectional study, thus longitudinal studies in oral lichen planus may present similar or different results. Conclusions The mechanism of malignant transformation in oral lichen planus is not defined. Previous analyses revealed that the aldehyde dehydrogenase 1 expression is significantly correlated with increased risk of transformation. This finding is consistent with our results because in the erosive and ulcerative forms of oral lichen planus, which have an increased risk of transformation, salivary aldehyde dehydrogenase 1 was overexpressed. A higher salivary aldehyde dehydrogenase level in non-reticular oral lichen planus can be a defensive mechanism against higher oxidative stress in these groups. Aldehyde dehydrogenase may be one of the malignant transformation markers in oral lichen planus. Further

  11. Coenzyme- and His-tag-induced crystallization of octopine dehydrogenase

    International Nuclear Information System (INIS)

    Smits, Sander H. J.; Mueller, Andre; Grieshaber, Manfred K.; Schmitt, Lutz

    2008-01-01

    The crystal structure of octopine dehydrogenase revealed a specific role of the His 5 tag in inducing the crystal contacts required for successful crystallization. Over the last decade, protein purification has become more efficient and standardized through the introduction of affinity tags. The choice and position of the tag, however, can directly influence the process of protein crystallization. Octopine dehydrogenase (OcDH) without a His tag and tagged protein constructs such as OcDH-His 5 and OcDH-LEHis 6 have been investigated for their crystallizability. Only OcDH-His 5 yielded crystals; however, they were multiple. To improve crystal quality, the cofactor NADH was added, resulting in single crystals that were suitable for structure determination. As shown by the structure, the His 5 tag protrudes into the cleft between the NADH and l-arginine-binding domains and is mainly fixed in place by water molecules. The protein is thereby stabilized to such an extent that the formation of crystal contacts can proceed. Together with NADH, the His 5 tag obviously locks the enzyme into a specific conformation which induces crystal growth

  12. High-pressure-induced water penetration into 3-isopropylmalate dehydrogenase

    International Nuclear Information System (INIS)

    Nagae, Takayuki; Kawamura, Takashi; Chavas, Leonard M. G.; Niwa, Ken; Hasegawa, Masashi; Kato, Chiaki; Watanabe, Nobuhisa

    2012-01-01

    Structures of 3-isopropylmalate dehydrogenase were determined at pressures ranging from 0.1 to 650 MPa. Comparison of these structures gives a detailed picture of the swelling of a cavity at the dimer interface and the generation of a new cleft on the molecular surface, which are accompanied by water penetration. Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH

  13. Idiopathic intracranial hypertension, hormones, and 11ß-hydroxysteroid dehydrogenases

    Directory of Open Access Journals (Sweden)

    Markey KA

    2016-04-01

    Full Text Available Keira A Markey,1 Maria Uldall,2 Hannah Botfield,1 Liam D Cato,1 Mohammed A L Miah,1 Ghaniah Hassan-Smith,1 Rigmor H Jensen,2 Ana M Gonzalez,1 Alexandra J Sinclair1 1Neurometabolism, Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, UK; 2Danish Headache Center, Clinic of Neurology, Rigshospitalet-Glostrup, University of Copenhagen, Glostrup, Denmark Abstract: Idiopathic intracranial hypertension (IIH results in raised intracranial pressure (ICP leading to papilledema, visual dysfunction, and headaches. Obese females of reproductive age are predominantly affected, but the underlying pathological mechanisms behind IIH remain unknown. This review provides an overview of pathogenic factors that could result in IIH with particular focus on hormones and the impact of obesity, including its role in neuroendocrine signaling and driving inflammation. Despite occurring almost exclusively in obese women, there have been a few studies evaluating the mechanisms by which hormones and adipokines exert their effects on ICP regulation in IIH. Research involving 11ß-hydroxysteroid dehydrogenase type 1, a modulator of glucocorticoids, suggests a potential role in IIH. Improved understanding of the complex interplay between adipose signaling factors such as adipokines, steroid hormones, and ICP regulation may be key to the understanding and future management of IIH. Keywords: 11beta-hydroxysteroid dehydrogenase type 1, steroid and adipokines, obesity, leptin

  14. The Alcohol Dehydrogenase Isoenzyme as a Potential Marker of Pancreatitis.

    Science.gov (United States)

    Jelski, Wojciech; Piechota, Joanna; Orywal, Karolina; Szmitkowski, Maciej

    2018-05-01

    Human pancreas parenchyma contains various alcohol dehydrogenase (ADH) isoenzymes and also possesses aldehyde dehydrogenase (ALDH) activity. The altered activities of ADH and ALDH in damaged pancreatic tissue in the course of pancreatitis are reflected in the human serum. The aim of this study was to investigate a potential role of ADH and ALDH as markers for acute (AP) and chronic pancreatitis (CP). Serum samples were collected for routine biochemical investigations from 75 patients suffering from acute pancreatitis and 70 patients with chronic pancreatitis. Fluorometric methods were used to measure the activity of class I and II ADH and ALDH activity. The total ADH activity and activity of class III and IV isoenzymes were measured by a photometric method. There was a significant increase in the activity of ADH III isoenzyme (15.06 mU/l and 14.62 mU/l vs. 11.82 mU/l; ppancreatitis or chronic pancreatitis compared to the control. The diagnostic sensitivity for ADH III was about 84%, specificity was 92 %, positive and negative predictive values were 93% and 87% respectively in acute pancreatitis. Area under the Receiver Operating Curve (ROC) curve for ADH III in AP and CP was 0.88 and 0.86 respectively. ADH III has a potential role as a marker of acute and chronic pancreatitis. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  15. Modulation of NADP(+)-dependent isocitrate dehydrogenase in aging.

    Science.gov (United States)

    Kil, In Sup; Lee, Young Sup; Bae, Young Seuk; Huh, Tae Lin; Park, Jeen-Woo

    2004-01-01

    NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose-6-phosphate dehydrogenase, malic enzyme, and NADP(+)-specific isocitrate dehydrogenases (ICDHs). Here, we investigated age-related changes in ICDH activity and protein expression in IMR-90 human diploid fibroblast cells and tissues from Fischer 344 rats. We found that in IMR-90 cells the activity of cytosolic ICDH (IDPc) gradually increased with age up to the 46-48 population doubling level (PDL) and then gradually decreased at later PDL. 2',7'-Dichloro-fluorescein fluorescence which reflects intracellular ROS generation was increased with aging in IMR-90 cells. In ad libitum-fed rats, we noted age-related, tissue-specific modulations of IDPc and mitochondrial ICDH (IDPm) activities and protein expression in the liver, kidney and testes. In contrast, ICDH activities and protein expression were not significantly modulated in diet-restricted rats. These data suggest that modulation of ICDH is an age-dependent and a tissue-specific phenomenon.

  16. Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins.

    Science.gov (United States)

    Lapierre, Catherine; Pilate, Gilles; Pollet, Brigitte; Mila, Isabelle; Leplé, Jean-Charles; Jouanin, Lise; Kim, Hoon; Ralph, John

    2004-02-01

    A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8-O-4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and beta-O-4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar.

  17. The radiation inactivation of glutamate and isocitrate dehydrogenases

    International Nuclear Information System (INIS)

    El Failat, R.R.A.

    1980-12-01

    The reaction of free radicals produced by ionizing radiation with the enzymes glutamate dehydrogenase (GDH) and NADP + -specific isocitrate dehydrogenase (ICDH) have been studied by steady-state and pulse radiolysis techniques. In de-aerated GDH solutions, hydroxyl radicals have been found to be the most efficient of the primary radicals generated from water in causing inactivation. The effect of reaction with the enzyme of selective free radicals (SCN) 2 - , (Br) 2 - and (I) 2 - on its activity has also been studied. In neutral solutions, the order of inactivating effectiveness is (I) 2 - > (Br) 2 - > (SCN) 2 - . In the case of the thiocyanate radical anion (SCN) 2 - , the inactivation efficiency is found to depend on KSCN concentration. The radiation inactivation of GDH at both neutral and alkaline pH is accompanied by the loss of sulphydryl groups. Pulse radiolysis was also used to determine the rate constants and the transient absorption spectra following the reaction of the free radicals with GDH. 60 Co-γ-radiolysis and pulse radiolysis were also used to study the effect of ionizing radiation on the activity of ICDH. The results obtained were similar to those of GDH. (author)

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

    Directory of Open Access Journals (Sweden)

    Erik Husin

    2013-07-01

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

  19. Inosine monophosphate dehydrogenase messenger RNA expression is correlated to clinical outcomes in mycophenolate mofetil-treated kidney transplant patients, whereas inosine monophosphate dehydrogenase activity is not

    NARCIS (Netherlands)

    Sombogaard, Ferdi; Peeters, Annemiek M. A.; Baan, Carla C.; Mathot, Ron A. A.; Quaedackers, Monique E.; Vulto, Arnold G.; Weimar, Willem; van Gelder, Teun

    2009-01-01

    Measurement of the pharmacodynamic biomarker inosine monophosphate dehydrogenase (IMPDH) activity in renal transplant recipients has been proposed to reflect the biological effect better than using pharmacokinetic parameters to monitor mycophenolate mofetil therapy. The IMPDH assays are however

  20. New recombinant bacterium comprises a heterologous gene encoding glycerol dehydrogenase and/or an up-regulated native gene encoding glycerol dehydrogenase, useful for producing ethanol

    DEFF Research Database (Denmark)

    2010-01-01

    dehydrogenase encoding region of the bacterium, or is inserted into a phosphotransacetylase encoding region of the bacterium, or is inserted into an acetate kinase encoding region of the bacterium. It is operably linked to an inducible, a regulated or a constitutive promoter. The up-regulated glycerol......TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preparation (claimed): Producing recombinant bacterium having enhanced ethanol production characteristics when cultivated in growth medium comprising glycerol comprises: (a) transforming a parental bacterium by (i) the insertion of a heterologous gene encoding...... glycerol dehydrogenase; and/or (ii) up-regulating a native gene encoding glycerol dehydrogenase; and (b) obtaining the recombinant bacterium. Preferred Bacterium: In the recombinant bacterium above, the inserted heterologous gene and/or the up-regulated native gene is encoding a glycerol dehydrogenase...

  1. Cloning and mRNA Expression of NADH Dehydrogenase during Ochlerotatus taeniorhynchus Development and Pesticide Response

    Science.gov (United States)

    NADH dehydrogenase, the largest of the respiratory complexes, is the first enzyme of the mitochondrial electron transport chain. We have cloned and sequenced cDNA of NADH dehydrogenase gene from Ochlerotatus (Ochlerotatus) taeniorhynchus (Wiedemann) adult (GeneBank Accession number: FJ458415). The ...

  2. Role and structural characterization of plant aldehyde dehydrogenases from family 2 and family 7

    Czech Academy of Sciences Publication Activity Database

    Končitíková, R.; Vigouroux, A.; Kopečná, M.; Andree, T.; Bartoš, Jan; Šebela, M.; Moréra, S.; Kopečný, D.

    2015-01-01

    Roč. 468, Part: 1 (2015), s. 109-123 ISSN 0264-6021 R&D Projects: GA ČR GA15-22322S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : aldehyde dehydrogenase 2 (ALDH2) * aldehyde dehydrogenase 7 (ALDH7) * benzaldehyde Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.562, year: 2015

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

    Science.gov (United States)

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

    2017-07-01

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

  4. Evaluation of alcohol dehydrogenase and aldehyde dehydrogenase enzymes as bi-enzymatic anodes in a membraneless ethanol microfluidic fuel cell

    Science.gov (United States)

    Galindo-de-la-Rosa, J.; Arjona, N.; Arriaga, L. G.; Ledesma-García, J.; Guerra-Balcázar, M.

    2015-12-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldH) enzymes were immobilized by covalent binding and used as the anode in a bi-enzymatic membraneless ethanol hybrid microfluidic fuel cell. The purpose of using both enzymes was to optimize the ethanol electro-oxidation reaction (EOR) by using ADH toward its direct oxidation and AldH for the oxidation of aldehydes as by-products of the EOR. For this reason, three enzymatic bioanode configurations were evaluated according with the location of enzymes: combined, vertical and horizontally separated. In the combined configuration, a current density of 16.3 mA cm-2, a voltage of 1.14 V and a power density of 7.02 mW cm-2 were obtained. When enzymes were separately placed in a horizontal and vertical position the ocp drops to 0.94 V and to 0.68 V, respectively. The current density also falls to values of 13.63 and 5.05 mA cm-2. The decrease of cell performance of bioanodes with separated enzymes compared with the combined bioanode was of 31.7% and 86.87% for the horizontal and the vertical array.

  5. Protein-bound NAD(P)H Lifetime is Sensitive to Multiple Fates of Glucose Carbon.

    Science.gov (United States)

    Sharick, Joe T; Favreau, Peter F; Gillette, Amani A; Sdao, Sophia M; Merrins, Matthew J; Skala, Melissa C

    2018-04-03

    While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ 2 ) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ 2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.

  6. Mitigation of waterlogging-induced damages to pepper by exogenous meja

    International Nuclear Information System (INIS)

    Jun, O.; Bin, L.; Zhi, Y.B.

    2017-01-01

    In this study, we studied the mitigation effects of exogenous Methyl jasmonate (MeJA) on waterlogging-induced damages to Xinyou No.5 wrinkled skin pepper cultivar by spraying MeJA on leave's surface at different waterlogging periods and investigated its underlying mechanisms. The results showed that administration of MeJA increased antioxidant enzymes' activities, proline and soluble sugar contents and alcohol dehydrogenase (ADH) activity, reduced relative conductivity, malondialdehyde (MDA) and hydroxyl free radical (.OH) accumulation, lactate dehydrogenase (LDH) activity and lactic acid and acetaldehyde accumulation, and maintained high root malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) activities and certain aerobic respiratory metabolism. The study also found that there were significant differences among exogenous MeJA treatments at different waterlogging periods. Peppers treated with exogenous MeJA 1 day and 2 days prior to waterlogging had optimal agronomic traits, higher chlorophyll content, enzymatic activities and osmolytic substances, as well as lower relative conductivity, MDA and ·OH accumulation. Overall, the results suggest that MeJA mitigates waterlogging-induced damages to pepper by adjusting osmolytic substances contents, antioxidant enzymatic activities and root respiration and metabolism and achieves better alleviation effects by spraying prior to waterlogging. (author)

  7. Calcium-regulation of mitochondrial respiration maintains ATP homeostasis and requires ARALAR/AGC1-malate aspartate shuttle in intact cortical neurons.

    Science.gov (United States)

    Llorente-Folch, Irene; Rueda, Carlos B; Amigo, Ignacio; del Arco, Araceli; Saheki, Takeyori; Pardo, Beatriz; Satrústegui, Jorgina

    2013-08-28

    Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse cortical neurons in basal conditions and in response to increased workload caused by increases in [Na+]cyt (veratridine, high-K+ depolarization) and/or [Ca2+]cyt (carbachol). Respiration in nonstimulated neurons on 2.5-5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in [Ca2+]mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads.

  8. Functional, structural and phylogenetic analysis of domains underlying the Al sensitivity of the aluminum-activated malate/anion transporter, TaALMT1.

    Science.gov (United States)

    Ligaba, Ayalew; Dreyer, Ingo; Margaryan, Armine; Schneider, David J; Kochian, Leon; Piñeros, Miguel

    2013-12-01

    Triticum aestivum aluminum-activated malate transporter (TaALMT1) is the founding member of a unique gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small sub-group of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (Al) resistance. TaALMT1 exhibits significant enhancement of transport activity in response to extracellular Al. In this study, we integrated structure-function analyses of structurally altered TaALMT1 proteins expressed in Xenopus oocytes with phylogenic analyses of the ALMT family. Our aim is to re-examine the role of protein domains in terms of their potential involvement in the Al-dependent enhancement (i.e. Al-responsiveness) of TaALMT1 transport activity, as well as the roles of all its 43 negatively charged amino acid residues. Our results indicate that the N-domain, which is predicted to form the conductive pathway, mediates ion transport even in the absence of the C-domain. However, segments in both domains are involved in Al(3+) sensing. We identified two regions, one at the N-terminus and a hydrophobic region at the C-terminus, that jointly contribute to the Al-response phenotype. Interestingly, the characteristic motif at the N-terminus appears to be specific for Al-responsive ALMTs. Our study highlights the need to include a comprehensive phylogenetic analysis when drawing inferences from structure-function analyses, as a significant proportion of the functional changes observed for TaALMT1 are most likely the result of alterations in the overall structural integrity of ALMT family proteins rather than modifications of specific sites involved in Al(3+) sensing. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  9. Oxaloacetate-to-malate conversion by mineral photoelectrochemistry: implications for the viability of the reductive tricarboxylic acid cycle in prebiotic chemistry

    Science.gov (United States)

    Guzman, Marcelo I.; Martin, Scot T.

    2008-10-01

    The carboxylic acids produced by the reductive tricarboxylic acid (rTCA) cycle are possibly a biosynthetic core of initial life, although several steps such as the reductive kinetics of oxaloacetate (OAA) to malate (MA) are problematic by conventional chemical routes. In this context, we studied the kinetics of this reaction as promoted by ZnS mineral photoelectrochemistry. The quantum efficiency φMA of MA production from the photoelectrochemical reduction of OAA followed φMA=0.13 [OAA] (2.1×10-3+[OAA])-1 and was independent of temperature (5 to 50°C). To evaluate the importance of this forward rate under a prebiotic scenario, we also studied the temperature-dependent rate of the backward thermal decarboxylation of OAA to pyruvate (PA), which followed an Arrhenius behavior as log (k-2)=11.74 4956/T, where k-2 is in units of s-1. These measured rates were employed in conjunction with the indirectly estimated carboxylation rate of PA to OAA to assess the possible importance of mineral photoelectrochemistry in the conversion of OAA to MA under several scenarios of prebiotic conditions on early Earth. As an example, our analysis shows that there is 90% efficiency with a forward velocity of 3 yr/cycle for the OAA→MA step of the rTCA cycle at 280 K. Efficiency and velocity both decrease for increasing temperature. These results suggest high viability for mineral photoelectrochemistry as an enzyme-free engine to drive the rTCA cycle through the early aeons of early Earth, at least for the investigated OAA→MA step.

  10. Direct Electron Transfer of Dehydrogenases for Development of 3rd Generation Biosensors and Enzymatic Fuel Cells

    Directory of Open Access Journals (Sweden)

    Paolo Bollella

    2018-04-01

    Full Text Available Dehydrogenase based bioelectrocatalysis has been increasingly exploited in recent years in order to develop new bioelectrochemical devices, such as biosensors and biofuel cells, with improved performances. In some cases, dehydrogeases are able to directly exchange electrons with an appropriately designed electrode surface, without the need for an added redox mediator, allowing bioelectrocatalysis based on a direct electron transfer process. In this review we briefly describe the electron transfer mechanism of dehydrogenase enzymes and some of the characteristics required for bioelectrocatalysis reactions via a direct electron transfer mechanism. Special attention is given to cellobiose dehydrogenase and fructose dehydrogenase, which showed efficient direct electron transfer reactions. An overview of the most recent biosensors and biofuel cells based on the two dehydrogenases will be presented. The various strategies to prepare modified electrodes in order to improve the electron transfer properties of the device will be carefully investigated and all analytical parameters will be presented, discussed and compared.

  11. Krebs cycle metabolite profiling for identification and stratification of pheochromocytomas/paragangliomas due to succinate dehydrogenase deficiency

    NARCIS (Netherlands)

    Richter, S; Peitzsch, M.; Rapizzi, E.; Lenders, J.W.M.; Qin, N.; Cubas, A.A. de; Schiavi, F.; Rao, J.U.; Beuschlein, F.; Quinkler, M.; Timmers, H.J.L.M.; Opocher, G.; Mannelli, M.; Pacak, K.; Robledo, M.; Eisenhofer, G.

    2014-01-01

    CONTEXT: Mutations of succinate dehydrogenase A/B/C/D genes (SDHx) increase susceptibility to development of pheochromocytomas and paragangliomas (PPGLs), with particularly high rates of malignancy associated with SDHB mutations. OBJECTIVE: We assessed whether altered succinate dehydrogenase

  12. Identification and Overexpression of a Bifunctional Aldehyde/Alcohol Dehydrogenase Responsible for Ethanol Production in Thermoanaerobacter mathranii

    DEFF Research Database (Denmark)

    Yao, Shuo; Just Mikkelsen, Marie

    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 (Adh....... 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...

  13. Lactate dehydrogenase inhibition: exploring possible applications beyond cancer treatment.

    Science.gov (United States)

    Di Stefano, Giuseppina; Manerba, Marcella; Di Ianni, Lorenza; Fiume, Luigi

    2016-04-01

    Lactate dehydrogenase (LDH) inhibition is considered a worthwhile attempt in the development of innovative anticancer strategies. Unfortunately, in spite of the involvement of several research institutions and pharma-companies, the discovery of LDH inhibitors with drug-like properties seems a hardly resolvable challenge. While awaiting new advancements, in the present review we will examine other pathologic conditions characterized by increased glycolysis and LDH activity, which could potentially benefit from LDH inhibition. The rationale for targeting LDH activity in these contexts is the same justifying the LDH-based approach in anticancer therapy: because of the enzyme position at the end of glycolytic pathway, LDH inhibitors are not expected to hinder glucose metabolism of normal cells. Moreover, we will summarize the latest contributions in the discovery of enzyme inhibitors and try to glance over the reasons underlying the complexity of this research.

  14. Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

    Science.gov (United States)

    Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

    2000-01-01

    Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For

  15. Idiopathic intracranial hypertension, hormones, and 11β-hydroxysteroid dehydrogenases

    Science.gov (United States)

    Markey, Keira A; Uldall, Maria; Botfield, Hannah; Cato, Liam D; Miah, Mohammed A L; Hassan-Smith, Ghaniah; Jensen, Rigmor H; Gonzalez, Ana M; Sinclair, Alexandra J

    2016-01-01

    Idiopathic intracranial hypertension (IIH) results in raised intracranial pressure (ICP) leading to papilledema, visual dysfunction, and headaches. Obese females of reproductive age are predominantly affected, but the underlying pathological mechanisms behind IIH remain unknown. This review provides an overview of pathogenic factors that could result in IIH with particular focus on hormones and the impact of obesity, including its role in neuroendocrine signaling and driving inflammation. Despite occurring almost exclusively in obese women, there have been a few studies evaluating the mechanisms by which hormones and adipokines exert their effects on ICP regulation in IIH. Research involving 11β-hydroxysteroid dehydrogenase type 1, a modulator of glucocorticoids, suggests a potential role in IIH. Improved understanding of the complex interplay between adipose signaling factors such as adipokines, steroid hormones, and ICP regulation may be key to the understanding and future management of IIH. PMID:27186074

  16. Lactate dehydrogenase (LDH isoenzymes patterns in ocular tumours

    Directory of Open Access Journals (Sweden)

    Singh Rajendra

    1991-01-01

    Full Text Available Estimation of lactate dehydrogenase (LDH isoenzymes in the serum and aqueous humor was carried out in 15 cases of benign ocular tumour, 15 cases of malignant tumor and 15 normal cases. Cases of both sexes aged between 1 year and 75 years were included. LDH, isoenzymes specially LDH4 and LDH5 are higher and LDH1 and LDH2 lower in sera of patients with malignant tumor specially retinoblastoma as compared to benign tumor cases and control cases. LDH isoenzymes in aqueous humor are significantly higher and show a characteristic pattern in retinoblastoma cases, the concentration was presumably too low in the control, malignant tumor other than retinoblastoma and benign tumor cases as its fractionation was not possible.

  17. Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase

    DEFF Research Database (Denmark)

    Madiraju, Anila K; Erion, Derek M; Rahimi, Yasmeen

    2014-01-01

    Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been...... prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered...... hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense...

  18. 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.

  19. 17 beta-hydroxysteroid dehydrogenase activity in canine pancreas

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  20. Pyruvate Dehydrogenase Kinase as a Novel Therapeutic Target in Oncology

    Directory of Open Access Journals (Sweden)

    Gopinath eSutendra

    2013-03-01

    Full Text Available Current drug development in oncology is non-selective as it typically focuses on pathways essential for the survival of all dividing cells. The unique metabolic profile of cancer, which is characterized by increased glycolysis and suppressed mitochondrial glucose oxidation provides cancer cells with a proliferative advantage, conducive with apoptosis resistance and even increased angiogenesis. Recent evidence suggests that targeting the cancer-specific metabolic and mitochondrial remodeling may offer selectivity in cancer treatment. Pyruvate dehydrogenase kinase (PDK is a mitochondrial enzyme that is activated in a variety of cancers and results in the selective inhibition of pyruvate dehydrogenase (PDH, a complex of enzymes that converts cytosolic pyruvate to mitochondrial acetyl-CoA, the substrate for the Krebs’ cycle. Inhibition of PDK with either small interfering RNAs or the orphan drug dichloroacetate (DCA shifts the metabolism of cancer cells from glycolysis to glucose oxidation and reverses the suppression of mitochondria-dependent apoptosis. In addition, this therapeutic strategy increases the production of diffusible Krebs’ cycle intermediates and mitochondria-derived reactive oxygen species (mROS, activating p53 or inhibiting pro-proliferative and pro-angiogenic transcription factors like nuclear factor of activated T-cells (NFAT and hypoxia-inducible factor 1α (HIF1α. These effects result in decreased tumor growth and angiogenesis in a variety of cancers with high selectivity. In a small but mechanistic clinical trial in patients with glioblastoma, a highly aggressive and vascular form of brain cancer, DCA decreased tumor angiogenesis and tumor growth, suggesting that metabolic targeting therapies can be translated directly to patients. Therefore, reversing the mitochondrial suppression with metabolic-modulating drugs, like PDK inhibitors holds promise in the rapidly expanding field of metabolic oncology.

  1. Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase.

    Science.gov (United States)

    Gerszon, Joanna; Serafin, Eligiusz; Buczkowski, Adam; Michlewska, Sylwia; Bielnicki, Jakub Antoni; Rodacka, Aleksandra

    2018-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the key redox-sensitive proteins whose activity is largely affected by oxidative modifications at its highly reactive cysteine residue in the enzyme's active site (Cys149). Prolonged exposure to oxidative stress may cause, inter alia, the formation of intermolecular disulfide bonds leading to accumulation of GAPDH aggregates and ultimately to cell death. Recently these anomalies have been linked with the pathogenesis of Alzheimer's disease. Novel evidences indicate that low molecular compounds may be effective inhibitors potentially preventing the GAPDH translocation to the nucleus, and inhibiting or slowing down its aggregation and oligomerization. Therefore, we decided to establish the ability of naturally occurring compound, piceatannol, to interact with GAPDH and to reveal its effect on functional properties and selected parameters of the dehydrogenase structure. The obtained data revealed that piceatannol binds to GAPDH. The ITC analysis indicated that one molecule of the tetrameric enzyme may bind up to 8 molecules of polyphenol (7.3 ± 0.9). Potential binding sites of piceatannol to the GAPDH molecule were analyzed using the Ligand Fit algorithm. Conducted analysis detected 11 ligand binding positions. We indicated that piceatannol decreases GAPDH activity. Detailed analysis allowed us to presume that this effect is due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149) which is directly involved in the catalytic reaction. Consequently, our studies strongly indicate that piceatannol would be an exceptional inhibitor thanks to its ability to break the aforementioned pathologic disulfide linkage, and therefore to inhibit GAPDH aggregation. We demonstrated that by binding with GAPDH piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation.

  2. Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase.

    Directory of Open Access Journals (Sweden)

    Joanna Gerszon

    Full Text Available Glyceraldehyde-3-phosphate dehydrogenase (GAPDH is one of the key redox-sensitive proteins whose activity is largely affected by oxidative modifications at its highly reactive cysteine residue in the enzyme's active site (Cys149. Prolonged exposure to oxidative stress may cause, inter alia, the formation of intermolecular disulfide bonds leading to accumulation of GAPDH aggregates and ultimately to cell death. Recently these anomalies have been linked with the pathogenesis of Alzheimer's disease. Novel evidences indicate that low molecular compounds may be effective inhibitors potentially preventing the GAPDH translocation to the nucleus, and inhibiting or slowing down its aggregation and oligomerization. Therefore, we decided to establish the ability of naturally occurring compound, piceatannol, to interact with GAPDH and to reveal its effect on functional properties and selected parameters of the dehydrogenase structure. The obtained data revealed that piceatannol binds to GAPDH. The ITC analysis indicated that one molecule of the tetrameric enzyme may bind up to 8 molecules of polyphenol (7.3 ± 0.9. Potential binding sites of piceatannol to the GAPDH molecule were analyzed using the Ligand Fit algorithm. Conducted analysis detected 11 ligand binding positions. We indicated that piceatannol decreases GAPDH activity. Detailed analysis allowed us to presume that this effect is due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149 which is directly involved in the catalytic reaction. Consequently, our studies strongly indicate that piceatannol would be an exceptional inhibitor thanks to its ability to break the aforementioned pathologic disulfide linkage, and therefore to inhibit GAPDH aggregation. We demonstrated that by binding with GAPDH piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation.

  3. 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

  4. Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum

    OpenAIRE

    Dai, Zongjie; Dong, Hongjun; Zhang, Yanping; Li, Yin

    2016-01-01

    Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from thes...

  5. Improved production of fatty acids by Saccharomyces cerevisiae through screening a cDNA library from the oleaginous yeast Yarrowia lipolytica

    DEFF Research Database (Denmark)

    Shi, Shuobo; Ji, Haichuan; Siewers, Verena

    2016-01-01

    , malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, FA hydroxylase, farnesyltransferase, anoctamin, dihydrolipoamide dehydrogenase and phosphatidylethanolamine-binding protein. The best enzyme resulted in a 2.5-fold improvement in production of free FAs. Our findings not only provide a novel...

  6. A molecular analysis of the Gelechiidae (Lepidoptera, Gelechioidea) with an interpretative grouping of its taxa

    DEFF Research Database (Denmark)

    Karsholt, Ole; Mutanen, Marko; Lee, Sangmi

    2013-01-01

    , Isocitrate dehydrogenase, Cytosolic malate dehydrogenase, Glyceraldehyde-3-phosphate dehydrogenase and Carbamoylphosphate synthase domain protein). Fifty-two taxa representing nearly all established subfamilies and tribes of Gelechiidae, and about 10% of described gelechiid genera, in addition to five...

  7. Development of an Amperometric Biosensor Platform for the Combined Determination of L-Malic, Fumaric, and L-Aspartic Acid.

    Science.gov (United States)

    Röhlen, Désirée L; Pilas, Johanna; Schöning, Michael J; Selmer, Thorsten

    2017-10-01

    Three amperometric biosensors have been developed for the detection of L-malic acid, fumaric acid, and L -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD + ) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for L-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM -1 (L-malate biosensor) and 0.4 μA mM -1 (fumarate biosensor). The L-aspartate detection system displayed a linear range of 1.0-10.0 mM with a sensitivity of 0.09 μA mM -1 . The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.

  8. Hexose-6-phosphate dehydrogenase contributes to skeletal muscle homeostasis independent of 11β-hydroxysteroid dehydrogenase type 1.

    LENUS (Irish Health Repository)

    Semjonous, Nina M

    2011-01-01

    Glucose-6-phosphate (G6P) metabolism by the enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the sarcoplasmic reticulum lumen generates nicotinamide adenine dinucleotide phosphate (reduced) to provide the redox potential for the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to activate glucocorticoid (GC). H6PDH knockout (KO) mice have a switch in 11β-HSD1 activity, resulting in GC inactivation and hypothalamic-pituitary-adrenal axis activation. Importantly, H6PDHKO mice develop a type II fiber myopathy with abnormalities in glucose metabolism and activation of the unfolded protein response (UPR). GCs play important roles in muscle physiology, and therefore, we have examined the importance of 11β-HSD1 and GC metabolism in mediating aspects of the H6PDHKO myopathy. To achieve this, we examined 11β-HSD1\\/H6PDH double-KO (DKO) mice, in which 11β-HSD1 mediated GC inactivation is negated. In contrast to H6PDHKO mice, DKO mice GC metabolism and hypothalamic-pituitary-adrenal axis set point is similar to that observed in 11β-HSD1KO mice. Critically, in contrast to 11β-HSD1KO mice, DKO mice phenocopy the salient features of the H6PDHKO, displaying reduced body mass, muscle atrophy, and vacuolation of type II fiber-rich muscle, fasting hypoglycemia, increased muscle glycogen deposition, and elevated expression of UPR genes. We propose that muscle G6P metabolism through H6PDH may be as important as changes in the redox environment when considering the mechanism underlying the activation of the UPR and the ensuing myopathy in H6PDHKO and DKO mice. These data are consistent with an 11β-HSD1-independent function for H6PDH in which sarcoplasmic reticulum G6P metabolism and nicotinamide adenine dinucleotide phosphate-(oxidized)\\/nicotinamide adenine dinucleotide phosphate (reduced) redox status are important for maintaining muscle homeostasis.

  9. Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Xu Guoqiang

    2012-02-01

    Full Text Available Abstract Background Fumaric acid is a commercially important component of foodstuffs, pharmaceuticals and industrial materials, yet the current methods of production are unsustainable and ecologically destructive. Results In this study, the fumarate biosynthetic pathway involving reductive reactions of the tricarboxylic acid cycle was exogenously introduced in S. cerevisiae by a series of simple genetic modifications. First, the Rhizopus oryzae genes for malate dehydrogenase (RoMDH and fumarase (RoFUM1 were heterologously expressed. Then, expression of the endogenous pyruvate carboxylase (PYC2 was up-regulated. The resultant yeast strain, FMME-001 ↑PYC2 + ↑RoMDH, was capable of producing significantly higher yields of fumarate in the glucose medium (3.18 ± 0.15 g liter-1 than the control strain FMME-001 empty vector. Conclusions The results presented here provide a novel strategy for fumarate biosynthesis, which represents an important advancement in producing high yields of fumarate in a sustainable and ecologically-friendly manner.

  10. Peripheral effects of FAAH deficiency on fuel and energy homeostasis: role of dysregulated lysine acetylation.

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    Bhavapriya Vaitheesvaran

    Full Text Available FAAH (fatty acid amide hydrolase, primarily expressed in the liver, hydrolyzes the endocannabinoids fatty acid ethanolamides (FAA. Human FAAH gene mutations are associated with increased body weight and obesity. In our present study, using targeted metabolite and lipid profiling, and new global acetylome profiling methodologies, we examined the role of the liver on fuel and energy homeostasis in whole body FAAH(-/- mice.FAAH(-/- mice exhibit altered energy homeostasis demonstrated by decreased oxygen consumption (Indirect calorimetry. FAAH(-/- mice are hyperinsulinemic and have adipose, skeletal and hepatic insulin resistance as indicated by stable isotope phenotyping (SIPHEN. Fed state skeletal muscle and liver triglyceride levels was increased 2-3 fold, while glycogen was decreased 42% and 57% respectively. Hepatic cholesterol synthesis was decreased 22% in FAAH(-/- mice. Dysregulated hepatic FAAH(-/- lysine acetylation was consistent with their metabolite profiling. Fasted to fed increases in hepatic FAAH(-/- acetyl-CoA (85%, p<0.01 corresponded to similar increases in citrate levels (45%. Altered FAAH(-/- mitochondrial malate dehydrogenase (MDH2 acetylation, which can affect the malate aspartate shuttle, was consistent with our observation of a 25% decrease in fed malate and aspartate levels. Decreased fasted but not fed dihydroxyacetone-P and glycerol-3-P levels in FAAH(-/- mice was consistent with a compensating contribution from decreased acetylation of fed FAAH(-/- aldolase B. Fed FAAH(-/- alcohol dehydrogenase (ADH acetylation was also decreased.Whole body FAAH deletion contributes to a pre-diabetic phenotype by mechanisms resulting in impairment of hepatic glucose and lipid metabolism. FAAH(-/- mice had altered hepatic lysine acetylation, the pattern sharing similarities with acetylation changes reported with chronic alcohol treatment. Dysregulated hepatic lysine acetylation seen with impaired FAA hydrolysis could support the liver

  11. Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels

    Energy Technology Data Exchange (ETDEWEB)

    Akduman, Begüm [Chemistry Department, Adnan Menderes University, Aydın (Turkey); Uygun, Murat [Koçarlı Vocational and Training School, Adnan Menderes University, Aydın (Turkey); Uygun, Deniz Aktaş, E-mail: daktas@adu.edu.tr [Chemistry Department, Adnan Menderes University, Aydın (Turkey); Akgöl, Sinan [Biochemistry Department, Ege University, İzmir (Turkey); Denizli, Adil [Chemistry Department, Hacettepe University, Ankara (Turkey)

    2013-12-01

    In this study, poly(2-hydroxyethyl methacrylate–glycidylmethacrylate) [poly(HEMA–GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA–GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30–50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA–GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA–GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0 mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH 5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0 M NaCI at pH 8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS–PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. - Highlights: • Poly(HEMA–GMA) cryogels were synthesized by radical cryocopolymerization technique. • Prepared cryogels were functionalized with IDA, then Zn(II) ions were chelated to the cryogel. • Zn(II) chelated poly

  12. Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels

    International Nuclear Information System (INIS)

    Akduman, Begüm; Uygun, Murat; Uygun, Deniz Aktaş; Akgöl, Sinan; Denizli, Adil

    2013-01-01

    In this study, poly(2-hydroxyethyl methacrylate–glycidylmethacrylate) [poly(HEMA–GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA–GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30–50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA–GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA–GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0 mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH 5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0 M NaCI at pH 8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS–PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. - Highlights: • Poly(HEMA–GMA) cryogels were synthesized by radical cryocopolymerization technique. • Prepared cryogels were functionalized with IDA, then Zn(II) ions were chelated to the cryogel. • Zn(II) chelated poly

  13. Two Members of the Aluminum-Activated Malate Transporter Family, SlALMT4 and SlALMT5, are Expressed during Fruit Development, and the Overexpression of SlALMT5 Alters Organic Acid Contents in Seeds in Tomato (Solanum lycopersicum).

    Science.gov (United States)

    Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Nakano, Ryohei; Ushijima, Koichiro; Kubo, Yasutaka; Mori, Izumi C; Higashiizumi, Emi; Galis, Ivan; Yamamoto, Yoko

    2016-11-01

    The aluminum-activated malate transporter (ALMT) family of proteins transports malate and/or inorganic anions across plant membranes. To demonstrate the possible role of ALMT genes in tomato fruit development, we focused on SlALMT4 and SlALMT5, the two major genes expressed during fruit development. Predicted proteins were classified into clade 2 of the family, many members of which localize to endomembranes. Tissue-specific gene expression was determined using transgenic tomato expressing the β-glucuronidase reporter gene controlled by their own promoters. Both the genes were expressed in vascular bundles connecting to developing seeds in fruit and in the embryo of mature seeds. Further, SlALMT5 was expressed in embryo in developing seeds in fruit. Subcellular localization of both proteins to the endoplasmic reticulum (ER) was established by transiently expressing the green fluorescent protein fusions in plant protoplasts. SlALMT5 probably localized to other endomembranes as well. Localization of SlALMT5 to the ER was also confirmed by immunoblot analysis. The transport function of both SlALMT proteins was investigated electrophysiologically in Xenopus oocytes. SlALMT5 transported malate and inorganic anions such as nitrate and chloride, but not citrate. SlALMT4 also transported malate, but the results were less consistent perhaps because it did not localize strongly to the plasma membrane. To elucidate the physiological role of SlALMT5 further, we overexpressed SlALMT5 in tomato. Compared with the wild type, overexpressors exhibited higher malate and citrate contents in mature seeds, but not in fruit. We conclude that the malate transport function of SlALMT5 expressed in developing fruit influences the organic acid contents in mature seeds. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  14. Adaptive Changes After 2 Weeks of 10-s Sprint Interval Training With Various Recovery Times

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    Robert A. Olek

    2018-04-01

    Full Text Available Purpose: The aim of this study was to compare the effect of applying two different rest recovery times in a 10-s sprint interval training session on aerobic and anaerobic capacities as well as skeletal muscle enzyme activities.Methods: Fourteen physically active but not highly trained male subjects (mean maximal oxygen uptake 50.5 ± 1.0 mlO2·kg−1·min−1 participated in the study. The training protocol involved a series of 10-s sprints separated by either 1-min (SIT10:1 or 4-min (SIT10:4 of recovery. The number of sprints progressed from four to six over six sessions separated by 1–2 days rest. Pre and post intervention anthropometric measurements, assessment of aerobic, anaerobic capacity and muscle biopsy were performed. In the muscle samples maximal activities of citrate synthase (CS, 3-hydroxyacylCoA dehydrogenase (HADH, carnitine palmitoyl-transferase (CPT, malate dehydrogenase (MDH, and its mitochondrial form (mMDH, as well as lactate dehydrogenase (LDH were determined. Analysis of variance was performed to determine changes between conditions.Results: Maximal oxygen uptake improved significantly in both training groups, by 13.6% in SIT10:1 and 11.9% in SIT10:4, with no difference between groups. Wingate anaerobic test results indicated main effect of time for total work, peak power output and mean power output, which increased significantly and similarly in both groups. Significant differences between training groups were observed for end power output, which increased by 10.8% in SIT10:1, but remained unchanged in SIT10:4. Both training protocols induced similar increase in CS activity (main effect of time p < 0.05, but no other enzymes.Conclusion: Sprint interval training protocols induce metabolic adaptation over a short period of time, and the reduced recovery between bouts may attenuate fatigue during maximal exercise.

  15. Dual enzymatic dynamic kinetic resolution by Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase and Candida antarctica lipase B

    KAUST Repository

    Karume, Ibrahim; Musa, Musa M.; Bsharat, Odey; Takahashi, Masateru; Hamdan, Samir; El Ali, Bassam

    2016-01-01

    The immobilization of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (TeSADH) using sol–gel method enables its use to racemize enantiopure alcohols in organic media. Here, we report the racemization of enantiopure phenyl

  16. Kernicterus by glucose-6-phosphate dehydrogenase deficiency: a case report and review of the literature

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    Cossio de Gurrola Gladys

    2008-05-01

    Full Text Available Abstract Introduction Glucose-6-phosphate dehydrogenase deficiency is an X-linked recessive disease that causes acute or chronic hemolytic anemia and potentially leads to severe jaundice in response to oxidative agents. This deficiency is the most common human innate error of metabolism, affecting more than 400 million people worldwide. Case presentation Here, we present the first documented case of kernicterus in Panama, in a glucose-6-phosphate dehydrogenase-deficient newborn clothed in naphthalene-impregnated garments, resulting in reduced psychomotor development, neurosensory hypoacousia, absence of speech and poor reflex of the pupil to light. Conclusion Mutational analysis revealed the glucose-6-phosphate dehydrogenase Mediterranean polymorphic variant, which explained the development of kernicterus after exposition of naphthalene. As the use of naphthalene in stored clothes is a common practice, glucose-6-phosphate dehydrogenase testing in neonatal screening could prevent severe clinical consequences.

  17. Mitochondrial type II NAD(PH dehydrogenases in fungal cell death

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    A. Pedro Gonçalves

    2015-03-01

    Full Text Available During aerobic respiration, cells produce energy through oxidative phosphorylation, which includes a specialized group of multi-subunit complexes in the inner mitochondrial membrane known as the electron transport chain. However, this canonical pathway is branched into single polypeptide alternative routes in some fungi, plants, protists and bacteria. They confer metabolic plasticity, allowing cells to adapt to different environmental conditions and stresses. Type II NAD(PH dehydrogenases (also called alternative NAD(PH dehydrogenases are non-proton pumping enzymes that bypass complex I. Recent evidence points to the involvement of fungal alternative NAD(PH dehydrogenases in the process of programmed cell death, in addition to their action as overflow systems upon oxidative stress. Consistent with this, alternative NAD(PH dehydrogenases are phylogenetically related to cell death - promoting proteins of the apoptosis-inducing factor (AIF-family.

  18. Structural characterization of a D-isomer specific 2-hydroxyacid dehydrogenase from Lactobacillus delbrueckii ssp. bulgaricus.

    Science.gov (United States)

    Holton, Simon J; Anandhakrishnan, Madhankumar; Geerlof, Arie; Wilmanns, Matthias

    2013-02-01

    Hydroxyacid dehydrogenases, responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids in lactic acid producing bacteria, have a range of biotechnology applications including antibiotic synthesis, flavor development in dairy products and the production of valuable synthons. The genome of Lactobacillus delbrueckii ssp. bulgaricus, a member of the heterogeneous group of lactic acid bacteria, encodes multiple hydroxyacid dehydrogenases whose structural and functional properties remain poorly characterized. Here, we report the apo and coenzyme NAD⁺ complexed crystal structures of the L. bulgaricusD-isomer specific 2-hydroxyacid dehydrogenase, D2-HDH. Comparison with closely related members of the NAD-dependent dehydrogenase family reveals that whilst the D2-HDH core fold is structurally conserved, the substrate-binding site has a number of non-canonical features that may influence substrate selection and thus dictate the physiological function of the enzyme. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Immobilisation and characterisation of glucose dehydrogenase immobilised on ReSyn: a proprietary polyethylenimine support matrix

    CSIR Research Space (South Africa)

    Twala, BV

    2010-01-01

    Full Text Available Immobilisation of enzymes is of considerable interest due to the advantages over soluble enzymes, including improved stability and recovery. Glucose Dehydrogenase (GDH) is an important biocatalytic enzyme due to is ability to recycle the biological...

  20. 11β-Hydroxysteroid Dehydrogenase 2 in Preeclampsia

    Directory of Open Access Journals (Sweden)

    Katarzyna Kosicka

    2016-01-01

    Full Text Available Preeclampsia is a serious medical problem affecting the mother and her child and influences their health not only during the pregnancy, but also many years after. Although preeclampsia is a subject of many research projects, the etiology of the condition remains unclear. One of the hypotheses related to the etiology of preeclampsia is the deficiency in placental 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2, the enzyme which in normal pregnancy protects the fetus from the excess of maternal cortisol. The reduced activity of the enzyme was observed in placentas from pregnancies complicated with preeclampsia. That suggests the overexposure of the developing child to maternal cortisol, which in high levels exerts proapoptotic effects and reduces fetal growth. The fetal growth restriction due to the diminished placental 11β-HSD2 function may be supported by the fact that preeclampsia is often accompanied with fetal hypotrophy. The causes of the reduced function of 11β-HSD2 in placental tissue are still discussed. This paper summarizes the phenomena that may affect the activity of the enzyme at various steps on the way from the gene to the protein.

  1. Retinol Dehydrogenases Regulate Vitamin A Metabolism for Visual Function

    Directory of Open Access Journals (Sweden)

    Bhubanananda Sahu

    2016-11-01

    Full Text Available The visual system produces visual chromophore, 11-cis-retinal from dietary vitamin A, all-trans-retinol making this vitamin essential for retinal health and function. These metabolic events are mediated by a sequential biochemical process called the visual cycle. Retinol dehydrogenases (RDHs are responsible for two reactions in the visual cycle performed in retinal pigmented epithelial (RPE cells, photoreceptor cells and Müller cells in the retina. RDHs in the RPE function as 11-cis-RDHs, which oxidize 11-cis-retinol to 11-cis-retinal in vivo. RDHs in rod photoreceptor cells in the retina work as all-trans-RDHs, which reduce all-trans-retinal to all-trans-retinol. Dysfunction of RDHs can cause inherited retinal diseases in humans. To facilitate further understanding of human diseases, mouse models of RDHs-related diseases have been carefully examined and have revealed the physiological contribution of specific RDHs to visual cycle function and overall retinal health. Herein we describe the function of RDHs in the RPE and the retina, particularly in rod photoreceptor cells, their regulatory properties for retinoid homeostasis and future therapeutic strategy for treatment of retinal diseases.

  2. Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum

    Science.gov (United States)

    Ding, Wei; Si, Meiru; Zhang, Weipeng; Zhang, Yaoling; Chen, Can; Zhang, Lei; Lu, Zhiqiang; Chen, Shaolin; Shen, Xihui

    2015-01-01

    Vanillin dehydrogenase (VDH) is a crucial enzyme involved in the degradation of lignin-derived aromatic compounds. Herein, the VDH from Corynebacterium glutamicum was characterized. The relative molecular mass (Mr) determined by SDS-PAGE was ~51kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity toward vanillin at pH 7.0 and 30C, and interestingly, it could utilize NAD+ and NADP+ as coenzymes with similar efficiency and showed no obvious difference toward NAD+ and NADP+. In addition to vanillin, this enzyme exhibited catalytic activity toward a broad range of substrates, including p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde. Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis. Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum. Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum. PMID:25622822

  3. Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase.

    Science.gov (United States)

    Kil, In Sup; Huh, Tae Lin; Lee, Young Sup; Lee, You Mie; Park, Jeen-Woo

    2006-01-01

    The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.

  4. Glutathionylation regulates cytosolic NADP+-dependent isocitrate dehydrogenase activity.

    Science.gov (United States)

    Shin, Seoung Woo; Oh, Chang Joo; Kil, In Sup; Park, Jeen-Woo

    2009-04-01

    Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) is susceptible to inactivation by numerous thiol-modifying reagents. This study now reports that Cys269 of IDPc is a target for S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by cytosolic glutaredoxin in the presence of GSH. Glutathionylated IDPc was significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion. Glutathionylation may play a protective role in the degradation of protein through the structural alterations of IDPc. HEK293 cells treated with diamide displayed decreased IDPc activity and accumulated glutathionylated enzyme. Using immunoprecipitation with an anti-IDPc IgG and immunoblotting with an anti-GSH IgG, we purified and positively identified glutathionylated IDPc from the kidneys of mice subjected to ischemia/reperfusion injury and from the livers of ethanol-administered rats. These results suggest that IDPc activity is modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.

  5. Salivary lactate dehydrogenase and aminotransferases in diabetic patients.

    Science.gov (United States)

    Malicka, Barbara; Skoskiewicz-Malinowska, Katarzyna; Kaczmarek, Urszula

    2016-11-01

    Diabetes mellitus (DM) is a group of metabolic diseases resulting from impaired insulin secretion and/or action. DM is characterized by hyperglycemia that can lead to the dysfunction or damage of organs, including the salivary glands.The aim of this study was to compare the levels of salivary lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in diabetic patients.The study was approved by the Bioethics Committee of Wroclaw Medical University (Poland). The study comprised 90 adults of both sexes, aged 21 to 57 years. The patients were divided into 3 groups: type 1 diabetics (D1), type 2 diabetics (D2), and a healthy control group (C). Each group consisted of 30 age- and sex-matched subjects. Total protein (P, by Lowry method), LDH, AST, ALT (with Alpha Diagnostics kits), and salivary flow rate were measured in unstimulated mixed saliva. The level of glycosylated hemoglobin (HbA1c) was measured with DCA 2000 Reagent Kit. The obtained data were analyzed using the Mann-Whitney U test and the Spearman rank at a significance level of P salivary LDH, AST, and ALT in D1 compared with D2 and C confirm that salivary glands of D1 might be attributed to autoimmunological damage associated with the pathomechanism of DM.

  6. Alcohol dehydrogenases from thermophilic and hyperthermophilic archaea and bacteria.

    Science.gov (United States)

    Radianingtyas, Helia; Wright, Phillip C

    2003-12-01

    Many studies have been undertaken to characterise alcohol dehydrogenases (ADHs) from thermophiles and hyperthermophiles, mainly to better understand their activities and thermostability. To date, there are 20 thermophilic archaeal and 17 thermophilic bacterial strains known to have ADHs or similar enzymes, including the hypothetical proteins. Some of these thermophiles are found to have multiple ADHs, sometimes of different types. A rigid delineation of amino acid sequences amongst currently elucidated thermophilic ADHs and similar proteins is phylogenetically apparent. All are NAD(P)-dependent, with one exception that utilises the cofactor F(420) instead. Within the NAD(P)-dependent group, the thermophilic ADHs are orderly clustered as zinc-dependent ADHs, short-chain ADHs, and iron-containing/activated ADHs. Distance matrix calculations reveal that thermophilic ADHs within one type are homologous, with those derived from a single genus often showing high similarities. Elucidation of the enzyme activity and stability, coupled with structure analysis, provides excellent information to explain the relationship between them, and thermophilic ADHs diversity.

  7. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D., E-mail: vappanna@laurentian.ca

    2014-11-07

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

  8. [Glucose-6-phosphate dehydrogenase deficiency in children: a case report].

    Science.gov (United States)

    Verdugo L, Patricia; Calvanese T, Marlene; Rodríguez V, Diego; Cárcamo C, Cassandra

    2014-02-01

    Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) is the most common red blood cell (RBC) enzyme disorder. The decrease as well as the absence of the enzyme increase RBC vulnerability to oxidative stress caused by exposure to certain medications or intake of fava beans. Among the most common clinical manifestations of this condition, acute hemolysis, chronic hemolysis, neonatal hyperbilirubinemia, and an asymptomatic form are observed. To analyze the case of a child who presented hemolytic crisis due to favism. A 2 year and 7 month old boy with a history of hyperbilirubinemia during the newborn period with no apparent cause, no family history of hemolytic anemia or parental consanguinity. He presented a prolonged neonatal jaundice and severe anemia requiring RBC transfusion. An intake of fava beans 48 h prior to onset of symptoms was reported. G6PD qualitative determination was compatible with this enzyme deficiency. G6PD deficiency can be highly variable in its clinical presentation, so it is necessary to keep it in mind during the diagnosis of hemolytic anemia at any age.

  9. Leucaena sp. recombinant cinnamyl alcohol dehydrogenase: purification and physicochemical characterization.

    Science.gov (United States)

    Patel, Parth; Gupta, Neha; Gaikwad, Sushama; Agrawal, Dinesh C; Khan, Bashir M

    2014-02-01

    Cinnamyl alcohol dehydrogenase is a broad substrate specificity enzyme catalyzing the final step in monolignol biosynthesis, leading to lignin formation in plants. Here, we report characterization of a recombinant CAD homologue (LlCAD2) isolated from Leucaena leucocephala. LlCAD2 is 80 kDa homo-dimer associated with non-covalent interactions, having substrate preference toward sinapaldehyde with Kcat/Km of 11.6×10(6) (M(-1) s(-1)), and a possible involvement of histidine at the active site. The enzyme remains stable up to 40 °C, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 0.002 and 5h, respectively. LlCAD2 showed optimal activity at pH 6.5 and 9 for reduction and oxidation reactions, respectively, and was stable between pH 7 and 9, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 7.5×10(-4) and 15 h, respectively. It is a Zn-metalloenzyme with 4 Zn(2+) per dimer, however, was inhibited in presence of externally supplemented Zn(2+) ions. The enzyme was resistant to osmolytes, reducing agents and non-ionic detergents. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase

    Science.gov (United States)

    Kohen, Amnon; Cannio, Raffaele; Bartolucci, Simonetta; Klinman, Judith P.; Klinman, Judith P.

    1999-06-01

    Biological catalysts (enzymes) speed up reactions by many orders of magnitude using fundamental physical processes to increase chemical reactivity. Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temperature. Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property. In reactions involving small molecules, the relative importance of tunnelling increases as the temperature is reduced. We have now investigated whether hydrogen tunnelling occurs at elevated temperatures in a biological system that functions physiologically under such conditions. Using a thermophilic alcohol dehydrogenase (ADH), we find that hydrogen tunnelling makes a significant contribution at 65°C this is analogous to previous findings with mesophilic ADH at 25°C ( ref. 5). Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temperature. These findings provide experimental evidence for a role of thermally excited enzyme fluctuations in modulating enzyme-catalysed bond cleavage.

  11. Cloning and Polymorphisms of Yak Lactate Dehydrogenase b Gene

    Directory of Open Access Journals (Sweden)

    Yaou Xu

    2013-06-01

    Full Text Available The main objective of this work was to study the unique polymorphisms of the lactate dehydrogenase-1 (LDH1 gene in yak (Bos grunniens. Native polyacrylamide gel electrophoresis revealed three phenotypes of LDH1 (a tetramer of H subunit in yak heart and longissimus muscle extracts. The corresponding gene, ldhb, encoding H subunits of three LDH1 phenotypes was obtained by RT-PCR. A total of six nucleotide differences were detected in yak ldhb compared with that of cattle, of which five mutations cause amino acid substitutions. Sequence analysis shows that the G896A and C689A, mutations of ldhb gene, result in alterations of differently charged amino acids, and create the three phenotypes (F, M, and S of yak LDH1. Molecular modeling of the H subunit of LDH indicates that the substituted amino acids are not located within NAD+ or substrate binding sites. PCR-RFLP examination of G896A mutation demonstrated that most LDH1-F samples are actually heterozygote at this site. These results help to elucidate the molecular basis and genetic characteristic of the three unique LDH1 phenotypes in yak.

  12. Undetected Toxicity Risk in Pharmacogenetic Testing for Dihydropyrimidine Dehydrogenase

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    Felicia Stefania Falvella

    2015-04-01

    Full Text Available Fluoropyrimidines, the mainstay agents for the treatment of colorectal cancer, alone or as a part of combination therapies, cause severe adverse reactions in about 10%–30% of patients. Dihydropyrimidine dehydrogenase (DPD, a key enzyme in the catabolism of 5-fluorouracil, has been intensively investigated in relation to fluoropyrimidine toxicity, and several DPD gene (DPYD polymorphisms are associated with decreased enzyme activity and increased risk of fluoropyrimidine-related toxicity. In patients carrying non-functional DPYD variants (c.1905+1G>A, c.1679T>G, c.2846A>T, fluoropyrimidines should be avoided or reduced according to the patients’ homozygous or heterozygous status, respectively. For other common DPYD variants (c.496A>G, c.1129-5923C>G, c.1896T>C, conflicting data are reported and their use in clinical practice still needs to be validated. The high frequency of DPYD polymorphism and the lack of large prospective trials may explain differences in studies’ results. The epigenetic regulation of DPD expression has been recently investigated to explain the variable activity of the enzyme. DPYD promoter methylation and its regulation by microRNAs may affect the toxicity risk of fluoropyrimidines. The studies we reviewed indicate that pharmacogenetic testing is promising to direct personalised dosing of fluoropyrimidines, although further investigations are needed to establish the role of DPD in severe toxicity in patients treated for colorectal cancer.

  13. Ebselen Reversibly Inhibits Human Glutamate Dehydrogenase at the Catalytic Site.

    Science.gov (United States)

    Jin, Yanhong; Li, Di; Lu, Shiying; Zhao, Han; Chen, Zhao; Hou, Wei; Ruan, Benfang Helen

    Human glutamate dehydrogenase (GDH) plays an important role in neurological diseases, tumor metabolism, and hyperinsulinism-hyperammonemia syndrome (HHS). However, there are very few inhibitors known for human GDH. Recently, Ebselen was reported to crosslink with Escherichia coli GDH at the active site cysteine residue (Cys321), but the sequence alignment showed that the corresponding residue is Ala329 in human GDH. To investigate whether Ebselen could be an inhibitor for human GDH, we cloned and expressed an N-terminal His-tagged human GDH in E. coli. The recombinant human GDH enzyme showed expected properties such as adenosine diphosphate activation and nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate dual recognition. Further, we developed a 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzenesulfonate sodium salt (EZMTT)-based assay for human GDH, which was highly sensitive and is suitable for high-throughput screening for potent GDH inhibitors. In addition, ForteBio binding assays demonstrated that Ebselen is a reversible active site inhibitor for human GDH. Since Ebselen is a multifunctional organoselenium compound in Phase III clinical trials for inflammation, an Ebselen-based GDH inhibitor might be valuable for future drug discovery for HHS patients.

  14. Interaction between alcohol dehydrogenase II gene, alcohol consumption, and risk for breast cancer

    OpenAIRE

    St?rmer, T; Wang-Gohrke, S; Arndt, V; Boeing, H; Kong, X; Kreienberg, R; Brenner, H

    2002-01-01

    MaeIII Restriction Fragment Length Polymorphism in exon 3 of the alcohol dehydrogenase II was assessed in serum from 467 randomly selected German women and 278 women with invasive breast cancer to evaluate the interaction between a polymorphism of the alcohol dehydrogenase II gene, alcohol consumption and risk for breast cancer. In both groups, usual consumption of different alcoholic beverages was asked for using semiquantitative food frequency questionnaires. We used multivariable logistic ...

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

    OpenAIRE

    Gideon C. Okpokwasili; Christian Okechukwu Nweke

    2010-01-01

    The toxicity of phenol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol on Pseudomonas, Bacillus and Escherichia species isolated from petroleum refinery wastewater was assessed via inhibition of dehydrogenase enzyme activity. At low concentrations, 2-nitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol stimulated dehydrogenase activity and at sufficient concentrations, phenolic compounds inhibi...

  16. Inhibiting sperm pyruvate dehydrogenase complex and its E3 subunit, dihydrolipoamide dehydrogenase affects fertilization in Syrian hamsters.

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    Archana B Siva

    Full Text Available BACKGROUND/AIMS: The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc and its E3 subunit, dihydrolipoamide dehydrogenase (DLD in hamster in vitro fertilization (IVF via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. METHODOLOGY AND PRINCIPAL FINDINGS: Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid. Oocytes fertilized with MICA-treated (MT [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. CONCLUSIONS: This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In

  17. Protective effect of maternal prenatal melatonin administration on rat pups born to mothers submitted to constant light during gestation

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    C.D. Cisternas

    2010-09-01

    Full Text Available We studied the effects of adverse conditions such as constant light (LL on the circadian rhythm of malate (MDH, EC 1.1.1.37 and lactate (LDH, EC 1.1.1.27 dehydrogenase activities of the testes of male Wistar rats on postnatal day 28 (PN28, anxiety-like behavior (elevated plus-maze test at PN60 and sexual behavior at PN120. The rats were assigned to mother groups on day 10 of pregnancy: control (12-h light/dark, LL (light from day 10 to 21 of pregnancy, and LL+Mel (LL and sc injection to the mothers of a daily dose of melatonin, 1 mg/kg body weight at circadian time 12, from day 17 to 21 of pregnancy. LL offspring did not show circadian rhythms of MDH (N = 62 and LDH (N = 63 activities (cosinor and ANOVA-LSD Fisher. They presented a 44.7% decrease in open-arm entries and a 67.9% decrease in time (plus-maze test, N = 15, P < 0.001, Mann-Whitney U-test and Kruskal-Wallis test, an increase in mounting (94.4%, intromission (94.5% and ejaculation (56.6% latencies (N = 12, P < 0.01, Mann-Whitney U-test and Kruskal-Wallis test and lower numbers of these events (61, 59 and 73%, respectively; P < 0.01, N = 12 compared to controls. The offspring of the LL+Mel group presented MDH and LDH circadian rhythms (P < 0.05, N = 50, cosinor and ANOVA-LSD Fisher, anxiety-like and sexual behaviors similar to control. These findings supported the importance of the melatonin signal and provide evidence for the protective effects of hormones on maternal programming during gestation. This protective action of melatonin is probably related to its entrainment capacity, favoring internal coupling of the fetal multioscillatory system.

  18. Molecular Basis for Converting (2S-Methylsuccinyl-CoA Dehydrogenase into an Oxidase

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    Simon Burgener

    2017-12-01

    Full Text Available Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases.

  19. Pyruvate dehydrogenase kinase inhibition: Reversing the Warburg effect in cancer therapy

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    Hayden Bell

    2016-06-01

    Full Text Available The poor efficacy of many cancer chemotherapeutics, which are often non-selective and highly toxic, is attributable to the remarkable heterogeneity and adaptability of cancer cells. The Warburg effect describes the up regulation of glycolysis as the main source of adenosine 5’-triphosphate in cancer cells, even under normoxic conditions, and is a unique metabolic phenotype of cancer cells. Mitochondrial suppression is also observed which may be implicated in apoptotic suppression and increased funneling of respiratory substrates to anabolic processes, conferring a survival advantage. The mitochondrial pyruvate dehydrogenase complex is subject to meticulous regulation, chiefly by pyruvate dehydrogenase kinase. At the interface between glycolysis and the tricarboxylic acid cycle, the pyruvate dehydrogenase complex functions as a metabolic gatekeeper in determining the fate of glucose, making pyruvate dehydrogenase kinase an attractive candidate in a bid to reverse the Warburg effect in cancer cells. The small pyruvate dehydrogenase kinase inhibitor dichloroacetate has, historically, been used in conditions associated with lactic acidosis but has since gained substantial interest as a potential cancer chemotherapeutic. This review considers the Warburg effect as a unique phenotype of cancer cells in-line with the history of and current approaches to cancer therapies based on pyruvate dehydrogenase kinase inhibition with particular reference to dichloroacetate and its derivatives.

  20. GENETIC IDENTIFICATION FOR TUNA AND RAINBOW RUNNER CAPTURE IN NORTH BALI WATERS

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    Gusti Ngurah Permana

    2006-06-01

    Full Text Available Gondol Research Institute for Mariculture identification of tuna and rainbow runner was an objective in this current study. Samples of five species were collected from territorial water of North Bali. The method used in this study was allozyme electrophoresis. The results showed that buffer of CAPM-6 (citric acid aminoprophylmorpholine resulted in a sharp and clear banding pattern. The species could be differentiated in six diagnostic isozyme patterns Idh* (isocitrate dehydrogenase, 6Pgd* (6 phosphogluconate dehydrogenase, Gpi* (glucose phosphate isomerase, Mdh* (malate dehydrogenase, Est* (esterase, and Sp* (sarcoplasmic protein. All species were in Hardy-weinberg equilibrium. Heterozygosities of species were ranged from 0.00 to 0.099. Yellowfin tuna has the highest heterozigosity compared with the other species. Clustering samples according to pairs revealed that genetic distance of Bullet tuna (A. rochei and Eastern little tuna (E. affinis had small value (0.001. By contrast, the largest value was observed between yellowfin tuna, T. albacares and rainbow runner, E. bipunnulata (0.007. This value indicated that Bullet tuna (A. rochei and Eastern little tuna (E. affinis closed relation, while among yellowfin tuna, skipjack tuna, and rainbow runner, were separated phylogenically.

  1. 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.

    2011-01-01

    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 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. PMID:22065761

  2. A novel strategy involved in [corrected] anti-oxidative defense: the conversion of NADH into NADPH by a metabolic network.

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    Ranji Singh

    Full Text Available The reduced nicotinamide adenine dinucleotide phosphate (NADPH is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH, a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC, malic enzyme (ME, malate dehydrogenase (MDH, malate synthase (MS, and isocitrate lyase (ICL that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK and the upregulation of pyruvate kinase (PK ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.

  3. Genetic variation in 15-hydroxyprostaglandin dehydrogenase and colon cancer susceptibility.

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    Cheryl L Thompson

    Full Text Available 15-Hydroxyprostaglandin dehydrogenase (15-PGDH is a metabolic antagonist of COX-2, catalyzing the degradation of inflammation mediator prostaglandin E2 (PGE2 and other prostanoids. Recent studies have established the 15-PGDH gene as a colon cancer suppressor.We evaluated 15-PDGH as a colon cancer susceptibility locus in a three-stage design. We first genotyped 102 single-nucleotide polymorphisms (SNPs in the 15-PGDH gene, spanning ∼50 kb up and down-stream of the coding region, in 464 colon cancer cases and 393 population controls. We then genotyped the same SNPs, and also assayed the expression levels of 15-PGDH in colon tissues from 69 independent patients for whom colon tissue and paired germline DNA samples were available. In the final stage 3, we genotyped the 9 most promising SNPs from stages 1 and 2 in an independent sample of 525 cases and 816 controls (stage 3.In the first two stages, three SNPs (rs1365611, rs6844282 and rs2332897 were statistically significant (p<0.05 in combined analysis of association with risk of colon cancer and of association with 15-PGDH expression, after adjustment for multiple testing. For one additional SNP, rs2555639, the T allele showed increased cancer risk and decreased 15-PGDH expression, but just missed statistical significance (p-adjusted = 0.063. In stage 3, rs2555639 alone showed evidence of association with an odds ratio (TT compared to CC of 1.50 (95% CI = 1.05-2.15, p = 0.026.Our data suggest that the rs2555639 T allele is associated with increased risk of colon cancer, and that carriers of this risk allele exhibit decreased expression of 15-PGDH in the colon.

  4. Very long-chain acyl-coenzyme A dehydrogenase deficiency

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    A. V. Degtyareva

    2014-01-01

    Full Text Available The paper describes a case of a baby with a severe infant form of very long-chain acyl-coenzyme A dehydrogenase deficiency, a very rare genetic disorder. The basis for the disease is a disorder of mitochondrial β-oxidation of long-chain fatty acids. Accumulation of acyl-CoA-derived fatty acids causes a toxic effect on the myocardium and cardiac conduction system, liver, skeletal muscles, and other organs. The development of hypoglycemia is typical. Treatment in the acute period involves the immediately ceased delivery of long-chain triglycerides, the provision of the body with medium-chain triglycerides, and the correction of glycemia. In our observation the baby was born at term with a satisfactory condition in a family with a poor history (the first baby had suddenly died at the age of 3,5 months. The disease manifested itself as bradyarrhythmia and cardiac arrest on day 2 of life. The clinical symptom complex also included hepatomegalia, hypoglycemic episodes, lactate acidosis, and elevated blood levels of cytolytic enzymes and creatine phosphokinase. The diagnosis was suspected on the basis of the high blood values of acylcarnitines (primarily C14:1 and verified by a molecular genetic examination. Syndrome therapy and dietotherapy resulted in the abolishment of the abnormality. At the age of 2 years of life, the infant’s physical, motor, mental, and speech development corresponded to his age although he had mild right-sided hemiparesis. Thus, timely therapy determines the favorable prognosis of the disease even in its severe infant forms. 

  5. Dysfunctional TCA-Cycle Metabolism in Glutamate Dehydrogenase Deficient Astrocytes.

    Science.gov (United States)

    Nissen, Jakob D; Pajęcka, Kamilla; Stridh, Malin H; Skytt, Dorte M; Waagepetersen, Helle S

    2015-12-01

    Astrocytes take up glutamate in the synaptic area subsequent to glutamatergic transmission by the aid of high affinity glutamate transporters. Glutamate is converted to glutamine or metabolized to support intermediary metabolism and energy production. Glutamate dehydrogenase (GDH) and aspartate aminotransferase (AAT) catalyze the reversible reaction between glutamate and α-ketoglutarate, which is the initial step for glutamate to enter TCA cycle metabolism. In contrast to GDH, AAT requires a concomitant interconversion of oxaloacetate and aspartate. We have investigated the role of GDH in astrocyte glutamate and glucose metabolism employing siRNA mediated knock down (KD) of GDH in cultured astrocytes using stable and radioactive isotopes for metabolic mapping. An increased level of aspartate was observed upon exposure to [U-(13) C]glutamate in astrocytes exhibiting reduced GDH activity. (13) C Labeling of aspartate and TCA cycle intermediates confirmed that the increased amount of aspartate is associated with elevated TCA cycle flux from α-ketoglutarate to oxaloacetate, i.e. truncated TCA cycle. (13) C Glucose metabolism was elevated in GDH deficient astrocytes as observed by increased de novo synthesis of aspartate via pyruvate carboxylation. In the absence of glucose, lactate production from glutamate via malic enzyme was lower in GDH deficient astrocytes. In conclusions, our studies reveal that metabolism via GDH serves an important anaplerotic role by adding net carbon to the TCA cycle. A reduction in GDH activity seems to cause the astrocytes to up-regulate activity in pathways involved in maintaining the amount of TCA cycle intermediates such as pyruvate carboxylation as well as utilization of alternate substrates such as branched chain amino acids. © 2015 Wiley Periodicals, Inc.

  6. Function of C-terminal hydrophobic region in fructose dehydrogenase

    International Nuclear Information System (INIS)

    Sugimoto, Yu; Kawai, Shota; Kitazumi, Yuki; Shirai, Osamu; Kano, Kenji

    2015-01-01

    Fructose dehydrogenase (FDH) catalyzes oxidation of D-fructose into 2-keto-D-fructose and is one of the enzymes allowing a direct electron transfer (DET)-type bioelectrocatalysis. FDH is a heterotrimeric membrane-bound enzyme (subunit I, II, and III) and subunit II has a C terminal hydrophobic region (CHR), which was expected to play a role in anchoring to membranes from the amino acid sequence. We have constructed a mutated FDH lacking of CHR (ΔchrFDH). Contrary to the expected function of CHR, ΔchrFDH is expressed in the membrane fraction, and subunit I/III subcomplex (ΔcFDH) is also expressed in a similar activity level but in the soluble fraction. In addition, the enzyme activity of the purified ΔchrFDH is about one twentieth of the native FDH. These results indicate that CHR is concerned with the binding between subunit I(/III) and subunit II and then with the enzyme activity. ΔchrFDH has clear DET activity that is larger than that expected from the solution activity, and the characteristics of the catalytic wave of ΔchrFDH are very similar to those of FDH. The deletion of CHR seems to increase the amounts of the enzyme with the proper orientation for the DET reaction at electrode surfaces. Gel filtration chromatography coupled with urea treatment shows that the binding in ΔchrFDH is stronger than that in FDH. It can be considered that the rigid binding between subunit I(/III) and II without CHR results in a conformation different from the native one, which leads to the decrease in the enzyme activity in solution

  7. Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Plapp, Bryce V.; Savarimuthu, Baskar Raj; Ferraro, Daniel J.; Rubach, Jon K.; Brown, Eric N.; Ramaswamy, S. (Iowa)

    2017-07-07

    During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2'-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme–NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ~1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD+ and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.

  8. Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis

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    Barretto O.C. de O.

    2006-01-01

    Full Text Available In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 ± 38 IU g-1 Hb-1 min-1 at 37ºC, compared to the human erythrocyte activity of 12 ± 2 IU g-1 Hb-1 min-1 at 37ºC. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa. The Michaelis-Menten constants (Km: 55 µM for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 µM were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively. A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.

  9. Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase.

    Science.gov (United States)

    Nisler, Jaroslav; Kopečný, David; Končitíková, Radka; Zatloukal, Marek; Bazgier, Václav; Berka, Karel; Zalabák, David; Briozzo, Pierre; Strnad, Miroslav; Spíchal, Lukáš

    2016-09-01

    Two new TDZ derivatives (HETDZ and 3FMTDZ) are very potent inhibitors of CKX and are promising candidates for in vivo studies. Cytokinin hormones regulate a wide range of essential processes in plants. Thidiazuron (N-phenyl-N'-1,2,3-thiadiazol-5-yl urea, TDZ), formerly registered as a cotton defoliant, is a well known inhibitor of cytokinin oxidase/dehydrogenase (CKX), an enzyme catalyzing the degradation of cytokinins. TDZ thus increases the lifetime of cytokinins and their effects in plants. We used in silico modeling to design, synthesize and characterize twenty new TDZ derivatives with improved inhibitory properties. Two compounds, namely 1-[1,2,3]thiadiazol-5-yl-3-(3-trifluoromethoxy-phenyl)urea (3FMTDZ) and 1-[2-(2-hydroxyethyl)phenyl]-3-(1,2,3-thiadiazol-5-yl)urea (HETDZ), displayed up to 15-fold lower IC 50 values compared with TDZ for AtCKX2 from Arabidopsis thaliana and ZmCKX1 and ZmCKX4a from Zea mays. Binding modes of 3FMTDZ and HETDZ were analyzed by X-ray crystallography. Crystal structure complexes, solved at 2.0 Å resolution, revealed that HETDZ and 3FMTDZ bound differently in the active site of ZmCKX4a: the thiadiazolyl ring of 3FMTDZ was positioned over the isoalloxazine ring of FAD, whereas that of HETDZ had the opposite orientation, pointing toward the entrance of the active site. The compounds were further tested for cytokinin activity in several cytokinin bioassays. We suggest that the combination of simple synthesis, lowered cytokinin activity, and enhanced inhibitory effects on CKX isoforms, makes 3FMTDZ and HETDZ suitable candidates for in vivo studies.

  10. Ebselen: Mechanisms of Glutamate Dehydrogenase and Glutaminase Enzyme Inhibition.

    Science.gov (United States)

    Yu, Yan; Jin, Yanhong; Zhou, Jie; Ruan, Haoqiang; Zhao, Han; Lu, Shiying; Zhang, Yue; Li, Di; Ji, Xiaoyun; Ruan, Benfang Helen

    2017-12-15

    Ebselen modulates target proteins through redox reactions with selenocysteine/cysteine residues, or through binding to the zinc finger domains. However, a recent contradiction in ebselen inhibition of kidney type glutaminase (KGA) stimulated our interest in investigating its inhibition mechanism with glutamate dehydrogenase (GDH), KGA, thioredoxin reductase (TrxR), and glutathione S-transferase. Fluorescein- or biotin-labeled ebselen derivatives were synthesized for mechanistic analyses. Biomolecular interaction analyses showed that only GDH, KGA, and TrxR proteins can bind to the ebselen derivative, and the binding to GDH and KGA could be competed off by glutamine or glutamate. From the gel shift assays, the fluorescein-labeled ebselen derivative could co-migrate with hexameric GDH and monomeric/dimeric TrxR in a dose-dependent manner; it also co-migrated with KGA but disrupted the tetrameric form of the KGA enzyme at a high compound concentration. Further proteomic analysis demonstrated that the ebselen derivative could cross-link with proteins through a specific cysteine at the active site of GDH and TrxR proteins, but for KGA protein, the binding site is at the N-terminal appendix domain outside of the catalytic domain, which might explain why ebselen is not a potent KGA enzyme inhibitor in functional assays. In conclusion, ebselen could inhibit enzyme activity by binding to the catalytic domain or disruption of the protein complex. In addition, ebselen is a relatively potent selective GDH inhibitor that might provide potential therapeutic opportunities for hyperinsulinism-hyperammonemia syndrome patients who have the mutational loss of GTP inhibition.

  11. Lactate dehydrogenase activity is inhibited by methylmalonate in vitro.

    Science.gov (United States)

    Saad, Laura O; Mirandola, Sandra R; Maciel, Evelise N; Castilho, Roger F

    2006-04-01

    Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism and oxidative stress. In the present work we studied the effect of MMA and two other inhibitors of mitochondrial respiratory chain complex II (malonate and 3-nitropropionate) on the activity of lactate dehydrogenase (LDH) in tissue homogenates from adult rats. MMA potently inhibited LDH-catalyzed conversion of lactate to pyruvate in liver and brain homogenates as well as in a purified bovine heart LDH preparation. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (K (i)=3.02+/-0.59 mM). Malonate and 3-nitropropionate also strongly inhibited LDH-catalyzed conversion of lactate to pyruvate in brain homogenates, while no inhibition was observed by succinate or propionate, when present in concentrations of up to 25 mM. We propose that inhibition of the lactate/pyruvate conversion by MMA contributes to lactate accumulation in blood, metabolic acidemia and inhibition of gluconeogenesis observed in patients with MMAemia. Moreover, the inhibition of LDH in the central nervous system may also impair the lactate shuttle between astrocytes and neurons, compromising neuronal energy metabolism.

  12. Acquired multiple Acyl-CoA dehydrogenase deficiency in 10 horses with atypical myopathy.

    Science.gov (United States)

    Westermann, C M; Dorland, L; Votion, D M; de Sain-van der Velden, M G M; Wijnberg, I D; Wanders, R J A; Spliet, W G M; Testerink, N; Berger, R; Ruiter, J P N; van der Kolk, J H

    2008-05-01

    The aim of the current study was to assess lipid metabolism in horses with atypical myopathy. Urine samples from 10 cases were subjected to analysis of organic acids, glycine conjugates, and acylcarnitines revealing increased mean excretion of lactic acid, ethylmalonic acid, 2-methylsuccinic acid, butyrylglycine, (iso)valerylglycine, hexanoylglycine, free carnitine, C2-, C3-, C4-, C5-, C6-, C8-, C8:1-, C10:1-, and C10:2-carnitine as compared with 15 control horses (12 healthy and three with acute myopathy due to other causes). Analysis of plasma revealed similar results for these predominantly short-chain acylcarnitines. Furthermore, measurement of dehydrogenase activities in lateral vastus muscle from one horse with atypical myopathy indeed showed deficiencies of short-chain acyl-CoA dehydrogenase (0.66 as compared with 2.27 and 2.48 in two controls), medium-chain acyl-CoA dehydrogenase (0.36 as compared with 4.31 and 4.82 in two controls) and isovaleryl-CoA dehydrogenase (0.74 as compared with 1.43 and 1.61 nmol min(-1) mg(-1) in two controls). A deficiency of several mitochondrial dehydrogenases that utilize flavin adenine dinucleotide as cofactor including the acyl-CoA dehydrogenases of fatty acid beta-oxidation, and enzymes that degrade the CoA-esters of glutaric acid, isovaleric acid, 2-methylbutyric acid, isobutyric acid, and sarcosine was suspected in 10 out of 10 cases as the possible etiology for a highly fatal and prevalent toxic equine muscle disease similar to the combined metabolic derangements seen in human multiple acyl-CoA dehydrogenase deficiency also known as glutaric acidemia type II.

  13. The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase

    Directory of Open Access Journals (Sweden)

    Rodrigues Valnês

    2009-01-01

    Full Text Available Abstract Background The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase. Findings We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1 is 68-fold larger than that for the mutant K69A (0.73 s-1. There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 μM; wild-type = 29 μM and NADPH (K69A = 30 μM; wild-type = 11 μM. The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4 μM and 134 (± 21, respectively. Conclusion Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.

  14. GENEL İMALAT MALİYETLERİNİN DAĞITIMINDA FAALİYET ESASINA DAYALI MALİYETLEME SİSTEMİ VE ÇEVRESEL MALİYETLERİN DEĞERLENDİRİLMESİ

    OpenAIRE

    Otlu, Fikret; Çukacı, Yusuf Cahit

    2010-01-01

    Özet: Bu çalışmada faaliyet esaslı maliyetleme sistemi ile gelenekselsistemlerin karşılaştırılması yapılmış olup, işletmelerin son yıllardakarşılaştıkları çevresel maliyetler hakkında bilgi verilerek, çevresel maliyetlerinbir genel imalat maliyeti olarak geleneksel ve faaliyet esasına dayalımaliyetleme sistemine göre dağıtımı bir örnek yardımıyla gösterilmiştir.Anahtar Kelimeler: Genel İmalat Maliyetleri, Geleneksel MaliyetlemeSistemleri, Faaliyet Esas...

  15. Technical Progress Report for UTSI/CFFF MDH Program Completion and Related Activity

    Energy Technology Data Exchange (ETDEWEB)

    None

    1998-04-01

    Routine preventive maintenance of the DOE Coal Fired Flow Facility (CFFF) is being performed. Modernization programs, funded under subcontract from Foster Wheeler Development Corporation by the DOE HIPPS Program, have been completed on the coal processing system, the data acquisition and control system and the control room. Environmental restoration actions continued with monitoring of groundwater wells and holding pond effluent. A plan for Groundwater Remediation was prepared, coordinated with DOE and approved by the State of Tennessee. Actions are underway to dispose of spent seed/ash mixtures and excess coal remaining from the MHD POC program. The status of five (5) High Temperature Superconductor (HTS) projects is reported.

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

    Science.gov (United States)

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-01-01

    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. PMID:23203056

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

    Directory of Open Access Journals (Sweden)

    Koji Sode

    2012-11-01

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

  18. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

    Science.gov (United States)

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

  19. Construction of mutant glucose oxidases with increased dye-mediated dehydrogenase activity.

    Science.gov (United States)

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-11-02

    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.

  20. A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD(+) regeneration.

    Science.gov (United States)

    Wang, Jizhong; Yang, Chengli; Chen, Xing; Bao, Bingxin; Zhang, Xuan; Li, Dali; Du, Xingfan; Shi, Ruofu; Yang, Junfang; Zhu, Ronghui

    2016-08-01

    To find an efficient and cheap system for NAD(+) regeneration A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD(+) regeneration was established. The system is attractive in that the O2 consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD(+) was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C CONCLUSION: The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD(+) regeneration.

  1. Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

    Science.gov (United States)

    Bomati, Erin K; Noel, Joseph P

    2005-05-01

    We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

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

    Directory of Open Access Journals (Sweden)

    Maira Kussainova

    2013-01-01

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

  3. Plasma Lactate Dehydrogenase Levels Predict Mortality in Acute Aortic Syndromes

    Science.gov (United States)

    Morello, Fulvio; Ravetti, Anna; Nazerian, Peiman; Liedl, Giovanni; Veglio, Maria Grazia; Battista, Stefania; Vanni, Simone; Pivetta, Emanuele; Montrucchio, Giuseppe; Mengozzi, Giulio; Rinaldi, Mauro; Moiraghi, Corrado; Lupia, Enrico

    2016-01-01

    Abstract In acute aortic syndromes (AAS), organ malperfusion represents a key event impacting both on diagnosis and outcome. Increased levels of plasma lactate dehydrogenase (LDH), a biomarker of malperfusion, have been reported in AAS, but the performance of LDH for the diagnosis of AAS and the relation of LDH with outcome in AAS have not been evaluated so far. This was a bi-centric prospective diagnostic accuracy study and a cohort outcome study. From 2008 to 2014, patients from 2 Emergency Departments suspected of having AAS underwent LDH assay at presentation. A final diagnosis was obtained by aortic imaging. Patients diagnosed with AAS were followed-up for in-hospital mortality. One thousand five hundred seventy-eight consecutive patients were clinically eligible, and 999 patients were included in the study. The final diagnosis was AAS in 201 (20.1%) patients. Median LDH was 424 U/L (interquartile range [IQR] 367–557) in patients with AAS and 383 U/L (IQR 331–460) in patients with alternative diagnoses (P < 0.001). Using a cutoff of 450 U/L, the sensitivity of LDH for AAS was 44% (95% confidence interval [CI] 37–51) and the specificity was 73% (95% CI 69–76). Overall in-hospital mortality for AAS was 23.8%. Mortality was 32.6% in patients with LDH ≥ 450 U/L and 16.8% in patients with LDH < 450 U/L (P = 0.006). Following stratification according to LDH quartiles, in-hospital mortality was 12% in the first (lowest) quartile, 18.4% in the second quartile, 23.5% in the third quartile, and 38% in the fourth (highest) quartile (P = 0.01). LDH ≥ 450 U/L was further identified as an independent predictor of death in AAS both in univariate and in stepwise logistic regression analyses (odds ratio 2.28, 95% CI 1.11–4.66; P = 0.025), in addition to well-established risk markers such as advanced age and hypotension. Subgroup analysis showed excess mortality in association with LDH ≥ 450 U/L in elderly, hemodynamically stable

  4. Glucose-6-phosphate dehydrogenase deficiency in Nigerian children.

    Directory of Open Access Journals (Sweden)

    Olatundun Williams

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD deficiency is the most common human enzymopathy and in Sub-Saharan Africa, is a significant cause of infection- and drug-induced hemolysis and neonatal jaundice. Our goals were to determine the prevalence of G6PD deficiency among Nigerian children of different ethnic backgrounds and to identify predictors of G6PD deficiency by analyzing vital signs and hematocrit and by asking screening questions about symptoms of hemolysis. We studied 1,122 children (561 males and 561 females aged 1 month to 15 years. The mean age was 7.4 ± 3.2 years. Children of Yoruba ethnicity made up the largest group (77.5% followed by those Igbo descent (10.6% and those of Igede (10.2% and Tiv (1.8% ethnicity. G6PD status was determined using the fluorescent spot method. We found that the overall prevalence of G6PD deficiency was 15.3% (24.1% in males, 6.6% in females. Yoruba children had a higher prevalence (16.9% than Igede (10.5%, Igbo (10.1% and Tiv (5.0% children. The odds of G6PD deficiency were 0.38 times as high in Igbo children compared to Yoruba children (p=0.0500. The odds for Igede and Tiv children were not significantly different from Yoruba children (p=0.7528 and 0.9789 respectively. Mean oxygen saturation, heart rate and hematocrit were not significantly different in G6PD deficient and G6PD sufficient children. The odds of being G6PD deficient were 2.1 times higher in children with scleral icterus than those without (p=0.0351. In conclusion, we determined the prevalence of G6PD deficiency in Nigerian sub-populations. The odds of G6PD deficiency were decreased in Igbo children compared to Yoruba children. There was no association between vital parameters or hematocrit and G6PD deficiency. We found that a history of scleral icterus may increase the odds of G6PD deficiency, but we did not exclude other common causes of icterus such as sickle cell disease or malarial infection.

  5. InterProScan Result: BJ984559 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available 557 L-lactate/malate dehydrogenase Biological Process: glycolysis (GO:0006096)|Biological Process: cellular ...carbohydrate metabolic process (GO:0044262)|Biological Process: oxidation reduction (GO:0055114) ...

  6. Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

    International Nuclear Information System (INIS)

    Castro, Miguel E.; Molina, Roberto C.; Diaz, Waldo A.; Pradenas, Gonzalo A.; Vasquez, Claudio C.

    2009-01-01

    Potassium tellurite (K 2 TeO 3 ) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.

  7. Construction of an integrated enzyme system consisting azoreductase and glucose 1-dehydrogenase for dye removal.

    Science.gov (United States)

    Yang, Yuyi; Wei, Buqing; Zhao, Yuhua; Wang, Jun

    2013-02-01

    Azo dyes are toxic and carcinogenic and are often present in industrial effluents. In this research, azoreductase and glucose 1-dehydrogenase were coupled for both continuous generation of the cofactor NADH and azo dye removal. The results show that 85% maximum relative activity of azoreductase in an integrated enzyme system was obtained at the conditions: 1U azoreductase:10U glucose 1-dehydrogenase, 250mM glucose, 1.0mM NAD(+) and 150μM methyl red. Sensitivity analysis of the factors in the enzyme system affecting dye removal examined by an artificial neural network model shows that the relative importance of enzyme ratio between azoreductase and glucose 1-dehydrogenase was 22%, followed by dye concentration (27%), NAD(+) concentration (23%) and glucose concentration (22%), indicating none of the variables could be ignored in the enzyme system. Batch results show that the enzyme system has application potential for dye removal. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Inactivation of pyruvate dehydrogenase kinase 2 by mitochondrial reactive oxygen species.

    Science.gov (United States)

    Hurd, Thomas R; Collins, Yvonne; Abakumova, Irina; Chouchani, Edward T; Baranowski, Bartlomiej; Fearnley, Ian M; Prime, Tracy A; Murphy, Michael P; James, Andrew M

    2012-10-12

    Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H(2)O(2)) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H(2)O(2) derives from superoxide (O(2)(.)), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O(2)(.) production, such as may occur under nutrient excess and low ATP demand, the increase in O(2)() and H(2)O(2) may provide feedback signals to modulate mitochondrial metabolism.

  9. Homology modelling and docking analysis of L-lactate dehydrogenase from Streptococcus thermopilus

    Directory of Open Access Journals (Sweden)

    Vukić Vladimir R.

    2016-01-01

    Full Text Available The aim of this research was to create a three-dimensional model of L-lactate dehydrogenase from the main yoghurt starter culture - Streptococcus thermopilus, to analyse its structural features and investigate substrate binding in the active site. NCBI BlastP was used against the Protein Data Bank database in order to identify the template for construction of homology models. Multiple sequence alignment was performed using the program MUSCULE within the UGENE 1.11.3 program. Homology models were constructed using the program Modeller v. 9.17. The obtained 3D model was verified by Ramachandran plots. Molecular docking simulations were performed using the program Surflex-Dock. The highest sequence similarity was observed with L-lactate dehydrogenase from Lactobacillus casei subsp. casei, with 69% identity. Therefore, its structure (PDB ID: 2ZQY:A was selected as a modelling template for homology modelling. Active residues are by sequence similarity predicted: S. thermophilus - HIS181 and S. aureus - HIS179. Binding energy of pyruvate to L-lactate dehydrogenase of S. thermopilus was - 7.874 kcal/mol. Pyruvate in L-lactate dehydrogenase of S. thermopilus makes H bonds with catalytic HIS181 (1.9 Å, as well as with THR235 (3.6 Å. Although our results indicate similar position of substrates between L-lactate dehydrogenase of S. thermopilus and S. aureus, differences in substrate distances and binding energy values could influence the reaction rate. Based on these results, the L-lactate dehydrogenase model proposed here could be used as a guide for further research, such as transition states of the reaction through molecular dynamics. [Projekat Ministarstva nauke Republike Srbije, br. III 46009

  10. Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

    Science.gov (United States)

    Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

    2015-12-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. 2-methylbutyryl-CoA dehydrogenase deficiency associated with autism and mental retardation

    DEFF Research Database (Denmark)

    Kanavin, Oivind J; Woldseth, Berit; Jellum, Egil

    2007-01-01

    BACKGROUND: 2-methylbutyryl-CoA dehydrogenase deficiency or short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) is caused by a defect in the degradation pathway of the amino acid L-isoleucine. METHODS: We report a four-year-old mentally retarded Somali boy with autism and a history...... cases with SBCADD, both originating from Somalia and Eritrea, indicating that it is relatively prevalent in this population. Autism has not previously been described with mutations in this gene, thus expanding the clinical spectrum of SBCADD....

  12. 2-methylbutyryl-CoA dehydrogenase deficiency associated with autism and mental retardation: a case report

    DEFF Research Database (Denmark)

    Kanavin, Øjvind; Woldseth, Berit; Jellum, Egil

    2007-01-01

    previously reported cases with SBCADD, both originating from Somalia and Eritrea, indicating that it is relatively prevalent in this population. Autism has not previously been described with mutations in this gene, thus expanding the clinical spectrum of SBCADD. PMID: 17883863 [PubMed - in process]......ABSTRACT: BACKGROUND: 2-methylbutyryl-CoA dehydrogenase deficiency or short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) is caused by a defect in the degradation pathway of the amino acid L-isoleucine. METHODS: We report a four-year-old mentally retarded Somali boy with autism...

  13. Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes.

    Science.gov (United States)

    Baggetto, L G; Lehninger, A L

    1987-05-29

    Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented.

  14. Studies on meat color, myoglobin content, enzyme activities, and genes associated with oxidative potential of pigs slaughtered at different growth stages

    Directory of Open Access Journals (Sweden)

    Qin Ping Yu

    2017-12-01

    Full Text Available Objective This experiment investigated meat color, myoglobin content, enzyme activities, and expression of genes associated with oxidative potential of pigs slaughtered at different growth stages. Methods Sixty 4-week-old Duroc×Landrace×Yorkshire pigs were assigned to 6 replicate groups, each containing 10 pigs. One pig from each group was sacrificed at day 35, 63, 98, and 161 to isolate longissimus dorsi and triceps muscles. Results Meat color scores were higher in pigs at 35 d than those at 63 d and 98 d (p<0.05, and those at 98 d were lower than those at 161 d (p<0.05. The total myoglobin was higher on 161 d compared with those at 63 d and 98 d (p<0.05. Increase in the proportions of metmyoglobin and deoxymyoglobin and a decrease in oxymyoglobin were observed between days 35 and 161 (p<0.05. Meat color scores were correlated to the proportion of oxymyoglobin (r = 0.59, p<0.01, and negatively correlated with deoxymyoglobin and metmyoglobin content (r = −0.48 and −0.62, p<0.05. Malate dehydrogenase (MDH activity at 35 d and 98 d was higher than that at 161 d (p<0.05. The highest lactate dehydrogenase/MDH ratio was achieved at 161 d (p<0.05. Calcineurin mRNA expression decreased at 35 d compared to that at 63 d and 98 d (p<0.05. Myocyte enhancer factor 2 mRNA results indicated a higher expression at 161 d than that at 63 d and 98 d (p<0.05. Conclusion Porcine meat color, myoglobin content, enzyme activities, and genes associated with oxidative potential varied at different stages.

  15. Identification of the human mitochondrial FAD transporter and its potential role in multiple acyl-CoA dehydrogenase deficiency

    NARCIS (Netherlands)

    Spaan, András N.; Ijlst, Lodewijk; van Roermund, Carlo W. T.; Wijburg, Frits A.; Wanders, Ronald J. A.; Waterham, Hans R.

    2005-01-01

    Multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric aciduria type II (GAII) is most often caused by mutations in the genes encoding the alpha- or beta-subunit of electron transfer flavoprotein (ETF) or electron transfer flavoprotein dehydrogenase (ETF-DH). Since not all patients have

  16. Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds

    OpenAIRE

    Wisselink, H. Wouter; Mars, Astrid E.; van der Meer, Pieter; Eggink, Gerrit; Jeroen Hugenholtz

    2004-01-01

    To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and 13C nuclear magnetic resonance analysis revealed that small amounts (

  17. Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds

    NARCIS (Netherlands)

    Wisselink, H.W.; Mars, A.E.; Meer, van der P.; Eggink, G.; Hugenholtz, J.

    2004-01-01

    To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance

  18. Kinetic and modelling studies of NAD+ and poly(ethylene glycol)-bound NAD+ in horse liver alcohol dehydrogenase

    NARCIS (Netherlands)

    Vanhommerig, S.A.M.; Sluyterman, L.A.A.E.; Meijer, E.M.

    1996-01-01

    Poly(ethylene glycol)-bound nicotinamide adenine dinucleotide (PEG-NAD+) has been successfully employed in the continuous production of L-amino acids from the corresponding alpha-keto acids by stereospecific reductive amination. Like many other dehydrogenases also horse liver alcohol dehydrogenase

  19. X-ray crystal structure and small-angle X-ray scattering of sheep liver sorbitol dehydrogenase

    DEFF Research Database (Denmark)

    Yennawar, Hemant; Møller, Magda; Gillilan, Richard

    2011-01-01

    The X-ray crystal structure of sheep liver sorbitol dehydrogenase (slSDH) has been determined using the crystal structure of human sorbitol dehydrogenase (hSDH) as a molecular-replacement model. slSDH crystallized in space group I222 with one monomer in the asymmetric unit. A conserved tetramer...

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

    NARCIS (Netherlands)

    Berg, A.

    1997-01-01

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

  1. Lactate dehydrogenase has no control on lactate production but has a strong negative control on formate production in Lactococcus lactis

    DEFF Research Database (Denmark)

    Andersen, H.W.; Pedersen, M.B.; Hammer, Karin

    2001-01-01

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

  2. Conversion of xanthine dehydrogenase into xanthine oxidase in rat liver and plasma at the onset of reperfusion after ischemia

    NARCIS (Netherlands)

    Kooij, A.; Schiller, H. J.; Schijns, M.; van Noorden, C. J.; Frederiks, W. M.

    1994-01-01

    The aim of this study was to test whether conversion of xanthine dehydrogenase into xanthine oxidase as induced by fasting, ischemia of the liver or both is an in vivo process or only occurs in vitro in homogenates. For this purpose, the conversion rate of xanthine dehydrogenase into xanthine

  3. Glucose-6-phosphate dehydrogenase activity decreases during storage of leukoreduced red blood cells

    NARCIS (Netherlands)

    Peters, Anna L.; van Bruggen, Robin; de Korte, Dirk; van Noorden, Cornelis J. F.; Vlaar, Alexander P. J.

    2016-01-01

    During storage, the activity of the red blood cell (RBC) antioxidant system decreases. Glucose-6-phosphate dehydrogenase (G6PD) is essential for protection against oxidative stress by producing NADPH. G6PD function of RBC transfusion products is reported to remain stable during storage, but activity

  4. Monitoring of fatty aldehyde dehydrogenase by formation of pyrenedecanoic acid from pyrenedecanal

    NARCIS (Netherlands)

    Keller, Markus A.; Watschinger, Katrin; Golderer, Georg; Maglione, Manuel; Sarg, Bettina; Lindner, Herbert H.; Werner-Felmayer, Gabriele; Terrinoni, Alessandro; Wanders, Ronald J. A.; Werner, Ernst R.

    2010-01-01

    Fatty aldehyde dehydrogenase (EC 1.2.1.48) converts long-chain fatty aldehydes to the corresponding acids. Deficiency in this enzyme causes the Sjogren Larsson Syndrome, a rare inherited disorder characterized by ichthyosis, spasticity, and mental retardation. Using a fluorescent aldehyde,

  5. 2-ethylhydracrylic aciduria in short/branched-chain acyl-CoA dehydrogenase deficiency

    DEFF Research Database (Denmark)

    Korman, Stanley H; Andresen, Brage S; Zeharia, Avraham

    2005-01-01

    BACKGROUND: Isolated excretion of 2-methylbutyrylglycine (2-MBG) is the hallmark of short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD), a recently identified defect in the proximal pathway of L-isoleucine oxidation. SBCADD might be underdiagnosed because detection and recognition...

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

    DEFF Research Database (Denmark)

    Nellemann, B.; Vendelbo, M.H.; Nielsen, Thomas Svava

    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....

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

    Directory of Open Access Journals (Sweden)

    Gideon C. Okpokwasili

    2010-04-01

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

  8. The Alcohol Dehydrogenase Kinetics Laboratory: Enhanced Data Analysis and Student-Designed Mini-Projects

    Science.gov (United States)

    Silverstein, Todd P.

    2016-01-01

    A highly instructive, wide-ranging laboratory project in which students study the effects of various parameters on the enzymatic activity of alcohol dehydrogenase has been adapted for the upper-division biochemistry and physical biochemistry laboratory. Our two main goals were to provide enhanced data analysis, featuring nonlinear regression, and…

  9. Posttranslational regulation of glucose-6-phosphate dehydrogenase activity in tongue epithelium

    NARCIS (Netherlands)

    Biagiotti, E.; Bosch, K. S.; Ninfali, P.; Frederiks, W. M.; van Noorden, C. J.

    2000-01-01

    Expression of glucose-6-phosphate dehydrogenase (G6PD) activity is high in tongue epithelium, but its exact function is still unknown, it may be related;either to the high proliferation rate of this tissue or to protection against oxidative stress. To elucidate its exact role, we localized

  10. Control of Glycolysis by Glyceraldehyde-3-Phosphate Dehydrogenase in Streptococcus cremoris and Streptococcus lactis

    NARCIS (Netherlands)

    POOLMAN, B; BOSMAN, B; KONINGS, WN

    1987-01-01

    The decreased response of the energy metabolism of lactose-starved Streptococcus cremoris upon readdition of lactose is caused by a decrease of the glycolytic activity. The decrease in glycolysis is accompanied by a decrease in the activities of glyceraldehyde-3-phosphate dehydrogenase and

  11. Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans

    DEFF Research Database (Denmark)

    Nguyen, Tien V; Andresen, Brage S; Corydon, Thomas J

    2002-01-01

    -CoA dehydrogenase. A single patient has previously been described whose fibroblasts exhibit a specific deficit in the oxidation of valine. Amplified ACAD8 cDNA made from patient fibroblast mRNA was homozygous for a single nucleotide change (905G>A) in the ACAD8 coding region compared to the sequence from control...... in a patient....

  12. Clinical variability in 3-hydroxy-2-methylbutyryl-CoA dehydrogenase deficiency

    NARCIS (Netherlands)

    Ensenauer, Regina; Niederhoff, Helmut; Ruiter, Jos P. N.; Wanders, Ronald J. A.; Schwab, K. Otfried; Brandis, Matthias; Lehnert, Willy

    2002-01-01

    We report the identification of two new 7-year-old patients with 3-hydroxy-2-methylbutyryl-CoA dehydrogenase deficiency, a recently described inborn error of isoleucine metabolism. The defect is localized one step above 3-ketothiolase, resulting in a urinary metabolite pattern similar to that seen

  13. Tissue carnitine homeostasis in very-long-chain acyl-CoA dehydrogenase-deficient mice

    NARCIS (Netherlands)

    Spiekerkoetter, Ute; Tokunaga, Chonan; Wendel, Udo; Mayatepek, Ertan; Ijlst, Lodewijk; Vaz, Frederic M.; van Vlies, Naomi; Overmars, Henk; Duran, Marinus; Wijburg, Frits A.; Wanders, Ronald J.; Strauss, Arnold W.

    2005-01-01

    Deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD) is the most common long-chain fatty acid oxidation defect and presents with heterogeneous clinical manifestations. Accumulation of long-chain acylcarnitines and deficiency of free carnitine have often been proposed to play an important

  14. Effects of sh-reagents on rat hepatic aldehyde dehydrogenase activity

    Energy Technology Data Exchange (ETDEWEB)

    Konoplitskaya, K.L.; Kuz' mina, G.I.; Grigor' yeva, M.V.; Poznyakova, T.N.

    The liver serves as the primary organ for the oxidation of ingested ethanol via a pathway involving alcohol- and aldehyde dehydrogenase. In view of the problem of alcoholism, three enzymes are of particular interest in understanding the biochemical mechanism that may be involved in alcohol addiction and in the formulation of therapeutic approaches. While alcohol dehydrogenase has been studied in considerable detail, current attention is centered on aldehyde dehydrogenase. A comparative analysis of the effects of a series of SH-active reagents - tetraethylthiuram disulfide (TETD), 5,5-dithiobisnitrobenzoic acid (DTNB), p-chloromercurybenzoate (PCMB), and N-ethylmaleimide (NEM) - were tested for their effects on the activity of aldehyde dehydrogenase of the hepatic mitochondrial (isozymes I and II) and microsomal (isozyme II) fractions of outbred albino rats. DTNB was found to be inhibited by 100 and 50% mitochondrial isozymes I and II, respectively, and by 20%, the microsomal enzyme under the conditions employed. DTNB and NEM inhibited by 30 and 50% isozymes I and II of the mitochondria, but had no effect on the microsomal isozyme. 24 references, 3 figures.

  15. Magnetic resonance and fluorescence studies on pyruvate dehydrogenase complexes and their small molecular weight constituents

    NARCIS (Netherlands)

    Grande, H.J.

    1976-01-01

    The articles presented in this thesis do not describe at first glance one well-defined subject. They are, however, in fact connected by one central theme: the study of large enzyme aggregates by molecular physical methods. Chosen was the pyruvate dehydrogenase complex (PDC) because of its

  16. The Effects of Fenarimol and Methyl Parathion on Glucose 6-Phosphate Dehydrogenase Enzyme Activity in Rats

    Directory of Open Access Journals (Sweden)

    Ferda ARI

    2017-10-01

    Full Text Available Fenarimol and methyl parathion are pesticides that have been used in agriculture for several years. These pesticides have significant effects on environmental and human health. Therefore, we investigated the effects of methyl parathion and fenarimol on glucose 6-phosphate dehydrogenase (EC 1.1.1.49 enzyme activity in rats. The glucose 6- phosphate dehydrogenase is the first enzyme of the pentose phosphate pathway and it is important in detoxifying reactions by NADPH generated. In this study, wistar albino rats administrated with methyl parathion (7 mg kg–1 and fenarimol (200 mg kg−1 by intraperitoneally for different periods (2, 4, 8, 16, 32, 64, and 72 h. The glucose 6-phosphate dehydrogenase enzyme activity was assayed in liver, kidney, brain, and small intestine in male and female rats. The exposure of fenarimol and methyl parathion caused increase of glucose 6-phosphate dehydrogenase enzyme activity in rat tissues, especially at last periods. We suggest that this increment of enzyme activity may be the reason of toxic effects of fenarimol and methyl parathion.

  17. Clinical aspects of short-chain acyl-CoA dehydrogenase deficiency

    NARCIS (Netherlands)

    Maldegem, B.T.; Wanders, R.J.A.; Wijburg, F.A.

    2010-01-01

    Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation. SCADD is biochemically characterized by increased C4-carnitine in plasma and ethylmalonic acid in urine. The diagnosis of SCADD is confirmed by DNA analysis showing

  18. Cytokinin oxidase/dehydrogenase genes in barley and wheat. Cloning and heterologous expression

    Czech Academy of Sciences Publication Activity Database

    Galuszka, P.; Frébortová, Jitka; Werner, T.; Yamada, M.; Strnad, Miroslav; Schmülling, T.; Frébort, I.

    2004-01-01

    Roč. 271, č. 20 (2004), s. 3990-4002 ISSN 0014-2956 Institutional research plan: CEZ:AV0Z5038910 Keywords : cereals * cloning * cytokinin oxidase/dehydrogenase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.260, year: 2004

  19. Assessing the stereoselectivity of Serratia marcescens CECT 977 2,3-butanediol dehydrogenase

    NARCIS (Netherlands)

    Medici, R.; Stammes, J.K.; Otten, L.G.; Hanefeld, U.; Kwakernaak, Stender

    2017-01-01

    α-Hydroxy ketones and vicinal diols constitute well-known building blocks in organic synthesis. Here we describe one enzyme that enables the enantioselective synthesis of both building blocks starting from diketones. The enzyme 2,3-butanediol dehydrogenase (BudC) from S. marcescens CECT 977 belongs

  20. Characterisation of recombinant human fatty aldehyde dehydrogenase: implications for Sjögren-Larsson syndrome

    NARCIS (Netherlands)

    Lloyd, Matthew D.; Boardman, Kieren D. E.; Smith, Andrew; van den Brink, Daan M.; Wanders, Ronald J. A.; Threadgill, Michael D.

    2007-01-01

    Fatty aldehyde dehydrogenase (FALDH) is an NAD+-dependent oxidoreductase involved in the metabolism of fatty alcohols. Enzyme activity has been implicated in the pathology of diabetes and cancer. Mutations in the human gene inactivate the enzyme and cause accumulation of fatty alcohols in

  1. Application of NAD(P)H oxidase for cofactor regeneration in dehydrogenase catalyzed oxidations

    DEFF Research Database (Denmark)

    Rehn, Gustav; Pedersen, Asbjørn Toftgaard; Woodley, John

    2016-01-01

    alcohol dehydrogenases. However, their effective use requires an effective regeneration of the oxidized nicotinamide cofactor (NAD(P)+), which is critical for the economic feasibility of the process. NAD(P)H oxidase is an enzyme class of particular interest for this cofactor regeneration since it enables...

  2. A new dawn for plant mitochondrial NAD(P)H dehydrogenases

    DEFF Research Database (Denmark)

    Møller, I.M.

    2002-01-01

    The expression of complex I and two homologues of bacterial and yeast NADH dehydrogenases, NDA and NDB, have been studied in potato leaf mitochondria. The mRNA level of NDA is completely light dependent and shows a diurnal rhythm with a sharp maximum just after dawn. NDA protein quantity and inte...

  3. Struktuur en interaktie analyse van NAD+ en NAD+ analoga in horse liver alcohol dehydrogenase

    NARCIS (Netherlands)

    Beijer, N.A.

    1988-01-01

    Dit verslag beschrijft een studie naar de relatie tussen struktuur en funktie voor het co-enzym NAn+ en zijn analoga in de aktieve holte van het enzym Horse Liver Alcohol Dehydrogenase (LADH). De rol van NAD+ in enzymgekatalyseerde oxidatie-reduktie reakties is die van het bewerkstelligen van een

  4. Biochemical and cytochemical evaluation of heterozygote individuals with glucose-6-phosphate dehydrogenase deficiency

    NARCIS (Netherlands)

    Gurbuz, Nilgun; Aksu, Tevfik Aslan; van Noorden, Cornelis J. F.

    2005-01-01

    The aim of this study was to diagnose heterozygous glucose-6-phosphate dehydrogenase (G6PD) deficient females by an inexpensive cytochemical G6PD staining method that is easy to perform, allowing diagnosis of G6PD deficiency without cumbersome genetic analysis. Three subject groups were included in

  5. 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

  6. Engineering of Class II Cellobiose Dehydrogenases for Improved Glucose Sensitivity and Reduced Maltose Affinity

    DEFF Research Database (Denmark)

    Ortiz, Roberto; Rahman, Mahbubur; Zangrilli, Beatrice

    2017-01-01

    The front cover artwork is provided by Prof. Lo Gorton from Lund University (Sweden) and his co-workers. The image shows mutated cellobiose dehydrogenase (CDH) immobilized on a graphite electrode and how preferentially glucose is oxidized by this enzyme. Read the full text of the Article at 10.1002...

  7. Transgenic barley overexpressing a cytokinin dehydrogenase gene shows greater tolerance to drought stress

    Czech Academy of Sciences Publication Activity Database

    Pospíšilová, H.; Jiskrová, E.; Vojta, P.; Mrízová, K.; Kokáš, F.; Majeská Čudějková, M.; Bergougnoux, V.; Plíhal, O.; Klimešová, J.; Novák, Ondřej; Dzurová, L.; Frébort, I.; Galuszka, P.

    2016-01-01

    Roč. 33, č. 5 (2016), s. 692-705 ISSN 1871-6784 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : ROOT-GROWTH * OXIDASE/DEHYDROGENASE GENES * BETA-GLUCOSIDASE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.813, year: 2016

  8. Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans

    DEFF Research Database (Denmark)

    Zameitat, E.; Gojkovic, Zoran; Knecht, Wolfgang

    2006-01-01

    Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine...

  9. Determination of Aldehyde Dehydrogenase (ALDH Isozymes in Human Cancer Samples - Comparison of Kinetic and Immunochemical Assays

    Directory of Open Access Journals (Sweden)

    Dorota Borecka

    2002-12-01

    Full Text Available A fluorimetric assay of aldehyde dehydrogenase isozymes, based on naphthaldehyde oxidation, is compared with Western Blotting analysis on several clinical samples obtained from surgery. The comparison reveals qualitatively good correlation of ALDH1A1 isozyme detection with two methods and somewhat worse on ALDH3A1 assay.

  10. Kinetic and chemical analyses of the cytokinin dehydrogenase-catalysed reaction: correlations with the crystal structure

    Czech Academy of Sciences Publication Activity Database

    Popelková, H.; Fraaije, M. W.; Novák, Ondřej; Frébortová, Jitka; Bilyeu, K. D.; Frébort, I.

    2006-01-01

    Roč. 398, č. 1 (2006), s. 113-124 ISSN 0264-6021 Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinin * cytokinin dehydrogenase (CKX) * flavoprotein Subject RIV: CE - Biochemistry Impact factor: 4.100, year: 2006

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  12. Galactonolactone Dehydrogenase Requires a Redox-Sensitive Thiol for Optimal Production of Vitamin C1.

    NARCIS (Netherlands)

    Leferink, N.G.H.; Duijn, van E.; Barendregt, A.; Heck, A.J.R.; Berkel, van W.J.H.

    2009-01-01

    The mitochondrial flavoenzyme L-galactono--lactone dehydrogenase (GALDH) catalyzes the ultimate step of vitamin C biosynthesis in plants. We found that recombinant GALDH from Arabidopsis (Arabidopsis thaliana) is inactivated by hydrogen peroxide due to selective oxidation of cysteine (Cys)-340,

  13. l-Galactono-gamma-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis.

    NARCIS (Netherlands)

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

    2008-01-01

    l-Galactono-1,4-lactone dehydrogenase (GALDH; ferricytochrome c oxidoreductase; EC 1.3.2.3) is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C (l-ascorbic acid) in plants. In the present study, we report on the biochemical properties of recombinant

  14. Newborn screening for dihydrolipoamide dehydrogenase deficiency: Citrulline as a useful analyte

    Directory of Open Access Journals (Sweden)

    Shane C. Quinonez

    2014-01-01

    Full Text Available Dihydrolipoamide dehydrogenase deficiency, also known as maple syrup urine disease (MSUD type III, is caused by the deficiency of the E3 subunit of branched chain alpha-ketoacid dehydrogenase (BCKDH, α-ketoglutarate dehydrogenase (αKGDH, and pyruvate dehydrogenase (PDH. DLD deficiency variably presents with either a severe neonatal encephalopathic phenotype or a primarily hepatic phenotype. As a variant form of MSUD, it is considered a core condition recommended for newborn screening. The detection of variant MSUD forms has proven difficult in the past with no asymptomatic DLD deficiency patients identified by current newborn screening strategies. Citrulline has recently been identified as an elevated dried blood spot (DBS metabolite in symptomatic patients affected with DLD deficiency. Here we report the retrospective DBS analysis and second-tier allo-isoleucine testing of 2 DLD deficiency patients. We show that an elevated citrulline and an elevated allo-isoleucine on second-tier testing can be used to successfully detect DLD deficiency. We additionally recommend that DLD deficiency be included in the “citrullinemia/elevated citrulline” ACMG Act Sheet and Algorithm.

  15. Alcohol dehydrogenase 3 genotype as a risk factor for upper aerodigestive tract cancers

    DEFF Research Database (Denmark)

    Nishimoto, Inês Nobuko; Pinheiro, Nidia A; Rogatto, Silvia R

    2004-01-01

    OBJECTIVE: To assess alcohol dehydrogenase 3 (ADH3) polymorphism at position Ile349Val as indicator of risk factor for upper aerodigestive tract (UADT) cancer to verify its association with UADT cancer in nonalcoholic or nonsmoking individuals. DESIGN: Cross-sectional study. SETTING: Primary care...

  16. Dihydropyrimidine Dehydrogenase Deficiency in Two Malaysian Siblings with Abnormal MRI Findings

    NARCIS (Netherlands)

    Chen, Bee Chin; Mohd Rawi, Rowani; Meinsma, Rutger; Meijer, Judith; Hennekam, Raoul C. M.; van Kuilenburg, André B. P.

    2014-01-01

    Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disorder of the pyrimidine metabolism. Deficiency of this enzyme leads to an accumulation of thymine and uracil and a deficiency of metabolites distal to the catabolic enzyme. The disorder presents with a wide clinical

  17. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase assay. 864.7360 Section 864.7360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages...

  18. Isolation and characterization of two cDNA clones encoding for glutamate dehydrogenase in Nicotiana plumbaginifolia.

    Science.gov (United States)

    Ficarelli, A; Tassi, F; Restivo, F M

    1999-03-01

    We have isolated two full length cDNA clones encoding Nicotiana plumbaginifolia NADH-glutamate dehydrogenase. Both clones share amino acid boxes of homology corresponding to conserved GDH catalytic domains and putative mitochondrial targeting sequence. One clone shows a putative EF-hand loop. The level of the two transcripts is affected differently by carbon source.

  19. Correlation of viral RNA biosynthesis with glucose-6-phosphate dehydrogenase activity and host resistance

    Czech Academy of Sciences Publication Activity Database

    Šindelář, Luděk; Šindelářová, Milada

    2002-01-01

    Roč. 215, - (2002), s. 862-869 ISSN 0032-0935 R&D Projects: GA ČR GA522/99/1264 Institutional research plan: CEZ:AV0Z5038910 Keywords : Glucose 6 phosphate dehydrogenase * Nicotiana (viral infection) * Plant viruses Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.960, year: 2002

  20. Fecal hydroxysteroid dehydrogenase activities in vegetarian Seventh-Day Adventists, control subjects, and bowel cancer patients.

    Science.gov (United States)

    Macdonald, I A; Webb, G R; Mahony, D E

    1978-10-01

    Cell-free extracts were prepared from mixed fecal anaerobic bacteria grown from stools of 14 vegetarian Seventh-Day Adventists, 16 omnivorous control subjects, and eight patients recently diagnosed with cancer of the large bowel. Preparations were assayed for NAD- and NADP-dependent 3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenases with bile salts and androsterone as substrates (eight substrate-cofactor combinations were tested). A significant intergroup difference was observed in the amounts of NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenase produced: bowel cancer patients exceeded controls, and controls exceeded Seventh-Day Adventists. Other enzyme activity comparisons were not significant. The pH values of the stools were significantly higher in cancer patients compared to Seventh-Day Adventists; values were 7.03 +/- 0.60 and 6.46 +/- 0.58 respectively. The pH value for controls was 6.66 +/- 0.62. A plot of pH value versus NADP-dependent 7alpha-hydroxysteroid dehydrogenase tended to separate the cancer patients from the other groups. Comparative data suggest that much of the 3alpha-hydroxysteroid dehydrogenase active against bile salt is also active against androsterone.

  1. Novel approaches for using dehydrogenases and ene-reductases for organic synthesis

    NARCIS (Netherlands)

    Gargiulo, S.

    2015-01-01

    Oxidation of alcohols is a reaction of major interest for organic chemistry. However, the most common chemical routes developed so far involve the use of toxic or hazardous reagents or catalysts that often lack good chemoselectivity. In this respect, alcohol dehydrogenases (ADHs) represent a very

  2. Sulfoacetate released during the assimilation of taurine-nitrogen by Neptuniibacter caesariensis: purification of sulfoacetaldehyde dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Krejčík, Zdeněk; Denger, K.; Weinitschke, S.; Hollemeyer, K.; Pačes, Václav; Cook, A.M.; Smits, T.H.M.

    2008-01-01

    Roč. 190, č. 2 (2008), s. 159-168 ISSN 0302-8933 Institutional research plan: CEZ:AV0Z50520514 Keywords : assimilation of taurine-nitrogen * sulfoacetaldehyde dehydrogenase * sulfoacetate exporter Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.975, year: 2008

  3. The Role of Mitochondrial NADPH-Dependent Isocitrate Dehydrogenase in Cancer Cells

    Czech Academy of Sciences Publication Activity Database

    Smolková, Katarína; Ježek, Petr

    2012-01-01

    Roč. 2012, č. 2012 (2012), ID273947 ISSN 1687-8876 R&D Projects: GA ČR GPP301/12/P381; GA ČR(CZ) GAP302/10/0346 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : isocitrate dehydrogenase 2 * Krebs cycle * cancer cells Subject RIV: ED - Physiology

  4. High energy electron beam inactivation of lactate dehydrogenase suspended in different aqueous media

    International Nuclear Information System (INIS)

    Hategan, A.; Oproiu, C.; Popescu, A.; Hategan, D.; Morariu, V.V.

    1998-01-01

    The direct and indirect effects of 5 MeV electron beam irradiation at various low temperatures, as well as the influence of the presence or absence of deuterium ions in the suspending medium of the enzyme, on the global enzymatic activity of lactate dehydrogenase have been studied. Frozen lactate dehydrogenase suspensions at 0 degC, -3 degC and -196 degC temperatures have been irradiated with the 5 MeV electron beam of a linear accelerator in the dose range 0-400 Gy. Liquid lactate dehydrogenase suspensions in D 2 O (99.98 %) and ultrapure water (17 ppm) at 0 degC have been irradiated in the dose range 0 -15 Gy. An exponential decrease was found in the enzymatic activity of irradiated lactate dehydrogenase, at all irradiation temperatures. The drastic decrease in the activity for the enzyme irradiated at 0 degC (total inhibition for a final dose of 100 Gy) indicate that at this temperature the indirect effects of radiation (due to the water radicals induced by radiation in the samples) are predominant. At -3 degC irradiation temperature the indirect effects of radiation are smaller but still present (a total decrease in the enzymatic activity for a dose of 250 Gy), while at -196 degC they are orders of magnitude reduced and the decrease in the enzymatic activity is due almost to the direct interaction of electrons with the macromolecules (70 % for a dose of 400 Gy)

  5. Interaction of benzoate pyrimidine analogues with class 1A dihydroorotate dehydrogenase from Lactococcus lactis

    DEFF Research Database (Denmark)

    Wolfe, Abigail E; Thymark, Majbritt; Gattis, Samuel G

    2007-01-01

    Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of dihydroorotate to orotate in the only redox reaction in pyrimidine biosynthesis. The pyrimidine binding sites are very similar in all structurally characterized DHODs, suggesting that the prospects for identifying a class-specific in......-system of the flavin, resulting in a green color....

  6. Characterization of immunoglobulin A kappa autoantibodies to human lactate dehydrogenase isoenzyme-3

    NARCIS (Netherlands)

    Weijers, R. N.; Oude Elferink, R. P.; Mulder, J.; Kruijswijk, H.

    1987-01-01

    We have purified with a cumulative recovery of 48% from the serum of a patient the immunoglobulin A kappa subunit of the lactate dehydrogenase-immunoglobulin A kappa (LD-IgA kappa) complex. It appears that the pI range of the complex is 5.4-5.8. The Ig part of the complex showed a monoclonal

  7. High-throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation.

    Science.gov (United States)

    Vasilchenko, Liliya G; Ludwig, Roland; Yershevich, Olga P; Haltrich, Dietmar; Rabinovich, Mikhail L

    2012-07-01

    Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe³⁺ (pathway 1) and reduction of ferric ions to Fe²⁺ reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. 3-cyanoindole-based inhibitors of inosine monophosphate dehydrogenase: synthesis and initial structure-activity relationships.

    Science.gov (United States)

    Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Chen, Ping; Norris, Derek; Watterson, Scott H; Ballentine, Shelley K; Fleener, Catherine A; Rouleau, Katherine A; Barrish, Joel C; Townsend, Robert; Hollenbaugh, Diane L; Iwanowicz, Edwin J

    2003-10-20

    A series of novel small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH), based upon a 3-cyanoindole core, were explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SAR), derived from in vitro studies, for this new series of inhibitors is given.

  9. A survey of cyclic replacements for the central diamide moiety of inhibitors of inosine monophosphate dehydrogenase.

    Science.gov (United States)

    Dhar, T G Murali; Liu, Chunjian; Pitts, William J; Guo, Junquing; Watterson, Scott H; Gu, Henry; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Barrish, Joel C; Hollenbaugh, Diane; Iwanowicz, Edwin J

    2002-11-04

    A series of heterocyclic replacements for the central diamide moiety of 1, a potent small molecule inhibitor of inosine monophosphate dehydrogenase (IMPDH) were explored The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for these new series of inhibitors is given.

  10. 5FU and oxaliplatin-containing chemotherapy in two dihydropyrimidine dehydrogenase-deficient patients

    NARCIS (Netherlands)

    Reerink, O; Mulder, NH; Szabo, BG; Hospers, GAP

    2004-01-01

    Patients with a germline mutation leading to a deficiency of the dihydropyrimidine dehydrogenase (DPD) enzyme are at risk from developing severe toxicity on the administration of 5FU-containing chemotherapy. We report on the implications of this inborn genetic error in two patients who received 5FU

  11. Prevalence of Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency in Estonia

    DEFF Research Database (Denmark)

    Joost, K; Ounap, K; Zordania, R

    2012-01-01

    The aim of our study was to evaluate the prevalence of long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) in the general Estonian population and among patients with symptoms suggestive of fatty acid oxidation (FAO) defects. We collected DNA from a cohort of 1,040 anonymous newborn blo...... prevalence of LCHADD in Estonia would be 1: 91,700....

  12. Mechanism-Based Inhibitors of Cytokinin Oxidase/Dehydrogenase Attack FAD Cofactor

    Czech Academy of Sciences Publication Activity Database

    Kopečný, D.; Šebela, M.; Briozzo, P.; Spíchal, Lukáš; Houba-Hérin, N.; Mašek, V.; Joly, N.; Madzak, C.; Anzenbacher, P.; Laloue, M.

    2008-01-01

    Roč. 380, č. 5 (2008), s. 886-899 ISSN 0022-2836 R&D Projects: GA ČR(CZ) GP522/08/P113 Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinin oxidase/dehydrogenase * cytokinin signaling * protein structure Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.146, year: 2008

  13. 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...

  14. Coupled reactions by coupled enzymes : alcohol to lactone cascade with alcohol dehydrogenase-cyclohexanone monooxygenase fusions

    NARCIS (Netherlands)

    Aalbers, Friso S; Fraaije, Marco W

    2017-01-01

    The combination of redox enzymes for redox-neutral cascade reactions has received increasing appreciation. An example is the combination of an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO). The ADH can use NADP(+) to oxidize cyclohexanol to form cyclohexanone and NADPH. Both

  15. Alcohol consumption and type 2 diabetes: Influence of genetic variation in alcohol dehydrogenase

    NARCIS (Netherlands)

    Beulens, J.W.J.; Rimm, E.B.; Hendriks, H.F.J.; Hu, F.B.; Manson, J.E.; Hunter, D.J.; Mukamal, K.J.

    2007-01-01

    OBJECTIVE - We sought to investigate whether a polymorphism in the alcohol dehydrogenase 1c (ADH1C) gene modifies the association between alcohol consumption and type 2 diabetes. RESEARCH DESIGN AND METHODS - In nested case-control studies of 640 women with incident diabetes and 1,000 control

  16. Alcohol consumption and type 2 diabetes - Influence of genetic variation in alcohol dehydrogenase

    NARCIS (Netherlands)

    Beulens, J.W.J.; Rimm, E.B.; Hendriks, H.F.J.; Hu, F.B.; Manson, J.E.; Hunter, D.J.; Mukamal, K.J.

    2007-01-01

    OBJECTIVE-We sought to investigate whether a polymorphism I in the alcohol dehydrogenase 1c (ADH1C) gene modifies the association between alcohol consumption and type 2 diabetes. RESEARCH DESIGN AND METHODS-In nested case-control studies of 640 women with incident diabetes and 1,000 control subjects

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

    Science.gov (United States)

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

    2017-01-15

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

  18. Intravenous immunoglobulin to treat hyperbilirubinemia in neonates with isolated Glucose-6-Phosphate dehydrogenase deficiency

    Directory of Open Access Journals (Sweden)

    Wadah Khriesat

    2017-04-01

    Full Text Available Background Glucose-6-phosphate dehydrogenase deficiency alone or concomitant with ABO isoimmunisation is a widespread indication for neonatal exchange transfusion. Aims To evaluate the effectiveness of Intravenous Immunoglobulin in the treatment of neonatal hyperbilirubinemia due to glucose-6-phosphate dehydrogenase deficiency. Methods A retrospective cohort study was conducted between 2006 and 2014 at the Jordan University of Science and technology. The medical records of 43 infants admitted to the neonatal intensive care unit for isolated glucose-6- phosphate dehydrogenase deficiency hemolytic disease of the newborns were reviewed. Patients were divided into two groups. Group I, a historical cohort, included newborns born between 2006 and 2010, Treatment included phototherapy and exchange transfusion. Group II included newborns born between 2011 and 2014, where, in addition to phototherapy, intravenous immunoglobulin was administered. The duration of phototherapy and number of exchange transfusions were evaluated. Results Of 412 newborns that were admitted with neonatal hyperbilirubinemia, Glucose-6-phosphate dehydrogenase deficiency was present in 43. Of these, 22, did not receive intravenous immunoglobulin and served as a control group. The other 21 newborns received intravenous immunoglobulin. There was no difference in the demographic characteristics between the two groups. Infants in the control group were significantly more likely to receive exchange blood transfusion than infants in the immunoglobulin treatment group, but were significantly less likely to need phototherapy. Conclusion Intravenous immunoglobulin is an effective alternative to exchange transfusion in infants with glucose-6-phosphate dehydrogenase deficiency hemolytic disease of the newborn. It is suggested that intravenous immunoglobulin may be beneficial as a prophylaxis for infants with hyperbilirubinemia.

  19. Physiological covalent regulation of rat liver branched-chain alpha-ketoacid dehydrogenase

    International Nuclear Information System (INIS)

    Harris, R.A.; Powell, S.M.; Paxton, R.; Gillim, S.E.; Nagae, H.

    1985-01-01

    A radiochemical assay was developed for measuring branched-chain alpha-ketoacid dehydrogenase activity of Triton X-100 extracts of freeze-clamped rat liver. The proportion of active (dephosphorylated) enzyme was determined by measuring enzyme activities before and after activation of the complex with a broad-specificity phosphoprotein phosphatase. Hepatic branched-chain alpha-ketoacid dehydrogenase activity in normal male Wistar rats was 97% active but decreased to 33% active after 2 days on low-protein (8%) diet and to 13% active after 4 days on the same diet. Restricting protein intake of lean and obese female Zucker rats also caused inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex. Essentially all of the enzyme was in the active state in rats maintained for 14 days on either 30 or 50% protein diets. This was also the case for rats maintained on a commercial chow diet (minimum 23% protein). However, maintaining rats on 20, 8, and 0% protein diets decreased the percentage of the active form of the enzyme to 58, 10, and 7% of the total, respectively. Fasting of chow-fed rats for 48 h had no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e., 93% of the enzyme remained in the active state compared to 97% for chow-fed rats. However, hepatic enzyme of rats maintained on 8% protein diet was 10% active before starvation and 83% active after 2 days of starvation. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain alpha-ketoacids

  20. Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese.

    Science.gov (United States)

    Broadbent, Jeffery R; Gummalla, Sanjay; Hughes, Joanne E; Johnson, Mark E; Rankin, Scott A; Drake, Mary Anne

    2004-08-01

    Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that alpha-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable alpha-keto acids to more-stable alpha-hydroxy acids via the action of alpha-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei d-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that alpha-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of d-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of alpha-keto acids, but sensory work indicated that this effect retarded cheese flavor development.

  1. Inducing the Alternative Oxidase Forms Part of the Molecular Strategy of Anoxic Survival in Freshwater Bivalves

    Directory of Open Access Journals (Sweden)

    Maria S. Yusseppone

    2018-02-01

    Full Text Available Hypoxia in freshwater ecosystems is spreading as a consequence of global change, including pollution and eutrophication. In the Patagonian Andes, a decline in precipitation causes reduced lake water volumes and stagnant conditions that limit oxygen transport and exacerbate hypoxia below the upper mixed layer. We analyzed the molecular and biochemical response of the North Patagonian bivalve Diplodon chilensis after 10 days of experimental anoxia (<0.2 mg O2/L, hypoxia (2 mg O2/L, and normoxia (9 mg O2/L. Specifically, we investigated the expression of an alternative oxidase (AOX pathway assumed to shortcut the regular mitochondrial electron transport system (ETS during metabolic rate depression (MRD in hypoxia-tolerant invertebrates. Whereas, the AOX system was strongly upregulated during anoxia in gills, ETS activities and energy mobilization decreased [less transcription of glycogen phosphorylase (GlyP and succinate dehydrogenase (SDH in gills and mantle]. Accumulation of succinate and induction of malate dehydrogenase (MDH activity could indicate activation of anaerobic mitochondrial pathways to support anoxic survival in D. chilensis. Oxidative stress [protein carbonylation, glutathione peroxidase (GPx expression] and apoptotic intensity (caspase 3/7 activity decreased, whereas an unfolded protein response (HSP90 was induced under anoxia. This is the first clear evidence of the concerted regulation of the AOX and ETS genes in a hypoxia-tolerant freshwater bivalve and yet another example that exposure to hypoxia and anoxia is not necessarily accompanied by oxidative stress in hypoxia-tolerant mollusks.

  2. Proteomic analysis of protective effects of polysaccharides from Salvia miltiorrhiza against immunological liver injury in mice.

    Science.gov (United States)

    Sun, Xue-Gang; Fu, Xiu-Qiong; Cai, Hong-Bing; Liu, Qiang; Li, Chun-Hua; Liu, Ya-Wei; Li, Ying-Jia; Liu, Zhi-Feng; Song, Yu-Hong; Lv, Zhi-Ping

    2011-07-01

    This study was designed to investigate mechanisms of the protective effects of Salvia miltiorrhiza polysaccharide (SMPS) against lipopolysaccharide (LPS)-induced immunological liver injury (ILI) in Bacille Calmette-Guérin (BCG)-primed mice. Two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis showed that three proteins are down-regulated and six proteins are up-regulated by SMPS. SMPS reduces the degree of liver injury by up-regulating the enzymes of the citric acid cycle, namely malate dehydrogenase (MDH) and 2-oxoglutarate dehydrogenase complex. LPS significantly increases nuclear factor kappa B (NF-κB) activation, inducible nitric oxide synthase (iNOS) expression and MDA level in BCG primed mice liver, whereas SMPS treatment protects against the immunological liver injury through inhibition of the NF-κB activation by up-regulation of PRDX6 and the subsequent attenuation of lipid peroxidation, iNOS expression and inflammation. Copyright © 2011 John Wiley & Sons, Ltd.

  3. Inducing the Alternative Oxidase Forms Part of the Molecular Strategy of Anoxic Survival in Freshwater Bivalves

    Science.gov (United States)

    Yusseppone, Maria S.; Rocchetta, Iara; Sabatini, Sebastian E.; Luquet, Carlos M.; Ríos de Molina, Maria del Carmen; Held, Christoph; Abele, Doris

    2018-01-01

    Hypoxia in freshwater ecosystems is spreading as a consequence of global change, including pollution and eutrophication. In the Patagonian Andes, a decline in precipitation causes reduced lake water volumes and stagnant conditions that limit oxygen transport and exacerbate hypoxia below the upper mixed layer. We analyzed the molecular and biochemical response of the North Patagonian bivalve Diplodon chilensis after 10 days of experimental anoxia (<0.2 mg O2/L), hypoxia (2 mg O2/L), and normoxia (9 mg O2/L). Specifically, we investigated the expression of an alternative oxidase (AOX) pathway assumed to shortcut the regular mitochondrial electron transport system (ETS) during metabolic rate depression (MRD) in hypoxia-tolerant invertebrates. Whereas, the AOX system was strongly upregulated during anoxia in gills, ETS activities and energy mobilization decreased [less transcription of glycogen phosphorylase (GlyP) and succinate dehydrogenase (SDH) in gills and mantle]. Accumulation of succinate and induction of malate dehydrogenase (MDH) activity could indicate activation of anaerobic mitochondrial pathways to support anoxic survival in D. chilensis. Oxidative stress [protein carbonylation, glutathione peroxidase (GPx) expression] and apoptotic intensity (caspase 3/7 activity) decreased, whereas an unfolded protein response (HSP90) was induced under anoxia. This is the first clear evidence of the concerted regulation of the AOX and ETS genes in a hypoxia-tolerant freshwater bivalve and yet another example that exposure to hypoxia and anoxia is not necessarily accompanied by oxidative stress in hypoxia-tolerant mollusks. PMID:29527172

  4. Increased feeding and nutrient excretion of adult Antarctic krill, Euphausia superba, exposed to enhanced carbon dioxide (CO₂.

    Directory of Open Access Journals (Sweden)

    Grace K Saba

    Full Text Available Ocean acidification has a wide-ranging potential for impacting the physiology and metabolism of zooplankton. Sufficiently elevated CO(2 concentrations can alter internal acid-base balance, compromising homeostatic regulation and disrupting internal systems ranging from oxygen transport to ion balance. We assessed feeding and nutrient excretion rates in natural populations of the keystone species Euphausia superba (Antarctic krill by conducting a CO(2 perturbation experiment at ambient and elevated atmospheric CO(2 levels in January 2011 along the West Antarctic Peninsula (WAP. Under elevated CO(2 conditions (∼672 ppm, ingestion rates of krill averaged 78 µg C individual(-1 d(-1 and were 3.5 times higher than krill ingestion rates at ambient, present day CO(2 concentrations. Additionally, rates of ammonium, phosphate, and dissolved organic carbon (DOC excretion by krill were 1.5, 1.5, and 3.0 times higher, respectively, in the high CO(2 treatment than at ambient CO(2 concentrations. Excretion of urea, however, was ∼17% lower in the high CO(2 treatment, suggesting differences in catabolic processes of krill between treatments. Activities of key metabolic enzymes, malate dehydrogenase (MDH and lactate dehydrogenase (LDH, were consistently higher in the high CO(2 treatment. The observed shifts in metabolism are consistent with increased physiological costs associated with regulating internal acid-base equilibria. This represents an additional stress that may hamper growth and reproduction, which would negatively impact an already declining krill population along the WAP.

  5. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific Opinion on the substantiation of a health claim related to citrulline-malate and faster recovery from muscle fatigue after exercise pursuant to Article 13(5) of Regulation (EC) No 1924/2006

    DEFF Research Database (Denmark)

    Tetens, Inge

    Following an application from Biocodex, submitted pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of Belgium, the Panel on Dietetic Products, Nutrition and Allergies was asked to deliver an opinion on the scientific substantiation of a health claim related...... to citrulline-malate and faster recovery from muscle fatigue after exercise. Citrulline-malate is sufficiently characterised. The claimed effect is “maintenance of ATP levels through reduction of lactates in excess for an improved recovery from muscle fatigue”. The target population proposed by the applicant...... is healthy children above six years of age and adults. The Panel considers that faster recovery from muscle fatigue after exercise contributing to the restoration of muscle function is a beneficial physiological effect. A total of 33 references were considered as pertinent to the claim by the applicant...

  6. Variabilidade isoenzimática entre linhagens de amendoim resistentes à seca Isoenzimatic variability between peanut lines resistant to drought

    Directory of Open Access Journals (Sweden)

    Roseane Cavalcanti dos Santos

    2000-04-01

    Full Text Available O uso da técnica de eletroforese para separar múltiplas formas moleculares de enzimas tem sido bastante explorada na área biológica, cujas diferenças detectadas nos tecidos podem ser eficientemente usadas para diferenciação de cultivares em qualquer fase de seu desenvolvimento fenológico. Nesse trabalho, procedeu-se ao estudo da variabilidade isoenzimática em seis linhagens de amendoim resistentes à seca, com o objetivo de se verificar as possíveis relações da variação encontrada na base desses descritores com essa aptidão no amendoim. Estudaram-se folíolos da parte apical com 5 dias após a germinação, utilizando-se a técnica de eletroforese em gel de poliacrilamida (7% sistema horizontal e contínuo de tampão. Os sistemas estudados foram fosfatase ácida (ACP, malato desidrogenase (MDH, leucina aminopeptidase (LAP, peroxidase (PO, e esterase (EST. A caracterização fenotípica dos genótipos permitiu a separação de quatro grupos para ACP, três para LAP, dois para MDH e seis para PO e EST. A partir da análise dos componentes principais dos grupos obtidos, observou-se que a cultivar IAC Tupã (sensível à seca foi separada das demais, especialmente da cultivar resistente Senegal 55437.The use of electrophoretic techniques to separate multiple molecular forms of enzymes has been used in the biological science, where differences in isozymes among tissues can be used efficiently on cultivar differentiation during any life cycle phase. In this paper, the variability of six drought resistant peanut lines was studied by isozymes analysis aiming to verify the possible relations between enzymatic descriptors and drought resistance character. Leaflets were analyzed by horizontal poliacrylamide gel electrophoresis technique and buffer continuos systems for the following systems: acid phosphatase (ACP, malate dehydrogenase (MDH, leucine aminopeptidase (LAP, peroxidase (POX and esterase (EST. The phenotypic characterization of the

  7. Structural and Thermodynamic Basis for Weak Interactions between Dihydrolipoamide Dehydrogenase and Subunit-binding Domain of the Branched-chain [alpha]-Ketoacid Dehydrogenase Complex

    Energy Technology Data Exchange (ETDEWEB)

    Brautigam, Chad A.; Wynn, R. Max; Chuang, Jacinta L.; Naik, Mandar T.; Young, Brittany B.; Huang, Tai-huang; Chuang, David T. (AS); (UTSMC)

    2012-02-27

    The purified mammalian branched-chain {alpha}-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain {alpha}-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-{angstrom} resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other {alpha}-ketoacid dehydrogenase complexes.

  8. 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...

  9. Purification and Characterization of a Novel NAD(P)+-Farnesol Dehydrogenase from Polygonum minus Leaves.

    Science.gov (United States)

    Ahmad-Sohdi, Nor-Ain-Shahajar; Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura; Hassan, Maizom

    2015-01-01

    Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with Km values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The Km values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control.

  10. Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.

    Science.gov (United States)

    Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Bao, Xiaoming; Shen, Yu

    2016-04-01

    Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described. Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their

  11. Dissociation of branched-chain alpha-keto acid dehydrogenase kinase (BDK) from branched-chain alpha-keto acid dehydrogenase complex (BCKDC) by BDK inhibitors.

    Science.gov (United States)

    Murakami, Taro; Matsuo, Masayuki; Shimizu, Ayako; Shimomura, Yoshiharu

    2005-02-01

    Branched-chain alpha-keto acid dehydrogenase kinase (BDK) phosphorylates and inactivates the branched-chain alpha-keto acid dehydrogenase complex (BCKDC), which is the rate-limiting enzyme in the branched-chain amino acid catabolism. BDK has been believed to be bound to the BCKDC. However, recent our studies demonstrated that protein-protein interaction between BDK and BCKDC is one of the factors to regulate BDK activity. Furthermore, only the bound form of BDK appears to have its activity. In the present study, we examined effects of BDK inhibitors on the amount of BDK bound to the BCKDC using rat liver extracts. The bound form of BDK in the extracts of liver from low protein diet-fed rats was measured by an immunoprecipitation pull down assay with or without BDK inhibitors. Among the BDK inhibitors. alpha-ketoisocaproate, alpha-chloroisocaproate, and a-ketoisovalerate released the BDK from the complex. Furthermore, the releasing effect of these inhibitors on the BDK appeared to depend on their inhibition constants. On the other hand, clofibric acid and thiamine pyrophosphate had no effect on the protein-protein interaction between two enzymes. These results suggest that the dissociation of the BDK from the BCKDC is one of the mechanisms responsible for the action of some inhibitors to BDK.

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

    Directory of Open Access Journals (Sweden)

    Binbin Sheng

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

  13. Alcohol drinking habits, alcohol dehydrogenase genotypes and risk of acute coronary syndrome

    DEFF Research Database (Denmark)

    Tolstrup, J.S.; Hansen, J.L.; Gronbaek, M.

    2010-01-01

    Aims: The risk of myocardial infarction is lower among light-to-moderate drinkers compared with abstainers. Results from some previous studies, but not all, suggest that this association is modified by variations in genes coding for alcohol dehydrogenase (ADH). We aimed to test this hypothesis......, including alcohol as both the amount of alcohol and the frequency of drinking. Methods: we conducted a nested case-cohort study within the Danish Diet, Cancer and Health study, including 1,645 men (770 incident cases of acute coronary syndrome from 1993-1997 through 2004 and 875 randomly selected controls......). Results: Higher alcohol intake (measured as amount or drinking frequency) was associated with lower risk of acute coronary syndrome; however, there was no evidence that these finding were modified by ADH1B or ADH1C genotypes. Conclusions: The importance of functional variation in alcohol dehydrogenase...

  14. Immobilization of alcohol dehydrogenase on ceramic silicon carbide membranes for enzymatic CH3 OH production

    DEFF Research Database (Denmark)

    Zeuner, Birgitte; Ma, Nicolaj; Berendt, Kasper

    2018-01-01

    BACKGROUND Alcohol dehydrogenase (ADH; EC 1.1.1.1) catalyzes oxidation of CH3OH to CHOH during NAD+ reduction to NADH. ADH can also accelerate the reverse reaction, which is studied as part of cascadic enzymatic conversion of CO2 to CH3OH. In the present study, immobilization of ADH onto macropor......BACKGROUND Alcohol dehydrogenase (ADH; EC 1.1.1.1) catalyzes oxidation of CH3OH to CHOH during NAD+ reduction to NADH. ADH can also accelerate the reverse reaction, which is studied as part of cascadic enzymatic conversion of CO2 to CH3OH. In the present study, immobilization of ADH onto......‐of‐concept for the use of NaOH‐treated SiC membranes for covalent enzyme immobilization and biocatalytic efficiency improvement of ADH during multiple reaction cycles. These data have implications for the development of robust extended enzymatic reactions....

  15. Cloning and characterization of the gene encoding IMP dehydrogenase from Arabidopsis thaliana.

    Science.gov (United States)

    Collart, F R; Osipiuk, J; Trent, J; Olsen, G J; Huberman, E

    1996-10-03

    We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Arabidopsis thaliana (At). The transcription unit of the At gene spans approximately 1900 bp and specifies a protein of 503 amino acids with a calculated relative molecular mass (M(r)) of 54,190. The gene is comprised of a minimum of four introns and five exons with all donor and acceptor splice sequences conforming to previously proposed consensus sequences. The deduced IMPDH amino-acid sequence from At shows a remarkable similarity to other eukaryotic IMPDH sequences, with a 48% identity to human Type II enzyme. Allowing for conservative substitutions, the enzyme is 69% similar to human Type II IMPDH. The putative active-site sequence of At IMPDH conforms to the IMP dehydrogenase/guanosine monophosphate reductase motif and contains an essential active-site cysteine residue.

  16. Cofactor specificity switch in Shikimate dehydrogenase by rational design and consensus engineering.

    Science.gov (United States)

    García-Guevara, Fernando; Bravo, Iris; Martínez-Anaya, Claudia; Segovia, Lorenzo

    2017-08-01

    Consensus engineering has been used to design more stable variants using the most frequent amino acid at each site of a multiple sequence alignment; sometimes consensus engineering modifies function, but efforts have mainly been focused on studying stability. Here we constructed a consensus Rossmann domain for the Shikimate dehydrogenase enzyme; separately we decided to switch the cofactor specificity through rational design in the Escherichia coli Shikimate dehydrogenase enzyme and then analyzed the effect of consensus mutations on top of our design. We found that consensus mutations closest to the 2' adenine moiety increased the activity in our design. Consensus engineering has been shown to result in more stable proteins and our findings suggest it could also be used as a complementary tool for increasing or modifying enzyme activity during design. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Myopathy in very-long-chain acyl-CoA dehydrogenase deficiency

    DEFF Research Database (Denmark)

    Scholte, H R; Van Coster, R N; de Jonge, P C

    1999-01-01

    was deficient in muscle and fibroblasts, consistent with deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD). The gene of this enzyme had a homozygous deletion of three base pairs in exon 9, skipping lysine residue 238. Fibroblasts oxidised myristate, palmitate and oleate at a rate of 129, 62 and 38......A 30-year-old man suffered since the age of 13 years from exercise induced episodes of intense generalised muscle pain, weakness and myoglobinuria. Fasting ketogenesis was low, while blood glucose remained normal. Muscle mitochondria failed to oxidise palmitoylcarnitine. Palmitoyl-CoA dehydrogenase......% of controls. In contrast to patients with cardiac VLCAD deficiency, our patient had no lipid storage, a normal heart function, a higher rate of oleate oxidation in fibroblasts and normal free carnitine in plasma and fibroblasts. 31P-nuclear magnetic resonance spectroscopy of muscle showed a normal oxidative...

  18. Promysalin Elicits Species-Selective Inhibition of Pseudomonas aeruginosa by Targeting Succinate Dehydrogenase.

    Science.gov (United States)

    Keohane, Colleen E; Steele, Andrew D; Fetzer, Christian; Khowsathit, Jittasak; Van Tyne, Daria; Moynié, Lucile; Gilmore, Michael S; Karanicolas, John; Sieber, Stephan A; Wuest, William M

    2018-02-07

    Natural products have served as an inspiration to scientists both for their complex three-dimensional architecture and exquisite biological activity. Promysalin is one such Pseudomonad secondary metabolite that exhibits narrow-spectrum antibacterial activity, originally isolated from the rhizosphere. We herein utilize affinity-based protein profiling (AfBPP) to identify succinate dehydrogenase (Sdh) as the biological target of the natural product. The target was further validated in silico, in vitro, in vivo, and through the selection, and sequencing, of a resistant mutant. Succinate dehydrogenase plays an essential role in primary metabolism of Pseudomonas aeruginosa as the only enzyme that is involved both in the tricarboxylic acid cycle (TCA) and in respiration via the electron transport chain. These findings add credence to other studies that suggest that the TCA cycle is an understudied target in the development of novel therapeutics to combat P. aeruginosa, a significant pathogen in clinical settings.

  19. Purification and characterization of the amine dehydrogenase from a facultative methylotroph.

    Science.gov (United States)

    Coleman, J P; Perry, J J

    1984-01-01

    Strain RA-6 is a pink-pigmented organism which can grow on a variety of substrates including methylamine. It can utilize methylamine as sole source of carbon via an isocitrate lyase negative serine pathway. Methylamine grown cells contain an inducible primary amine dehydrogenase [primary amine: (acceptor) oxidoreductase (deaminating)] which is not present in succinate grown cells. The amine dehydrogenase was purified to over 90% homogeneity. It is an acidic protein (isoelectric point of 5.37) with a molecular weight of 118,000 containing subunits with approximate molecular weights of 16,500 and 46,000. It is active on an array of primary terminal amines and is strongly inhibited by carbonyl reagents. Cytochrome c or artificial electron acceptors are required for activity; neither NAD nor NADP can serve as primary electron acceptor.

  20. Post-irradiation repairing processes of glucose-6-phosphate dehydrogenase and catalase from Hansenula Polymorpha yeast

    International Nuclear Information System (INIS)

    Postolache, Carmen; Postolache, Cristian; Dinu, Diana; Dinischiotu, Anca; Sahini, Victor Emanuel

    2002-01-01

    The post-irradiation repairing mechanisms of two Hansenula Polymorpha yeast enzymes, glucose-6-phosphate dehydrogenase and catalase, were studied. The kinetic parameters of the selected enzymes were investigated over one month since the moment of γ-irradiation with different doses in the presence of oxygen. Dose dependent decrease of initial reaction rates was noticed for both enzymes. Small variation of initial reaction rate was recorded for glucose-6-phosphate dehydrogenase over one month, with a decreasing tendency. No significant electrophoretic changes of molecular forms of this enzyme were observed after irradiation. Continuous strong decrease of catalase activity was evident for the first 20 days after irradiation. Partial recovery process of the catalytic activity was revealed by this study. (authors)

  1. Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor

    International Nuclear Information System (INIS)

    Savkur, Rajesh S.; Bramlett, Kelli S.; Michael, Laura F.; Burris, Thomas P.

    2005-01-01

    The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression

  2. Structural organization of the human short-chain acyl-CoA dehydrogenase gene

    DEFF Research Database (Denmark)

    Corydon, M J; Andresen, B S; Bross, P

    1997-01-01

    Short-chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric mitochondrial flavoenzyme that catalyzes the initial reaction in short-chain fatty acid beta-oxidation. Defects in the SCAD enzyme are associated with failure to thrive, often with neuromuscular dysfunction and elevated urinary excretion...... shown to be associated with ethylmalonic aciduria. From analysis of 18 unrelated Danish families, we show that the four SCAD gene polymorphisms constitute five allelic variants of the SCAD gene, and that the 625A variant together with the less frequent variant form of the three other polymorphisms (321C....... The evolutionary relationship between SCAD and five other members of the acyl-CoA dehydrogenase family was investigated by two independent approaches that gave similar phylogenetic trees....

  3. Calcium inhibition of the NAD+-linked isocitrate dehydrogenase from blowfly flight muscle mitochondria.

    Science.gov (United States)

    Bulos, B A; Thomas, B J; Sacktor, B

    1984-08-25

    Free Ca2+ was shown to inhibit the NAD+-isocitrate dehydrogenase from blowfly flight muscle mitochondria. Inhibition by free Ca2+ concentrations of 40 microM or greater was found in the absence or presence of ADP and citrate, two known activators of the enzyme. Calcium decreased the affinity of the enzyme for its substrate, the magnesium DL-isocitrate chelate; no change in the apparent V of the reaction was observed. Calcium was inhibitory when activity was measured in the presence of fixed concentrations of magnesium DL-isocitrate chelate in the presence of several fixed concentrations of either free isocitrate3-, an activator, or free Mg2+, an inhibitor of the enzyme. That NAD+-isocitrate dehydrogenase from blowfly flight muscle mitochondria was not activated by micromolar free Ca2+ is consistent with the view that calcium does not play a role in regulating the flux through the tricarboxylate cycle in this species.

  4. Effects of accelerated electrons and microwaves on frozen enzyme lactate dehydrogenase

    International Nuclear Information System (INIS)

    Hategan, A.; Martin, D.; Popescu, L.M.; Butan, C.

    2000-01-01

    Results on the influence of 6 MeV electron beam irradiation and 2.45 GHz 565 W microwaves as well as the effects of the combined electron and microwave irradiation, at - 21 deg. C, on enzyme lactate dehydrogenase are presented. The microwave irradiated macromolecules exhibited a non-linear behaviour (successive activation and inactivation of the enzyme molecules) suggesting the major influence of the nonthermal component of microwave radiation. The combined electron and microwave irradiation lead to a similar decrease of the activity as the electron beam irradiation, the microwave influence being apparently insignificant in the dose, power and time ranges used. Radiation target analysis of the enzymatic decrease due to electron irradiation indicated very large aggregation of the enzyme molecules. Our data suggest that radiation target analysis is not suitable to measure the molecular mass of lactate dehydrogenase, when irradiating frozen enzyme suspensions. (authors)

  5. A novel 3-hydroxysteroid dehydrogenase that regulates reproductive development and longevity.

    Directory of Open Access Journals (Sweden)

    Joshua Wollam

    Full Text Available Endogenous small molecule metabolites that regulate animal longevity are emerging as a novel means to influence health and life span. In C. elegans, bile acid-like steroids called the dafachronic acids (DAs regulate developmental timing and longevity through the conserved nuclear hormone receptor DAF-12, a homolog of mammalian sterol-regulated receptors LXR and FXR. Using metabolic genetics, mass spectrometry, and biochemical approaches, we identify new activities in DA biosynthesis and characterize an evolutionarily conserved short chain dehydrogenase, DHS-16, as a novel 3-hydroxysteroid dehydrogenase. Through regulation of DA production, DHS-16 controls DAF-12 activity governing longevity in response to signals from the gonad. Our elucidation of C. elegans bile acid biosynthetic pathways reveals the possibility of novel ligands as well as striking biochemical conservation to other animals, which could illuminate new targets for manipulating longevity in metazoans.

  6. Application of a radioimmunoassay to the induction of the 20β hydroxy steroid dehydrogenases with streptomyces hydrogenans

    International Nuclear Information System (INIS)

    Lotz, B.

    1978-01-01

    An antiserum has been prepared against crystallized 20β-hydroxysteroid dehydrogenate of streptomyces hydrogenous and used for different immunodiffusion and immunoprecipitation tests. A de novo synthesis of the 20β-hydroxysteroid dehydrogenase with streptomyces hydrogenous after cultivation of the cells in the presence of diene diol was hence found. The halflife of the 20β-hydroxysteroid dehydrogenase synthetizing mRNA in induced cells and that of the total mRNA in non-induced cells were calculated to be 126 sec and 66 sec respectively. The 20β-hydroxysteroid dehydrogenase in vivo appears to consist of four identical subunits. The monomers with a molecular weight of 27 350 exhibited a strong tendency to form diners and tetrameric complexes in the absence of dissociation agents. The synthesis rates of the 20β-hydroxysteroid dehydrogenase under induction conditions was 8.33%, the percentage of the total protein after induction 1.6%. (orig.) [de

  7. Mutations in ALDH6A1 encoding methylmalonate semialdehyde dehydrogenase are associated with dysmyelination and transient methylmalonic aciduria

    NARCIS (Netherlands)

    Marcadier, Julien L.; Smith, Amanda M.; Pohl, Daniela; Schwartzentruber, Jeremy; Al-Dirbashi, Osama Y.; Majewski, Jacek; Ferdinandusse, Sacha; Wanders, Ronald J. A.; Bulman, Dennis E.; Boycott, Kym M.; Chakraborty, Pranesh; Geraghty, Michael T.; Boycott, Kym; Friedman, Jan; Michaud, Jacques; Bernier, Francois; Brudno, Michael; Fernandez, Bridget; Knoppers, Bartha; Samuels, Mark; Scherer, Steve

    2013-01-01

    Methylmalonate semialdehyde dehydrogenase (MMSDH) deficiency is a rare autosomal recessive disorder with varied metabolite abnormalities, including accumulation of 3-hydroxyisobutyric, 3-hydroxypropionic, 3-aminoisobutyric and methylmalonic acids, as well as β-alanine. Existing reports describe a

  8. Oxidation of aromatic alcohols by purified methanol dehydrogenase from Methylosinus trichosporium.

    OpenAIRE

    Mountfort, D O

    1990-01-01

    Methanol dehydrogenase was found to be present in subcellular preparations of methanol-grown Methylosinus trichosporium and occurred almost wholly in the soluble fraction of the cell. The enzyme, purified by DEAE-Sephadex and Sephadex G-100 chromatography, showed broad specificity toward different substrates and oxidized the aromatic alcohols benzyl, vanillyl, and veratryl alcohols in addition to a range of aliphatic primary alcohols. No enzyme activity was found toward the corresponding alde...

  9. Stabilizing Effects of Deep Eutectic Solvents on Alcohol Dehydrogenase Mediated Systems

    OpenAIRE

    Fatima Zohra Ibn Majdoub Hassani; Ivan Lavandera; Joseph Kreit

    2016-01-01

    This study explored the effects of different organic solvents, temperature, and the amount of glycerol on the alcohol dehydrogenase (ADH)-catalysed stereoselective reduction of different ketones. These conversions were then analyzed by gas chromatography. It was found that when the amount of deep eutectic solvents (DES) increases, it can improve the stereoselectivity of the enzyme although reducing its ability to convert the substrate into the corresponding alcohol. Moreover, glycerol was fou...

  10. Kinetic isotope effect studies on milk xanthine oxidase and on chicken liver xanthine dehydrogenase

    International Nuclear Information System (INIS)

    D'Ardenne, S.C.; Edmondson, D.E.

    1990-01-01

    The effect of isotopic substitution of the 8-H of xanthine (with 2 H and 3 H) on the rate of oxidation by bovine xanthine oxidase and by chicken xanthine dehydrogenase has been measured. V/K isotope effects were determined from competition experiments. No difference in H/T (V/K) values was observed between xanthine oxidase and xanthine dehydrogenase. Xanthine dehydrogenase exhibited a larger T/D (V/K) value than that observed for xanthine oxidase. Observed H/T (V/K) values for either enzyme are less than those H/T (V/K) values calculated with D/T (V/K) data. These discrepancies are suggested to arise from the presence of a rate-limiting step(s) prior to the irreversible C-H bond cleavage step in the mechanistic pathways of both enzymes. These kinetic complexities preclude examination of whether tunneling contributes to the reaction coordinate for the H-transfer step in each enzyme. No observable exchange of tritium with solvent is observed during the anaerobic incubation of [8- 3 H]xanthine with either enzyme, which suggests the reverse commitment to catalysis (C r ) is essentially zero. With the assumption of adherence to reduced mass relationships, the intrinsic deuterium isotope effect ( D k) for xanthine oxidation is calculated. By the use of these values and steady-state kinetic data, the minimal rate for the hydrogen-transfer step is calculated to be ∼75-fold faster than k cat for xanthine oxidase and ∼10-fold faster than k cat for xanthine dehydrogenase. Values calculated for each enzyme were found to be identical within experimental uncertainty

  11. Role of cytosolic NADP+-dependent isocitrate dehydrogenase in ischemia-reperfusion injury in mouse kidney

    OpenAIRE

    Kim, Jinu; Kim, Ki Young; Jang, Hee-Seong; Yoshida, Takumi; Tsuchiya, Ken; Nitta, Kosaku; Park, Jeen-Woo; Bonventre, Joseph V.; Park, Kwon Moo

    2008-01-01

    Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expressio...

  12. Measurement of the enzymes lactate dehydrogenase and creatine kinase using reflectance spectroscopy and reagent strips.

    OpenAIRE

    Stevens, J F; Tsang, W; Newall, R G

    1983-01-01

    Two new methods for the assay of total activities of lactate dehydrogenase and creatine kinase are described, in which the enzyme activities are measured from a solid-state reagent strip during a kinetic reaction, the reaction being monitored in the ultra-violet region of the spectrum by reflectance spectroscopy. The performances of these methods are evaluated, and compared to conventional "wet" chemistry methods. The solid-phase reagent methods demonstrated precision and accuracy acceptable ...

  13. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris

    OpenAIRE

    Sygmund, Christoph; Staudigl, Petra; Klausberger, Miriam; Pinotsis, Nikos; Djinovic-Carugo, Kristina; Gorton, Lo; Haltrich, Dietmar; Ludwig, Roland

    2011-01-01

    Abstract Background FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spr...

  14. Interdomain electron transfer in cellobiose dehydrogenase is governed by surface electrostatics

    Czech Academy of Sciences Publication Activity Database

    Kádek, Alan; Kavan, Daniel; Marcoux, J.; Stojko, J.; Felice, A.K.G.; Cianférani, S.; Ludwig, R.; Halada, Petr; Man, Petr

    2017-01-01

    Roč. 1861, č. 2 (2017), s. 157-167 ISSN 0304-4165 R&D Projects: GA ČR GAP206/12/0503; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) LQ1604 Institutional support: RVO:61388971 Keywords : Cellobiose dehydrogenase * Direct electron transfer * Electrostatic interaction Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.702, year: 2016

  15. Sorbitol Can Fuel Mouse Sperm Motility and Protein Tyrosine Phosphorylation via Sorbitol Dehydrogenase1

    OpenAIRE

    Cao, Wenlei; Aghajanian, Haig K.; Haig-Ladewig, Lisa A.; Gerton, George L.

    2009-01-01

    Energy sources that can be metabolized to yield ATP are essential for normal sperm functions such as motility. Two major monosaccharides, sorbitol and fructose, are present in semen. Furthermore, sorbitol dehydrogenase (SORD) can convert sorbitol to fructose, which can then be metabolized via the glycolytic pathway in sperm to make ATP. Here we characterize Sord mRNA and SORD expression during mouse spermatogenesis and examine the ability of sorbitol to support epididymal sperm motility and t...

  16. Cytokinin metabolism of pathogenic fungus Leptosphaeria maculans involves isopentenyltransferase, adenosine kinase and cytokinin oxidase/dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Trdá, Lucie; Barešová, Monika; Šašek, Vladimír; Nováková, Miroslava; Zahajská, Lenka; Dobrev, Petre; Motyka, Václav; Burketová, Lenka

    2017-01-01

    Roč. 8, JUL 21 (2017), č. článku 1374. ISSN 1664-302X R&D Projects: GA ČR GA13-26798S; GA ČR(CZ) GA16-14649S Institutional support: RVO:61389030 Keywords : Adenosine kinase * Cytokinin * Cytokinin oxidase/dehydrogenase * Isopentenyltransferase * Leptosphaeria maculans * Zeatin cis/trans isomerase Subject RIV: GF - Plant Pathology, Vermin, Weed, Plant Protection OBOR OECD: Microbiology Impact factor: 4.076, year: 2016

  17. Different Routes for Conifer- and Sinapaldehyde and Higher Saccharification upon Deficiency in the Dehydrogenase CAD1

    OpenAIRE

    Van Acker, Rebecca; Dejardin, Annabelle; Desmet, Sandrien; Hoengenaert, Lennart; Vanholme, Ruben; Morreel, Kris; Laurans, Françoise; Kim, Hoon; Santoro, Nicholas; Foster, Cliff; Goeminne, Geert; Legée, Frédéric; Lapierre, Catherine; Pilate, Gilles; Ralph, John

    2017-01-01

    In the search for renewable energy sources, genetic engineering is a promising strategy to improve plant cell wall composition for biofuel and bioproducts generation. Lignin is a major factor determining saccharification efficiency and, therefore, is a prime target to engineer. Here, lignin content and composition were modified in poplar (Populus tremula 3 Populus alba) by specifically down-regulating CINNAMYL ALCOHOL DEHYDROGENASE1 (CAD1) by a hairpin-RNA-mediated silencing approach, which r...

  18. Syringyl lignin is unaltered by severe sinapyl alcohol dehydrogenase suppression in tobacco

    OpenAIRE

    Barakate, Abdellah; Stephens, Jennifer; Goldie, Alison; Hunter, William N.; Marshall, David; Hancock, Robert D.; Lapierre, Catherine; Morreele, Kris; Boerjane, Wout

    2011-01-01

    The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was cha...

  19. Glucose 6 phosphatase dehydrogenase (G6PD) and neurodegenerative disorders: Mapping diagnostic and therapeutic opportunities

    OpenAIRE

    Manju Tiwari

    2017-01-01

    Glucose 6 phosphate dehydrogenase (G6PD) is a key and rate limiting enzyme in the pentose phosphate pathway (PPP). The physiological significance of enzyme is providing reduced energy to specific cells like erythrocyte by maintaining co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH). There are preponderance research findings that demonstrate the enzyme (G6PD) role in the energy balance, and it is associated with blood-related diseases and disorders, primarily the anemia resulted f...

  20. ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Mráček, Tomáš; Holzerová, Eliška; Drahota, Zdeněk; Kovářová, Nikola; Vrbacký, Marek; Ješina, Pavel; Houštěk, Josef

    2014-01-01

    Roč. 1837, č. 1 (2014), s. 98-111 ISSN 0005-2728 R&D Projects: GA ČR(CZ) GPP303/10/P227; GA MŠk(CZ) LL1204 Grant - others:Univerzita Karlova(CZ) 750213 Institutional support: RVO:67985823 Keywords : mitochondrial glycerol-3-phosphate dehydrogenase * ROS production * supercomplex * in-gel ROS detection Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.353, year: 2014

  1. 11beta-hydroxysteroid dehydrogenase type 1 regulates glucocorticoid-induced insulin resistance in skeletal muscle.

    LENUS (Irish Health Repository)

    Morgan, Stuart A

    2009-11-01

    Glucocorticoid excess is characterized by increased adiposity, skeletal myopathy, and insulin resistance, but the precise molecular mechanisms are unknown. Within skeletal muscle, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone (11-dehydrocorticosterone in rodents) to active cortisol (corticosterone in rodents). We aimed to determine the mechanisms underpinning glucocorticoid-induced insulin resistance in skeletal muscle and indentify how 11beta-HSD1 inhibitors improve insulin sensitivity.

  2. Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study

    Czech Academy of Sciences Publication Activity Database

    Kopečný, D.; Briozzo, P.; Popelková, H.; Šebela, M.; Končitíková, R.; Spíchal, Lukáš; Nisler, Jaroslav; Madzak, C.; Frébort, Ivo; Laloue, M.; Houba-Herin, N.

    2010-01-01

    Roč. 92, č. 8 (2010), s. 1052-1062 ISSN 0300-9084 R&D Projects: GA ČR GA522/08/0555; GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : benzylurea * crystal structure * cytokinin oxidase/dehydrogenase Subject RIV: CE - Biochemistry Impact factor: 3.787, year: 2010

  3. Aldehyde dehydrogenase polymorphism in North American, South American, and Mexican Indian populations.

    Science.gov (United States)

    Goedde, H W; Agarwal, D P; Harada, S; Rothhammer, F; Whittaker, J O; Lisker, R

    1986-01-01

    While about 40% of the South American Indian populations (Atacameños, Mapuche, Shuara) were found to be deficient in aldehyde dehydrogenase isozyme I (ALDH2 or E2), preliminary investigations showed very low incidence of isozyme deficiency among North American natives (Sioux, Navajo) and Mexican Indians (mestizo). Possible implications of such trait differences on cross-cultural behavioral response to alcohol drinking are discussed. PMID:3953578

  4. Structural and functional characterization of plant aminoaldehyde dehydrogenase from Pisum sativum with a broad specificity for natural and synthetic aminoaldehydes

    Czech Academy of Sciences Publication Activity Database

    Tylichová, M.; Kopečný, D.; Moréra, S.; Briozzo, P.; Lenobel, René; Snégaroff, J.; Šebela, M.

    2010-01-01

    Roč. 396, č. 4 (2010), s. 870-882 ISSN 0022-2836 R&D Projects: GA ČR GA522/08/0555; GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : aminoaldehyde dehydrogenase * betaine aldehyde dehydrogenase * NAD+ complex Subject RIV: CE - Biochemistry Impact factor: 4.008, year: 2010

  5. Glutamate dehydrogenase affects resistance to cell wall antibiotics in Bacillus subtilis.

    Science.gov (United States)

    Lee, Yong Heon; Kingston, Anthony W; Helmann, John D

    2012-03-01

    The glutamate dehydrogenase RocG of Bacillus subtilis is a bifunctional protein with both enzymatic and regulatory functions. Here we show that the rocG null mutant is sensitive to β-lactams, including cefuroxime (CEF), and to fosfomycin but that resistant mutants arise due to gain-of-function mutations in gudB, which encodes an otherwise inactive glutamate dehydrogenase. In the presence of CEF, ΔrocG ΔgudB mutant cells exhibit growth arrest when they reach mid-exponential phase. Using microarray-based transcriptional profiling, we found that the σ(W) regulon was downregulated in the ΔrocG ΔgudB null mutant. A survey of σ(W)-controlled genes for effects on CEF resistance identified both the NfeD protein YuaF and the flotillin homologue YuaG (FloT). Notably, overexpression of yuaFG in the rocG null mutant prevents the growth arrest induced by CEF. The YuaG flotillin has been shown previously to localize to defined lipid microdomains, and we show here that the yuaFGI operon contributes to a σ(W)-dependent decrease in membrane fluidity. We conclude that glutamate dehydrogenase activity affects the expression of the σ(W) regulon, by pathways that are yet unclear, and thereby influences resistance to CEF and other antibiotics.

  6. A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity.

    Science.gov (United States)

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

    2015-02-27

    The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. A Novel Cofactor-binding Mode in Bacterial IMP Dehydrogenases Explains Inhibitor Selectivity*

    Science.gov (United States)

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R.; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

    2015-01-01

    The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5′-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. PMID:25572472

  8. XoxF Is Required for Expression of Methanol Dehydrogenase in Methylobacterium extorquens AM1 ▿

    Science.gov (United States)

    Skovran, Elizabeth; Palmer, Alexander D.; Rountree, Austin M.; Good, Nathan M.; Lidstrom, Mary E.

    2011-01-01

    In Gram-negative methylotrophic bacteria, the first step in methylotrophic growth is the oxidation of methanol to formaldehyde in the periplasm by methanol dehydrogenase. In most organisms studied to date, this enzyme consists of the MxaF and MxaI proteins, which make up the large and small subunits of this heterotetrameric enzyme. The Methylobacterium extorquens AM1 genome contains two homologs of MxaF, XoxF1 and XoxF2, which are ∼50% identical to MxaF and ∼90% identical to each other. It was previously reported that xoxF is not required for methanol growth in M. extorquens AM1, but here we show that when both xoxF homologs are absent, strains are unable to grow in methanol medium and lack methanol dehydrogenase activity. We demonstrate that these defects result from the loss of gene expression from the mxa promoter and suggest that XoxF is part of a complex regulatory cascade involving the 2-component systems MxcQE and MxbDM, which are required for the expression of the methanol dehydrogenase genes. PMID:21873495

  9. Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase.

    Science.gov (United States)

    Munawar, Nayla; Engel, Paul C

    2013-01-01

    Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting L-methionine, L-norleucine and L-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.

  10. Very long chain acyl-coenzyme A dehydrogenase deficiency with adult onset

    DEFF Research Database (Denmark)

    Smelt, A H; Poorthuis, B J; Onkenhout, W

    1998-01-01

    Very long chain acyl-coenzyme A (acyl-CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial beta-oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified strongly elevated levels of tetradecenoic acid, 14:1(n-9), tetrade......Very long chain acyl-coenzyme A (acyl-CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial beta-oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified strongly elevated levels of tetradecenoic acid, 14:1(n-9......), tetradecadienoic acid, 14:2(n-6), and hexadecadienoic acid, 16:2(n-6). Palmitoyl-CoA and behenoyl-CoA dehydrogenase in fibroblasts were deficient. Muscle VLCAD activity was very low. DNA analysis revealed compound heterozygosity for two missense mutations in the VLCAD gene. The relatively mild clinical course may...... be due to residual enzyme activity as a consequence of the two missense mutations. Treatment with L-carnitine and medium chain triglycerides in the diet did not reduce the attacks of rhabdomyolysis....

  11. Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

    Science.gov (United States)

    Fredslund, Folmer; Otten, Harm; Gemperlein, Sabrina; Poulsen, Jens Christian N; Carius, Yvonne; Kohring, Gert Wieland; Lo Leggio, Leila

    2016-11-01

    Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the T m for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

  12. A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase.

    Science.gov (United States)

    Logemann, E; Reinold, S; Somssich, I E; Hahlbrock, K

    1997-08-01

    We describe an aromatic alcohol dehydrogenase with properties indicating a novel type of function in the defense response of plants to pathogens. To obtain the enzyme free of contamination with possible isoforms, a parsley (Petroselinum crispum) cDNA comprising the entire coding region of the elicitor-responsive gene, ELI3, was expressed in Escherichia coli. In accord with large amino acid sequence similarities with established cinnamyl and benzyl alcohol dehydrogenases from other plants, the enzyme efficiently reduced various cinnamyl and benzyl aldehydes using NADPH as a co-substrate. Highest substrate affinities were observed for cinnamaldehyde, 4-coumaraldehyde and coniferaldehyde, whereas sinapaldehyde, one of the most efficient substrates of several previously analyzed cinnamyl alcohol dehydrogenases and a characteristic precursor molecule of angiosperm lignin, was not converted. A single form of ELI3 mRNA was strongly and rapidly induced in fungal elicitor-treated parsley cells. These results, together with earlier findings that the ELI3 gene is strongly activated both in elicitor-treated parsley cells and at fungal infection sites in parsley leaves, but not in lignifying tissue, suggest a specific role of this enzyme in pathogen defense-related phenylpropanoid metabolism.

  13. GOLD HULL AND INTERNODE2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice.

    Science.gov (United States)

    Zhang, Kewei; Qian, Qian; Huang, Zejun; Wang, Yiqin; Li, Ming; Hong, Lilan; Zeng, Dali; Gu, Minghong; Chu, Chengcai; Cheng, Zhukuan

    2006-03-01

    Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis.

  14. GOLD HULL AND INTERNODE2 Encodes a Primarily Multifunctional Cinnamyl-Alcohol Dehydrogenase in Rice1

    Science.gov (United States)

    Zhang, Kewei; Qian, Qian; Huang, Zejun; Wang, Yiqin; Li, Ming; Hong, Lilan; Zeng, Dali; Gu, Minghong; Chu, Chengcai; Cheng, Zhukuan

    2006-01-01

    Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis. PMID:16443696

  15. 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. Copyright © Physiologia Plantarum 2012.

  16. NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860: Structural and Functional Features

    Directory of Open Access Journals (Sweden)

    Ekaterina Yu. Bezsudnova

    2016-01-01

    Full Text Available We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution, three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å, and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å. The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

  17. Five isozymograms of Trichomonas vaginalis and Trichomonas tenax: A comparative study%阴道毛滴虫和口腔毛滴虫5种同工酶谱对比分析

    Institute of Scientific and Technical Information of China (English)

    梁裕芬; 韦俊彬; 运晨霞; 黄庶识

    2010-01-01

    目的 分析阴道毛滴虫(Trichomonas vaginalis,Tv)和口腔毛滴虫(Trichomonas tenax,TT)的同工谱特征及其亲缘关系,寻找同工酶谱鉴别指标.方法 采用聚丙烯酰胺凝胶垂直板电泳,分别进行苹果酸脱氢酶(malate dehydrogenase, MDH)、谷氨酸脱氢酶(glutamic dehydrogenase, GLDH)、6-磷酸-葡萄糖脱氧酶(glucose-6-phosphate dehydrogenase, G6PD)、乙醇脱氢酶(alcohol dehydrogenase, ADH)和过氧化氢酶(Catalase)5种同工酶染色,比较分析2种滴虫的酶谱.结果 MDH同工酶:2种滴虫只有相对迁移率(Rm) 52、85 2条区带相同,而Rm 34、42、94、100 4条区带在2种滴虫之间明显不同;GLDH同工酶:TV无显带,TT共分离出6条酶带;G6PD同工酶:TV只有Rm35、42、52 3条区带,而TT除了显示这3条区带外,还显示出其他迁移率更大的区带;ADH和Catalase均无区带显示.结论 MDH、GLDH和G6PD3种同工酶均具有鉴别意义.

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

    Directory of Open Access Journals (Sweden)

    María Agustina Domínguez-Martín

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

  19. Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

    Science.gov (United States)

    Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

    1995-08-01

    Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

  20. 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