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Sample records for activity lactate dehydrogenase

  1. Lactate Dehydrogenase B Controls Lysosome Activity and Autophagy in Cancer.

    PubMed

    Brisson, Lucie; Bański, Piotr; Sboarina, Martina; Dethier, Coralie; Danhier, Pierre; Fontenille, Marie-Joséphine; Van Hée, Vincent F; Vazeille, Thibaut; Tardy, Morgane; Falces, Jorge; Bouzin, Caroline; Porporato, Paolo E; Frédérick, Raphaël; Michiels, Carine; Copetti, Tamara; Sonveaux, Pierre

    2016-09-12

    Metabolic adaptability is essential for tumor progression and includes cooperation between cancer cells with different metabolic phenotypes. Optimal glucose supply to glycolytic cancer cells occurs when oxidative cancer cells use lactate preferentially to glucose. However, using lactate instead of glucose mimics glucose deprivation, and glucose starvation induces autophagy. We report that lactate sustains autophagy in cancer. In cancer cells preferentially to normal cells, lactate dehydrogenase B (LDHB), catalyzing the conversion of lactate and NAD(+) to pyruvate, NADH and H(+), controls lysosomal acidification, vesicle maturation, and intracellular proteolysis. LDHB activity is necessary for basal autophagy and cancer cell proliferation not only in oxidative cancer cells but also in glycolytic cancer cells. PMID:27622334

  2. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling.

    PubMed

    Ahn, Heejin; Lee, Kyunghee; Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

  3. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling

    PubMed Central

    Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

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

    PubMed

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

    2011-11-22

    Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates. PMID:22065761

  5. Studies on the active center of D- and L-lactate dehydrogenases using oxamate-diaminohexyl-Sepharose affinity chromatography.

    PubMed Central

    Tuengler, P; Stein, T N; Long, G L

    1980-01-01

    Vertebrate and invertebrate L-lactate dehydrogenases (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) are effectively bound to oxamate-diaminohexyl-Sepharose, whereas several D-lactate dehydrogenases (D-lactate:NAD+ oxidoreductase, EC 1.1.1.28) do not bind to the same Sepharose. One explanation for our findings is that the enzymes' substrate is oriented in a reversed manner in the active center of the D- and L-lactate dehydrogenases. PMID:6934514

  6. Age-dependent variations of lactate dehydrogenase and creatine kinase activities in water buffalo calf serum.

    PubMed

    Avallone, L; Lombardi, P; Florio, S; d'Angelo, A; Bogin, E

    1996-12-01

    The electrophoretic patterns of the serum enzymes lactate dehydrogenase and creatine kinase from water buffalo calves are described. Differences in total activities as well as their relative distribution were seen at ages ranging from 1 to 10 weeks. While total lactate dehydrogenase activity increased by over 100%, total creatine kinase increased by almost 400%. The relative activities of lactate dehydrogenase 1 and 5 decreased with age. Lactate dehydrogenase 2 and 3 increased and lactate dehydrogenase 4 did not change. In relation to creatine kinase, the prevalent isoenzyme was creatine kinase-MM, but it's relative activity gradually decreased in comparison to the other two isoenzymes (creatine kinase-MB and creatine kinase-BB). Creatine kinase-BB was completely absent until the 3rd week of age. The percentage modifications of creatine kinase isoenzymes were correlated to age. The results suggest that isoenzymatic separation and characterization of lactate dehydrogenase and creatine kinase in relation to the various tissues can significantly contribute to the diagnosis of diseases which are linked to tissue damage.

  7. Relationship of lactate dehydrogenase activity to body measurements of Angus x Charolais cows and calves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives were to examine 1) relationships between lactate dehydrogenase (LDH) activity and body measurements of grazing beef cows, and 2) the association between maternal LDH activity in late gestation and subsequent calf birth weight (BRW), hip height (HH) at weaning, and adjusted weaning weight ...

  8. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  9. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  10. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  11. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

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

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase isoenzymes test system is a device intended to measure the activity of lactate dehydrogenase isoenzymes (a group of enzymes with similar biological activity) in serum. Measurements of...

  13. Multichannel Simultaneous Determination of Activities of Lactate Dehydrogenase

    SciTech Connect

    Ma, L.

    2000-09-12

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

  14. Fructose 1,6-diphosphate-activated L-lactate dehydrogenase from Streptococcus lactis: kinetic properties and factors affecting activation.

    PubMed Central

    Crow, V L; Pritchard, G G

    1977-01-01

    The L-(+)-lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) of Streptococcus lactis C10, like that of other streptococci, was activated by fructose 1,6-diphosphate (FDP). The enzyme showed some activity in the absence of FDP, with a pH optimum of 8.2; FDP decreased the Km for both pyruvate and reduced nicotinamide adenine dinucleotide (NADH) and shifted the pH optimum to 6.9. Enzyme activity showed a hyperbolic response to both NADH and pyruvate in all the buffers tried except phosphate buffer, in which the response to increasing NADH was sigmoidal. The FDP concentration required for half-maximal velocity (FDP0.5V) was markedly influenced by the nature of the assay buffer used. Thus the FDP0.5V was 0.002 mM in 90 mM triethanolamine buffer, 0.2 mM in 90 mM tris(hydroxymethyl)aminomethanemaleate buffer, and 4.4 mM in 90 mM phosphate buffer. Phosphate inhibition of FDP binding is not a general property of streptococcal lactate dehydrogenase, since the FDP0.5V value for S. faecalis 8043 lactate dehydrogenase was not increased by phosphate. The S. faecalis and S. lactis lactate dehydrogenases also differed in that Mn2+ enhanced FDP binding in S. faecalis but had no effect on the S. lactis dehydrogenase. The FDP concentration (12 to 15 mM) found in S. lactis cells during logarithmic growth on a high-carbohydrate (3% lactose) medium would be adequate to give almost complete activation of the lactate dehydrogenase even if the high FDP0.5V value found in 90 mM phosphate were similar to the FDP requirement in vivo. PMID:17595

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

  16. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed Central

    Kelly, N; Delaney, M; O'Carra, P

    1978-01-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  17. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed

    Kelly, N; Delaney, M; O'Carra, P

    1978-06-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  18. [Lactate dehydrogenase and Krebs cycle enzyme activity in rat liver during the growth of transplanted and spontaneous tumors].

    PubMed

    Morozkina, T S

    1978-03-01

    Certain distinctions in the mouse and rat liver responses to transplanted and spontaneous tumours have been discovered at the initial periods of their growth. The most pronounced changes (the mosaic distribution of enzymatic activity in the lobe) are observed in the case of spontaneous tumours. Activities the Krebs cycle enzymes, especially of NAD-dependent enzymes are seen inhibited in the tumour-bearing liver at the terminal periods of growth of both spontaneous and transplanted tumours; lactate dehydrogenase activity increases (with the exception of mitochondrial lactate dehydrogenase in the rat liver with transplanted sarcomas). PMID:684845

  19. Prostate cancer cells metabolize d-lactate inside mitochondria via a D-lactate dehydrogenase which is more active and highly expressed than in normal cells.

    PubMed

    de Bari, Lidia; Moro, Loredana; Passarella, Salvatore

    2013-03-01

    Although D-lactate metabolism has been shown to occur in a variety of mitochondria, the metabolic fate of D-lactate in cancer cells has never been investigated, as it is believed to be exported to the extracellular phase. We show that mitochondria from both cancer (PC-3) and normal (PNT1A) prostate cells can metabolize D-lactate in an energy competent manner. This is due to the mitochondrial D-lactate dehydrogenase, a membrane flavoprotein, the activity and protein level of which are higher in PC-3 than in PNT1A cells, as detected by both kinetic and immunological analysis. D-Lactate can enter prostate mitochondria and cause the export of newly synthesized malate in a carrier-mediated manner, with the rate of malate efflux from mitochondria twofold higher in cancer. PMID:23333299

  20. The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.

    PubMed Central

    Zammit, V A; Newsholme, E A

    1976-01-01

    Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is

  1. A comparison of potato and vertebrate lactate dehydrogenases.

    PubMed Central

    Poerio, E; Davies, D D

    1980-01-01

    A 2000-fold purification of L(+)-lactate dehydrogenase from potatoes is reported. Five isoenzymes of lactate dehydrogenase can be detected in crude extracts of potato, and three of these are present in the purified preparation. The enzyme (mol.wt. 150 000), which is composed of four subunits (mol.wt. 37 500), is active with the same oxo acids and hydroxy acids that have been reported as substrates with the same oxo acids and hydroxy acids that have been reported as substrates for vertebrate lactate dehydrogenases. These similarities between potato and vertebrate lactate dehydrogenases contrast sharply with some other reports on potato lactate dehydrogenase. These discrepancies are discussed in relation to the proposition that vertebrate and potato lactate dehydrogenases share a common evolutionary origin. PMID:7236200

  2. Muscular cholinesterase and lactate dehydrogenase activities in deep-sea fish from the NW Mediterranean.

    PubMed

    Koenig, Samuel; Solé, Montserrat

    2014-03-01

    Organisms inhabiting submarine canyons can be potentially exposed to higher inputs of anthropogenic chemicals than their counterparts from the adjacent areas. To find out to what extend this observation applies to a NW Mediterranean canyon (i.e. Blanes canyon) off the Catalan coast, four deep-sea fish species were collected from inside the canyon (BC) and the adjacent open slope (OS). The selected species were: Alepocephalus rostratus, Lepidion lepidion, Coelorinchus mediterraneus and Bathypterois mediterraneus. Prior to the choice of an adequate sentinel species, the natural variation of the selected parameters (biomarkers) in relation to factors such as size, sex, sampling depth and seasonality need to be characterised. In this study, the activities of cholinesterases (ChEs) and lactate dehydrogenase (LDH) enzymes were determined in the muscle of the four deep-sea fish. Of all ChEs, acetylcholinesterase (AChE) activity was dominant and selected for further monitoring. Overall, AChE activity exhibited a significant relationship with fish size whereas LDH activity was mostly dependent on the sex and gonadal development status, although in a species-dependent manner. The seasonal variability of LDH activity was more marked than for AChE activity, and inside-outside canyon (BC-OS) differences were not consistent in all contrasted fish species, and in fact they were more dependent on biological traits. Thus, they did not suggest a differential stress condition between sites inside and outside the canyon. PMID:24296242

  3. Muscular cholinesterase and lactate dehydrogenase activities in deep-sea fish from the NW Mediterranean.

    PubMed

    Koenig, Samuel; Solé, Montserrat

    2014-03-01

    Organisms inhabiting submarine canyons can be potentially exposed to higher inputs of anthropogenic chemicals than their counterparts from the adjacent areas. To find out to what extend this observation applies to a NW Mediterranean canyon (i.e. Blanes canyon) off the Catalan coast, four deep-sea fish species were collected from inside the canyon (BC) and the adjacent open slope (OS). The selected species were: Alepocephalus rostratus, Lepidion lepidion, Coelorinchus mediterraneus and Bathypterois mediterraneus. Prior to the choice of an adequate sentinel species, the natural variation of the selected parameters (biomarkers) in relation to factors such as size, sex, sampling depth and seasonality need to be characterised. In this study, the activities of cholinesterases (ChEs) and lactate dehydrogenase (LDH) enzymes were determined in the muscle of the four deep-sea fish. Of all ChEs, acetylcholinesterase (AChE) activity was dominant and selected for further monitoring. Overall, AChE activity exhibited a significant relationship with fish size whereas LDH activity was mostly dependent on the sex and gonadal development status, although in a species-dependent manner. The seasonal variability of LDH activity was more marked than for AChE activity, and inside-outside canyon (BC-OS) differences were not consistent in all contrasted fish species, and in fact they were more dependent on biological traits. Thus, they did not suggest a differential stress condition between sites inside and outside the canyon.

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

    PubMed

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

    2015-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  6. Lactate dehydrogenase activity in Bacteroides fragilis group strains with induced resistance to metronidazole.

    PubMed

    Presečki Stanko, Aleksandra; Sóki, Jozsef; Varda Brkić, Dijana; Plečko, Vanda

    2016-06-01

    The aims of this study were to induce in vitro metronidazole resistance in nim-negative Bacteroides fragilis group strains and to determine the lactate dehydrogenase (LDH) activity of the induced strains. A collection of B. fragilis group strains were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Minimum inhibitory concentrations (MICs) for metronidazole were determined by the agar dilution technique. The presence of nim genes was screened by PCR. A sample of 52 nim-negative metronidazole-susceptible strains were selected at random and were exposed to metronidazole in the resistance induction experiment. LDH activity was measured by spectrophotometry. Of the 52 selected strains, 12 (23.1%) acquired resistance to metronidazole. MICs ranged from 8mg/L to 96mg/L. Eight of the twelve induced strains displayed decreased LDH activity, whilst only one expressed a significant increase in LDH activity with LDH values of 49.1U/mg and 222.0U/mg, respectively. In conclusion, in vitro induction of metronidazole resistance could be selected in nim-negative B. fragilis group strains. A statistically significant decrease in LDH activity was in contrast to previous findings in which, underlying higher metronidazole MICs, an increase in LDH activity compensated for the decreased activity of pyruvate-ferredoxin oxidoreductase (PFOR). These findings could be explained if the induction caused only physiological and not genetic changes. We believe that genetic mutations in the B. fragilis strain that demonstrated an emergent increase in LDH activity were responsible for the increased activity. PMID:27436459

  7. Genetics Home Reference: lactate dehydrogenase deficiency

    MedlinePlus

    ... dehydrogenase-B pieces (subunits) of the lactate dehydrogenase enzyme. This enzyme is found throughout the body and is important ... cells. There are five different forms of this enzyme, each made up of four protein subunits. Various ...

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2013-07-19

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

  10. Estimating the number of viable animal cells in multi-well cultures based on their lactate dehydrogenase activities.

    PubMed

    Haslam, G; Wyatt, D; Kitos, P A

    2000-01-01

    A method is described for estimating the numbers ofanimal cells in multi-well culture by simultaneouslymeasuring the lactate dehydrogenase activity of thetotal culture and the medium. The difference betweenthe two reflects the dehydrogenase content of thecells and correlates with cell number. This LDH/INTmethod was tested using several lines of normal andtransformed suspension and adherent cells. Thelactate dehydrogenase activities of duplicate cultureswere determined colourimetrically using reactioncocktails containing lactate, NAD(+), diaphorase,and p-iodonitrotetrazolium violet, with and withoutTriton X-100. The difference in absorbance at 490 nm(DeltaA(490) = A(490, test) - A(490, control)) was used to calculate the lactatedehydrogenase activity of the total culture (+ Triton)and the medium (- Triton). The cellular lactatedehydrogenase activity (difference between totaland medium dehydrogenaseactivities) was proportional to viable cell number. The effects on cell growth of four metabolicinhibitors, sodium azide, actinomycin D,cycloheximide, and taxol, were determined using theLDH/INT assay and direct cell counting. The inhibitorconcentrations that caused decreases in the LDHactivity and cell number by 50% were similar. TheLDH/INT assay is quick and sensitive, works equallywell for adherent and suspension cells, and providesinformation about LDH activities of both the mediumand cells. It is particularly useful for screeningpotential cell-growth inhibitors. PMID:19002967

  11. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes.

  12. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes. PMID:25542170

  13. Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase

    PubMed Central

    Wu, Gang; Fiser, András; ter Kuile, Benno; Šali, Andrej; Müller, Miklós

    1999-01-01

    Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently. PMID:10339579

  14. Structure-Function Relationships in Lactate Dehydrogenase

    PubMed Central

    Adams, Margaret J.; Buehner, Manfred; Chandrasekhar, K.; Ford, Geoffrey C.; Hackert, Marvin L.; Liljas, Anders; Rossmann, Michael G.; Smiley, Ira E.; Allison, William S.; Everse, Johannes; Kaplan, Nathan O.; Taylor, Susan S.

    1973-01-01

    The binding of coenzyme and substrate are considered in relation to the known primary and tertiary structure of lactate dehydrogenase (EC 1.1.1.27). The adenine binds in a hydrophobic crevice, and the two coenzyme phosphates are oriented by interactions with the protein. The positively charged guanidinium group of arginine 101 then folds over the negatively charged phosphates, collapsing the loop region over the active center and positioning the unreactive B side of the nicotinamide in a hydrophobic protein environment. Collapse of the loop also introduces various charged groups into the vicinity of the substrate binding site. The substrate is situated between histidine 195 and the C4 position on the nicotinamide ring, and is partially oriented by interactions between its carboxyl group and arginine 171. The spatial arrangements of these groups may provide the specificity for the L-isomer of lactate. PMID:4146647

  15. Properties of lactate dehydrogenase in a psychrophilic marine bacterium.

    PubMed Central

    Mitchell, P; Yen, H C; Mathemeier, P F

    1985-01-01

    Lactate dehydrogenase (EC 1.1.1.27) from Vibrio marinus MP-1 was purified 15-fold and ammonium activated. The optimum pH for pyruvate reduction was 7.4. Maximum lactate dehydrogenase activity occurred at 10 to 15 degrees C, and none occurred at 40 degrees C. The crude-extract enzyme was stable between 15 and 20 degrees C and lost 50% of its activity after 60 min at 45 degrees C. The partially purified enzyme was stable between 8 and 15 degrees C and lost 50% of its activity after 60 min at 30 degrees C. The thermal stability of lactate dehydrogenase was increased by mercaptoethanol, with 50% remaining activity at 42 degrees C. Images PMID:4004236

  16. Protein Conformational Landscapes and Catalysis. Influence of Active Site Conformations in the Reaction Catalyzed by L-Lactate Dehydrogenase

    PubMed Central

    Świderek, Katarzyna; Tuñón, Iñaki; Martí, Sergio; Moliner, Vicent

    2015-01-01

    In the last decade L-Lactate Dehydrogenase (LDH) has become an extremely useful marker in both clinical diagnosis and in monitoring the course of many human diseases. It has been assumed from the 80s that the full catalytic process of LDH starts with the binding of the cofactor and the substrate followed by the enclosure of the active site by a mobile loop of the protein before the reaction to take place. In this paper we show that the chemical step of the LDH catalyzed reaction can proceed within the open loop conformation, and the different reactivity of the different protein conformations would be in agreement with the broad range of rate constants measured in single molecule spectrometry studies. Starting from a recently solved X-ray diffraction structure that presented an open loop conformation in two of the four chains of the tetramer, QM/MM free energy surfaces have been obtained at different levels of theory. Depending on the level of theory used to describe the electronic structure, the free energy barrier for the transformation of pyruvate into lactate with the open conformation of the protein varies between 12.9 and 16.3 kcal/mol, after quantizing the vibrations and adding the contributions of recrossing and tunneling effects. These values are very close to the experimentally deduced one (14.2 kcal·mol−1) and ~2 kcal·mol−1 smaller than the ones obtained with the closed loop conformer. Calculation of primary KIEs and IR spectra in both protein conformations are also consistent with our hypothesis and in agreement with experimental data. Our calculations suggest that the closure of the active site is mainly required for the inverse process; the oxidation of lactate to pyruvate. According to this hypothesis H4 type LDH enzyme molecules, where it has been propose that lactate is transformed into pyruvate, should have a better ability to close the mobile loop than the M4 type LDH molecules. PMID:25705562

  17. The Arabidopsis KS-type dehydrin recovers lactate dehydrogenase activity inhibited by copper with the contribution of His residues.

    PubMed

    Hara, Masakazu; Monna, Shuhei; Murata, Takae; Nakano, Taiyo; Amano, Shono; Nachbar, Markus; Wätzig, Hermann

    2016-04-01

    Dehydrin, which is one of the late embryogenesis abundant (LEA) proteins, is involved in the ability of plants to tolerate the lack of water. Although many reports have indicated that dehydrins bind heavy metals, the physiological role of this metal binding has not been well understood. Here, we report that the Arabidopsis KS-type dehydrin (AtHIRD11) recovered the lactate dehydrogenase (LDH) activity denatured by Cu(2+). The LDH activity was partially inhibited by 0.93 μM Cu(2+) but totally inactivated by 9.3 μM Cu(2+). AtHIRD11 recovered the activity of LDH treated with 9.3 μM Cu(2+) in a dose-dependent manner. The recovery activity of AtHIRD11 was significantly higher than those of serum albumin and lysozyme. The conversion of His residues to Ala in AtHIRD11 resulted in the loss of the Cu(2+) binding of the protein as well as the disappearance of the conformational change induced by Cu(2+) that is observed by circular dichroism spectroscopy. The mutant protein showed lower recovery activity than the original AtHIRD11. These results indicate that AtHIRD11 can reactivate LDH inhibited by Cu(2+) via the His residues. This function may prevent physiological damage to plants due to heavy-metal stress. PMID:26940498

  18. Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

    PubMed Central

    Kolk, A H; van Kuyk, L; Boettcher, B

    1978-01-01

    Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein. Images Fig. 2. Fig. 3. PMID:213050

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

    PubMed Central

    Baker, J. L.; Derr, A. M.; Faustoferri, R. C.

    2015-01-01

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

  20. Peafowl lactate dehydrogenase: problem of isoenzyme identification.

    PubMed

    Rose, R G; Wilson, A C

    1966-09-16

    Peafowl, like other vertebrates, contain multiple forms of lactate dehydrogenase. The electrophoretic properties of the peafowl isoenzymes are unusual in that the isoenzyme from heart tissue can be either more or less anodic than that of muscle, depending on the pH. This finding focuses attention on the problem of isoenzyme identification. It is suggested that isoenzymes be identified on the basis of properties that are chemically and biologically more significant than electrophoretic mobility.

  1. Peafowl lactate dehydrogenase: problem of isoenzyme identification.

    PubMed

    Rose, R G; Wilson, A C

    1966-09-16

    Peafowl, like other vertebrates, contain multiple forms of lactate dehydrogenase. The electrophoretic properties of the peafowl isoenzymes are unusual in that the isoenzyme from heart tissue can be either more or less anodic than that of muscle, depending on the pH. This finding focuses attention on the problem of isoenzyme identification. It is suggested that isoenzymes be identified on the basis of properties that are chemically and biologically more significant than electrophoretic mobility. PMID:5917779

  2. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase

    SciTech Connect

    Mat-Jan, F.; Alam, K.Y.; Clark, D.P. )

    1989-01-01

    Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.

  3. Elevated lactate dehydrogenase activity and increased cardiovascular mortality in the arsenic-endemic areas of southwestern Taiwan

    SciTech Connect

    Liao, Ya-Tang; Chen, Chien-Jen; Li, Wan-Fen; Hsu, Ling-I; Tsai, Li-Yu; Huang, Yeou-Lih; Sun, Chien-Wen; Chen, Wei J.; Wang, Shu-Li

    2012-08-01

    Arsenic ingestion has been linked to increasing global prevalence of and mortality from cardiovascular disease (CVD); arsenic can be removed from drinking water to reduce related health effects. Lactate dehydrogenase (LDH) is used for the evaluation of acute arsenic toxicity in vivo and in vitro, but it is not validated for the evaluation of long-term, chronic arsenic exposure. The present study examined the long-term effect of chronic arsenic exposure on CVD and serum LDH levels, after consideration of arsenic metabolism capacity. A total of 380 subjects from an arseniasis-endemic area and 303 from a non-endemic area of southwestern Taiwan were recruited in 2002. Various urinary arsenic species were analyzed using high-performance liquid chromatography (HPLC) and hydride generation systems. Fasting serum was used for quantitative determination of the total LDH activity. A significant dose–response relationship was observed between arsenic exposure and LDH elevation, independent of urinary arsenic profiles (P < 0.001). Furthermore, abnormal LDH elevation was associated with CVD mortality after adjustment for Framingham risk scores for 10-year CVD and arsenic exposure (hazard ratio, 3.98; 95% confidence interval, 1.07–14.81). LDH was elevated in subjects with arsenic exposure in a dose-dependent manner. LDH is a marker of arsenic toxicity associated with CVD mortality. Results of this study have important implications for use in ascertaining long-term arsenic exposure risk of CVD. -- Highlights: ► We showed that arsenic exposure was correlated with LDH elevation. ► LDH elevation was related to arsenic methylation capacity. ► Abnormal LDH elevation can be a marker of susceptibility to CVD mortality.

  4. Elevated Plasma Activity of Lactate Dehydrogenase Isoenzyme-3 (LDH3) in Experimentally Induced Immunologic Lung Injury

    PubMed Central

    Hagadorn, J. E.; Bloor, C. M.; Yang, M. S.

    1971-01-01

    Normal rats injected intravenously with rabbit antiserum to rat lung develop acute pulmonary lesions characterized by an altered vascular permeability. In the present study, an increase in plasma LDH3 activity is shown to be positively correlated with the different levels of circulating antilung antibodies and with the morphologic severity of lung injury elicited by these pathogenic immunoglobulins. Within 24 hours, the acute lung changes are resolved, accompanied by a return of the activities of plasma LDH isoenzymes to normal. It is proposed that the plasma LDH3 isoenzymes are released into the circulation from injured alveolar capillary endothelial cells. ImagesFig 1 PMID:5133518

  5. NADP+-Preferring D-Lactate Dehydrogenase from Sporolactobacillus inulinus.

    PubMed

    Zhu, Lingfeng; Xu, Xiaoling; Wang, Limin; Dong, Hui; Yu, Bo; Ma, Yanhe

    2015-09-01

    Hydroxy acid dehydrogenases, including l- and d-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD(+) as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d-lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn(174) was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.

  6. Lactate dehydrogenase in two digenetic trematodes and their host.

    PubMed

    Haque, M; Siddiqi, A H; Siddiqui, J

    1990-12-01

    Polyacrylamide gel electrophoresis of the two digenetic trematodes, Gigantocotyle explanatum from the liver and Gastrothylax crumenifer from the rumen of the water buffalo, Bubalus bubalis revealed the presence of at least six and seven isoenzymes of lactate dehydrogenase (LDH), respectively in a partially purified enzyme preparation. The respective host tissues showed five isoenzymes of LDH, which are characteristic to the vertebrates. Both parachloromercuribenzoate and iodoacetate affected the LDH activity of the parasites and host tissues differently. Spectrophotometric analysis also showed different specific activity and susceptibility to the action of thiol inhibitors. The host LDH was quite stable at 57 degrees C for 30 min, but that of the parasites was less stable.

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

    PubMed

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

    2013-01-01

    The intertidal marine snail, Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH) is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD(+), which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 1.1.1.28) from foot muscle of aerobic control versus 24 h anoxia-exposed L. littorea. Anoxic LDH displayed a near 50% decrease in V max (pyruvate-reducing direction) as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves. PMID:24233354

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

    PubMed Central

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

    2013-01-01

    The intertidal marine snail, Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH) is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD+, which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 1.1.1.28) from foot muscle of aerobic control versus 24 h anoxia-exposed L. littorea. Anoxic LDH displayed a near 50% decrease in Vmax (pyruvate-reducing direction) as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves. PMID:24233354

  9. [Human semen lactate dehydrogenase isoenzymes in fertility studies (author's transl)].

    PubMed

    Gonzalez Buitrago, J M; García Díez, L C; de Castro, S

    1981-01-01

    The lactate dehydrogenase isoenzyme pattern has been obtained in the semen of 87 males undergoing fertility studies. The proportion of LDH-X, the isoenzyme specific to the spermatozoa, is reduced in proportion to the reduction of the sperm density and motility. LDH-X is the most abundant isoenzyme in the semen of normospermic subjects. As to the other isoenzymes, the most abundant ones are the LDH-2 and the LDH-3. The results obtained lead us to conclude that the measurement of the lactate dehydrogenase isoenzymes may be useful in studies of fertility as an indicative parameter of the quality of the semen.

  10. Macromolecular crowding effect upon in vitro enzyme kinetics: mixed activation-diffusion control of the oxidation of NADH by pyruvate catalyzed by lactate dehydrogenase.

    PubMed

    Balcells, Cristina; Pastor, Isabel; Vilaseca, Eudald; Madurga, Sergio; Cascante, Marta; Mas, Francesc

    2014-04-17

    Enzyme kinetics studies have been usually designed as dilute solution experiments, which differ substantially from in vivo conditions. However, cell cytosol is crowded with a high concentration of molecules having different shapes and sizes. The consequences of such crowding in enzymatic reactions remain unclear. The aim of the present study is to understand the effect of macromolecular crowding produced by dextran of different sizes and at diverse concentrations in the well-known reaction of oxidation of NADH by pyruvate catalyzed by L-lactate dehydrogenase (LDH). Our results indicate that the reaction rate is determined by both the occupied volume and the relative size of dextran obstacles with respect to the enzyme present in the reaction. Moreover, we analyzed the influence of macromolecular crowding on the Michaelis-Menten constants, vmax and Km. The obtained results show that only high concentrations and large sizes of dextran reduce both constants suggesting a mixed activation-diffusion control of this enzymatic reaction due to the dextran crowding action. From our knowledge, this is the first experimental study that depicts mixed activation-diffusion control in an enzymatic reaction due to the effect of crowding.

  11. Assessment of freshness and freeze-thawing of sea bream fillets (Sparus aurata) by a cytosolic enzyme: Lactate dehydrogenase.

    PubMed

    Diop, Mamadou; Watier, Denis; Masson, Pierre-Yves; Diouf, Amadou; Amara, Rachid; Grard, Thierry; Lencel, Philippe

    2016-11-01

    The evaluation of freshness and freeze-thawing of fish fillets was carried out by assessment of autolysis of cells using a cytosolic enzyme lactate dehydrogenase. Autolysis plays an important role in spoilage of fish and postmortem changes in fish tissue are due to the breakdown of the cellular structures and release of cytoplasmic contents. The outflow of a cytosolic enzyme, lactate dehydrogenase, was studied in sea bream fillets and the Sparus aurata fibroblasts (SAF-1) cell-line during an 8day storage period at +4°C. A significant increase of lactate dehydrogenase release was observed, especially after 5days of storage. The ratio between the free and the total lactate dehydrogenase activity is a promising predictive marker to measure the quality of fresh fish fillets. The effect of freeze-thawing on cytosolic lactate dehydrogenase and lysosomal α-d-glucosidase activities was also tested. Despite the protecting effect of the tissue compared to the cell-line, a loss of lactate dehydrogenase activity, but not of α-d-glucosidase, was observed. In conclusion, lactate dehydrogenase may be used as a marker to both assess freshness of fish and distinguish between fresh and frozen-thawed fish fillets.

  12. Assessment of freshness and freeze-thawing of sea bream fillets (Sparus aurata) by a cytosolic enzyme: Lactate dehydrogenase.

    PubMed

    Diop, Mamadou; Watier, Denis; Masson, Pierre-Yves; Diouf, Amadou; Amara, Rachid; Grard, Thierry; Lencel, Philippe

    2016-11-01

    The evaluation of freshness and freeze-thawing of fish fillets was carried out by assessment of autolysis of cells using a cytosolic enzyme lactate dehydrogenase. Autolysis plays an important role in spoilage of fish and postmortem changes in fish tissue are due to the breakdown of the cellular structures and release of cytoplasmic contents. The outflow of a cytosolic enzyme, lactate dehydrogenase, was studied in sea bream fillets and the Sparus aurata fibroblasts (SAF-1) cell-line during an 8day storage period at +4°C. A significant increase of lactate dehydrogenase release was observed, especially after 5days of storage. The ratio between the free and the total lactate dehydrogenase activity is a promising predictive marker to measure the quality of fresh fish fillets. The effect of freeze-thawing on cytosolic lactate dehydrogenase and lysosomal α-d-glucosidase activities was also tested. Despite the protecting effect of the tissue compared to the cell-line, a loss of lactate dehydrogenase activity, but not of α-d-glucosidase, was observed. In conclusion, lactate dehydrogenase may be used as a marker to both assess freshness of fish and distinguish between fresh and frozen-thawed fish fillets. PMID:27211667

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

    PubMed Central

    Dave, Khyati K.; Punekar, Narayan S.

    2015-01-01

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

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

    PubMed

    Dave, Khyati K; Punekar, Narayan S

    2015-01-01

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

  15. Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis).

    PubMed

    Guha, Anirban; Gera, Sandeep; Sharma, Anshu

    2012-03-01

    Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis (SCM) with the aim of developing suitable diagnostic kit for SCM. Trace elements and enzyme activity in milk were estimated with Atomic absorption Spectrophotometer, GBC 932 plus and biochemical methods, respectively. Somatic cell count (SCC) was done microscopically. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. A statistically significant (p<0.01) increase in SCC, Fe, Zn, Co and LDH occurred in SCM milk containing gram positive bacterial agents only. ALP was found to be elevated in milk infected by both gram positive and negative bacteria. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×10(5) cells/ml of milk as the benchmark. Only ALP and Zn, the former being superior, were found to be suitable for diagnosis of SCM irrespective of etiological agents. LDH, Co and Fe can be introduced in the screening programs where Gram positive bacteria are omnipresent. It is recommended that both ALP and Zn be measured together in milk to diagnose buffalo SCM, irrespective of etiology. PMID:25049573

  16. Production of natural antimicrobial compound D-phenyllactic acid using Leuconostoc mesenteroides ATCC 8293 whole cells involving highly active D-lactate dehydrogenase.

    PubMed

    Li, L; Shin, S-Y; Lee, K W; Han, N S

    2014-10-01

    Phenyllactic acid (PLA) is an antimicrobial compound naturally synthesized in various fermented foods and its D-form of PLA is known to be more active than the L-isomer. In this study, Leuconostoc mesenteroides ATCC 8293 cells, elaborating D-lactate dehydrogenase (D-ldh) were used to produce D-PLA from phenylpyruvic acid (PPA). When cultured in the presence of PPA (≤50 mmol l(-1)), growing cells produced a maximum yield of 35 mmol l(-1) of D-PLA, and the yields were between 75·2 and 83·3%. Higher conversion yields were obtained at pH 6·0-7·0 when growing cells were used, while the optimum pH range was broader for resting cells. The time required for the complete conversion of PPA into PLA could be shortened to 3 h using resting cells. D-ldh, an enzyme encoded by the LEUM_1756 gene of Leuc. mesenteroides ATCC 8293, was found to be responsible for the conversion of PPA into PLA. The Km and kcat values of the enzyme for PPA were found to be 15·4 mmol l(-1) and 5645 s(-1), respectively. The conditions required for the efficient production of D-PLA were optimized for both growing and resting cells of Leuc. mesenteroides, with special emphasis on achieving high stereoselectivity and conversion yield. Significance and impact of the study: This is the first study on the production of D-phenyllactic acid, which is a natural antimicrobial compound, from phenylpyruvate using Leuconostoc mesenteroides cells. The strain, ATCC 8293, that was used in the study, possesses high stereoselectivity and delivers a high yield. Therefore, it might be a promising candidate for use in large-scale production facilities and in fermented foods.

  17. Functional Replacement of the Escherichia coli d-(−)-Lactate Dehydrogenase Gene (ldhA) with the l-(+)-Lactate Dehydrogenase Gene (ldhL) from Pediococcus acidilactici†

    PubMed Central

    Zhou, Shengde; Shanmugam, K. T.; Ingram, L. O.

    2003-01-01

    The microbial production of l-(+)-lactic acid is rapidly expanding to allow increased production of polylactic acid (PLA), a renewable, biodegradable plastic. The physical properties of PLA can be tailored for specific applications by controlling the ratio of l-(+) and d-(−) isomers. For most uses of PLA, the l-(+) isomer is more abundant. As an approach to reduce costs associated with biocatalysis (complex nutrients, antibiotics, aeration, product purification, and waste disposal), a recombinant derivative of Escherichia coli W3110 was developed that contains five chromosomal deletions (focA-pflB frdBC adhE ackA ldhA). This strain was constructed from a d-(−)-lactic acid-producing strain, SZ63 (focA-pflB frdBC adhE ackA), by replacing part of the chromosomal ldhA coding region with Pediococcus acidilactici ldhL encoding an l-lactate dehydrogenase. Although the initial strain (SZ79) grew and fermented poorly, a mutant (SZ85) was readily isolated by selecting for improved growth. SZ85 exhibited a 30-fold increase in l-lactate dehydrogenase activity in comparison to SZ79, functionally replacing the native d-lactate dehydrogenase activity. Sequencing revealed mutations in the upstream, coding, and terminator regions of ldhL in SZ85, which are presumed to be responsible for increased l-lactate dehydrogenase activity. SZ85 produced l-lactic acid in M9 mineral salts medium containing glucose or xylose with a yield of 93 to 95%, a purity of 98% (based on total fermentation products), and an optical purity greater than 99%. Unlike other recombinant biocatalysts for l-lactic acid, SZ85 remained prototrophic and is devoid of plasmids and antibiotic resistance genes. PMID:12676706

  18. Membrane-bound, pyridine nucleotide-independent L-lactate dehydrogenase of Rhodopseudomonas sphaeroides.

    PubMed Central

    Markwell, J P; Lascelles, J

    1978-01-01

    Rhodopseudomonas sphaeroides has a pyridine nucleotide-independent L-lactate dehydrogenase associated with the membrane fraction of cells grown either aerobically or phototrophically. The dehydrogenase is present in cells grown on a variety of carbon sources, but at levels less than 20% of that found in cells grown with DL-lactate. The dehydrogenase has been purified 45-fold from membranes of strain L-57, a non-photosynthetic mutant, by steps involving solubilization with lauryl dimethylamine oxide and three anion-exchange chromatography steps. The purified enzyme was specific for the L-isomer of lactate. The Km of the purified enzyme for L-lactate is 1.4 mM, whereas that of the membrane-associated enzyme is 0.5 mM. The enzyme activity was inhibited competitively by D-lactate and non-competitively by oxalate and oxamate. Quinacrine, a flavin analog, also inhibited the activity. The inducible enzyme may serve as a marker of membrane protein in studies of membrane development. PMID:304854

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

    PubMed

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

    1976-06-01

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

  20. Metabolic engineering of lactate dehydrogenase rescues mice from acidosis.

    PubMed

    Acharya, Abhinav P; Rafi, Mohammad; Woods, Elliot C; Gardner, Austin B; Murthy, Niren

    2014-06-05

    Acidosis causes millions of deaths each year and strategies for normalizing the blood pH in acidosis patients are greatly needed. The lactate dehydrogenase (LDH) pathway has great potential for treating acidosis due to its ability to convert protons and pyruvate into lactate and thereby raise blood pH, but has been challenging to develop into a therapy because there are no pharmaceutical-based approaches for engineering metabolic pathways in vivo. In this report we demonstrate that the metabolic flux of the LDH pathway can be engineered with the compound 5-amino-2-hydroxymethylphenyl boronic acid (ABA), which binds lactate and accelerates the consumption of protons by converting pyruvate to lactate and increasing the NAD(+)/NADH ratio. We demonstrate here that ABA can rescue mice from metformin induced acidosis, by binding lactate, and increasing the blood pH from 6.7 to 7.2 and the blood NAD(+)/NADH ratio by 5 fold. ABA is the first class of molecule that can metabolically engineer the LDH pathway and has the potential to have a significant impact on medicine, given the large number of patients that suffer from acidosis.

  1. Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals

    PubMed Central

    Schueren, Fabian; Lingner, Thomas; George, Rosemol; Hofhuis, Julia; Dickel, Corinna; Gärtner, Jutta; Thoms, Sven

    2014-01-01

    Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes. DOI: http://dx.doi.org/10.7554/eLife.03640.001 PMID:25247702

  2. Control of Lactate Dehydrogenase, Lactate Glycolysis, and α-Amylase by O2 Deficit in Barley Aleurone Layers 1

    PubMed Central

    Hanson, Andrew D.; Jacobsen, John V.

    1984-01-01

    After 4 days in an atmosphere of N2, aleurone layers of barley (Hordeum vulgare L. cv Himalaya) remained viable as judged by their ability to produce near normal amounts of α-amylases when incubated with gibberellic acid (GA3) in air. However, layers did not produce α-amylase when GA3 was supplied under N2, apparently because α-amylase mRNA failed to accumulate. When an 8-hour pulse of [U-14C]glucose was supplied under N2 to freshly prepared aleurone layers, both [14C]lactate and [14C]ethanol accumulated; the [14C]lactate/[14C]ethanol ratio was about 0.3. Prior incubation of layers for 1 day under N2 changed this ratio to about 0.8, indicating an increase in the relative importance of the lactate branch of glycolysis. l(+)Lactate dehydrogenase (LDH) activity was low in freshly prepared aleurone layers and increased 10-fold during 2 days under N2, whereas alcohol dehydrogenase activity (ADH) was high initially and rose by 60%. The responses of LDH and ADH activities to O2 tension were dissimilar; when layers were incubated in various O2/N2 mixtures, LDH activity peaked at 2 to 5% O2 whereas ADH activity was highest at 0% O2. The LDH activity was resolved into several enzymically active bands by native polyacrylamide gel electrophoresis. We conclude that barley aleurone layers are highly adapted to O2 deficiency, that they possess an inducible LDH system as well as an ADH system, and we infer that the LDH and ADH systems are independently regulated. Images Fig. 2 Fig. 5 PMID:16663667

  3. The lactate dehydrogenase of the icefish heart: biochemical adaptations to hypoxia tolerance.

    PubMed

    Feller, G; Pauly, J P; Smal, A; O'Carra, P; Gerday, C

    1991-09-20

    Cardiac lactate dehydrogenase from the hemoglobin- and myoglobin-free antarctic icefish has been purified by affinity chromatography. Structural and kinetic properties of the enzyme were found close or identical to those of its skeletal muscle counterpart and other M-type lactate dehydrogenases. A model involving a dual oxidative-anaerobic metabolism of the icefish heart is proposed. PMID:1911860

  4. Lactate dehydrogenase (LD), alkaline phosphatase (ALP) isoenzymatic patterns in Iraqi children with visceral leishmaniasis before and after treatment with stibogluconate.

    PubMed

    Taher, Jasim Hameed; Al-Mulla Hummadi, Yassir Mustafa Kamal; Al-Bashir, Nada Muhammed Taha; Al-Araji, Ali Shaalan

    2016-06-01

    The mean levels of alkaline phosphatase (ALP), lactate dehydrogenase enzymes exhibited a significant elevation in visceral leishmaniasis (VL) patients compared to the control. There was no significant change in relation to the sex and age. ALP isoenzymes revealed three banding patterns which differ from the three zymodems which were obtained from control group. These differences may be due to isoenzymes activity of patients with VL before and after therapy. Lactate dehydrogenase (LD) isoenzymes revealed five banding patterns differ from the five normal zymodems. These differences mainly occurred due to LD isoenzymes activity in patients with VL before and after therapy. PMID:27413293

  5. Lactate dehydrogenase concentration in nasal wash fluid indicates severity of rhinovirus-induced wheezy bronchitis in preschool children.

    PubMed

    Cangiano, Giulia; Proietti, Elena; Kronig, Marie Noelle; Kieninger, Elisabeth; Sadeghi, Christine D; Gorgievski, Meri; Barbani, Maria Teresa; Midulla, Fabio; Tapparel, Caroline; Kaiser, Laurent; Alves, Marco P; Regamey, Nicolas

    2014-12-01

    The clinical course of rhinovirus (RV)-associated wheezing illnesses is difficult to predict. We measured lactate dehydrogenase concentrations, RV load, antiviral and proinflammatory cytokines in nasal washes obtained from 126 preschool children with RV wheezy bronchitis. lactate dehydrogenase values were inversely associated with subsequent need for oxygen therapy. lactate dehydrogenase may be a useful biomarker predicting disease severity in RV wheezy bronchitis.

  6. Identification of lactate dehydrogenase as a mammalian pyrroloquinoline quinone (PQQ)-binding protein.

    PubMed

    Akagawa, Mitsugu; Minematsu, Kenji; Shibata, Takahiro; Kondo, Tatsuhiko; Ishii, Takeshi; Uchida, Koji

    2016-05-27

    Pyrroloquinoline quinone (PQQ), a redox-active o-quinone, is an important nutrient involved in numerous physiological and biochemical processes in mammals. Despite such beneficial functions, the underlying molecular mechanisms remain to be established. In the present study, using PQQ-immobilized Sepharose beads as a probe, we examined the presence of protein(s) that are capable of binding PQQ in mouse NIH/3T3 fibroblasts and identified five cellular proteins, including l-lactate dehydrogenase (LDH) A chain, as potential mammalian PQQ-binding proteins. In vitro studies using a purified rabbit muscle LDH show that PQQ inhibits the formation of lactate from pyruvate in the presence of NADH (forward reaction), whereas it enhances the conversion of lactate to pyruvate in the presence of NAD(+) (reverse reaction). The molecular mechanism underlying PQQ-mediated regulation of LDH activity is attributed to the oxidation of NADH to NAD(+) by PQQ. Indeed, the PQQ-bound LDH oxidizes NADH, generating NAD(+), and significantly catalyzes the conversion of lactate to pyruvate. Furthermore, PQQ attenuates cellular lactate release and increases intracellular ATP levels in the NIH/3T3 fibroblasts. Our results suggest that PQQ, modulating LDH activity to facilitate pyruvate formation through its redox-cycling activity, may be involved in the enhanced energy production via mitochondrial TCA cycle and oxidative phosphorylation.

  7. Identification of lactate dehydrogenase as a mammalian pyrroloquinoline quinone (PQQ)-binding protein

    PubMed Central

    Akagawa, Mitsugu; Minematsu, Kenji; Shibata, Takahiro; Kondo, Tatsuhiko; Ishii, Takeshi; Uchida, Koji

    2016-01-01

    Pyrroloquinoline quinone (PQQ), a redox-active o-quinone, is an important nutrient involved in numerous physiological and biochemical processes in mammals. Despite such beneficial functions, the underlying molecular mechanisms remain to be established. In the present study, using PQQ-immobilized Sepharose beads as a probe, we examined the presence of protein(s) that are capable of binding PQQ in mouse NIH/3T3 fibroblasts and identified five cellular proteins, including l-lactate dehydrogenase (LDH) A chain, as potential mammalian PQQ-binding proteins. In vitro studies using a purified rabbit muscle LDH show that PQQ inhibits the formation of lactate from pyruvate in the presence of NADH (forward reaction), whereas it enhances the conversion of lactate to pyruvate in the presence of NAD+ (reverse reaction). The molecular mechanism underlying PQQ-mediated regulation of LDH activity is attributed to the oxidation of NADH to NAD+ by PQQ. Indeed, the PQQ-bound LDH oxidizes NADH, generating NAD+, and significantly catalyzes the conversion of lactate to pyruvate. Furthermore, PQQ attenuates cellular lactate release and increases intracellular ATP levels in the NIH/3T3 fibroblasts. Our results suggest that PQQ, modulating LDH activity to facilitate pyruvate formation through its redox-cycling activity, may be involved in the enhanced energy production via mitochondrial TCA cycle and oxidative phosphorylation. PMID:27230956

  8. NAD-Independent l-Lactate Dehydrogenase Required for l-Lactate Utilization in Pseudomonas stutzeri A1501

    PubMed Central

    Gao, Chao; Wang, Yujiao; Zhang, Yingxin; Lv, Min; Dou, Peipei; Xu, Ping

    2015-01-01

    ABSTRACT NAD-independent l-lactate dehydrogenases (l-iLDHs) play important roles in l-lactate utilization of different organisms. All of the previously reported l-iLDHs were flavoproteins that catalyze the oxidation of l-lactate by the flavin mononucleotide (FMN)-dependent mechanism. Based on comparative genomic analysis, a gene cluster with three genes (lldA, lldB, and lldC) encoding a novel type of l-iLDH was identified in Pseudomonas stutzeri A1501. When the gene cluster was expressed in Escherichia coli, distinctive l-iLDH activity was detected. The expressed l-iLDH was purified by ammonium sulfate precipitation, ion-exchange chromatography, and affinity chromatography. SDS-PAGE and successive matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis of the purified l-iLDH indicated that it is a complex of LldA, LldB, and LldC (encoded by lldA, lldB, and lldC, respectively). Purified l-iLDH (LldABC) is a dimer of three subunits (LldA, LldB, and LldC), and the ratio between LldA, LldB, and LldC is 1:1:1. Different from the FMN-containing l-iLDH, absorption spectra and elemental analysis suggested that LldABC might use the iron-sulfur cluster for the l-lactate oxidation. LldABC has narrow substrate specificity, and only l-lactate and dl-2-hydrobutyrate were rapidly oxidized. Mg2+ could activate l-iLDH activity effectively (6.6-fold). Steady-state kinetics indicated a ping-pong mechanism of LldABC for the l-lactate oxidation. Based on the gene knockout results, LldABC was confirmed to be required for the l-lactate metabolism of P. stutzeri A1501. LldABC is the first purified and characterized l-iLDH with different subunits that uses the iron-sulfur cluster as the cofactor. IMPORTANCE Providing new insights into the diversity of microbial lactate utilization could assist in the production of valuable chemicals and understanding microbial pathogenesis. An NAD-independent l-lactate dehydrogenase (l-iLDH) encoded by the

  9. Acute overexpression of lactate dehydrogenase-A perturbs beta-cell mitochondrial metabolism and insulin secretion.

    PubMed

    Ainscow, E K; Zhao, C; Rutter, G A

    2000-07-01

    Islet beta-cells express low levels of lactate dehydrogenase and have high glycerol phosphate dehydrogenase activity. To determine whether this configuration favors oxidative glucose metabolism via mitochondria in the beta-cell and is important for beta-cell metabolic signal transduction, we have determined the effects on glucose metabolism and insulin secretion of acute overexpression of the skeletal muscle isoform of lactate dehydrogenase (LDH)-A. Monitored in single MIN6 beta-cells, LDH hyperexpression (achieved by intranuclear cDNA microinjection or adenoviral infection) diminished the response to glucose of both phases of increases in mitochondrial NAD(P)H, as well as increases in mitochondrial membrane potential, cytosolic free ATP, and cystolic free Ca2+. These effects were observed at all glucose concentrations, but were most pronounced at submaximal glucose levels. Correspondingly, adenoviral vector-mediated LDH-A overexpression reduced insulin secretion stimulated by 11 mmol/l glucose and the subsequent response to stimulation with 30 mmol/l glucose, but it was without significant effect when the concentration of glucose was raised acutely from 3 to 30 mmol/l. Thus, overexpression of LDH activity interferes with normal glucose metabolism and insulin secretion in the islet beta-cell type, and it may therefore be directly responsible for insulin secretory defects in some forms of type 2 diabetes. The results also reinforce the view that glucose-derived pyruvate metabolism in the mitochondrion is critical for glucose-stimulated insulin secretion in the beta-cell.

  10. Cloning and Polymorphisms of Yak Lactate Dehydrogenase b Gene

    PubMed Central

    Wang, Guosheng; Zhao, Xingbo; Zhong, Juming; Cao, Meng; He, Qinghua; Liu, Zhengxin; Lin, Yaqiu; Xu, Yaou; Zheng, Yucai

    2013-01-01

    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. PMID:23739677

  11. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    SciTech Connect

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D.

    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.

  12. Lactate Dehydrogenase in Hepatocellular Carcinoma: Something Old, Something New

    PubMed Central

    Faloppi, Luca; Bianconi, Maristella; Memeo, Riccardo; Casadei Gardini, Andrea; Giampieri, Riccardo; Bittoni, Alessandro; Andrikou, Kalliopi; Del Prete, Michela; Cascinu, Stefano; Scartozzi, Mario

    2016-01-01

    Hepatocellular carcinoma (HCC) is the most common primary liver tumour (80–90%) and represents more than 5.7% of all cancers. Although in recent years the therapeutic options for these patients have increased, clinical results are yet unsatisfactory and the prognosis remains dismal. Clinical or molecular criteria allowing a more accurate selection of patients are in fact largely lacking. Lactic dehydrogenase (LDH) is a glycolytic key enzyme in the conversion of pyruvate to lactate under anaerobic conditions. In preclinical models, upregulation of LDH has been suggested to ensure both an efficient anaerobic/glycolytic metabolism and a reduced dependence on oxygen under hypoxic conditions in tumour cells. Data from several analyses on different tumour types seem to suggest that LDH levels may be a significant prognostic factor. The role of LDH in HCC has been investigated by different authors in heterogeneous populations of patients. It has been tested as a potential biomarker in retrospective, small, and nonfocused studies in patients undergoing surgery, transarterial chemoembolization (TACE), and systemic therapy. In the major part of these studies, high LDH serum levels seem to predict a poorer outcome. We have reviewed literature in this setting trying to resume basis for future studies validating the role of LDH in this disease. PMID:27314036

  13. SERUM VALUES OF ALKALINE PHOSPHATASE AND LACTATE DEHYDROGENASE IN OSTEOSARCOMA

    PubMed Central

    ZUMÁRRAGA, JUAN PABLO; BAPTISTA, ANDRÉ MATHIAS; ROSA, LUIS PABLO DE LA; CAIERO, MARCELO TADEU; CAMARGO, OLAVO PIRES DE

    2016-01-01

    ABSTRACT Objective: To study the relationship between the pre and post chemotherapy (CT) serum levels of alkaline phosphatase (AP) and lactate dehydrogenase (LDH), and the percentage of tumor necrosis (TN) found in specimens after the pre surgical CT in patients with osteosarcoma. Methods: Series of cases with retrospective evaluation of patients diagnosed with osteosarcoma. Participants were divided into two groups according to serum values of both enzymes. The values of AP and LDH were obtained before and after preoperative CT. The percentage of tumor necrosis (TN) of surgical specimens of each patient was also included. Results: One hundred and thirty seven medical records were included from 1990 to 2013. Both the AP as LDH decreased in the patients studied, being the higher in pre CT than post CT. The average LHD decrease was 795.12U/L and AP decrease was 437.40 U/L. The average TN was 34.10 %. There was no statistically significant correlation between the serums values and the percentage of tumoral necrosis. Conclusion: The serum levels values of AP and LDH are not good predictors for the chemotherapy-induced necrosis in patients with osteosarcoma. Level of Evidence IV, Case Series. PMID:27217815

  14. Acetate Utilization in Lactococcus lactis Deficient in Lactate Dehydrogenase: a Rescue Pathway for Maintaining Redox Balance

    PubMed Central

    Hols, Pascal; Ramos, Ana; Hugenholtz, Jeroen; Delcour, Jean; de Vos, Willem M.; Santos, Helena; Kleerebezem, Michiel

    1999-01-01

    Acetate was shown to improve glucose fermentation in Lactococcus lactis deficient in lactate dehydrogenase. 13C and 1H nuclear magnetic resonance studies using [2-13C]glucose and [2-13C]acetate as substrates demonstrated that acetate was exclusively converted to ethanol. This novel pathway provides an alternative route for NAD+ regeneration in the absence of lactate dehydrogenase. PMID:10464231

  15. Evaluation of the anti-tumor effects of lactate dehydrogenase inhibitor galloflavin in endometrial cancer cells.

    PubMed

    Han, Xiaoyun; Sheng, Xiugui; Jones, Hannah M; Jackson, Amanda L; Kilgore, Joshua; Stine, Jessica E; Schointuch, Monica N; Zhou, Chunxiao; Bae-Jump, Victoria L

    2015-01-29

    High rates of aerobic glycolysis represent a key mechanism by which endometrial cancer cells consume glucose as its primary energy source. The up-regulated glycolytic pathway is a common therapeutic target whose inhibition has implications for anti-tumor activity in cancer cells. The present study was aimed at evaluating the potential of a novel lactate dehydrogenase (LDH) inhibitor, Galloflavin, as a therapeutic agent for endometrial cancer. Our results revealed that Galloflavin effectively inhibited cell growth in endometrial cancer cell lines and primary cultures of human endometrial cancer through its involvement in multiple signaling pathways that regulate metabolism, cell cycle, apoptosis, cell stress and metastasis.

  16. Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast L-lactate dehydrogenase).

    PubMed

    Mowat, C G; Beaudoin, I; Durley, R C; Barton, J D; Pike, A D; Chen, Z W; Reid, G A; Chapman, S K; Mathews, F S; Lederer, F

    2000-03-28

    Flavocytochrome b(2) from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction. The crystal structure of the native yeast enzyme has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863] as well as that of the sulfite adduct of the recombinant enzyme produced in Escherichia coli [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]; several key active site residues were identified. In the sulfite adduct crystal structure, Arg289 adopts two alternative conformations. In one of them, its side chain is stacked against that of Arg376, which interacts with the substrate; in the second orientation, the R289 side chain points toward the active site. This residue has now been mutated to lysine and the mutant enzyme, R289K-b(2), characterized kinetically. Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotope effect) indicate the mutation affects k(cat) by a factor of about 10 and k(cat)/K(M) by up to nearly 10(2). Pre-steady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirely limited by flavin reduction. Inhibition studies on R289K-b(2) with a range of compounds show a general rise in K(i) values relative to that of wild-type enzyme, in line with the elevation of the K(M) for L-lactate in R289K-b(2); they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a loss of the inhibition by excess substrate. Altogether, the kinetic studies indicate that the mutation has altered the first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both in Michaelis complex and transition-state stabilization, as well as in ligand binding to the active site when the flavin is in the semiquinone state. In addition, it appears that the mutation has not affected electron transfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step

  17. Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR.

    PubMed

    Neves, A R; Ramos, A; Shearman, C; Gasson, M J; Almeida, J S; Santos, H

    2000-06-01

    The metabolism of glucose by nongrowing cells of Lactococcus lactis strain FI7851, constructed from the wild-type L. lactis strain MG1363 by disruption of the lactate dehydrogenase (ldh) gene [Gasson, M.J., Benson, K., Swindel, S. & Griffin, H. (1996) Lait 76, 33-40] was studied in a noninvasive manner by 13C-NMR. The kinetics of the build-up and consumption of the pools of intracellular intermediates mannitol 1-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate, and phosphoenolpyruvate as well as the utilization of [1-13C]glucose and formation of products (lactate, acetate, mannitol, ethanol, acetoin, 2,3-butanediol) were monitored in vivo with a time resolution of 30 s. The metabolism of glucose by the parental wild-type strain was also examined for comparison. A clear shift from typical homolactic fermentation (parental strain) to a mixed acid fermentation (lactate dehdydrogenase deficient; LDHd strain) was observed. Furthermore, high levels of mannitol were transiently produced and metabolized once glucose was depleted. Mannitol 1-phosphate accumulated intracellularly up to 76 mM concentration. Mannitol was formed from fructose 6-phosphate by the combined action of mannitol-1-phosphate dehydrogenase and phosphatase. The results show that the formation of mannitol 1-phosphate by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient production of mannitol 1-phosphate serving as a regeneration pathway for NAD+ regeneration. Oxygen availability caused a drastic change in the pattern of intermediates and end-products, reinforcing the key-role of the fulfilment of the redox balance. The flux control coefficients for the step catalysed by mannitol-1-phosphate dehydrogenase were calculated and the implications in the design of metabolic engineering strategies are discussed.

  18. Diammonium phosphate stimulates transcription of L-lactate dehydrogenase leading to increased L-lactate production in the thermotolerant Bacillus coagulans strain.

    PubMed

    Sun, Lifan; Li, Yanfeng; Wang, Limin; Wang, Yanping; Yu, Bo

    2016-08-01

    Exploration of cost-effective fermentation substrates for efficient lactate production is an important economic objective. Although some organic nitrogen sources are also cheaper, inorganic nitrogen salts for lactate fermentation have additional advantages in facilitating downstream procedures and significantly improving the commercial competitiveness of lactate production. In this study, we first established an application of diammonium phosphate to replace yeast extract with a reduced 90 % nitrogen cost for a thermotolerant Bacillus coagulans strain. In vivo enzymatic and transcriptional analyses demonstrated that diammonium phosphate stimulates the gene expression of L-lactate dehydrogenase, thus providing higher specific enzyme activity in vivo and increasing L-lactic acid production. This new information provides a foundation for establishing a cost-effective process for polymer-grade L-lactic acid production in an industrial setting. PMID:26883345

  19. Prognostic value of preoperative serum lactate dehydrogenase in thymic carcinoma

    PubMed Central

    Yuan, Zu-Yang; Gao, Shu-Geng; Mu, Ju-Wei; Xue, Qi; Mao, You-Sheng; Wang, Da-Li; Zhao, Jun; Gao, Yu-Shun; Huang, Jin-Feng

    2016-01-01

    Background The prognostic value of serum lactate dehydrogenase (LDH) has been demonstrated in various solid tumors. We attempted to determine whether serum LDH was predictive of survival in thymic carcinoma after surgical resection. Methods Ninety-five patients with thymic carcinoma treated in our hospital between January 2005 and December 2015 were retrospectively enrolled. Serum LDH was measured before surgery and categorized as low or high relative to the upper limit of normal (ULN) (225 U/L). The relationships of serum LDH level and other clinical variables with survival were estimated by Cox regression and Kaplan-Meier survival analysis. Results Serum LDH levels were found to be significantly associated with overall survival (OS) and progression-free survival (PFS) of these patients. The 1-, 3-, and 5-year PFS were 76%, 51%, and 38%, and the 1-, 3- and 5-year OS were 97%, 75%, and 46%, respectively. Univariate analysis found that high serum LDH (>225 U/L) was associated with both lower OS [hazard ratio (HR) =2.710; 95% confidence interval (CI): 1.363–1.5.391; P=0.004] and PFS (HR =3.365; 95% CI: 1.776–6.374; P<0.001). Multivariate analysis found that high serum LDH was associated with lower PFS (HR =2.122; 95% CI: 1.056–4.267; P=0.035). Moreover, high LDH was significantly associated with advanced Masaoka stage (P=0.001). Conclusions High serum LDH (>225 U/L) was an independent predictor of decreased PFS in thymic carcinoma patients. It was also significantly associated with reduced OS, but was not an independent predictor of death in those patients. PMID:27746998

  20. Phylogenetic analysis of vertebrate lactate dehydrogenase (LDH) multigene families.

    PubMed

    Li, Yi-Ju; Tsoi, Stephen C-M; Mannen, Hideyuka; Shoei-lung Li, Steven

    2002-05-01

    In this paper we analyzed 49 lactate dehydrogenase (LDH) sequences, mostly from vertebrates. The amino acid sequence differences were found to be larger for a human-killifish pair than a human-lamprey pair. This indicates that some protein sequence convergence may occur and reduce the sequence differences in distantly related species. We also examined transitions and transversions separately for several species pairs and found that the transitions tend to be saturated in the distantly related species pair, while transversions are increasing. We conclude that transversions maintain a conservative rate through the evolutionary time. Kimura's two-parameter model for multiple-hit correction on transversions only was used to derive a distance measure and then construct a neighbor-joining (NJ) tree. Three findings were revealed from the NJ tree: (i) the branching order of the tree is consistent with the common branch pattern of major vertebrates; (ii) Ldh-A and Ldh-B genes were duplicated near the origin of vertebrates; and (iii) Ldh-C and Ldh-A in mammals were produced by an independent gene duplication in early mammalian history. Furthermore, a relative rate test showed that mammalian Ldh-C evolved more rapidly than mammalian Ldh-A. Under a two-rate model, this duplication event was calibrated to be approximately 247 million years ago (mya), dating back to the Triassic period. Other gene duplication events were also discovered in Xenopus, the first duplication occurring approximately 60-70 mya in both Ldh-A and Ldh-B, followed by another recent gene duplication event, approximately 20 mya, in Ldh-B. PMID:11965434

  1. Phylogenetic analysis of vertebrate lactate dehydrogenase (LDH) multigene families.

    PubMed

    Li, Yi-Ju; Tsoi, Stephen C-M; Mannen, Hideyuka; Shoei-lung Li, Steven

    2002-05-01

    In this paper we analyzed 49 lactate dehydrogenase (LDH) sequences, mostly from vertebrates. The amino acid sequence differences were found to be larger for a human-killifish pair than a human-lamprey pair. This indicates that some protein sequence convergence may occur and reduce the sequence differences in distantly related species. We also examined transitions and transversions separately for several species pairs and found that the transitions tend to be saturated in the distantly related species pair, while transversions are increasing. We conclude that transversions maintain a conservative rate through the evolutionary time. Kimura's two-parameter model for multiple-hit correction on transversions only was used to derive a distance measure and then construct a neighbor-joining (NJ) tree. Three findings were revealed from the NJ tree: (i) the branching order of the tree is consistent with the common branch pattern of major vertebrates; (ii) Ldh-A and Ldh-B genes were duplicated near the origin of vertebrates; and (iii) Ldh-C and Ldh-A in mammals were produced by an independent gene duplication in early mammalian history. Furthermore, a relative rate test showed that mammalian Ldh-C evolved more rapidly than mammalian Ldh-A. Under a two-rate model, this duplication event was calibrated to be approximately 247 million years ago (mya), dating back to the Triassic period. Other gene duplication events were also discovered in Xenopus, the first duplication occurring approximately 60-70 mya in both Ldh-A and Ldh-B, followed by another recent gene duplication event, approximately 20 mya, in Ldh-B.

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

    PubMed

    Thitiprasert, Sitanan; Sooksai, Sarintip; Thongchul, Nuttha

    2011-08-01

    Rhizopus oryzae is becoming more important due to its ability to produce an optically pure L: -lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4'-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways. PMID:21416338

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

    PubMed

    Thitiprasert, Sitanan; Sooksai, Sarintip; Thongchul, Nuttha

    2011-08-01

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

  4. Myristica fragrans Suppresses Tumor Growth and Metabolism by Inhibiting Lactate Dehydrogenase A.

    PubMed

    Kim, Eun-Yeong; Choi, Hee-Jung; Park, Mi-Ju; Jung, Yeon-Seop; Lee, Syng-Ook; Kim, Keuk-Jun; Choi, Jung-Hye; Chung, Tae-Wook; Ha, Ki-Tae

    2016-01-01

    Most cancer cells predominantly produce ATP by maintaining a high rate of lactate fermentation, rather than by maintaining a comparatively low rate of tricarboxylic acid cycle, i.e., Warburg's effect. In the pathway, the pyruvate produced by glycolysis is converted to lactic acid by lactate dehydrogenase (LDH). Here, we demonstrated that water extracts from the seeds of Myristica fragrans Houtt. (MF) inhibit the in vitro enzymatic activity of LDH. MF effectively suppressed cell growth and the overall Warburg effect in HT29 human colon cancer cells. Although the expression of LDH-A was not changed by MF, both lactate production and LDH activity were decreased in MF-treated cells under both normoxic and hypoxic conditions. In addition, intracellular ATP levels were also decreased by MF treatment, and the uptake of glucose was also reduced by MF treatment. Furthermore, the experiment on tumor growth in the in vivo mice model revealed that MF effectively reduced the growth of allotransplanted Lewis lung carcinoma cells. Taken together, these results suggest that MF effectively inhibits cancer growth and metabolism by inhibiting the activity of LDH, a major enzyme responsible for regulating cancer metabolism. These results implicate MF as a potential candidate for development into a novel drug against cancer through inhibition of LDH activity.

  5. Myristica fragrans Suppresses Tumor Growth and Metabolism by Inhibiting Lactate Dehydrogenase A.

    PubMed

    Kim, Eun-Yeong; Choi, Hee-Jung; Park, Mi-Ju; Jung, Yeon-Seop; Lee, Syng-Ook; Kim, Keuk-Jun; Choi, Jung-Hye; Chung, Tae-Wook; Ha, Ki-Tae

    2016-01-01

    Most cancer cells predominantly produce ATP by maintaining a high rate of lactate fermentation, rather than by maintaining a comparatively low rate of tricarboxylic acid cycle, i.e., Warburg's effect. In the pathway, the pyruvate produced by glycolysis is converted to lactic acid by lactate dehydrogenase (LDH). Here, we demonstrated that water extracts from the seeds of Myristica fragrans Houtt. (MF) inhibit the in vitro enzymatic activity of LDH. MF effectively suppressed cell growth and the overall Warburg effect in HT29 human colon cancer cells. Although the expression of LDH-A was not changed by MF, both lactate production and LDH activity were decreased in MF-treated cells under both normoxic and hypoxic conditions. In addition, intracellular ATP levels were also decreased by MF treatment, and the uptake of glucose was also reduced by MF treatment. Furthermore, the experiment on tumor growth in the in vivo mice model revealed that MF effectively reduced the growth of allotransplanted Lewis lung carcinoma cells. Taken together, these results suggest that MF effectively inhibits cancer growth and metabolism by inhibiting the activity of LDH, a major enzyme responsible for regulating cancer metabolism. These results implicate MF as a potential candidate for development into a novel drug against cancer through inhibition of LDH activity. PMID:27430914

  6. Catabolism of circulating enzymes: plasma clearance, endocytosis, and breakdown of lactate dehydrogenase-1 in rabbits

    SciTech Connect

    Smit, M.J.; Beekhuis, H.; Duursma, A.M.; Bouma, J.M.; Gruber, M.

    1988-12-01

    Lactate dehydrogenase-1, intravenously injected into rabbits, was cleared with first-order kinetics (half-life 27 min), until at least 80% of the injected activity had disappeared from plasma. Radioactivity from injected SVI-labeled enzyme disappeared at this same rate. Trichloroacetic-acid-soluble breakdown products started to appear in the circulation shortly after injection of the labeled enzyme. Body scans of the rabbits for 80 min after injection of T I-labeled enzyme revealed rapid accumulation of label in the liver, peaking 10-20 min after injection. Subsequently, activity in the liver declined and radioactivity (probably labeled breakdown products of low molecular mass) steadily accumulated in the bladder. Tissue fractionation of liver, 19 min after injection of labeled enzyme, indicated that the radioactivity was present both in endosomes and in lysosomes, suggesting uptake by endocytosis, followed by breakdown in the lysosomes. Measurements of radioactivity in liver and plasma suggest that the liver is responsible for the breakdown of at least 75% of the injected enzyme. Radioautography of tissue sections of liver and spleen showed accumulated radioactivity in sinusoidal liver cells and red pulpa, respectively. These results are very similar to those for lactate dehydrogenase-5, creatine kinase MM, and several other enzymes that we have previously studied in rats.

  7. Effect of osmolytes on protein dynamics in the lactate dehydrogenase-catalyzed reaction.

    PubMed

    Zhadin, Nickolay; Callender, Robert

    2011-03-15

    Laser-induced temperature jump relaxation spectroscopy was used to probe the effect of osmolytes on the microscopic rate constants of the lactate dehydrogenase-catalyzed reaction. NADH fluorescence and absorption relaxation kinetics were measured for the lactate dehydrogenase (LDH) reaction system in the presence of varying amounts of trimethylamine N-oxide (TMAO), a protein-stabilizing osmolyte, or urea, a protein-destabilizing osmolyte. Trimethylamine N-oxide (TMAO) at a concentration of 1 M strongly increases the rate of hydride transfer, nearly nullifies its activation energy, and also slightly increases the enthalpy of hydride transfer. In 1 M urea, the hydride transfer enthalpy is almost nullified, but the activation energy of the step is not affected significantly. TMAO increases the preference of the closed conformation of the active site loop in the LDH·NAD(+)·lactate complex; urea decreases it. The loop opening rate in the LDH·NADH·pyruvate complex changes its temperature dependence to inverse Arrhenius with TMAO. In this complex, urea accelerates the loop motion, without changing the loop opening enthalpy. A strong, non-Arrhenius decrease in the pyruvate binding rate in the presence of TMAO offers a decrease in the fraction of the open loop, pyruvate binding competent form at higher temperatures. The pyruvate off rate is not affected by urea but decreases with TMAO. Thus, the osmolytes strongly affect the rates and thermodynamics of specific events along the LDH-catalyzed reaction: binding of substrates, loop closure, and the chemical event. Qualitatively, these results can be understood as an osmolyte-induced change in the energy landscape of the protein complexes, shifting the conformational nature of functional substates within the protein ensemble.

  8. Plasma Lactate Dehydrogenase Levels Predict Mortality in Acute Aortic Syndromes

    PubMed Central

    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

  9. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  10. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial...

  11. [THE LEVEL OF LACTATE DEHYDROGENASE AS A MARKER OF RENAL DYSFUNCTION IN NEONATES WITH ASPHYXIA].

    PubMed

    Loboda, A M

    2015-01-01

    The article examines the possibility of determining the level of lactate dehydrogenase (LD) in biological fluids as a marker of renal dysfunction and energy supply in neonates with asphyxia. Investigation included 200 full-term newborns with disturbance kidney function: 100 infants who had severe asphyxia, and 100--with moderate asphyxia. LD activity was determined by kinetic spectrophotometric method. Determination of the activity of LD in the urine in the early neonatal period it is advisable to use as a non-invasive marker for the diagnosis of renal dysfunction in neonates with asphyxia. The content of LD in the blood serum can be used as one of the early markers of kidney damage in newborns with asphyxia. PMID:27491157

  12. l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria.

    PubMed

    Pizzuto, Roberto; Paventi, Gianluca; Porcile, Carola; Sarnataro, Daniela; Daniele, Aurora; Passarella, Salvatore

    2012-09-01

    As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M.

  13. Lactate dehydrogenase as a marker of Plasmodium infection in malaria vector Anopheles.

    PubMed

    Riandey, M F; Sannier, C; Peltre, G; Monteny, N; Cavaleyra, M

    1996-06-01

    Lactate dehydrogenase (Ldh) electrophoresis showed the presence of Plasmodium yoelii yoelii in Anopheles stephensi and An. gambiae. The Ldh appeared as an additional band (pLdh) whose activity was more intense with 3-acetyl pyridine adenine dinucleotide as coenzyme than with beta nicotin-amide adenine dinucleotide. Several allelic forms occurred both in the vector and the host. The isoelectric point of Ldh, similar in the vector and host, differed from those of Ldh from mosquito and mouse. The presence of pLdh was detected from the 2nd to the 28th day of infection. The pLdh appeared to be proportional to the number of sporozoites present in infected salivary glands. However, pLdh was not found in salivary glands or midguts, but it was detected in the rest of the corresponding mosquito. The origin and use of pLdh as a marker of Plasmodium in its vector is discussed. PMID:8827592

  14. Metabolic and cellular profile of wether goats: protein fractions and lactate dehydrogenase isoenzymes- reference values.

    PubMed

    Blackwell, J G; Libby, D W

    1982-06-01

    Blood serum concentrations of electrolytes; mineral, serum protein, and biochemical components; serum lactate dehydrogenase (LDH) activity; and blood cellular components were determined in 8 wether goats over a 59-day feeding period. The blood was collected on 6 sampling days, the blood cellular components were analyzed on collection days. The biochemical components were analyzed from frozen samples approximately 3 weeks after collection. The serum protein fractions and LDH isoenzymes were separated electrophoretically from frozen samples. The purpose was to determine the variability and changes in these values, using sample-day intervals over the feeding period. The blood serum concentrations of many of the biochemical and blood cellular components that were measured or calculated varied considerably, showing fluctuations over some portion of the period and ending with an erratic peak at period's end. The WBC count, neutrophil, and band neutrophil values were sharply decreased on sampling days 24 and 35, followed by fluctuations over the last half of the period.

  15. Purification and properties of a monomeric lactate dehydrogenase from yak Hypoderma sinense larva.

    PubMed

    Li, Pengfei; Jin, Suyu; Huang, Lin; Liu, Haohao; Huang, Zhihong; Lin, Yaqiu; Zheng, Yucai

    2013-06-01

    The objective of the present study was to study the characteristics of lactate dehydrogenase (LDH) from Hypoderma sinense larva. H. sinense larvae were collected from yak (Bos grunniens) and identified by a PCR-RFLP method. Analysis of LDH activity showed that the total LDH activity in H. sinense larva was negatively correlated with the length of larva. Polyacrylamide gel electrophoresis of the extracts of H. sinense larvae revealed one band of LDH, which was then purified by affinity chromatography and gel filtration. This enzyme showed an approximately 36 kDa band on SDS-gel under both reducing and non-reducing conditions, in addition, size exclusion chromatography analysis showed that its molecular weight was smaller than bovine serum albumin (67 kDa), indicating that it contains only one subunit. Michaelis constants (Km) values assay revealed that LDH from H. sinense larva showed significantly lower Km for lactate than other animals. LDH of H. sinense larva was stable at 60 °C for 15 min, and also exhibited high catalytic efficiency in a wide range of pH. HgCl₂ at the concentration of 0.1mM significantly decreased the activity of LDH from H. sinense larva but not at the concentration of 0.01 mM. The results of the present study demonstrate that LDH from H. sinense larva is a thermal stable and pH insensitive enzyme suitable for catalyzing both forward and reverse reactions.

  16. Surface modification of silicon dioxide, silicon nitride and titanium oxynitride for lactate dehydrogenase immobilization.

    PubMed

    Saengdee, Pawasuth; Chaisriratanakul, Woraphan; Bunjongpru, Win; Sripumkhai, Witsaroot; Srisuwan, Awirut; Jeamsaksiri, Wutthinan; Hruanun, Charndet; Poyai, Amporn; Promptmas, Chamras

    2015-05-15

    Three different types of surface, silicon dioxide (SiO2), silicon nitride (Si3N4), and titanium oxynitride (TiON) were modified for lactate dehydrogenase (LDH) immobilization using (3-aminopropyl)triethoxysilane (APTES) to obtain an amino layer on each surface. The APTES modified surfaces can directly react with LDH via physical attachment. LDH can be chemically immobilized on those surfaces after incorporation with glutaraldehyde (GA) to obtain aldehyde layers of APTES-GA modified surfaces. The wetting properties, chemical bonding composition, and morphology of the modified surface were determined by contact angle (CA) measurement, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM), respectively. In this experiment, the immobilized protein content and LDH activity on each modified surface was used as an indicator of surface modification achievement. The results revealed that both the APTES and APTES-GA treatments successfully link the LDH molecule to those surfaces while retaining its activity. All types of tested surfaces modified with APTES-GA gave better LDH immobilizing efficiency than APTES, especially the SiO2 surface. In addition, the SiO2 surface offered the highest LDH immobilization among tested surfaces, with both APTES and APTES-GA modification. However, TiON and Si3N4 surfaces could be used as alternative candidate materials in the preparation of ion-sensitive field-effect transistor (ISFET) based biosensors, including lactate sensors using immobilized LDH on the ISFET surface.

  17. Energy Landscape of the Michaelis Complex of Lactate Dehydrogenase: Relationship to Catalytic Mechanism

    PubMed Central

    2015-01-01

    Lactate dehydrogenase (LDH) catalyzes the interconversion between pyruvate and lactate with nicotinamide adenine dinucleotide (NAD) as a cofactor. Using isotope-edited difference Fourier transform infrared spectroscopy on the “live” reaction mixture (LDH·NADH·pyruvate ⇌ LDH·NAD+·lactate) for the wild-type protein and a mutant with an impaired catalytic efficiency, a set of interconverting conformational substates within the pyruvate side of the Michaelis complex tied to chemical activity is revealed. The important structural features of these substates include (1) electronic orbital overlap between pyruvate’s C2=O bond and the nicotinamide ring of NADH, as shown from the observation of a delocalized vibrational mode involving motions from both moieties, and (2) a characteristic hydrogen bond distance between the pyruvate C2=O group and active site residues, as shown by the observation of at least four C2=O stretch bands indicating varying degrees of C2=O bond polarization. These structural features form a critical part of the expected reaction coordinate along the reaction path, and the ability to quantitatively determine them as well as the substate population ratios in the Michaelis complex provides a unique opportunity to probe the structure–activity relationship in LDH catalysis. The various substates have a strong variance in their propensity toward on enzyme chemistry. Our results suggest a physical mechanism for understanding the LDH-catalyzed chemistry in which the bulk of the rate enhancement can be viewed as arising from a stochastic search through an available phase space that, in the enzyme system, involves a restricted ensemble of more reactive conformational substates as compared to the same chemistry in solution. PMID:24576110

  18. Energy landscape of the Michaelis complex of lactate dehydrogenase: relationship to catalytic mechanism.

    PubMed

    Peng, Huo-Lei; Deng, Hua; Dyer, R Brian; Callender, Robert

    2014-03-25

    Lactate dehydrogenase (LDH) catalyzes the interconversion between pyruvate and lactate with nicotinamide adenine dinucleotide (NAD) as a cofactor. Using isotope-edited difference Fourier transform infrared spectroscopy on the "live" reaction mixture (LDH·NADH·pyruvate ⇌ LDH·NAD(+)·lactate) for the wild-type protein and a mutant with an impaired catalytic efficiency, a set of interconverting conformational substates within the pyruvate side of the Michaelis complex tied to chemical activity is revealed. The important structural features of these substates include (1) electronic orbital overlap between pyruvate's C2═O bond and the nicotinamide ring of NADH, as shown from the observation of a delocalized vibrational mode involving motions from both moieties, and (2) a characteristic hydrogen bond distance between the pyruvate C2═O group and active site residues, as shown by the observation of at least four C2═O stretch bands indicating varying degrees of C2═O bond polarization. These structural features form a critical part of the expected reaction coordinate along the reaction path, and the ability to quantitatively determine them as well as the substate population ratios in the Michaelis complex provides a unique opportunity to probe the structure-activity relationship in LDH catalysis. The various substates have a strong variance in their propensity toward on enzyme chemistry. Our results suggest a physical mechanism for understanding the LDH-catalyzed chemistry in which the bulk of the rate enhancement can be viewed as arising from a stochastic search through an available phase space that, in the enzyme system, involves a restricted ensemble of more reactive conformational substates as compared to the same chemistry in solution.

  19. Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.

    PubMed

    Yip, Shirley S M; Zhou, Meixia; Joly, John; Snedecor, Bradley; Shen, Amy; Crawford, Yongping

    2014-09-01

    Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3. PMID:24841241

  20. [Lactate dehydrogenase isoenzymes in the eye, cardiac and skeletal muscles of several decapods].

    PubMed

    Chernyĭ, V G; Chizhevich, E P; Shukoliukov, S A

    1976-01-01

    Properties of lactate dehydrogenase (LDH) in the eye, heart and muscles of Hemigrapsus sanguineus, Paralithodes camtschatica, Erimacrus isenbeckii, Pandalus latirastrus, Pagurus brachiomastus have been studied with acrylamide gel electrophoresis and kinetics analysis. LDH in all the tissues of all the representatives studied was found to be specific for L-pyruvate and lactate; it migrated in electrophoresis as a single band revealing low mobility towards anode. The isoenzyme from P. camtschatica and P. latirastrus differed from the isoenzymes of other animals studied by higher mobility towards anode that reflected higher negative value of its total charge. The LDH isoenzymes in all the animals studied resembled the A4 (LDH5) of the vertebrates being unstable to the denaturing action of high temperature and being unaffected by high concentrations of pyruvate up to 1.0.10-3M. On the other hand, in conrast to the A4 of mammals, the LDH in question displayed enhancement of the reaction rate and decrease of the Km values upon increase in the NAD+ and NAD.H concentrations both in the presence of high or low lactate and pyruvate concentrations. The isoenzymes displayed catalytic activity also in the presence of NADP, the Km values for pyruvate in the presence of equimolar (2.25 mM) concentrations of NAD.H or NADP.H were practically identical and were found to be within the limits of 14-26.10-5 M. Molecular weight of the LDH studied assessed by the gel filtration method was found to be 130-140,000. It is suggested that the LDH isoenzyme from the representatives of the decapod crayfish studied is homologous in its certain properties to the homotetrameric A4 form of the vertebrates.

  1. Cellular localization of D-lactate dehydrogenase and NADH oxidase from Archaeoglobus fulgidus

    PubMed Central

    Reddy Pagala, Vishwajeeth; Park, Joohye; Reed, David W.; Hartzell, Patricia L.

    2002-01-01

    Members of the genus Archaeoglobus are hyperthermophilic sulfate reducers with an optimal growth temperature of 83 °C. Archaeoglobus fulgidus can utilize simple compounds including D-lactate, L-lactate and pyruvate as the sole substrate for carbon and electrons for dissimilatory sulfate reduction. Previously we showed that this organism makes a D-lactate dehydrogenase (Dld) that requires FAD and Zn2+ for activity. To determine the cellular location and topology of Dld and to identify proteins that interact with Dld, an antibody directed against Dld was prepared. Immunocytochemical studies using gold particle-coated secondary antibodies show that more than 85% of Dld is associated with the membrane. A truncated form of Dld was detected in immunoblots of whole cells treated with protease, showing that Dld is an integral membrane protein and that a significant portion of Dld, including part of the FAD-binding pocket, is outside the membrane facing the S-layer. The gene encoding Dld is part of an operon that includes noxA2, which encodes one of several NADH oxidases in A. fulgidus. Previous studies have shown that NoxA2 remains bound to Dld during purification. Thin sections of A. fulgidus probed simultaneously with antibodies against Dld and NoxA2 show that both proteins co-localized to the same sites in the membrane. Although these data show a tight interaction between NoxA2 and Dld, the role of NoxA2 in electron transport reactions is unknown. Rather, NoxA2 may protect proteins involved in electron transfer by reducing O2 to H2O2 or H2O. PMID:15803647

  2. Metabolic Imaging: A link between Lactate Dehydrogenase A, Lactate and Tumor Phenotype

    PubMed Central

    Thakur, Sunitha B.; Vider, Jelena; Russell, James; Blasberg, Ronald; Koutcher, Jason A.

    2014-01-01

    Purpose We compared the metabolic profiles and the association between LDH-A expression and lactate production in two isogenic murine breast cancer cell lines and tumors (67NR and 4T1). These cell lines were derived from a single mammary tumor and have different growth and metabolic phenotypes. Experimental Design LDH-A expression, lactate concentration, glucose utilization and oxygen consumption were measured in cells, and the potential relationship between tumor lactate levels (measured by magnetic resonance spectroscopic imaging (MRSI)) and tumor glucose utilization (measured by [18F] 2-deoxy-2-fluoro-D-glucose positron emission tomography ([18F]FDG-PET)) was assessed in orthotopic breast tumors derived from these cell lines. Results We show a substantial difference in LDH-A expression between 67NR and 4T1 cells under normoxia and hypoxia. We also show that small orthotopic 4T1 tumors generate tenfold more lactate than corresponding 67NR tumors. The high lactate levels in small primary 4T1 tumors are associated with intense pimonidazole staining (a hypoxia indicator). Less intense hypoxia staining was observed in the larger 67NR tumors, and is consistent with the gradual increase and plateau of lactate concentration in enlarging 67NR tumors. Conclusions Lactate-MRSI has a greater dynamic range than [18F]FDG-PET and may be a more sensitive measure with which to evaluate the aggressive and metastatic potential of primary breast tumors. PMID:21844011

  3. Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae)

    PubMed Central

    Wang, Yang; Wei, Lian; Wei, Dengbang; Li, Xiao; Xu, Lina; Wei, Linna

    2016-01-01

    Testis-specific lactate dehydrogenase (LDH-C4) is one of the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4 in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000–5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A4 (LDH-A4), Lactate Dehydrogenase B4 (LDH-B4), and LDH-C4 were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km) of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L), LDH-B4 (Ki = 23.800 mmol/L), and LDH-C4 (Ki = 65.500 mmol/L). These data suggest that inhibition of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4. PMID:26751442

  4. Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria.

    PubMed

    Doelle, H W

    1971-12-01

    Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.

  5. Effect of Intramuscular or Intrahepatic Injections of Clostridium perfringens on Rabbit Serum Lactate Dehydrogenase 1

    PubMed Central

    Thomason, Dwayne

    1970-01-01

    Serum lactate dehydrogenase (LDH) activity, LDH isoenzyme pattern, phospholipase C activity, phosphorous level, hemoglobin, and erythrocyte osmotic fragility were followed in rabbits after intramuscular (IM) or intrahepatic (IH) injections of Clostridium perfringens. On the first day after IM injection, there was a drop in LDH activity; this was followed by an increase of LDH activity on the third and sixth day. On the seventh day, LDH activity began to decline, and by the ninth day it had almost returned to normal. On the sixth day after IM injection, there was an increase in serum LDH isoenzyme 5, hemoglobin, and erythrocyte osmotic fragility, but the increase of erythrocyte osmotic fragility and serum hemoglobin could not be attributed to phospholipase C activity since that enzyme was not detected nor was there an increase in serum phosphorus. C. perfringens was recovered by culturing the wound of IM-injected rabbits but not recovered from IH-injected rabbits. Rabbits injected IH showed no change from normal values in any of the tests performed. PMID:16557808

  6. Gradual neofunctionalization in the convergent evolution of trichomonad lactate and malate dehydrogenases

    PubMed Central

    Steindel, Phillip A.; Chen, Emily H.; Wirth, Jacob D.

    2016-01-01

    Abstract Lactate and malate dehydrogenases (LDH and MDH) are homologous, core metabolic enzymes common to nearly all living organisms. LDHs have evolved convergently from MDHs at least four times, achieving altered substrate specificity by a different mechanism each time. For instance, the LDH of anaerobic trichomonad parasites recently evolved independently from an ancestral trichomonad MDH by gene duplication. LDH plays a central role in trichomonad metabolism by catalyzing the reduction of pyruvate to lactate, thereby regenerating the NAD+ required for glycolysis. Using ancestral reconstruction methods, we identified the biochemical and evolutionary mechanisms responsible for this convergent event. The last common ancestor of these enzymes was a highly specific MDH, similar to modern trichomonad MDHs. In contrast, the LDH lineage evolved promiscuous activity by relaxing specificity in a gradual process of neofunctionalization involving one highly detrimental substitution at the “specificity residue” (R91L) and many additional mutations of small effect. L91 has different functional consequences in LDHs and in MDHs, indicating a prominent role for epistasis. Crystal structures of modern‐day and ancestral enzymes show that the evolution of substrate specificity paralleled structural changes in dimerization and α‐helix orientation. The relatively small “specificity residue” of the trichomonad LDHs can accommodate a range of substrate sizes and may permit solvent to access the active site, both of which promote substrate promiscuity. The trichomonad LDHs present a multi‐faceted counterpoint to the independent evolution of LDHs in other organisms and illustrate the diverse mechanisms by which protein function, structure, and stability coevolve. PMID:26889885

  7. Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer.

    PubMed

    Cheung, Yee-Wai; Kwok, Jane; Law, Alan W L; Watt, Rory M; Kotaka, Masayo; Tanner, Julian A

    2013-10-01

    DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson-Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics.

  8. Role of lactate dehydrogenase in metmyoglobin reduction and color stability of different bovine muscles.

    PubMed

    Kim, Y H; Keeton, J T; Smith, S B; Berghman, L R; Savell, J W

    2009-11-01

    The role of lactate dehydrogenase (LDH) in metmyoglobin reducing activity (MRA) and color stability of different bovine muscles was studied in two consecutive experiments. In experiment 1, three different bovine muscles -M. longissimus lumborum (LL), M. semimembranosus (SM), and M. psoas major (PM) - were obtained (n=7, respectively), cut into steaks, PVC packaged, and then displayed for 7days at 1°C. The LL was the most red over display time and had more (P<0.05) LDH-B activity (catalyzing toward NADH generation), LDH1 isoform expression, NADH, and higher (P<0.05) MRA than the other two muscles studied. The PM had the least color stability and lowest MRA. In experiment 2, LL steaks (n=8) were cut in half, one side syringe-injected with oxamate, and the other injected with distilled water. Inclusion of oxamate decreased (P<0.05) LDH-B activity, NADH, and a* values after 10days display at 1°C. These results suggest that variation in color stability of physiologically different muscles is regulated by different replenishment rates of NADH via different LDH isozymes. PMID:20416707

  9. Kinetics and thermodynamics of lactate dehydrogenases from beef heart, beef muscle, and flounder muscle.

    PubMed

    Borgmann, U; Laidler, K J; Moon, T W

    1975-11-01

    The eight rate constants for a four-step ordered ternary-complex mechanism have been compared for lactate dehydrogenases (EC1.1.1.27) from three sources, beef heart, beef muscle, and flounder muscle. The rate constants were determined at temperatures ranging from 5 degrees C to 50 degrees C, and the corresponding activation parameters deltaG not equal to, deltaH not equal to, and deltaS not equal to were calculated. Significant differences are noted for the values for the three types of enzyme. The relative heights of the activation barriers are much the same in all three cases, differences in kinetic behavior resulting mainly from differences in the stable binary and ternary enzyme-substrate complexes. These complexes are, in general, at lower free-energy and enthalpy levels of the beef-heart and beef-muscle enzymes than for the flounder-muscle enzyme. A high degree of compensation is found between the enthalpies and entropies of activation, resulting in relatively small differences between the free energies (and rates) for homologous steps with different enzymes. Analysis of the results, on the assumption that the compensation effect is due to weak-bonding effects, suggests that there are fewer weak bonds in the stable complexes of the muscle enzymes.

  10. Amino acid sequence homology among the 2-hydroxy acid dehydrogenases: mitochondrial and cytoplasmic malate dehydrogenases form a homologous system with lactate dehydrogenase.

    PubMed Central

    Birktoft, J J; Fernley, R T; Bradshaw, R A; Banaszak, L J

    1982-01-01

    The amino acid sequence of porcine heart mitochondrial malate dehydrogenase (mMDH; L-malate: NAD+ oxidoreductase, EC 1.1.1.37) has been compared with the sequences of six different lactate dehydrogenases (LDH; L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) and with the "x-ray" sequence of cytoplasmic malate dehydrogenase (sMDH). The main points are that (i) all three enzymes are homologous; (ii) invariant residues in the catalytic center of these enzymes include a histidine and an internally located aspartate that function as a proton relay system; (iii) numerous residues important to coenzyme binding are conserved, including several glycines and charged residues; and (iv) amino acid side chains present in the subunit interface common to the MDHs and LDHs appear to be better conserved than those in the protein interior. It is concluded that LDH, sMDH, and mMDH are derived from a common ancestral gene and probably have similar catalytic mechanisms. PMID:6959107

  11. The determination of lactate dehydrogenase isoenzymes in normal human muscle and other tissues

    PubMed Central

    Emery, A. E. H.

    1967-01-01

    1. A technique has been developed, based on preferential inhibition by urea, for determining the amounts and proportions of the M and H sub-units of lactate dehydrogenase (referred to as LDH-M and LDH-H respectively) in human tissues, including muscle. 2. There was good agreement between the results obtained with urea inhibition and those obtained with starch-gel electrophoresis. 3. With increasing age there was a significant decrease in the total amount of lactate dehydrogenase and the amount of LDH-M in skeletal muscle. This could not be accounted for by the replacement of functioning muscle tissue by fibrous connective tissue. 4. The proportion of LDH-M was less in certain muscles (e.g. soleus and extra-ocular) than in other muscles (e.g. gastrocnemius and rectus abdominis). 5. The proportions of LDH-M and LDH-H did not differ significantly in different superficial limb muscles and were not significantly affected by either age or sex. 6. Specimens of muscle from 86 different individuals (all Europeans) have been subjected to electrophoresis, but no variants of lactate dehydrogenase isoenzymes have been found. PMID:5584002

  12. The Contribution of Electrostatic and van der Waals Interactions to the Stereospecificity of the Reaction Catalyzed by Lactate Dehydrogenase

    PubMed Central

    van Beek, Jeroen; Callender, Robert; Gunner, M. R.

    1997-01-01

    Continuum electrostatic calculations in conjunction with molecular dynamics simulations have been used to investigate the source of the stereospecificity in the hydride transfer reaction catalyzed by lactate dehydrogenase (LDH). These studies show that favorable electrostatic interactions between the carboxamide group of the reduced nicotinamide adenine dinucleotide coenzyme and protein residues of the active site of LDH can account for much if not all of the stereospecificity of the LDH-catalyzed reaction, with A-side hydride transfer more than 107 times greater than B-side transfer. Unfavorable steric interactions within the binding complex for B-side transfer are not found. ImagesFIGURE 2 PMID:9017191

  13. Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli.

    PubMed Central

    LeVan, K M; Goldberg, E

    1991-01-01

    The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable. Images Fig. 2. Fig. 3. PMID:1996957

  14. The rodent malaria lactate dehydrogenase assay provides a high throughput solution for in vivo vaccine studies.

    PubMed

    Otsuki, Hitoshi; Yokouchi, Yuki; Iyoku, Natsumi; Tachibana, Mayumi; Tsuboi, Takafumi; Torii, Motomi

    2015-08-01

    Rodent malaria is a useful model for evaluating the efficacy of malaria vaccine candidates; however, labor-intensive microscopic parasite counting hampers the use of an in vivo parasite challenge in high-throughput screening. The measurement of malaria parasite lactate dehydrogenase (pLDH) activity, which is commonly used in the in vitro growth inhibition assay of Plasmodium falciparum, may be the cheapest and simplest alternative to microscopic parasite counting. However, the pLDH assay has not been applied in the in vivo rodent malaria model. Here, we showed that the pLDH assay is reliable and accurately determines parasitemia in the rodent malaria model. pLDH activity measured using a chromogenic substrate reflects the parasite number in the blood; it allows fast and easy assessment using a conventional microplate reader. To validate this approach, we synthesized recombinant PyMSP1-19 protein (rPyMSP1-19) using a wheat germ cell-free protein synthesis system and immunized mice with rPyMSP1-19. The antisera showed specific reactivity on the surface of the Plasmodium yoelii merozoite and immunized mice were protected against a lethal P. yoelii 17 XL challenge. The pLDH assay quickly and easily demonstrated a significant reduction of the parasite numbers in the immunized mice. Accordingly, the pLDH assay proved to be an efficient alternative to rodent malaria parasite counting, and may therefore accelerate in vivo vaccine candidate screening.

  15. An atomic-resolution view of neofunctionalization in the evolution of apicomplexan lactate dehydrogenases

    PubMed Central

    Boucher, Jeffrey I; Jacobowitz, Joseph R; Beckett, Brian C; Classen, Scott; Theobald, Douglas L

    2014-01-01

    Malate and lactate dehydrogenases (MDH and LDH) are homologous, core metabolic enzymes that share a fold and catalytic mechanism yet possess strict specificity for their substrates. In the Apicomplexa, convergent evolution of an unusual LDH from MDH produced a difference in specificity exceeding 12 orders of magnitude. The mechanisms responsible for this extraordinary functional shift are currently unknown. Using ancestral protein resurrection, we find that specificity evolved in apicomplexan LDHs by classic neofunctionalization characterized by long-range epistasis, a promiscuous intermediate, and few gain-of-function mutations of large effect. In canonical MDHs and LDHs, a single residue in the active-site loop governs substrate specificity: Arg102 in MDHs and Gln102 in LDHs. During the evolution of the apicomplexan LDH, however, specificity switched via an insertion that shifted the position and identity of this ‘specificity residue’ to Trp107f. Residues far from the active site also determine specificity, as shown by the crystal structures of three ancestral proteins bracketing the key duplication event. This work provides an unprecedented atomic-resolution view of evolutionary trajectories creating a nascent enzymatic function. DOI: http://dx.doi.org/10.7554/eLife.02304.001 PMID:24966208

  16. Cryoprotection mechanisms of polyethylene glycols on lactate dehydrogenase during freeze-thawing.

    PubMed

    Mi, Yanli; Wood, George; Thoma, Laura

    2004-01-01

    The purpose of this study was to explore the cryoprotection mechanisms of high molecular weight polyethylene glycols (PEGs) (eg, PEG 4000 and PEG 8000) on lactate dehydrogenase (LDH). Ultraviolet activity assays, circular dichroism (CD) spectroscopy, gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), (14)C-PEG 4000 labeling and binding, and cryostage microscopic study were conducted. Different molecular weights and concentrations of PEGs in LDH formulations were treated by freeze-thawing. Higher molecular weights and concentrations of PEGs in LDH-PEG formulations obtained better activity and secondary structure recoveries of LDH after freeze-thawing. Insoluble aggregation of LDH was not observed in gel filtration studies. SDS-PAGE results suggested surface characteristic modifications of LDH by the larger molecular weight PEGs. The 14C-PEG 4000 labeling and binding study showed extensive nonspecific interactions between the PEG 4000 and LDH molecules in a concentration-dependent manner. The bound LDH-PEG 4000/free PEG 4000 ratio increased when LDH or PEG 4000 concentrations increased. Cryostage microscopic study showed that PEG 8000 delayed the ice crystallization and eutectic transition of LDH formulation. It appeared that multiple mechanisms were at work during PEGs' cryoprotection of LDH. It was unclear whether the delayed eutectic characteristics of PEGs contributed to LDH cryoprotection. The favorable interaction, rather than preferential exclusion, between LDH and PEGs (eg, 4000) cryoprotected LDH. PMID:15760107

  17. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    SciTech Connect

    Li, Fenglei

    2006-08-09

    , evaporation rate can be controlled or adjusted in this method during the crystallization process to favor either nucleation or growing processes for optimizing crystallization process. The protein crystals gotten by this method were experimentally proven to possess high x-ray diffraction qualities. Finally, we crystallized human lactate dehydrogenase 1 (H4) complexed with NADH and determined its structure by x-ray crystallography. The structure of LDH/NADH displays a significantly different structural feature, compared with LDH/NADH/inhibitor ternary complex structure, that subunits in LDH/NADH complex show open conformation or two conformations on the active site while the subunits in LDH/NADH/inhibitor are all in close conformation. Multiple LDH/NADH crystals were obtained and used for x-ray diffraction experiments. Difference in subunit conformation was observed among the structures independently solved from multiple individual LDH/NADH crystals. Structural differences observed among crystals suggest the existence of multiple conformers in solution.

  18. Identification of proteins interacting with lactate dehydrogenase in claw muscle of the porcelain crab Petrolisthes cinctipes

    PubMed Central

    Cayenne, Andrea P.; Gabert, Beverly; Stillman, Jonathon H.

    2011-01-01

    Biochemical adaptation of enzymes involves conservation of activity, stability and affinity across a wide range of intracellular and environmental conditions. Enzyme adaptation by alteration of primary structure is well known, but the roles of protein-protein interactions in enzyme adaptation are less well understood. Interspecific differences in thermal stability of lactate dehydrogenase (LDH) in porcelain crabs (genus Petrolisthes) are related to intrinsic differences among LDH molecules and by interactions with other stabilizing proteins. Here, we identified proteins that interact with LDH in porcelain crab claw muscle tissue using co-immunoprecipitation, and showed LDH exists in high molecular weight complexes using size exclusion chromatography and Western blot analyses. Co-immunoprecipitated proteins were separated using 2D SDS PAGE and analyzed by LC/ESI using peptide MS/MS. Peptide MS/MS ions were compared to an EST database for Petrolisthes cinctipes to identify proteins. Identified proteins included cytoskeletal elements, glycolytic enzymes, a phosphagen kinase, and the respiratory protein hemocyanin. Our results support the hypothesis that LDH interacts with glycolytic enzymes in a metabolon structured by cytoskeletal elements that may also include the enzyme for transfer of the adenylate charge in glycolytically produced ATP. Those interactions may play specific roles in biochemical adaptation of glycolytic enzymes. PMID:21968246

  19. Establishment of permanent chimerism in a lactate dehydrogenase-deficient mouse mutant with hemolytic anemia

    SciTech Connect

    Datta, T.; Doermer, P.

    1987-12-01

    Pluripotent hemopoietic stem cell function was investigated in the homozygous muscle type lactate dehydrogenase (LDH-A) mutant mouse using bone marrow transplantation experiments. Hemopoietic tissues of LDH-A mutants showed a marked decreased in enzyme activity that was associated with severe hemolytic anemia. This condition proved to be transplantable into wild type mice (+/+) through total body irradiation (TBI) at a lethal dose of 8.0 Gy followed by engraftment of mutant bone marrow cells. Since the mutants are extremely radiosensitive (lethal dose50/30 4.4 Gy vs 7.3 Gy in +/+ mice), 8.0-Gy TBI followed by injection of even high numbers of normal bone marrow cells did not prevent death within 5-6 days. After a nonlethal dose of 4.0 Gy and grafting of normal bone marrow cells, a transient chimerism showing peripheral blood characteristics of the wild type was produced that returned to the mutant condition within 12 weeks. The transfusion of wild type red blood cells prior to and following 8.0-Gy TBI and reconstitution with wild type bone marrow cells prevented the early death of the mutants and permanent chimerism was achieved. The chimeras showed all hematological parameters of wild type mice, and radiosensitivity returned to normal. It is concluded that the mutant pluripotent stem cells are functionally comparable to normal stem cells, emphasizing the significance of this mouse model for studies of stem cell regulation.

  20. Lactate dehydrogenase isoenzyme patterns upon chronic exposure to cigarette smoke: Protective effect of bacoside A.

    PubMed

    Anbarasi, Kothandapani; Sabitha, Kuruvimalai Ekambaram; Devi, Chennam Srinivasulu Shyamala

    2005-09-01

    Despite a strong association between cigarette smoking and alarming increase in mortality rate from smoking-related diseases, around 35-40% of the world's population continues to smoke and many more are being exposed to environmental tobacco smoke. Since the role of free radicals and oxidative damage in the pathogenesis of smoking-related diseases has been suggested, bacoside A, a potent antioxidant was tested for its ability to protect against cigarette smoking-induced toxicity in terms of lactate dehydrogenase (LDH) and its isoenzymes. Rats were exposed to cigarette smoke and simultaneously administered with bacoside A, for a period of 12 weeks. Total LDH activity was assayed in serum, lung, heart, brain, liver and kidney, and serum LDH isoforms were separated electrophoretically. Cigarette smoke exposure resulted in significant increase in serum LDH and its isoenzymes with a concomitant decrease in these organs. These alterations were prevented by administration of bacoside A. Excessive oxidants from cigarette smoke is known to cause peroxidation of membrane lipids leading to cellular damage, thereby resulting in the leakage of LDH into the circulation. Bacoside A could have rendered protection to the organs by stabilizing their cell membranes and prevented the release of LDH, probably through its free radical scavenging and anti-lipid peroxidative effect.

  1. The enzymatic reaction catalyzed by lactate dehydrogenase exhibits one dominant reaction path

    PubMed Central

    Masterson, Jean E.; Schwartz, Steven D.

    2014-01-01

    Enzymes are the most efficient chemical catalysts known, but the exact nature of chemical barrier crossing in enzymes is not fully understood. Application of transition state theory to enzymatic reactions indicates that the rates of all possible reaction paths, weighted by their relative probabilities, must be considered in order to achieve an accurate calculation of the overall rate. Previous studies in our group have shown a single mechanism for enzymatic barrier passage in human heart lactate dehydrogenase (LDH). To ensure that this result was not due to our methodology insufficiently sampling reactive phase space, we implement high-perturbation transition path sampling in both microcanonical and canonical regimes for the reaction catalyzed by human heart LDH. We find that, although multiple, distinct paths through reactive phase space are possible for this enzymatic reaction, one specific reaction path is dominant. Since the frequency of these paths in a canonical ensemble is inversely proportional to the free energy barriers separating them from other regions of phase space, we conclude that the rarer reaction paths are likely to have a negligible contribution. Furthermore, the non-dominate reaction paths correspond to altered reactive conformations and only occur after multiple steps of high perturbation, suggesting that these paths may be the result of non-biologically significant changes to the structure of the enzymatic active site. PMID:25368440

  2. Early diagnosis of radiodermatitis using lactate dehydrogenase isozymes in hairless mice (SKH1-hr)

    PubMed Central

    Kim, Won-Dong

    2012-01-01

    In this study, we evaluate a method for the early diagnosis of radiodermatitis for use in the prevention and therapy of this condition. Hairless mice (SKH1-hr) were used to study the early diagnosis of radiodermatitis. Lactate dehydrogenase (LDH, EC 1.1.1.27) isozymes were analyzed using native-polyacrylamide gel electrophoresis and western blotting of blood serum and tissues collected from SKH1-hr mice. Radiodermatitis developed 24 days after the first X-irradiation. Reduced spleen weight was observed after the last X-irradiation (P<0.05). Thereafter the weight increased until 24 days after the first irradiation, finally reaching levels comparable to those in the sham-irradiated control group. LDH activity was the highest in skeletal muscle and lowest in blood serum. LDH C4, A4, A3B, A2B2, AB3, and B4 isozymes were detected, in the mentioned order, from the cathode. This result was similar in other mouse strains. In the irradiated group, LDH A4 isozyme levels were reduced in the serum until inflammation occurred, whereas those of B4 isozyme were elevated. The subunits A and B followed a similar trend to that of LDH A4 and B4 isozyme, respectively. Importantly, antibodies against LDH B4 isozyme could prove useful in the early diagnosis of radiodermatitis. PMID:23326284

  3. Detection of an intermediate late in the unfolding pathway of bacillus stearothermophilus lactate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Sleigh, Roger N.; Halsall, David J.; Clarke, Anthony R.; Behan-Martin, Moira; Holbrook, J. J.

    1994-08-01

    In vivo proteins fold to form one active structure in minutes or seconds, ruling out the possibility that a polypeptide samples all possible conformational space during folding. We have used site directed mutagenesis to produce 15 single tryptophan containing mutants of Bacillus stearothermophilus lactate dehydrogenase (BS LDH) thus enabling the equilibria of unfolding to be seen from 15 defined positions. These mutant versions of BS LDH have the same X-ray structure as the wild type protein8. Previously Smith et al.11 had detected and assigned structures to 4 folding states. The first intermediate, a monomer with secondary and super secondary structure largely intact, is formed after the dimer dissociates at 0.55 M guanidinium hydrochloride (GuHCl). The second intermediate on the unfolding pathway is stable at 2.2 M GuHCl. It had been assumed previously that the transition from this molten-globule structure to the fully denatured form occurred as a single process. We have now identified a core folding motif. In this, helix (alpha) -1F forms a helix-sheet interaction with (beta) -K and (beta) -K has interactions with both (alpha) -2G and (alpha) -3G. This super secondary interaction forms the most stable folding motif in BS LDH and is lost at 2.8 M GuHCl leaving helix (alpha) -1F, (alpha) -2G, and (alpha) -3G which are stable until 3 M GuHCl.

  4. Lactate dehydrogenase regulation in aged skeletal muscle: Regulation by anabolic steroids and functional overload.

    PubMed

    Washington, Tyrone A; Healey, Julie M; Thompson, Raymond W; Lowe, Larry L; Carson, James A

    2014-09-01

    Aging alters the skeletal muscle response to overload-induced growth. The onset of functional overload is characterized by increased myoblast proliferation and an altered muscle metabolic profile. The onset of functional overload is associated with increased energy demands that are met through the interconversion of lactate and pyruvate via the activity of lactate dehydrogenase (LDH). Testosterone targets many of the processes activated at the onset of functional overload. However, the effect of aging on this metabolic plasticity at the onset of functional overload and how anabolic steroid administration modulates this response is not well understood. The purpose of this study was to determine if aging would alter overload-induced LDH activity and expression at the onset of functional overload and whether anabolic steroid administration would modulate this response. Five-month and 25-month male Fischer 344xF1 BRN were given nandrolone decanoate (ND) or sham injections for 14days and then the plantaris was functionally overloaded (OV) for 3days by synergist ablation. Aging reduced muscle LDH-A & LDH-B activity 70% (p<0.05). Aging also reduced LDH-A mRNA abundance, however there was no age effect on LDH-B mRNA abundance. In 5-month muscle, both ND and OV decreased LDH-A and LDH-B activity. However, there was no synergistic or additive effect. In 5-month muscle, ND and OV decreased LDH-A mRNA expression with no change in LDH-B expression. In 25-month muscle, ND and OV increased LDH-A and LDH-B activity. LDH-A mRNA expression was not altered by ND or OV in aged muscle. However, there was a main effect of OV to decrease LDH-B mRNA expression. There was also an age-induced LDH isoform shift. ND and OV treatment increased the "fast" LDH isoforms in aged muscle, whereas ND and OV increased the "slow" isoforms in young muscle. Our study provides evidence that aging alters aspects of skeletal muscle metabolic plasticity normally induced by overload and anabolic steroid

  5. Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production.

    PubMed

    Jung, Moo-Young; Ng, Chiam Yu; Song, Hyohak; Lee, Jinwon; Oh, Min-Kyu

    2012-07-01

    2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.

  6. An electrochemical biosensor with nanointerface for lactate detection based on lactate dehydrogenase immobilized on zinc oxide nanorods.

    PubMed

    Nesakumar, Noel; Thandavan, Kavitha; Sethuraman, Swaminathan; Krishnan, Uma Maheswari; Rayappan, John Bosco Balaguru

    2014-01-15

    Hepatic immaturity is observed particularly in children whose age is under three, when the lactate concentration is greater than the normal level in blood. An electrochemical lactate biosensor was developed by immobilizing lactate dehydrogenase (LDH) on to ZnO nanorods at pH 7.4 via chitosan. Growth of polycrystalline ZnO nanorods towards (101) plane was confirmed using XRD. The FE-SEM study revealed the formation of ZnO nanorods with an aspect ratio of 3.24. Immobilization of LDH on ZnO nanorods was confirmed using FTIR spectra and surface coverage. Electrochemical studies were carried out through cyclic voltammetry and amperometry using three electrode system with Au/NanoZnO/LDH as working electrode, Ag/AgCl in 0.1 M KCl as reference electrode and Pt wire as counter electrode. The sensitivity of the biosensor was found to be 1.832 μA μmol(-1) L exhibiting linearity 0.2-0.8 μmol L(-1) with the detection and quantification limits of 4.73 and 15.75 nmol L(-1) respectively. The response time of Au/NanoZnO/LDH bioelectrode was found to be <1 s. Prediction band for net current was framed to enhance specificity. Michaelis-Menten constant (KM(app)) and maximum rate (Imax) values for immobilized LDH were found to be 0.38 μmol L(-1) and 2.798 μA respectively. Repeatability and reproducibility of LDH biosensor were also reported. PMID:24231089

  7. Purification and radioimmunoassay of rat lactate dehydrogenase A and B subunits.

    PubMed Central

    Beebee, T J; Carty, D S

    1982-01-01

    We have developed procedures for purifying lactate dehydrogenase isoenzymes from rat tissues that involve two affinity-chromatography steps and that facilitate the isolation of milligram quantities of highly purified proteins within 2--3 days. Antibodies raised against pure A and B subunits in rabbits and hens were used in radioimmunoassays and showed negligible cross-reactivity with heterologous subunits. The radioimmunoassays provide a sensitive method for measuring nanogram amounts of A-subunit and B-subunit polypeptides in tissue homogenates and were employed to characterize the enzyme purification procedures. Images Fig. 1. Fig. 2. PMID:7138505

  8. Resting oxygen consumption varies among lactate dehydrogenase genotypes in the sow bug, Porcellio scaber

    PubMed Central

    Mitton, J. B.; Carter, P. A.; DiGiacomo, A.

    1997-01-01

    Laboratory studies of respiration in the sow bug, Porcellio scaber, reveal that respiration rates are related to genetic variation at the lactate dehydrogenase (Ldh) locus. In population samples taken from Burlington, North Carolina and Pacific Grove, California, respiration rates differed among Ldh genotypes, but not among genotypes at the other enzyme polymorphisms. In both population samples, the respiration rate of the common Ldh homozygote exceeded the respiration rate of the heterozygote by more than 50 per cent. The differences in respiration rates are consistent with previously reported viability differentials at the Ldh polymorphism.

  9. Glucose metabolism in perfused skeletal muscle. Pyruvate dehydrogenase activity in starvation, diabetes and exercise.

    PubMed Central

    Hagg, S A; Taylor, S I; Ruberman, N B

    1976-01-01

    1. The interconversion of pyruvate dehydrogenase between its inactive phosphorylated and active dephosphorylated forms was studied in skeletal muscle. 2. Exercise, induced by electrical stimulation of the sciatic nerve (5/s), increased the measured activity of (active) pyruvate dehydrogenase threefold in intact anaesthetized rated within 2 min. No further increase was seen after 15 min of stimulation. 3. In the perfused rat hindquarter, (active) pyruvate dehydrogenase activity was decreased by 50% in muscle of starved and diabetic rats. Exercise produced a twofold increase in its activity in all groups; however, the relative differences between fed, starved and diabetic groups persisted. 4. Perfusion of muslce with acetoacetate (2 mM) decreased (active) pyruvate dehydrogenase activity by 50% at rest but not during exercise. 5. Whole-tissue concentrations of pyruvate and citrate, inhibitors of (active) pyruvate dehydrogenase kinase and (inactive) pyruvate dehydrogenase phosphate phosphatase respectively, were not altered by excerise. A decrease in the ATP/ADP ratio was observed, but did not appear to be sufficient to account for the increase in (active) pyruvate dehydrogenase activity. 6. The results suggest that interconversion of the phosphorylated and dephosphorylated forms of pyruvate dehydrogenase plays a major role in the regulation of pyruvate oxidation by eomparison of enzyme activity with measurements of lactate oxidation in the perfused hindquarter [see the preceding paper, Berger et al. (1976)] suggest that pyruvate oxidation is also modulated by the concentrations of substrates, cofactors and inhibitors of (active) pyruvate dehydrogenase activity. PMID:825112

  10. Evaluation of D-dimer and lactate dehydrogenase plasma levels in patients with relapsed acute leukemia

    PubMed Central

    HU, WANGQIANG; WANG, XIAOXIA; YANG, RONGRONG

    2016-01-01

    Despite the outstanding advances made over the past decade regarding our knowledge of acute leukemia (AL), relapsed AL remains to be associated with a dismal prognosis. A better understanding of AL relapse and monitoring of the D-dimer and lactate dehydrogenase (LDH) plasma levels following chemotherapy may aid clinicians in determining whether relapse may occur in the subsequent phases of the disease. The present study evaluated D-dimer and LDH levels in 204 patients with relapsed AL. Data were collected at the initial onset of AL, at complete remission (CR) and in patients with relapsed AL. D-dimer plasma levels were significantly increased in patients with initial AL and in patients with relapsed AL (P=0.005 and P=0.007, respectively) but not in those with CR. LDH levels were significantly increased in AL patients at the initial onset of disease and at relapse compared with patients achieving CR, irrespective of cell type. Plasma prothrombin time, activated partial thromboplastin time and fibrinogen levels were not significantly different across patients (with the exception of acute promyelocytic leukemia patients) at the initial onset, relapsed AL or CR. Routine hematological parameters (white blood cell count, hemoglobin, platelet count) were significantly different at the initial onset of AL (P=0.002, P<0.001 and P=0.001, respectively) and during relapsed AL (P=0.009, P=0.003 and P<0.001, respectively) compared with patients achieving CR, suggesting an association between D-dimer, LDH and relapsed AL. These results also indicate that determination of D-dimer and LDH levels may be useful for predicting the probability of relapse during chemotherapy, but should also be combined with routine hematological parameters. PMID:27347185

  11. Reactivity of the essential thiol group of lactate dehydrogenase and substrate binding

    PubMed Central

    Holbrook, J. J.; Stinson, R. A.

    1970-01-01

    1. The preparation of a derivative of pig heart lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group is described. The derivative has unchanged s20,w and is catalytically inactive. 2. The rate of reaction of the essential thiol group is controlled by a system with a pK>9. 3. The essential thiol group is protected by NADH against reaction with maleimide. 4. Lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group or alkylated with maleimide still binds one molecule of NADH/subunit but with a three- to four-fold diminished affinity. 5. The inhibited enzymes also bind one molecule of NAD+–sulphite complex/subunit but with affinity decreased 103–104-fold. 6. The inhibited enzymes fail to bind C2 and C3 molecules to give the ternary complexes enzyme–NAD+–pyruvate, enzyme–NADH–oxamate and enzyme–NADH–oxalate. The 1:1:1 stoicheiometry of the last-mentioned complex with the native enzyme was established by gel filtration. 7. Structures that account for these results are discussed. PMID:4321894

  12. Crystal structure and thermodynamic properties of d-lactate dehydrogenase from Lactobacillus jensenii.

    PubMed

    Kim, Sangwoo; Gu, Sol-A; Kim, Yong Hwan; Kim, Kyung-Jin

    2014-07-01

    The thermostable d-lactate dehydrogenase from Lactobacillus jensenii (Ljd-LDH) is a key enzyme in the production of the d-form of lactic acid from pyruvate concomitant with the oxidation of NADH to NAD(+). The polymers of d-lactic acid are used as biodegradable bioplastics. The crystal structures of Ljd-LDH and in complex with NAD(+) were determined at 2.13 and 2.60Å resolutions, respectively. The Ljd-LDH monomer consists of the N-terminal substrate-binding domain and the C-terminal NAD-binding domain. The Ljd-LDH forms a homodimeric structure, and the C-terminal NAD-binding domain mostly enables the dimerization of the enzyme. The NAD cofactor is bound to the GxGxxG NAD-binding motif located between the two domains. Structural comparisons of Ljd-LDH with other d-LDHs reveal that Ljd-LDH has unique amino acid residues at the linker region, which indicates that the open-close dynamics of Ljd-LDH might be different from that of other d-LDHs. Moreover, thermostability experiments showed that the T50(10) value of Ljd-LDH (54.5°C) was much higher than the commercially available d-lactate dehydrogenase (42.7°C). In addition, Ljd-LDH has at least a 7°C higher denaturation temperature compared to commercially available d-LDHs. PMID:24794195

  13. Printed paper sensors for serum lactate dehydrogenase using pullulan-based inks to immobilize reagents.

    PubMed

    Kannan, Balamurali; Jahanshahi-Anbuhi, Sana; Pelton, Robert H; Li, Yingfu; Filipe, Carlos D M; Brennan, John D

    2015-09-15

    In this study, a paper-based point-of-care (POC) colorimetric biosensor was developed for the detection of lactate dehydrogenase in serum using a nonporous, oxygen impermeable reversibly gelling polysaccharide material based on pullulan. The pullulan could be printed onto paper surfaces along with all required assay reagents, providing a means for high-stability immobilization of all reagents on paper. Serum containing lactate dehydrogenase (LDH) was directly spotted on to the pullulan-coated bioactive paper and provided quantitative colorimetric data that was comparable to that obtained with a conventional plate-reader method. The paper strip was found to be highly stable and could be stored at 4 °C for at least 10 weeks with no loss in performance, as compared to a complete loss in performance within 1 day when the reagents were printed without the stabilizing polysaccharide. The ease of fabrication coupled with the high stability of the printed reagents provides a facile platform for easily manufactured POC sensors.

  14. Comparative enzymology-new insights from studies of an "old" enzyme, lactate dehydrogenase.

    PubMed

    Storey, Kenneth B

    2016-09-01

    Comparative enzymology explores the molecular mechanisms that alter the properties of enzymes to best fit and adapt them to the biotic demands and abiotic stresses that affect the cellular environment in which these protein catalysts function. For many years, comparative enzymology was primarily concerned with analyzing enzyme functional properties (e.g. substrate affinities, allosteric effectors, responses to temperature or pH, stabilizers, denaturants, etc.) in order to determine how enzyme properties were optimized to function under changing conditions. More recently it became apparent that posttranslational modifications of enzymes play a huge role in metabolic regulation. At first, such modifications appeared to target just crucial regulatory enzymes but recent work is showing that many dehydrogenases are also targets of posttranslational modification leading to substantial changes in enzyme properties. The present article focuses in particular on lactate dehydrogenase (LDH) showing that stress-induced changes in enzyme properties can be linked with reversible posttranslational modifications; e.g. changes in the phosphorylation state of LDH occur in response to dehydration stress in frogs and anoxia exposure of turtles and snails. Furthermore, these studies show that LDH is also a target of other posttranslational modifications including acetylation, methylation and ubiquitination that change in response to anoxia or dehydration stress. Selected new methods for exploring posttranslational modifications of dehydrogenases are discussed and new challenges for the future of comparative enzymology are presented that will help to achieve a deeper understanding of biochemical adaptation through enzyme regulation. PMID:26688543

  15. The utility of lactate dehydrogenase in the follow up of patients with diffuse large B-cell lymphoma

    PubMed Central

    William, Basem Magdy; Bongu, Navneeth Rao; Bast, Martin; Bociek, Robert Gregory; Bierman, Philip Jay; Vose, Julie Marie; Armitage, James Olen

    2013-01-01

    Background Serum lactate dehydrogenase is a non-specific marker for lymphoma whose prognostic significance is well established for both indolent and aggressive lymphomas at the time of diagnosis. The performance characteristics of this enzyme in predicting relapse in patients with diffuse large B-cell lymphoma has not been well studied. Methods This study compared serum lactate dehydrogenase levels in 27 patients with diffuse large B-cell lymphoma who relapsed after sustaining a complete response versus 87 patients who did not relapse. For relapsed patients, the serum lactate dehydrogenase level at relapse was compared with the level three months before (considered baseline). For non-relapsed patients, the last two levels during follow-up were compared. For statistical analysis the T-test was used to compare differences in mean values between groups. The sensitivity, specificity, positive and negative predictive values for serum lactate dehydrogenase in detecting relapse compared to confirmatory imaging were calculated. Results At relapse, only 33% patients had increases in serum lactate dehydrogenase above the upper limit of normal. The mean increase was 1.2-fold above the upper limit of normal for relapsed vs. 0.83 for those who did not relapse (p-value = 0.59). The mean increase in serum lactate dehydrogenase, from baseline, was 1.1-fold in non-relapsed vs. 1.3 in relapsed patients (p-value = 0.3). The likelihood ratio of relapse was 4.65 for patients who had 1.5-fold increases in serum lactate dehydrogenase above baseline (p-value = 0.03). The sensitivity, specificity, positive and negative predictive values of 1.5-fold increases for detecting relapse, compared to clinical and imaging findings were 0.18, 0.95, 0.55, and 0.79, respectively. Conclusion A 1.5-fold increase in serum lactate dehydrogenase, over a period of 3 months, is associated with increased likelihood of relapse from diffuse large B-cell lymphoma. PMID:23904809

  16. [Lactate dehydrogenase isoenzymatic makeup of the skeletal muscles of rats after a flight on the Kosmos-690 biosatellite].

    PubMed

    Petrova, N V

    1978-01-01

    The isoenzyme composition of lactate dehydrogenase in the soleus and plantaris muscles of rats which had flown for 20.5 days onboard the biosatellite Cosmos-690 equipped with a radiation source was studied. Difference in the isoenzyme composition of lactate dehydrogenase in flight and synchronous rats disappeared 27 days after the experiments; however, some changes persisted as compared with vivarium controls. The data obtained give evidence that irradiation-induced effects in skeletal muscles manifested themselves at a far later stage than weightlessness-induced changes.

  17. IFCC methods for the measurement of catalytic concentration of enzymes. Part 8. IFCC method for lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27)

    PubMed Central

    Bais, Renze; Philcox, Margaret

    1994-01-01

    Human lactate dehydrogenase is a tetramer made up of two types of subunits, either H (heart) or M (muscle). Combination of these subunits gives rise to the five isoenzymes of lactate dehydrogenase which are found in mammalian tissues. The relative proportions of the individual isoenzymes found in serum of patients is related to the severity of the lesion in the organ or tissue from which they originate and the half-life of the individual tissue-specific enzymes. Thus, one cannot predict the relative proportions of the different isoenzymes in any one patient sample. Lactate dehydrogenase catalyses the reversible oxidation of lactate to pyruvate and either reaction can be measured readily. However, in this method, the lactate to pyruvate reaction has been selected because of the following reasons; the time-course of the reaction is more linear, the reaction results in an increase in absorbance and optimization of substrates is possible (see appendix A). The principles applied in the selection of the conditions of measurement are those stated in previous publications by the IFCC’s Committee on Enzymes [1]. Human serum and tissue extracts have been used as the sources of enzymes. The final concentration of substrates and the pH have been selected on the basis of experiments and empirical optimization techniques and have been confirmed by calculation from rate equations. The catalytic and physical properties of the isoenzymes differ, but because of the importance of the heart specific isoenzyme (LD1) in the assessment of coronary heart disease and as a tumour marker, this method has been optimized for this isoenzyme. However, the method is also suitable, although less optimally, for the determination of the other isoenzymes of lactate dehydrogenase which may be present in serum. PMID:18924988

  18. Isolation and characterization of two cDNA clones of anaerobically induced lactate dehydrogenase from barley roots

    SciTech Connect

    Hondred, D.; Hanson, A.D. )

    1990-05-01

    In barley roots during hypoxia, five lactate dehydrogenase (LDH) isozymes accumulate with a concomitant increase in enzyme activity ({approximately}20-fold). These isozymes are thought to be tetramers resulting from the random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH have been isolated from a {lambda}gt11 library using antiserum raised against barley LDH purified {approximately}3,000-fold and using nucleic acid probes synthesized by the polymerase chain reaction. Two cDNA clones were obtained (1,305 and 1,166 bp). The deduced amino acid sequences of the two barley LDHs are 96% identical to each other, and 50% and 40% identical to vertebrate and bacterial LDHs, respectively. Northern blots identified a single mRNA band ({approximately}1.5 kb) whose level rose 8-fold during hypoxia.

  19. "Flipped" patterns of lactate dehydrogenase isoenzymes in serum of elite college basketball players.

    PubMed

    Rotenberg, Z; Seip, R; Wolfe, L A; Bruns, D E

    1988-11-01

    We kinetically measured total lactate dehydrogenase (LD, EC 1.1.1.27), total creatine kinase (CK, EC 2.7.3.2), and aspartate aminotransferase (AST, EC 2.6.1.1.) in 16 elite college basketball players, before the competition season and not in close temporal relation to near-maximal exercise, and in 17 healthy non-athlete controls. LD isoenzymes were determined by both electrophoretic and immunoprecipitation methods. CK-MB isoenzyme was measured electrophoretically. We found significantly higher mean LD-1 values and LD-1/LD-2 ratios in the players than the controls: 31.6 (SD 3.7)% vs 25.8 (SD 3.2)% (P less than 0.005) and 1.1 (SD 0.13) vs 0.87 (SD 0.16) (P less than 0.001), respectively. A "flipped" LD pattern (LD-1 greater than LD-2) was found in half the players and in six of the eight black athletes, but in only two of the control group and in none of the black controls. Mean CK activity in serum exceeded normal values in the serum of the athletes and was higher in comparison with the control group [274 (SD 156) vs 103 (SD 82) U/L]. Mean CK was significantly higher in the eight athletes with the flipped LD pattern than in those with LD-1 less than LD-2 [322 (SD 163) vs 180 (SD 98) U/L; P = 0.05], and also in comparison with CK in the two controls with flipped LD pattern. We saw no significant difference in mean CK between the nine players with normal immunochemical LD-1/LD ratios and the seven players with above-normal ratios. CK-MB was not detected in either athletes or controls. None of the players had any clinical or electrocardiographic evidence for myocardial ischemia or infarction. Evidently the flipped LD pattern usually found in patients with acute myocardial infarction and reported in some athletes after extreme exercise such as ultra-marathon running may also be found in athletes who are in their "basal fitness shape" but who are not involved in competitive physical activity.

  20. Purification and Electrophoretic Characterization of Lactate Dehydrogenase from Mammalian Blood: A Different Twist on a Classic Experiment

    ERIC Educational Resources Information Center

    Brunauer, Linda S.

    2016-01-01

    A multiweek protein purification suite, suitable for upper-division biochemistry or biotechnology undergraduate students, is described. Students work in small teams to isolate the enzyme lactate dehydrogenase (LDH) from a nontraditional tissue source, mammalian blood, using a sequence of three column chromatographic procedures: ion-exchange, size…

  1. Development of L-lactate dehydrogenase biosensor based on porous silicon resonant microcavities as fluorescence enhancers.

    PubMed

    Jenie, S N Aisyiyah; Prieto-Simon, Beatriz; Voelcker, Nicolas H

    2015-12-15

    The up-regulation of L-lactate dehydrogenase (LDH), an intracellular enzyme present in most of all body tissues, is indicative of several pathological conditions and cellular death. Herein, we demonstrate LDH detection using porous silicon (pSi) microcavities as a luminescence-enhancing optical biosensing platform. Non-fluorescent resazurin was covalently attached onto the pSi surface via thermal hydrocarbonisation, thermal hydrosylilation and acylation. Each surface modification step was confirmed by means of FTIR and the optical shifts of the resonance wavelength of the microcavity. Thermal hydrocarbonisation also afforded excellent surface stability, ensuring that the resazurin was not reduced on the pSi surface. Using a pSi microcavity biosensor, the fluorescence signal upon detection of LDH was amplified by 10 and 5-fold compared to that of a single layer and a detuned microcavity, respectively, giving a limit of detection of 0.08 U/ml. The biosensor showed a linear response between 0.16 and 6.5 U/ml, covering the concentration range of LDH in normal as well as damaged tissues. The biosensor was selective for LDH and did not produce a signal upon incubation with another NAD-dependant enzyme L-glutamic dehydrogenase. The use of the pSi microcavity as a sensing platform reduced reagent usage by 30% and analysis time threefold compared to the standard LDH assay in solution.

  2. Free energy landscape of the Michaelis complex of lactate dehydrogenase: A network analysis of atomistic simulations

    NASA Astrophysics Data System (ADS)

    Pan, Xiaoliang; Schwartz, Steven

    2015-03-01

    It has long been recognized that the structure of a protein is a hierarchy of conformations interconverting on multiple time scales. However, the conformational heterogeneity is rarely considered in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD+). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they are catalytic competent at different reaction rates. In this study, millisecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network the Michaelis complex and the structures of the substates at atomistic scale. It also shed some light on understanding the complete picture of the catalytic mechanism of LDH.

  3. Direct Evidence of Catalytic Heterogeneity in Lactate Dehydrogenase by Temperature Jump Infrared Spectroscopy

    PubMed Central

    2015-01-01

    Protein conformational heterogeneity and dynamics are known to play an important role in enzyme catalysis, but their influence has been difficult to observe directly. We have studied the effects of heterogeneity in the catalytic reaction of pig heart lactate dehydrogenase using isotope edited infrared spectroscopy, laser-induced temperature jump relaxation, and kinetic modeling. The isotope edited infrared spectrum reveals the presence of multiple reactive conformations of pyruvate bound to the enzyme, with three major reactive populations having substrate C2 carbonyl stretches at 1686, 1679, and 1674 cm–1, respectively. The temperature jump relaxation measurements and kinetic modeling indicate that these substates form a heterogeneous branched reaction pathway, and each substate catalyzes the conversion of pyruvate to lactate with a different rate. Furthermore, the rate of hydride transfer is inversely correlated with the frequency of the C2 carbonyl stretch (the rate increases as the frequency decreases), consistent with the relationship between the frequency of this mode and the polarization of the bond, which determines its reactivity toward hydride transfer. The enzyme does not appear to be optimized to use the fastest pathway preferentially but rather accesses multiple pathways in a search process that often selects slower ones. These results provide further support for a dynamic view of enzyme catalysis where the role of the enzyme is not just to bring reactants together but also to guide the conformational search for chemically competent interactions. PMID:25149276

  4. Lactate dehydrogenase-A inhibition induces human glioblastoma multiforme stem cell differentiation and death

    PubMed Central

    Daniele, Simona; Giacomelli, Chiara; Zappelli, Elisa; Granchi, Carlotta; Trincavelli, Maria Letizia; Minutolo, Filippo; Martini, Claudia

    2015-01-01

    Therapies that target the signal transduction and metabolic pathways of cancer stem cells (CSCs) are innovative strategies to effectively reduce the recurrence and significantly improve the outcome of glioblastoma multiforme (GBM). CSCs exhibit an increased rate of glycolysis, thus rendering them intrinsically more sensitive to prospective therapeutic strategies based on the inhibition of the glycolytic pathway. The enzyme lactate dehydrogenase-A (LDH-A), which catalyses the interconversion of pyruvate and lactate, is up-regulated in human cancers, including GBM. Although several papers have explored the benefits of targeting cancer metabolism in GBM, the effects of direct LDH-A inhibition in glial tumours have not yet been investigated, particularly in the stem cell subpopulation. Here, two representative LDH-A inhibitors (NHI-1 and NHI-2) were studied in GBM-derived CSCs and compared to differentiated tumour cells. LDH-A inhibition was particularly effective in CSCs isolated from different GBM cell lines, where the two compounds blocked CSC formation and elicited long-lasting effects by triggering both apoptosis and cellular differentiation. These data demonstrate that GBM, particularly the stem cell subpopulation, is sensitive to glycolytic inhibition and shed light on the therapeutic potential of LDH-A inhibitors in this tumour type. PMID:26494310

  5. Lactate does not activate NF-κB in oxidative tumor cells

    PubMed Central

    Van Hée, Vincent F.; Pérez-Escuredo, Jhudit; Cacace, Andrea; Copetti, Tamara; Sonveaux, Pierre

    2015-01-01

    The lactate anion is currently emerging as an oncometabolite. Lactate, produced and exported by glycolytic and glutaminolytic cells in tumors, can be recycled as an oxidative fuel by oxidative tumors cells. Independently of hypoxia, it can also activate transcription factor hypoxia-inducible factor-1 (HIF-1) in tumor and endothelial cells, promoting angiogenesis. These protumoral activities of lactate depend on lactate uptake, a process primarily facilitated by the inward, passive lactate-proton symporter monocarboxylate transporter 1 (MCT1); the conversion of lactate and NAD+ to pyruvate, NADH and H+ by lactate dehydrogenase-1 (LDH-1); and a competition between pyruvate and α-ketoglutarate that inhibits prolylhydroxylases (PHDs). Endothelial cells do not primarily use lactate as an oxidative fuel but, rather, as a signaling agent. In addition to HIF-1, lactate can indeed activate transcription factor nuclear factor-κB (NF-κB) in these cells, through a mechanism not only depending on PHD inhibition but also on NADH alimenting NAD(P)H oxidases to generate reactive oxygen species (ROS). While NF-κB activity in endothelial cells promotes angiogenesis, NF-κB activation in tumor cells is known to stimulate tumor progression by conferring resistance to apoptosis, stemness, pro-angiogenic and metastatic capabilities. In this study, we therefore tested whether exogenous lactate could activate NF-κB in oxidative tumor cells equipped for lactate signaling. We report that, precisely because they are oxidative, HeLa and SiHa human tumor cells do not activate NF-κB in response to lactate. Indeed, while lactate-derived pyruvate is well-known to inhibit PHDs in these cells, we found that NADH aliments oxidative phosphorylation (OXPHOS) in mitochondria rather than NAD(P)H oxidases in the cytosol. These data were confirmed using oxidative human Cal27 and MCF7 tumor cells. This new information positions the malate-aspartate shuttle as a key player in the oxidative metabolism

  6. Activity of select dehydrogenases with Sepharose-immobilized N6-carboxymethyl-NAD

    PubMed Central

    Beauchamp, Justin; Vieille, Claire

    2015-01-01

    N6-carboxymethyl-NAD (N6-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N6-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N6-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N6-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N6-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N6-amine group on NAD. PMID:25611453

  7. Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.

    PubMed

    Beauchamp, Justin; Vieille, Claire

    2015-01-01

    N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N(6)-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N(6)-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N(6)-amine group on NAD.

  8. An atypical distribution of lactate dehydrogenase isoenzymes in the hooded seal (Cystophora cristata) brain may reflect a biochemical adaptation to diving.

    PubMed

    Hoff, Mariana Leivas Müller; Fabrizius, Andrej; Folkow, Lars P; Burmester, Thorsten

    2016-04-01

    The brains of some diving mammals can withstand periods of severe hypoxia without signs of deleterious effects. This may in part be due to an enhanced cerebral capacity for anaerobic energy production. Here, we have tested this hypothesis by comparing various parameters of the lactate dehydrogenase (LDH) in the brain of the hooded seal (Cystophora cristata) with those in the brains of the ferret (Mustela putorius furo) and mouse (Mus musculus). We found that mRNA and protein expression of lactate dehydrogenase a (LDHA) and lactate dehydrogenase b (LDHB), and also the LDH activity were significantly higher in the ferret brain than in brains of the hooded seal and the mouse (p < 0.0001). No conspicuous differences in the LDHA and LDHB sequences were observed. There was also no difference in the buffering capacities of the brains. Thus, an enhanced capacity for anaerobic energy production likely does not explain the higher hypoxia tolerance of the seal brain. However, the brain of the hooded seal had higher relative levels of LDHB isoenzymes (LDH1 and LDH2) compared to the non-diving mammals. Moreover, immunofluorescence studies showed more pronounced co-localization of LDHB and glial fibrillary acidic protein in the cortex of the hooded seal. Since LDHB isoenzymes primarily catalyze the conversion of lactate to pyruvate, this finding suggests that the contribution of astrocytes to the brain aerobic metabolism is higher in the hooded seal than in non-diving species. The cerebral tolerance of the hooded seal to hypoxia may therefore partly rely on different LDH isoenzymes distribution. PMID:26820264

  9. A new technique for the precise location of lactate and malate dehydrogenases in goat, boar and water buffalo spermatozoa using gel incubation film.

    PubMed

    Kohsaka, T; Takahara, H; Tagami, S; Sasada, H; Masaki, J

    1992-05-01

    Gel incubation film, which contained gelatin to prevent the diffusion of enzyme during chemical reaction and phenazine methosulfate to operate as a hydrogen acceptor between NADH and tetrazolium, was used and light microscopy revealed that lactate dehydrogenase was located in the head and tail of the spermatozoa as well as in the midpiece, whereas malate dehydrogenase was confined to the midpiece in spermatozoa of the animals examined. In goat spermatozoa, lactate dehydrogenase was associated mainly with the inner acrosomal membrane in the head, the mitochondrial matrix in the midpiece and with flagellar fibrils in the tail, whereas malate dehydrogenase was present only in the mitochondrial matrix.

  10. Prognostic significance of pretreated serum lactate dehydrogenase level in nasopharyngeal carcinoma among Chinese population

    PubMed Central

    Zhang, Mingwei; Wei, Shushan; Su, Li; Lv, Wenlong; Hong, Jinsheng

    2016-01-01

    Abstract Background: A large number of studies have investigated the prognostic value of pretreated lactate dehydrogenase (LDH) level in nasopharyngeal carcinoma (NPC) patients while the role of it was inconsistent and inconclusive. Hence, the aim of the current study was to conduct a meta-analysis of all published studies to quantify the prognostic impact of pretreated serum LDH in NPC for Chinese population. Objectives: The aim of the current study was to conduct a meta-analysis of all published studies to quantify the prognostic impact of pretreated serum lactate dehydrogenase (LDH) in nasopharyngeal carcinoma (NPC) for Chinese population. Methods: The PubMed, Medline, Embase, and Web of Science databases were searched for studies that assessed survival outcome and LDH in NPC. Overall survival (OS) was the primary survival outcome. Distant metastasis-free survival (DMFS) and disease-free survival (DFS) were secondary outcomes. The pooled hazard ratios (HRs), associated with 95% confidence intervals (95% CIs), were combined to calculate overall effects. The Cochran Q and I2 statistics were used to assess heterogeneity. When apparent heterogeneity was observed, sensitivity and meta-regression analyses were performed to explore its origin. Results: Sixteen studies, which included 14,803 patients, were enrolled in the current meta-analysis to yield statistics. Overall, the pooled HR for OS in 11 eligible studies with high LDH level was 1.79 (95% CI = 1.47–2.12), and the pooled HR for DMFS in 9 eligible studies with high LDH level was 1.85 (95% CI = 1.48–2.22). Meanwhile, the pooled HR for DFS in 5 eligible studies with high LDH level was 1.63 (95% CI = 1.34–1.91). Egger test and funnel plots revealed that the publication bias in the current meta-analysis was insignificant. Conclusions: The present meta-analysis demonstrated that high pretreated LDH level is significantly associated with poorer OS, DMFS, and DFS, suggesting that pretreated LDH could

  11. Hypoxically inducible barley lactate dehydrogenase: cDNA cloning and molecular analysis

    SciTech Connect

    Hondred, D. ); Hanson, A.D. Univ. de Montreal, Quebec )

    1990-09-01

    In the roots of barley and other cereals, hypoxia induces a set of five isozymes of L-lactate dehydrogenase (LDH; (S)-lactate:NADH oxidoreductase, EC 1.1.1.27). Biochemical and genetic data indicate that the five LDH isozymes are tetramers that arise from random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH were isolated from a {lambda}gt11 cDNA library derived from hypoxically treated barley roots. The library was screened with antiserum raised against barley LDH purified {approx}3,000-fold by an improved three-step procedure. Immunopositive clones were rescreened with a cDNA probe synthesized by the polymerase chain reaction using primers modeled from the amino acid sequences of two tryptic LDH peptides. Two types of LDH clones were found. Nucleotide sequence analysis of one representative insert of each type (respectively, 1,305 and 1,166 base pairs) revealed open reading framed encoding 10 peptide fragments of LDH. The 1,305-base-pair insert included the entire coding region of a 356-residue LDH monomer. The nucleotide sequences of the two LDH cDNAs were 92% identical in the coding region, but highly divergent in the 3{prime} noncoding region, and thus probably correspond to the two postulated Ldh loci. The deduced amino acid sequences of the two barley LDHs were 96% identical to each other and very similar to those from vertebrate and bacterial LDHs. RNA blot hybridization showed a single mRNA band of 1.5 kilobases whose level rose about 8-fold in roots during hypoxic induction, as did the level of translatable LDH message.

  12. Pressure-adaptive differences in lactate dehydrogenases of three hagfishes: Eptatretus burgeri, Paramyxine atami and Eptatretus okinoseanus.

    PubMed

    Nishiguchi, Yoshikazu; Miwa, Tetsuya; Abe, Fumiyoshi

    2008-05-01

    The tolerance of abyssal pressures likely depends on adaptive modifications of fish proteins. However, structural modifications of proteins which allow functioning at high pressure remain unclear. We compared the activities of lactate dehydrogenase (LDH), an important enzyme in glycolytic reaction, in three hagfishes inhabiting different depths under increased pressure. LDH in Eptatretus okinoseanus, found at a depth of 1,000 m, was highly active at high pressure of 100 MPa maintaining the activity at 70% of that at 0.1 MPa. In contrast, LDH activity in Paramyxine atami, found at 250-400 m, decreased to 55% at 15 MPa, and that in Eptatretus burgeri, found at 45-60 m, was completely absent at 5 MPa. The result suggests that subunit interaction of the LDH-tetramer is more stable in E. okinoseanus than that in P. atami and E. burgeri under high-pressure conditions. We found six amino acid substitutions between the three LDH primary structures. Accordingly, these amino acid residues are likely to contribute to the stability of the E. okinoseanus LDH under high-pressure conditions. PMID:18299796

  13. Gene Expression Variation in Duplicate Lactate dehydrogenase Genes: Do Ecological Species Show Distinct Responses?

    PubMed Central

    Cristescu, Melania E.; Demiri, Bora; Altshuler, Ianina; Crease, Teresa J.

    2014-01-01

    Lactate dehydrogenase (LDH) has been shown to play an important role in adaptation of several aquatic species to different habitats. The genomes of Daphnia pulex, a pond species, and Daphnia pulicaria, a lake inhabitant, encode two L-LDH enzymes, LDHA and LDHB. We estimated relative levels of Ldh gene expression in these two closely related species and their hybrids in four environmental settings, each characterized by one of two temperatures (10°C or 20°C), and one of two concentrations of dissolved oxygen (DO; 6.5–7 mg/l or 2–3 mg/l). We found that levels of LdhA expression were 4 to 48 times higher than LdhB expression (p<0.005) in all three groups (the two parental species and hybrids). Moreover, levels of LdhB expression differed significantly (p<0.05) between D. pulex and D. pulicaria, but neither species differed from the hybrid. Consistently higher expression of LdhA relative to LdhB in both species and the hybrid suggests that the two isozymes could be performing different functions. No significant differences in levels of gene expression were observed among the four combinations of temperature and dissolved oxygen (p>0.1). Given that Daphnia dwell in environments characterized by fluctuating conditions with long periods of low dissolved oxygen concentration, we suggest that these species could employ regulated metabolic depression to survive in such environments. PMID:25080082

  14. Synaptosomal lactate dehydrogenase isoenzyme composition is shifted toward aerobic forms in primate brain evolution.

    PubMed

    Duka, Tetyana; Anderson, Sarah M; Collins, Zachary; Raghanti, Mary Ann; Ely, John J; Hof, Patrick R; Wildman, Derek E; Goodman, Morris; Grossman, Lawrence I; Sherwood, Chet C

    2014-01-01

    With the evolution of a relatively large brain size in haplorhine primates (i.e. tarsiers, monkeys, apes, and humans), there have been associated changes in the molecular machinery that delivers energy to the neocortex. Here we investigated variation in lactate dehydrogenase (LDH) expression and isoenzyme composition of the neocortex and striatum in primates using quantitative Western blotting and isoenzyme analysis of total homogenates and synaptosomal fractions. Analysis of isoform expression revealed that LDH in synaptosomal fractions from both forebrain regions shifted towards a predominance of the heart-type, aerobic isoform LDH-B among haplorhines as compared to strepsirrhines (i.e. lorises and lemurs), while in the total homogenate of the neocortex and striatum there was no significant difference in LDH isoenzyme composition between the primate suborders. The largest increase occurred in synapse-associated LDH-B expression in the neocortex, with an especially remarkable elevation in the ratio of LDH-B/LDH-A in humans. The phylogenetic variation in the ratio of LDH-B/LDH-A was correlated with species-typical brain mass but not the encephalization quotient. A significant LDH-B increase in the subneuronal fraction from haplorhine neocortex and striatum suggests a relatively higher rate of aerobic glycolysis that is linked to synaptosomal mitochondrial metabolism. Our results indicate that there is a differential composition of LDH isoenzymes and metabolism in synaptic terminals that evolved in primates to meet increased energy requirements in association with brain enlargement. PMID:24686273

  15. The Regulation and Function of Lactate Dehydrogenase A: Therapeutic Potential in Brain Tumor.

    PubMed

    Valvona, Cara J; Fillmore, Helen L; Nunn, Peter B; Pilkington, Geoffrey J

    2016-01-01

    There are over 120 types of brain tumor and approximately 45% of primary brain tumors are gliomas, of which glioblastoma multiforme (GBM) is the most common and aggressive with a median survival rate of 14 months. Despite progress in our knowledge, current therapies are unable to effectively combat primary brain tumors and patient survival remains poor. Tumor metabolism is important to consider in therapeutic approaches and is the focus of numerous research investigations. Lactate dehydrogenase A (LDHA) is a cytosolic enzyme, predominantly involved in anaerobic and aerobic glycolysis (the Warburg effect); however, it has multiple additional functions in non-neoplastic and neoplastic tissues, which are not commonly known or discussed. This review summarizes what is currently known about the function of LDHA and identifies areas that would benefit from further exploration. The current knowledge of the role of LDHA in the brain and its potential as a therapeutic target for brain tumors will also be highlighted. The Warburg effect appears to be universal in tumors, including primary brain tumors, and LDHA (because of its involvement with this process) has been identified as a potential therapeutic target. Currently, there are, however, no suitable LDHA inhibitors available for tumor therapies in the clinic.

  16. Synaptosomal lactate dehydrogenase isoenzyme composition is shifted toward aerobic forms in primate brain evolution.

    PubMed

    Duka, Tetyana; Anderson, Sarah M; Collins, Zachary; Raghanti, Mary Ann; Ely, John J; Hof, Patrick R; Wildman, Derek E; Goodman, Morris; Grossman, Lawrence I; Sherwood, Chet C

    2014-01-01

    With the evolution of a relatively large brain size in haplorhine primates (i.e. tarsiers, monkeys, apes, and humans), there have been associated changes in the molecular machinery that delivers energy to the neocortex. Here we investigated variation in lactate dehydrogenase (LDH) expression and isoenzyme composition of the neocortex and striatum in primates using quantitative Western blotting and isoenzyme analysis of total homogenates and synaptosomal fractions. Analysis of isoform expression revealed that LDH in synaptosomal fractions from both forebrain regions shifted towards a predominance of the heart-type, aerobic isoform LDH-B among haplorhines as compared to strepsirrhines (i.e. lorises and lemurs), while in the total homogenate of the neocortex and striatum there was no significant difference in LDH isoenzyme composition between the primate suborders. The largest increase occurred in synapse-associated LDH-B expression in the neocortex, with an especially remarkable elevation in the ratio of LDH-B/LDH-A in humans. The phylogenetic variation in the ratio of LDH-B/LDH-A was correlated with species-typical brain mass but not the encephalization quotient. A significant LDH-B increase in the subneuronal fraction from haplorhine neocortex and striatum suggests a relatively higher rate of aerobic glycolysis that is linked to synaptosomal mitochondrial metabolism. Our results indicate that there is a differential composition of LDH isoenzymes and metabolism in synaptic terminals that evolved in primates to meet increased energy requirements in association with brain enlargement.

  17. Computational analyses of mammalian lactate dehydrogenases: human, mouse, opossum and platypus LDHs.

    PubMed

    Holmes, Roger S; Goldberg, Erwin

    2009-10-01

    Computational methods were used to predict the amino acid sequences and gene locations for mammalian lactate dehydrogenase (LDH) genes and proteins using genome sequence databanks. Human LDHA, LDHC and LDH6A genes were located in tandem on chromosome 11, while LDH6B and LDH6C genes were on chromosomes 15 and 12, respectively. Opossum LDHC and LDH6B genes were located in tandem with the opossum LDHA gene on chromosome 5 and contained 7 (LDHA and LDHC) or 8 (LDH6B) exons. An amino acid sequence prediction for the opossum LDH6B subunit gave an extended N-terminal sequence, similar to the human and mouse LDH6B sequences, which may support the export of this enzyme into mitochondria. The platypus genome contained at least 3 LDH genes encoding LDHA, LDHB and LDH6B subunits. Phylogenetic studies and sequence analyses indicated that LDHA, LDHB and LDH6B genes are present in all mammalian genomes examined, including a monotreme species (platypus), whereas the LDHC gene may have arisen more recently in marsupial mammals.

  18. SYNAPTOSOMAL LACTATE DEHYDROGENASE ISOENZYME COMPOSITION IS SHIFTED TOWARD AEROBIC FORMS IN PRIMATE BRAIN EVOLUTION

    PubMed Central

    Duka, Tetyana; Anderson, Sarah M.; Collins, Zachary; Raghanti, Mary Ann; Ely, John J.; Hof, Patrick R.; Wildman, Derek E.; Goodman, Morris; Grossman, Lawrence I.; Sherwood, Chet C.

    2014-01-01

    With the evolution of a relatively large brain size in haplorhine primates (i.e., tarsiers, monkeys, apes and humans), there have been associated changes in the molecular machinery that delivers energy to the neocortex. Here we investigated variation in lactate dehydrogenase (LDH) expression and isoenzyme composition of the neocortex and striatum in primates using quantitative Western blotting and isoenzyme analysis of total homogenates and synaptosomal fractions. Analysis of isoform expression revealed that LDH in the synaptosomal fraction from both forebrain regions shifted towards a predominance of the heart-type, aerobic isoforms, LDHB, among haplorhines as compared to strepsirrhines (i.e., lorises and lemurs), while in total homogenate of neocortex and striatum there was no significant difference in the LDH isoenzyme composition between the primate suborders. The largest increase occurred in synapse-associated LDH-B expression in the neocortex, displaying an especially remarkable elevation in the ratio of LDH-B to LDH-A in humans. The phylogenetic variation in LDH-B to LDH-A ratio was correlated with species typical brain mass, but not encephalization quotient. A significant LDHB increase in the sub-neuronal fraction from haplorhine neocortex and striatum suggests a relatively higher rate of aerobic glycolysis that is linked to synaptosomal mitochondrial metabolism. Our results indicate that there is differential composition of LDH isoenzymes and metabolism in synaptic terminals that evolved in primates to meet increased energy requirements in association with brain enlargement. PMID:24686273

  19. Common and rare variants associating with serum levels of creatine kinase and lactate dehydrogenase

    PubMed Central

    Kristjansson, Ragnar P.; Oddsson, Asmundur; Helgason, Hannes; Sveinbjornsson, Gardar; Arnadottir, Gudny A.; Jensson, Brynjar O.; Jonasdottir, Aslaug; Jonasdottir, Adalbjorg; Bragi Walters, G.; Sulem, Gerald; Oskarsdottir, Arna; Benonisdottir, Stefania; Davidsson, Olafur B.; Masson, Gisli; Th Magnusson, Olafur; Holm, Hilma; Sigurdardottir, Olof; Jonsdottir, Ingileif; Eyjolfsson, Gudmundur I.; Olafsson, Isleifur; Gudbjartsson, Daniel F.; Thorsteinsdottir, Unnur; Sulem, Patrick; Stefansson, Kari

    2016-01-01

    Creatine kinase (CK) and lactate dehydrogenase (LDH) are widely used markers of tissue damage. To search for sequence variants influencing serum levels of CK and LDH, 28.3 million sequence variants identified through whole-genome sequencing of 2,636 Icelanders were imputed into 63,159 and 98,585 people with CK and LDH measurements, respectively. Here we describe 13 variants associating with serum CK and 16 with LDH levels, including four that associate with both. Among those, 15 are non-synonymous variants and 12 have a minor allele frequency below 5%. We report sequence variants in genes encoding the enzymes being measured (CKM and LDHA), as well as in genes linked to muscular (ANO5) and immune/inflammatory function (CD163/CD163L1, CSF1, CFH, HLA-DQB1, LILRB5, NINJ1 and STAB1). A number of the genes are linked to the mononuclear/phagocyte system and clearance of enzymes from the serum. This highlights the variety in the sources of normal diversity in serum levels of enzymes. PMID:26838040

  20. Lactate Dehydrogenase Is an Important Prognostic Indicator for Hepatocellular Carcinoma after Partial Hepatectomy12

    PubMed Central

    Zhang, Jing-Ping; Wang, Hong-Bo; Lin, Yue-Hao; Xu, Jing; Wang, Jun; Wang, Kai; Liu, Wan-Li

    2015-01-01

    Preoperative serum lactate dehydrogenase (LDH) has been used as a prognostic indicator for patients with hepatocellular carcinoma (HCC) treated with sorafenib or undergoing transcatheter arterial chemoembolization, but its significance in predicting survival of HCC patients who received curative resection remains undefined. A total of 683 patients with histopathologically confirmed HCC were enrolled in this study. The prognostic significance of preoperative serum LDH was determined by Kaplan-Meier analysis and a Cox proportional hazards regression model. The association between the preoperative serum LDH and clinicopathological parameters was evaluated by the χ2 test or linear regression analysis when appropriate. Higher preoperative serum LDH level was associated with worse prognosis. In a multivariate Cox proportional hazards analysis, the preoperative serum LDH level could predict overall survival and recurrence independently. Higher preoperative serum LDH level is associated with the elevated serum alpha-fetoprotein, the presence of hepatitis B surface antigen, larger tumor size, the presence of macrovascular invasion, the advanced tumor–lymph node–metastasis stage, worse tumor differentiation, and Child-Pugh B. Preoperative serum LDH level was an inexpensive, simple, convenient, and routinely measured biomarker exhibiting a potential to select patients at high risk with poor clinical outcome for appropriate treatment strategies. PMID:26692531

  1. Using molecular beacons to probe molecular interactions between lactate dehydrogenase and single-stranded DNA.

    PubMed

    Fang, X; Li, J J; Tan, W

    2000-07-15

    The interactions between two key macromolecular species, nucleic acids and proteins, control many important biological processes. There have been limited effective methodologies to study these interactions in real time. In this work, we have applied a newly developed molecular beacon (MB) DNA probe for the analysis of an enzyme, lactate dehydrogenase (LDH), and for the investigation of its properties of binding with single-stranded DNA. Molecular beacons are single-stranded oligonucleotide probes designed to report the presence of specific complementary nucleic acids by fluorescence detection. The interaction between LDH and MB has resulted in a significant fluorescence signal enhancement, which is used for the elucidation of MB/LDH binding properties. The processes of binding between MB and different isoenzymes of LDH have been studied. The results show that the stoichiometry of LDH-5/MB binding is 1:1, and the binding constant is 1.9 x 10(-7) M(-1). We have also studied salt effects, binding sites, temperature effects, pH effects, and the binding specificities for different isoenzymes. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has a signal transduction mechanism built within the molecule and can thus be used for the development of rapid protein assays and for real-time measurements.

  2. Glycoconjugates Influence Caspase Release and Minimize Production of Lactate Dehydrogenase upon Pathogen Exposure

    NASA Astrophysics Data System (ADS)

    Eassa, Souzan; Tarasenko, Olga

    2010-04-01

    Many pathogens stimulate cell death of immune cells while promoting survival of pathogens. Early cell death is characterized by the release of mediators, namely Caspases (Cas). Infections caused by pathogens can be eradicated if immune cells could resist cell death and kill pathogens upon exposure. In this research, we studied whether glycoconjugates (GCs) influence Cas release and cytotoxicity upon pathogen damage. GC1 and GC3 constituted samples studied principally. Bacterial spores were used as a pathogen model. GC effects were determined "prior to," "during," and "following" pathogen exposure throughout phagocytosis. Cytotoxic damage was assessed by measuring lactate dehydrogenase (LDH) production. Our data show that GC3 was more effective than GC1 during phagocytosis. GC3 controls Cas release under all three exposure conditions. Minimum production of LDH was noticed in the "following" exposure condition compared to the "prior to" and "during" exposure conditions for GC1 and GC3. The present study provided the selection method of GC ligands bearing anti-cytotoxic and anti-apoptotic properties.

  3. Effects and Mechanism of Atmospheric-Pressure Dielectric Barrier Discharge Cold Plasma on Lactate Dehydrogenase (LDH) Enzyme

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Xu, Zimu; Shen, Jie; Li, Xu; Ding, Lili; Ma, Jie; Lan, Yan; Xia, Weidong; Cheng, Cheng; Sun, Qiang; Zhang, Zelong; Chu, Paul K.

    2015-05-01

    Proteins are carriers of biological functions and the effects of atmospheric-pressure non-thermal plasmas on proteins are important to applications such as sterilization and plasma-induced apoptosis of cancer cells. Herein, we report our detailed investigation of the effects of helium-oxygen non-thermal dielectric barrier discharge (DBD) plasmas on the inactivation of lactate dehydrogenase (LDH) enzyme solutions. Circular dichroism (CD) and dynamic light scattering (DLS) indicate that the loss of activity stems from plasma-induced modification of the secondary molecular structure as well as polymerization of the peptide chains. Raising the treatment intensity leads to a reduced alpha-helix content, increase in the percentage of the beta-sheet regions and random sequence, as well as gradually decreasing LDH activity. However, the structure of the LDH plasma-treated for 300 seconds exhibits a recovery trend after storage for 24 h and its activity also increases slightly. By comparing direct and indirect plasma treatments, plasma-induced LDH inactivation can be attributed to reactive species (RS) in the plasma, especially ones with a long lifetime including hydrogen peroxide, ozone, and nitrate ion which play the major role in the alteration of the macromolecular structure and molecular diameter in lieu of heat, UV radiation, and charged particles.

  4. Effects and Mechanism of Atmospheric-Pressure Dielectric Barrier Discharge Cold Plasma on Lactate Dehydrogenase (LDH) Enzyme

    PubMed Central

    Zhang, Hao; Xu, Zimu; Shen, Jie; Li, Xu; Ding, Lili; Ma, Jie; Lan, Yan; Xia, Weidong; Cheng, Cheng; Sun, Qiang; Zhang, Zelong; Chu, Paul K.

    2015-01-01

    Proteins are carriers of biological functions and the effects of atmospheric-pressure non-thermal plasmas on proteins are important to applications such as sterilization and plasma-induced apoptosis of cancer cells. Herein, we report our detailed investigation of the effects of helium-oxygen non-thermal dielectric barrier discharge (DBD) plasmas on the inactivation of lactate dehydrogenase (LDH) enzyme solutions. Circular dichroism (CD) and dynamic light scattering (DLS) indicate that the loss of activity stems from plasma-induced modification of the secondary molecular structure as well as polymerization of the peptide chains. Raising the treatment intensity leads to a reduced alpha-helix content, increase in the percentage of the beta-sheet regions and random sequence, as well as gradually decreasing LDH activity. However, the structure of the LDH plasma-treated for 300 seconds exhibits a recovery trend after storage for 24 h and its activity also increases slightly. By comparing direct and indirect plasma treatments, plasma-induced LDH inactivation can be attributed to reactive species (RS) in the plasma, especially ones with a long lifetime including hydrogen peroxide, ozone, and nitrate ion which play the major role in the alteration of the macromolecular structure and molecular diameter in lieu of heat, UV radiation, and charged particles. PMID:25992482

  5. Effects and Mechanism of Atmospheric-Pressure Dielectric Barrier Discharge Cold Plasma on Lactate Dehydrogenase (LDH) Enzyme.

    PubMed

    Zhang, Hao; Xu, Zimu; Shen, Jie; Li, Xu; Ding, Lili; Ma, Jie; Lan, Yan; Xia, Weidong; Cheng, Cheng; Sun, Qiang; Zhang, Zelong; Chu, Paul K

    2015-01-01

    Proteins are carriers of biological functions and the effects of atmospheric-pressure non-thermal plasmas on proteins are important to applications such as sterilization and plasma-induced apoptosis of cancer cells. Herein, we report our detailed investigation of the effects of helium-oxygen non-thermal dielectric barrier discharge (DBD) plasmas on the inactivation of lactate dehydrogenase (LDH) enzyme solutions. Circular dichroism (CD) and dynamic light scattering (DLS) indicate that the loss of activity stems from plasma-induced modification of the secondary molecular structure as well as polymerization of the peptide chains. Raising the treatment intensity leads to a reduced alpha-helix content, increase in the percentage of the beta-sheet regions and random sequence, as well as gradually decreasing LDH activity. However, the structure of the LDH plasma-treated for 300 seconds exhibits a recovery trend after storage for 24 h and its activity also increases slightly. By comparing direct and indirect plasma treatments, plasma-induced LDH inactivation can be attributed to reactive species (RS) in the plasma, especially ones with a long lifetime including hydrogen peroxide, ozone, and nitrate ion which play the major role in the alteration of the macromolecular structure and molecular diameter in lieu of heat, UV radiation, and charged particles.

  6. Isocitrate dehydrogenases and oxoglutarate dehydrogenase activities of baker's yeast grown in a variety of hypoxic conditions.

    PubMed

    Machado, A; Nuñez de Castro, I; Mayor, F

    1975-02-28

    The activities of isocitrate dehydrogenase (NAD), isocitrate dehydrogenase (NADP) and oxoglutarate dehydrogenase have been investigated in Saccharomyces cerevisiae grown in a variety of aerobic and hypoxic conditions, the latter including oxygen deprivation, high glucose concentration, addition of inhibitors of mitochondrial protein synthesis, respiratory inhibition by azide, and impaired respiration mutants. All hypoxic conditions led to a marked decrease of oxoglutarate dehydrogenase and significant decreases of the two isocitrate dehydrogenases. According to its kinetic properties, the NAD-isocitrate dehydrogenase will not be operative in hypoxia "in vivo". From these and other related facts it is concluded that hypoxic conditions in yeast generally lead to a splitting of the tricarboxylic acid cycle and that glutamate synthesis in these conditions takes place through the coupling of the NADP-linked isocitrate and glutamate dehydrogenases.

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2016-06-01

    Chiral α-hydroxy acids (AHAs) are rapidly becoming important synthetic building blocks, in particular for the production of pharmaceuticals and other fine chemicals. Chiral compounds of a variety of functionalities are now often derived using enzymes, and L-lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (bsLDH) has the potential to be employed for the industrial synthesis of chiral α-hydroxy acids. Despite the thorough characterization of this enzyme, generation of variants with high activity on non-natural substrates has remained difficult and therefore limits the use of bsLDH in industry. Here, we present the engineering of bsLDH using semi-rational design as a method of focusing screening in a small and smart library for novel biocatalysts. In this study, six mutant libraries were designed in an effort to expand the substrate range of bsLDH. The eight variants identified as having enhanced activity toward the selected α-keto acids belonged to the same library, which targeted two positions simultaneously. These new variants now may be useful biocatalysts for chiral synthesis of α-hydroxy acids. PMID:26852025

  9. Amperometric lactate biosensor for flow injection analysis based on a screen-printed carbon electrode containing Meldola's Blue-Reinecke salt, coated with lactate dehydrogenase and NAD+.

    PubMed

    Piano, M; Serban, S; Pittson, R; Drago, G A; Hart, J P

    2010-06-30

    A biosensor for the measurement of lactate in serum has been developed, which is based on a screen-printed carbon electrode, modified with Meldola's Blue-Reinecke Salt (MBRS-SPCE), coated with the enzyme lactate dehydrogenase NAD(+) dependent (from Porcine heart), and NAD(+). A cellulose acetate layer was deposited on the top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V vs. Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer pH 10 containing 0.1 M potassium chloride solution; a flow rate of 0.8 ml min(-1) was used throughout the investigation. The biosensor response was linear over the range 0.55-10 mM lactate; the former represents the detection limit. The precision of the system was determined by carrying out 10 repeat injections of 10 mM l(+)lactic acid standard; the calculated coefficient of variation was 4.28%. It was demonstrated that this biosensor system could be applied to the direct measurement of lactate in serum without pre-treatment; therefore, this would allow high throughput-analysis, at low cost, for this clinically important analyte. PMID:20685431

  10. Synbiotics suppress the release of lactate dehydrogenase, promote non-specific immunity and integrity of jejunum mucosa in piglets.

    PubMed

    Andrejčáková, Zuzana; Sopková, Drahomíra; Vlčková, Radoslava; Kulichová, Lucia; Gancarčíková, Soňa; Almášiová, Viera; Holovská, Katarína; Petrilla, Vladimír; Krešáková, Lenka

    2016-09-01

    The aim of our experiment was to study how synbiotics are able to deal with the problems of post-weaning piglets. Lactobacillus plantarum - Biocenol(TM) LP96 (CCM 7512), Lactobacillus fermentum - Biocenol(TM) LF99 (CCM 7514) and flaxseed (rich in n-3 polyunsaturated fatty acids) were administered to 36 conventional piglets from a problematic breed with confirmed presence of enterotoxigenic Escherichia coli and Coronavirus. The experimental piglets were supplied with probiotic cheeses and crushed flax-seed in the period starting 10 days before weaning and lasting up to 14 days post-weaning. Piglets in the control group were supplied only control cheese. The impact of such additives on the release of lactate dehydrogenase (LDH; spectroscopic and electrophoretic assay), alteration of immunity (index of metabolic activity), jejunum histology (light microscopy), and health of conventional piglets from a problematic breed (monitoring of hematology, consistency and moisture of feces and body temperature) were examined. We found significant decrease in LDH leakage in the blood serum and tissue extracts, indicating better cell membrane integrity in the individual organs of animals. Probiotics and flaxseed applied together seem to be a good source of nutrients to improve the immune status and the integrity of jejunum mucosa during infection. © 2015 Japanese Society of Animal Science. PMID:27581561

  11. Cryptosporidium Lactate Dehydrogenase Is Associated with the Parasitophorous Vacuole Membrane and Is a Potential Target for Developing Therapeutics

    PubMed Central

    Zhu, Guan

    2015-01-01

    Abstract The apicomplexan, Cryptosporidium parvum, possesses a bacterial-type lactate dehydrogenase (CpLDH). This is considered to be an essential enzyme, as this parasite lacks the Krebs cycle and cytochrome-based respiration, and mainly–if not solely, relies on glycolysis to produce ATP. Here, we provide evidence that in extracellular parasites (e.g., sporozoites and merozoites), CpLDH is localized in the cytosol. However, it becomes associated with the parasitophorous vacuole membrane (PVM) during the intracellular developmental stages, suggesting involvement of the PVM in parasite energy metabolism. We characterized the biochemical features of CpLDH and observed that, at lower micromolar levels, the LDH inhibitors gossypol and FX11 could inhibit both CpLDH activity (Ki = 14.8 μM and 55.6 μM, respectively), as well as parasite growth in vitro (IC50 = 11.8 μM and 39.5 μM, respectively). These observations not only reveal a new function for the poorly understood PVM structure in hosting the intracellular development of C. parvum, but also suggest LDH as a potential target for developing therapeutics against this opportunistic pathogen, for which fully effective treatments are not yet available. PMID:26562790

  12. Cryptosporidium Lactate Dehydrogenase Is Associated with the Parasitophorous Vacuole Membrane and Is a Potential Target for Developing Therapeutics.

    PubMed

    Zhang, Haili; Guo, Fengguang; Zhu, Guan

    2015-01-01

    The apicomplexan, Cryptosporidium parvum, possesses a bacterial-type lactate dehydrogenase (CpLDH). This is considered to be an essential enzyme, as this parasite lacks the Krebs cycle and cytochrome-based respiration, and mainly-if not solely, relies on glycolysis to produce ATP. Here, we provide evidence that in extracellular parasites (e.g., sporozoites and merozoites), CpLDH is localized in the cytosol. However, it becomes associated with the parasitophorous vacuole membrane (PVM) during the intracellular developmental stages, suggesting involvement of the PVM in parasite energy metabolism. We characterized the biochemical features of CpLDH and observed that, at lower micromolar levels, the LDH inhibitors gossypol and FX11 could inhibit both CpLDH activity (Ki = 14.8 μM and 55.6 μM, respectively), as well as parasite growth in vitro (IC50 = 11.8 μM and 39.5 μM, respectively). These observations not only reveal a new function for the poorly understood PVM structure in hosting the intracellular development of C. parvum, but also suggest LDH as a potential target for developing therapeutics against this opportunistic pathogen, for which fully effective treatments are not yet available. PMID:26562790

  13. Plasmodium falciparum and Plasmodium vivax specific lactate dehydrogenase: genetic polymorphism study from Indian isolates.

    PubMed

    Keluskar, Priyadarshan; Singh, Vineeta; Gupta, Purva; Ingle, Sanjay

    2014-08-01

    Control and eradication of malaria is hindered by the acquisition of drug resistance by Plasmodium species. This has necessitated a persistent search for novel drugs and more efficient targets. Plasmodium species specific lactate dehydrogenase is one of the potential therapeutic and diagnostic targets, because of its indispensable role in endoerythrocytic stage of the parasite. A target molecule that is highly conserved in the parasite population can be more effectively used in diagnostics and therapeutics, hence, in the present study polymorphism in PfLDH (Plasmodiumfalciparum specific LDH) and PvLDH (Plasmodiumvivax specific LDH) genes was analyzed using PCR-single strand confirmation polymorphism (PCR-SSCP) and sequencing. Forty-six P. falciparum and thirty-five P. vivax samples were screened from different states of India. Our findings have revealed presence of a single PfLDH genotype and six PvLDH genotypes among the studied samples. Interestingly, along with synonymous substitutions, nonsynonymous substitutions were reported to be present for the first time in the PvLDH genotypes. Further, through amino acid sequence alignment and homology modeling studies we observed that the catalytic residues were conserved in all PvLDH genotypes and the nonsynonymous substitutions have not altered the enzyme structure significantly. Evolutionary genetics studies have confirmed that PfLDH and PvLDH loci are under strong purifying selection. Phylogenetic analysis of the pLDH gene sequences revealed that P. falciparum compared to P. vivax, has recent origin. The study therefore supports PfLDH and PvLDH as suitable therapeutic and diagnostic targets as well as phylogenetic markers to understand the genealogy of malaria species.

  14. Changes in antibody specificities and cytokine release after infection with lactate dehydrogenase-elevating virus.

    PubMed

    Aparicio, José L; Saxena, Anubha; Coutelier, Jean-Paul; Van Snick, Jacques; Retegui, Lilia A

    2013-03-01

    Lactate dehydrogenase-elevating virus (LDV) is an apparently innocuous and persistent virus that can modify mouse immune reactions. We have shown that LDV-infected mice immunized with human growth hormone (hGH) showed a deep modification of the specificity of the anti-hGH antibodies (Ab) in CBA/Ht mice but not BALB/c animals. The aim of this work was to extend the previous observations to another mouse strain, C57BL/6, as well as to an antigen unrelated to hGH, ovalbumin (OVA), and to explore at the same time the production of various cytokines at serum and cellular levels. The amount of Ab directed to hGH or OVA native antigenic determinants versus the concentration of Ab to cryptic epitopes was evaluated by ELISA competition experiments. Results indicated that LDV infection affected Ab specificity solely in CBA/Ht mice. In CBA/Ht the virus infection was associated with a reduction of the Ab titers to hGH native epitopes and with a decrease of IL-13 and IL-17 serum levels, but Ab to native OVA epitopes were increased with a simultaneous increase of IL-17. Accordingly, only lymph node cells from infected CBA/Ht mice immunized with OVA were found to produce INF-γ, IL-13 and IL-17. Thus, a correlation of cytokine production with a change in Ab specificity after a viral infection was found, although this phenomenon was restricted to a given antigen and to the genetic background of immunized animals. These observations suggest that an apparent harmless virus can affect some immunological mechanisms, which could lead, for example, to inflammatory or autoimmune disorders.

  15. Preliminary Study on Serum Lactate Dehydrogenase (LDH)-Prognostic Biomarker in Carcinoma Breast

    PubMed Central

    Gandhe, Mahendra Bhauraoji; Gupta, Dilip; Reddy, M.V.R.

    2016-01-01

    Introduction Serum Lactate Dehydrogenase (LDH) is one of the biochemical markers for breast cancer. Serum LDH is enzyme required for anaerobic glycolysis. One of its isoenzyme is increased in breast cancer due to up-regulation in its gene. It leads to increase in serum LDH level in breast cancer patients. Serum LDH is economical, easily available and easy to estimate. Aim In the present study, we evaluated the LDH levels in circulation of newly diagnosed patients of breast cancer and tried to correlate it with different TNM staging of carcinoma breast before interventions and after adjuvant therapy of these patients. Materials and Methods This prospective study was done on 83 diagnosed patients of breast cancer was conducted among poor patients in rural area. This study was conducted in the Department of Surgery between October 2008 to October 2010, at MGIMS, Sevagram, Maharashtra, a rural medical college located in Central India. Out of total 83 participants, 10 participants were having adverse events following surgery and remaining 73 participants were without adverse events following surgery. The significant difference in serum LDH levels between two groups, with and without adverse surgical outcome was calculated by Mann-Whitney U test. Results Patients with higher clinical TNM staging were having higher serum LDH levels. The serum LDH levels at sixth months following surgery showed a trend of statistically significant difference between patients with and without adverse events. As increased serum LDH levels in breast cancer patients shows poor prognosis, surgical outcome or advanced metastases. Conclusion Serum LDH monitoring can be used as a prognostic biomarker in patients of breast cancer. For confirmation of this finding, we require further more studies on larger sample size and long-term follow-up in patients specifically with higher serum LDH levels. PMID:27134855

  16. Histopathological differentiation of oral squamous cell carcinoma and salivary lactate dehydrogenase: A biochemical study

    PubMed Central

    D’Cruz, Audrey M.; Pathiyil, Varsha

    2015-01-01

    Context: Early diagnosis of oral cancer is a priority health objective, in which oral health professionals may play a pivotal role. Detection should lead to less damage from cancer therapy and to a better prognosis. Aims: The aim was to estimate and compare the salivary lactate dehydrogenase (LDH) levels in various histological differentiation of oral squamous cell carcinoma (OSCC) patients and normal subjects. Settings and Design: Hospital-based setting, case-control study. Subjects and Methods: A case–control study was undertaken comprising 30 OSCC patients and 30 healthy controls. The OSCC patients were grouped into well-differentiated, moderately differentiated and poorly differentiated OSCC based on their histological tumor differentiation. Unstimulated whole saliva was collected, assayed for LDH levels using a standard kit and measured spectrophotometrically at 340 nm. Statistical Analysis Used: The results obtained were subjected to statistical analysis using Kruskal–Wallis and Mann–Whitney U-tests. Spearman's correlation was used to correlate the histological tumor differentiation with the salivary LDH levels. Results: The mean salivary LDH levels in the control group, well-differentiated OSCC group, moderately differentiated OSCC group, and poorly differentiated OSCC group were 117.33 ± 19.37 IU/L, 355.83 ± 16.73 IU/L, 484.18 ± 25.84 IU/L, and 620.35 ± 18.69 IU/L, respectively. The difference in the mean salivary LDH levels was statistically significant among the various groups (Kruskal–Wallis χ2 = 50.820, P < 0.001). Spearman's correlation showed significant difference between salivary LDH levels and histological differentiation of OSCC (r = −0.689, P < 0.01). Conclusions: The salivary LDH levels were higher in OSCC patients when compared to the healthy controls. The salivary LDH levels were found to be the highest among the poorly differentiated OSCC. PMID:25992341

  17. Plasmodium falciparum and Plasmodium vivax specific lactate dehydrogenase: genetic polymorphism study from Indian isolates.

    PubMed

    Keluskar, Priyadarshan; Singh, Vineeta; Gupta, Purva; Ingle, Sanjay

    2014-08-01

    Control and eradication of malaria is hindered by the acquisition of drug resistance by Plasmodium species. This has necessitated a persistent search for novel drugs and more efficient targets. Plasmodium species specific lactate dehydrogenase is one of the potential therapeutic and diagnostic targets, because of its indispensable role in endoerythrocytic stage of the parasite. A target molecule that is highly conserved in the parasite population can be more effectively used in diagnostics and therapeutics, hence, in the present study polymorphism in PfLDH (Plasmodiumfalciparum specific LDH) and PvLDH (Plasmodiumvivax specific LDH) genes was analyzed using PCR-single strand confirmation polymorphism (PCR-SSCP) and sequencing. Forty-six P. falciparum and thirty-five P. vivax samples were screened from different states of India. Our findings have revealed presence of a single PfLDH genotype and six PvLDH genotypes among the studied samples. Interestingly, along with synonymous substitutions, nonsynonymous substitutions were reported to be present for the first time in the PvLDH genotypes. Further, through amino acid sequence alignment and homology modeling studies we observed that the catalytic residues were conserved in all PvLDH genotypes and the nonsynonymous substitutions have not altered the enzyme structure significantly. Evolutionary genetics studies have confirmed that PfLDH and PvLDH loci are under strong purifying selection. Phylogenetic analysis of the pLDH gene sequences revealed that P. falciparum compared to P. vivax, has recent origin. The study therefore supports PfLDH and PvLDH as suitable therapeutic and diagnostic targets as well as phylogenetic markers to understand the genealogy of malaria species. PMID:24953504

  18. An alternative allosteric regulation mechanism of an acidophilic l-lactate dehydrogenase from Enterococcus mundtii 15-1A.

    PubMed

    Matoba, Yasuyuki; Miyasako, Masashi; Matsuo, Koichi; Oda, Kosuke; Noda, Masafumi; Higashikawa, Fumiko; Kumagai, Takanori; Sugiyama, Masanori

    2014-01-01

    A plant-derived Enterococcus mundtii 15-1A, that has been previously isolated from Brassica rapa L. subsp. nipposinica (L.H. Bailey) Hanelt var. linearifolia by our group, possesses two kinds of l-lactate dehydrogenase (l-LDH): LDH-1 and LDH-2. LDH-1 was activated under low concentration of fluctose-1,6-bisphosphate (FBP) at both pH 5.5 and 7.5. Although LDH-2 was also activated under the low concentration of FBP at pH 5.5, a high concentration of FBP is necessary to activate it at pH 7.5. The present study shows the crystal structures of the acidophilic LDH-2 in a complex with and without FBP and NADH. Although the tertiary structure of the ligands-bound LDH-2 is similar to that of the active form of other bacterial l-LDHs, the structure without the ligands is different from that of any other previously determined l-LDHs. Major structural alterations between the two structures of LDH-2 were observed at two regions in one subunit. At the N-terminal parts of the two regions, the ligands-bound form takes an α-helical structure, while the form without ligands displays more disordered and extended structures. A vacuum-ultraviolet circular dichroism analysis showed that the α-helix content of LDH-2 in solution is approximately 30% at pH 7.5, which is close to that in the crystal structure of the form without ligands. A D241N mutant of LDH-2, which was created by us to easily form an α-helix at one of the two parts, exhibited catalytic activity even in the absence of FBP at both pH 5.5 and 7.5.

  19. [Characteristics of the lactate dehydrogenase isoenzyme spectrum of the skin in the presence of thermal lesions].

    PubMed

    Nosova, I M; Zaets, T L; Kotkina, T I

    1977-09-01

    Experiments were conducted on rats; a study was made of the activity of lactic dehydrogenase (LDH) and its isoenzymes in the zone of affection and in the adjacent areas of the skin at various periods after the burn infliction; on the 1st--8th day there occurred a reduction of the sum total LDH activity in the scab zone and the underlying tissue by 70--80%, and in the margin and the intact skin--by 50%. These changes were accompanied by shifts in the isoenzymatic LDH spectrum in the affected tissue; the activity of fraction 5 displayed a sharp rise on the 1st day and that of fractions 2 and 3--a reduction, on the contrary, by the 8th day there was seen some diminution of fraction 5 activity and an elevation of fractions 2 and 3 activity. The following picture is observed on the 14th--22nd day after the burn; the sum total LDH activity remains low, the isoenzymatic LDH spectrum in the margin and the scab is largely normalized, whereas in the underlying tissue there persist changes in the LDH enzyme ratio (a reduction of fractions 1--3 activity, and a rise of fraction 5 activity). PMID:912082

  20. Recent Update on Human Lactate Dehydrogenase Enzyme 5 (hLDH5) Inhibitors: A Promising Approach for Cancer Chemotherapy.

    PubMed

    Rani, Reshma; Kumar, Vinit

    2016-01-28

    Human lactate dehydrogenase (hLDH5), a glycolytic enzyme responsible for the conversion of pyruvate to lactate coupled with oxidation of NADH to NAD(+), plays a crucial role in the promotion of glycolysis in invasive tumor cells. Recently, hLDH5 has been considered a vital therapeutic target for invasive cancers. Selective inhibition of hLDH5 using small molecules holds potential prospects for the treatment of cancer and associated diseases. Consequently, significant progress has been made in the discovery of selective small-molecule hLDH5 inhibitors displaying remarkable inhibitory potencies. The purpose of this review is to discuss briefly the roles of hLDH isoforms and to compile small hLDH5 inhibitors into groups based on their chemical classes and pharmacological applications.

  1. Impact of probiotic-supplemented diet on the expression level of lactate dehydrogenase in the leukocytes of rabbits.

    PubMed

    Ghoneim, Magdy A E; Moselhy, Said S

    2014-04-01

    Probiotics are known as living, nonpathogenic microorganisms that colonize the intestine and provide benefit to the host. The present study aims to measure one important energy metabolism-related enzyme activity in blood of rabbits fed on probiotics of recommended concentration. In addition, it also aims for the evaluation of the expression level of lactate dehydrogenase (LDH) enzyme using reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Two groups of rabbits are used: control group receiving normal standardized diet and the other probiotic-supplemented group receiving the same diet containing probiotic, namely, Mega acidophilus (200 million cfu/kg body weight/day) for 4 weeks. The obtained results revealed that the rabbits supplemented with probiotics showed a significant decrease in the levels of serum total cholesterol (TC), triacylglycerol, high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) when compared with control group. Risk factors detected by measuring TC/HDL-c and LDL-c/HDL-c ratios showed statistically significant decrease in probiotic-supplemented rabbits when compared with control group. In addition, blood glucose and total LDH activity were elevated in probiotic-supplemented rabbits when compared with control group. RT-PCR products of LDH-M gene produced two specific amplicons. One amplicon has the expected size of 243 bp from all samples of rabbits as revealed by GelPro software. The level of LDH-M expression was found to be increased in the probiotic-supplemented group. However, unexpected amplicons are produced at 586 bp in all the samples, which may be a dimeric form of the amplified region. It was concluded that this probiotic blend is beneficiary for the metabolic reactions of lipids in the body. Moreover, LDH expression level can be considered as a biomarker for the effect of probiotic and hence monitoring the metabolic changes as reflected from its administration.

  2. Effect of neem limonoids on lactate dehydrogenase (LDH) of the rice leaffolder, Cnaphalocrocis medinalis (Guenée) (Insecta: Lepidoptera: Pyralidae).

    PubMed

    Senthil Nathan, Sengottayan; Kalaivani, Kandaswamy; Chung, Paul Gene; Murugan, Kadarkarai

    2006-03-01

    Neem is derived from the neem tree Azadirachta indica A. Juss. (Meliaceae), and its primary insecticidal component is the tetranortriterpenoid azadirachtin and other limonoids. The effect of neem limonoids azadirachtin, salannin, deacetylgedunin, gedunin, 17-hydroxyazadiradione and deacetylnimbin on enzyme lactate dehydrogenase (LDH) activity of the rice leaffolder (RLF) Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) larvae was investigated. There was a decrease in enzyme activity relative to the control at all concentrations tested. When fed a diet of rice leaves treated with neem limonoids in bioassays, gut tissue enzyme, LDH levels in rice leaffolder larvae are affected. These results indicate neem limonoids affect LDH activity. These effects are most pronounced in early instar larvae. Azadirachtin was the most potent in of all the limonoids in all experiments indicating strong enzyme inhibition. Clear dose-response relationships were established with respect to LDH activity. PMID:16154614

  3. Direct electrochemistry of lactate dehydrogenase immobilized on silica sol-gel modified gold electrode and its application.

    PubMed

    Di, Junwei; Cheng, Jiongjia; Xu, Quan; Zheng, Huie; Zhuang, Jingyue; Sun, Yongbo; Wang, Keyu; Mo, Xiangyin; Bi, Shuping

    2007-12-15

    The direct electrochemistry of lactate dehydrogenase (LDH) immobilized in silica sol-gel film on gold electrode was investigated, and an obvious cathodic peak at about -200 mV (versus SCE) was found for the first time. The LDH-modified electrode showed a surface controlled irreversible electrode process involving a one electron transfer reaction with the charge-transfer coefficient (alpha) of 0.79 and the apparent heterogeneous electron transfer rate constant (K(s)) of 3.2 s(-1). The activated voltammetric response and decreased charge-transfer resistance of Ru(NH(3))(6)(2+/3+) on the LDH-modified electrode provided further evidence. The surface morphologies of silica sol-gel and the LDH embedded in silica sol-gel film were characterized by SEM. A potential application of the LDH-modified electrode as a biosensor for determination of lactic acid was also investigated. The calibration range of lactic acid was from 2.0 x 10(-6) to 3.0 x 10(-5) mol L(-1) and the detection limit was 8.0 x 10(-7) mol L(-1) at a signal-to-noise ratio of 3. Finally, the effect of environmental pollutant resorcinol on the direct electrochemical behavior of LDH was studied. The experimental results of voltammetry indicated that the conformation of LDH molecule was altered by the interaction between LDH and resorcinol. The modified electrode can be applied as a biomarker to study the pollution effect in the environment.

  4. Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography.

    PubMed Central

    Brinkworth, R I; Masters, C J; Winzor, D J

    1975-01-01

    Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 x 10 (4)M-1 for the interaction of enzyme with NADH at 5 degrees C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 x 10(5)M-1, 3 x 10(5)M-1, 4 x 10(5)M-1, 7 x 10(5)M-1 and 2 x 10(6)M-1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase. PMID:175784

  5. Purification of the fructose 1,6-bisphosphate-dependent lactate dehydrogenase from Streptococcus uberis and an investigation of its existence in different forms.

    PubMed Central

    Williams, R A; Andrews, P

    1986-01-01

    The fructose 1,6-bisphosphate [Fru(1,6)P2]-dependent lactate dehydrogenase in cells of Streptococcus uberis N.C.D.O. 2039 was purified by a procedure that included chromatography on DEAE-cellulose and Blue Sepharose CL-6B in phosphate buffers. The enzyme appeared to interact with Blue Sepharose through NADH-binding sites. The homogeneous enzyme had catalytic properties that were generally similar to those of other Fru(1,6)P2-dependent lactate dehydrogenases, and it had no catalytic activity in the absence of Fru(1,6)P2. Its existence in different forms, depending on conditions, was investigated by ultracentrifugation, analytical gel filtration and activity measurements. It consisted of subunits with Mr 35,900 +/- 500 and, in the presence of adequate concentrations of Fru(1,6)P2, phosphate or NADH, it existed as a tetramer, whereas when these ligands were in lower concentrations or absent, the subunits were in a concentration-dependent association-dissociation equilibrium. Dissociation occurred slowly and inactivated the enzyme, and although added ligands reversed the dissociation, the lost activity was at best only partly restored. An exception occurred when dissociation was caused by a decrease in temperature, in which case the lost activity was fully restored at the original temperature. The tetramer also lost activity at certain ligand concentrations without dissociating. The results together indicated the presence on the enzyme of two classes of binding site for both Fru(1,6)P2 and NADH, and the likelihood that phosphate bound at the same sites as Fru(1,6)P2. Two different ligands together were much more effective at preventing inactivation and dissociation than was expected from their effectiveness when present separately. It was concluded that tetrameric forms of the enzyme rather than the enzyme in association-dissociation equilibrium were involved in the regulation of its activity in vivo. PMID:3790089

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

    PubMed

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

    2014-01-01

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

  7. Disruption of lactate dehydrogenase and alcohol dehydrogenase for increased hydrogen production and its effect on metabolic flux in Enterobacter aerogenes.

    PubMed

    Zhao, Hongxin; Lu, Yuan; Wang, Liyan; Zhang, Chong; Yang, Cheng; Xing, Xinhui

    2015-10-01

    Hydrogen production by Enterobacter aerogenes from glucose was enhanced by deleting the targeted ldhA and adh genes responsible for two NADH-consuming pathways which consume most NADH generated from glycolysis. Compared with the wild-type, the hydrogen yield of IAM1183-ΔldhA increased 1.5 fold. Metabolic flux analysis showed both IAM1183-ΔldhA and IAM1183-Δadh exhibited significant changes in flux, including enhanced flux towards the hydrogen generation. The lactate production of IAM1183-ΔldhA significantly decreased by 91.42%, while the alcohol yield of IAM1183-Δadh decreased to 30%. The mutant IAM1183-ΔldhA with better hydrogen-producing performance was selected for further investigation in a 5-L fermentor. The hydrogen production of IAM1183-ΔldhA was 2.3 times higher than the wild-type. Further results from the fermentation process showed that the pH decreased to 5.39 levels, then gradually increased to 5.96, indicating that some acidic metabolites might be degraded or uptaken by cells.

  8. Biochemical and in silico Characterization of Recombinant L-Lactate Dehydrogenase of Theileria annulata.

    PubMed

    Nural, Belma; Erdemir, Aysegul; Mutlu, Ozal; Yakarsonmez, Sinem; Danis, Ozkan; Topuzogullari, Murat; Turgut-Balik, Dilek

    2016-04-01

    Theileria annulata is a parasite that causes theileriosis in cattle. Reports about drug resistance made essential to develop new drug. LDH of Theileria schizonts is the vital enzyme for its anaerobic metabolism. TaLDH gene was first cloned into pGEM-T cloning vector with two introns in our previous study. Here we report cloning of TaLDH without introns into pLATE 31 vector in E. coli BL21(DE3). Protein was in an inactive form. Two mutations were fixed to express the active protein. Protein was purified by affinity chromatography and evaluated by SDS-PAGE and size exclusion chromatography. Optimum pH of enzyme was performed in pH 7.5, and enzyme was stabilized at 20-40 °C. Enzyme kinetics of recombinant TaLDH were found to be in the direction of pyruvate to lactate K m 0.1324 and K i 4.295 mM, k cat, 44.55/s and k cat /K m, 3.3693 × 10(5)/M/s. 3D structure of TaLDH was predicted, and possible drug binding sites were determined by homology modelling. PMID:26921192

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

    PubMed Central

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

    2014-01-01

    Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production—NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)—were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6. PMID:25217009

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

    PubMed

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

    2014-12-01

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

  11. Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.

    PubMed

    Jungmann, Richard A; Kiryukhina, Olga

    2005-07-01

    Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA

  12. Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.

    PubMed

    Jungmann, Richard A; Kiryukhina, Olga

    2005-07-01

    Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA

  13. Cationic surfactant-based colorimetric detection of Plasmodium lactate dehydrogenase, a biomarker for malaria, using the specific DNA aptamer.

    PubMed

    Lee, Seonghwan; Manjunatha, D H; Jeon, Weejeong; Ban, Changill

    2014-01-01

    A simple, sensitive, and selective colorimetric biosensor for the detection of the malarial biomarkers Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum LDH (PfLDH) was demonstrated using the pL1 aptamer as the recognition element and gold nanoparticles (AuNPs) as probes. The proposed method is based on the aggregation of AuNPs using hexadecyltrimethylammonium bromide (CTAB). The AuNPs exhibited a sensitive color change from red to blue, which could be seen directly with the naked eye and was monitored using UV-visible absorption spectroscopy and transmission electron microscopy (TEM). The reaction conditions were optimized to obtain the maximum color intensity. PvLDH and PfLDH were discernible with a detection limit of 1.25 pM and 2.94 pM, respectively. The applicability of the proposed biosensor was also examined in commercially available human serum.

  14. Molecular Characterization of Two Lactate Dehydrogenase Genes with a Novel Structural Organization on the Genome of Lactobacillus sp. Strain MONT4

    PubMed Central

    Weekes, Jennifer; Yüksel, Gülhan Ü.

    2004-01-01

    Two lactate dehydrogenase (ldh) genes from Lactobacillus sp. strain MONT4 were cloned by complementation in Escherichia coli DC1368 (ldh pfl) and were sequenced. The sequence analysis revealed a novel genomic organization of the ldh genes. Subcloning of the individual ldh genes and their Northern blot analyses indicated that the genes are monocistronic. PMID:15466577

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

    SciTech Connect

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

    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

  16. Lactate dehydrogenase A negatively regulated by miRNAs promotes aerobic glycolysis and is increased in colorectal cancer.

    PubMed

    Wang, Jian; Wang, Hui; Liu, Aifen; Fang, Changge; Hao, Jianguo; Wang, Zhenghui

    2015-08-14

    Reprogramming metabolism of tumor cells is a hallmark of cancer. Lactate dehydrogenase A (LDHA) is frequently overexpressed in tumor cells. Previous studies has shown higher levels of LDHA is related with colorectal cancer (CRC), but its role in tumor maintenance and underlying molecular mechanisms has not been established. Here, we investigated miRNAs-induced changes in LDHA expression. We reported that colorectal cancer express higher levels of LDHA compared with adjacent normal tissue. Knockdown of LDHA resulted in decreased lactate and ATP production, and glucose uptake. Colorectal cancer cells with knockdown of LDHA had much slower growth rate than control cells. Furthermore, we found that miR-34a, miR-34c, miR-369-3p, miR-374a, and miR-4524a/b target LDHA and regulate glycolysis in cancer cells. There is a negative correlation between these miRNAs and LDHA expression in colorectal cancer tissues. More importantly, we identified a genetic loci newly associated with increased colorectal cancer progression, rs18407893 at 11p15.4 (in 3'-UTR of LDHA), which maps to the seed sequence recognized by miR-374a. Cancer cells overexpressed miR-374a has decreased levels of LDHA compared with miR-374a-MUT (rs18407893 at 11p15.4). Taken together, these novel findings provide more therapeutic approaches to the Warburg effect and therapeutic targets of cancer energy metabolism. PMID:26062441

  17. Discovery of N-hydroxyindole-based inhibitors of human lactate dehydrogenase isoform A (LDH-A) as starvation agents against cancer cells.

    PubMed

    Granchi, Carlotta; Roy, Sarabindu; Giacomelli, Chiara; Macchia, Marco; Tuccinardi, Tiziano; Martinelli, Adriano; Lanza, Mario; Betti, Laura; Giannaccini, Gino; Lucacchini, Antonio; Funel, Nicola; León, Leticia G; Giovannetti, Elisa; Peters, Godefridus J; Palchaudhuri, Rahul; Calvaresi, Emilia C; Hergenrother, Paul J; Minutolo, Filippo

    2011-03-24

    Highly invasive tumor cells are characterized by a metabolic switch, known as the Warburg effect, from "normal" oxidative phosphorylation to increased glycolysis even under sufficiently oxygenated conditions. This dependence on glycolysis also confers a growth advantage to cells present in hypoxic regions of the tumor. One of the key enzymes involved in glycolysis, the muscle isoform of lactate dehydrogenase (LDH-A), is overexpressed by metastatic cancer cells and is linked to the vitality of tumors in hypoxia. This enzyme may be considered as a potential target for new anticancer agents, since its inhibition cuts cancer energetic and anabolic supply, thus reducing the metastatic and invasive potential of cancer cells. We have discovered new and efficient N-hydroxyindole-based inhibitors of LDH-A, which are isoform-selective (over LDH-B) and competitive with both the substrate (pyruvate) and the cofactor (NADH). The antiproliferative activity of these compounds was confirmed on a series of cancer cell lines, and they proved to be particularly effective under hypoxic conditions. Moreover, NMR experiments showed that these compounds are able to reduce the glucose-to-lactate conversion inside the cell.

  18. The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying.

    PubMed

    Popova, Antoaneta V; Rausch, Saskia; Hundertmark, Michaela; Gibon, Yves; Hincha, Dirk K

    2015-10-01

    The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses. PMID:25988244

  19. The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying.

    PubMed

    Popova, Antoaneta V; Rausch, Saskia; Hundertmark, Michaela; Gibon, Yves; Hincha, Dirk K

    2015-10-01

    The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses.

  20. Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation

    SciTech Connect

    Gupta, S.; Clark, D.P. )

    1989-07-01

    Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of adh pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed terms of redox balance.

  1. Oligosaccharide-based Surfactant/Citric Acid Buffer System Stabilizes Lactate Dehydrogenase during Freeze-drying and Storage without the Addition of Natural Sugar.

    PubMed

    Ogawa, Shigesaburo; Kawai, Ryuichiro; Koga, Maito; Asakura, Kouichi; Takahashi, Isao; Osanai, Shuichi

    2016-06-01

    Experiments were conducted to assess the maintenance effects of oligosaccharide-based surfactants on the enzymatic activity of a model protein, lactate dehydrogenase (LDH), during freeze-drying and room temperature storage using the citric acid buffer system. Oligosaccharide-based surfactants, which exhibit a high glass transition temperature (Tg), promoted the eminent retention of enzymatic activity during these protocols, whereas monosaccharide-based surfactants with a low Tg displayed poor performance at high concentration, albeit much better than that of Tween 80 at middle concentration. The increase in the alkyl chain length did not exert positive effects as observed for the maintenance effect during freeze-thawing, but an amphiphilic nature and a glass forming ability were crucial for the effective stabilization at a low excipient concentration during freeze-drying. Even a low oligosaccharide-based surfactant content (0.1 mg mL(-1)) could maintain LDH activity during freeze-drying, but a high surfactant content (1.0 mg mL(-1)) was required to prevent buffer precipitation and retain high LDH activity on storage. Regarding storage, glass formation restricted molecular mobility in the lyophilized matrix, and LDH activity was effectively retained. The present results describe a strategy based on the glass-forming ability of surfactant-type excipients that affords a natural sugar-free formulation or an alternative use for polysorbate-type surfactants.

  2. Insulin, CCAAT/Enhancer-Binding Proteins and Lactate Regulate the Human 11β-Hydroxysteroid Dehydrogenase Type 2 Gene Expression in Colon Cancer Cell Lines

    PubMed Central

    Alikhani-Koupaei, Rasoul; Ignatova, Irena D.; Guettinger, Andreas; Frey, Felix J.; Frey, Brigitte M.

    2014-01-01

    11β-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon. PMID:25133511

  3. Investigation of drying stresses on proteins during lyophilization: differentiation between primary and secondary-drying stresses on lactate dehydrogenase using a humidity controlled mini freeze-dryer.

    PubMed

    Luthra, Sumit; Obert, Jean-Philippe; Kalonia, Devendra S; Pikal, Michael J

    2007-01-01

    This article describes the design, performance testing, and application of a controlled humidity mini-freeze-dryer in studying the physical stability of lactate dehydrogenase during lyophilization. Performance evaluation of the mini-freeze-dryer was conducted with tests, namely water sublimation, radiation heat exchange, lowest achievable temperature, and leak testing. Protein stability studies were conducted by comparing protein activity at various stages of lyophilization with the initial activity. The shelf and condenser temperature were stable at <-40 degrees C, wall temperature was within 2 degrees C of the shelf temperature, and the leak rate was small. The chamber pressure was controlled by the ice on the condenser and the product temperature during sublimation was equal to the shelf temperature. Addition of Tween 80 prevented activity loss in solution and after freeze-thaw. No activity loss was observed after primary-drying even in absence of lyoprotectants and with collapse of cake structure. Five percent (w/w) sucrose concentration was required to achieve full stabilization. In conclusion, performance testing established that the mini-freeze-dryer was suitable for mechanistic freeze-drying studies. Secondary-drying was the critical step for protein stability. The concentration of sucrose required to stabilize the protein completely was several orders of magnitude higher than that required to satisfy the direct interaction requirement of the protein. PMID:17031859

  4. Measurements of C-reactive protein in serum and lactate dehydrogenase in serum and synovial fluid of patients with osteoarthritis.

    PubMed

    Hurter, K; Spreng, D; Rytz, U; Schawalder, P; Ott-Knüsel, F; Schmökel, H

    2005-03-01

    Diagnosis of osteoarthritis (OA) is based upon the clinical orthopaedic examination and the radiographic assessment, both of which can be non-specific and insensitive in early joint disease. The aim of our study was to investigate if there is an increase in serum levels of C-reactive protein (CRP) in degenerative joint disease (DJD) and if CRP could be used to help diagnose OA. We also wished to investigate whether it was possible to distinguish a joint with clinically and radiographically confirmed OA from a healthy joint by comparing lactate dehydrogenase (LDH) levels within the synovial fluid and the serum. We have shown a difference in synovial LDH levels between diseased and healthy joints (P<0.0001). There was also a significant difference between LDH in arthritic synovial fluid and serum, with no correlation between the values. Despite the fact that the values of our clinical patients tended to be higher than the values of our control group (P=0.05) all measured values were within the normal limits of previous publications. From these data, we conclude that single measurements of serum CRP do not permit detection of OA in clinical patients and that serum LDH is not a reliable marker for osteoarthritis. LDH levels in the synovial fluid could be of diagnostic value for identifying osteoarthritis. PMID:15727922

  5. Importance of serum sialic acid and lactate dehydrogenase in diagnosis and treatment monitoring of cervical cancer patients.

    PubMed

    Patel, P S; Rawal, G N; Balar, D B

    1993-09-01

    In an attempt to establish a blood-based biochemical index for diagnosis of cervical cancer and treatment monitoring of patients suffering from the disease, serum levels of total sialic acid (TSA), lipid-bound sialic acid (LSA), and lactate dehydrogenase were estimated by highly specific spectrophotometric methods. Serum concentrations of the markers in 108 untreated cervical cancer patients were compared with the levels of the biomarkers in 125 healthy, age-matched female individuals (controls). The alterations in serum levels of the markers after radiotherapy in cervical cancer patients were also observed. The levels of all markers were significantly higher (P < 0.001) in untreated cervical cancer patients compared to the controls. TSA was found to be the most sensitive (90.74%) marker for diagnosis of cervical cancer. Combined use of the markers revealed maximum (100%) sensitivity. In comparison between early (stage I+II) and advanced (stage III+IV) malignant disease, the markers showed insignificant changes. TSA and LSA values in patients who did not respond to radiotherapy were significantly higher (P < 0.05 and P < 0.001, respectively) than those of the responders. The results suggest that combined evaluation of the markers is helpful for diagnosis as well as for treatment monitoring of cervical carcinoma patients.

  6. Regulation of transplacental virus infection by developmental and immunological factors: studies with lactate dehydrogenase-elevating virus.

    PubMed

    Haven, T R; Rowland, R R; Plagemann, P G; Wong, G H; Bradley, S E; Cafruny, W A

    1996-04-01

    Placental and fetal infections with lactate dehydrogenase-elevating virus (LDV) were determined by virus titration, indirect fluorescence antibody (IFA), and in situ hybridization with cDNA probes. Experiments were designed to determine the effects of gestational age, timing of maternal LDV infection, and immunological (antibody and cytokine) factors on mouse placental and fetal LDV infection. Virus infection of the placenta was detected at high levels (almost all placentas infected) within 24 h post-maternal infection (p.m.i.), whereas fetal LDV infection was detected only at a low level by 24 h p.m.i. The percentage of fetuses becoming LDV infected progressively increased between 24 and 72 h p.m.i. When fetal infection was studied at 72 h p.m.i., earlier gestational ages (9-11 days) were associated with fetal resistance to infection, whereas between 12.5 and 15 days of gestation, virus infection was detected in 50-71% of fetuses. Maternal treatment with interferon-gamma (IFN-gamma) or anti-LDV monoclonal antibodies was associated with reduced rates of fetal, but not placental, LDV infection. These results demonstrate that both developmental and immunological factors are important in the regulation of transplacental LDV infection.

  7. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of D-lactate dehydrogenase from Lactobacillus jensenii.

    PubMed

    Kim, Sangwoo; Kim, Yong Hwan; Kim, Kyung-Jin

    2014-08-01

    The thermostable D-lactate dehydrogenase from Lactobacillus jensenii (LjD-LDH) is a key enzyme for the production of the D-form of lactic acid from pyruvate concomitant with the oxidation of NADH to NAD(+). The polymers of lactic acid are used as biodegradable bioplastics. The LjD-LDH protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 28%(w/v) polyethylene glycol 400, 100 mM Tris-HCl pH 9, 200 mM magnesium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 Å. The crystal belonged to space group P3121, with unit-cell parameters a = b = 90.5, c = 157.8 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.58 Å(3) Da(-1), which corresponds to a solvent content of approximately 52.3%. The structure was solved by single-wavelength anomalous dispersion using a selenomethionine derivative. PMID:25084378

  8. The value of lactate dehydrogenase serum levels as a prognostic and predictive factor for advanced pancreatic cancer patients receiving sorafenib

    PubMed Central

    Faloppi, Luca; Bianconi, Maristella; Giampieri, Riccardo; Sobrero, Alberto; Labianca, Roberto; Ferrari, Daris; Barni, Sandro; Aitini, Enrico; Zaniboni, Alberto; Boni, Corrado; Caprioni, Francesco; Mosconi, Stefania; Fanello, Silvia; Berardi, Rossana; Bittoni, Alessandro; Andrikou, Kalliopi; Cinquini, Michela; Torri, Valter; Scartozzi, Mario; Cascinu, Stefano

    2015-01-01

    Although lactate dehydrogenase (LDH) serum levels, indirect markers of angiogenesis, are associated with a worse outcome in several tumours, their prognostic value is not defined in pancreatic cancer. Moreover, high levels are associated even with a lack of efficacy of tyrosine kinase inhibitors, contributing to explain negative results in clinical trials. We assessed the role of LDH in advanced pancreatic cancer receiving sorafenib. Seventy-one of 114 patients included in the randomised phase II trial MAPS (chemotherapy plus or not sorafenib) and with available serum LDH levels, were included in this analysis. Patients were categorized according to serum LDH levels (LDH ≤vs.> upper normal rate). A significant difference was found in progression free survival (PFS) and in overall survival (OS) between patients with LDH values under or above the cut-off (PFS: 5.2 vs. 2.7 months, p = 0.0287; OS: 10.7 vs. 5.9 months, p = 0.0021). After stratification according to LDH serum levels and sorafenib treatment, patients with low LDH serum levels treated with sorafenib showed an advantage in PFS (p = 0.05) and OS (p = 0.0012). LDH appears to be a reliable parameter to assess the prognosis of advanced pancreatic cancer patients, and it may be a predictive parameter to select patients candidate to receive sorafenib. PMID:26397228

  9. Changes in Protein Architecture and Sub-Picosecond Protein Dynamics Impact the Reaction Catalyzed by Lactate Dehydrogenase

    PubMed Central

    Masterson, Jean E.; Schwartz, Steven D.

    2013-01-01

    We have previously established the importance of a promoting vibration, a sub-picosecond protein motion that propagates through a specific axis of residues, in the reaction coordinate of lactate dehydrogenase (LDH). To test the effect that perturbation of this motion would have on the enzymatic reaction, we employ transition path sampling to obtain transition path ensembles for four independent LDH enzymatic systems: the wild type enzyme, a version of the enzyme expressing heavy isotopic substitution, and two enzymes with mutations in the promoting vibration axis. We show that even slight changes in the promoting vibration of LDH result in dramatic changes in enzymatic chemistry. In the “heavy” version of the enzyme, we find that the dampening of the sub-picosecond dynamics from heavy isotopic substitution leads to a drastic increase in the time of barrier crossing. Furthermore, we see that mutation of the promoting vibration axis causes a decrease in the variability of transition paths available to the enzymatic reaction. The combined results reveal the importance of the protein architecture of LDH in enzymatic catalysis by establishing how the promoting vibration is finely tuned to facilitate chemistry. PMID:23441954

  10. The value of lactate dehydrogenase serum levels as a prognostic and predictive factor for advanced pancreatic cancer patients receiving sorafenib.

    PubMed

    Faloppi, Luca; Bianconi, Maristella; Giampieri, Riccardo; Sobrero, Alberto; Labianca, Roberto; Ferrari, Daris; Barni, Sandro; Aitini, Enrico; Zaniboni, Alberto; Boni, Corrado; Caprioni, Francesco; Mosconi, Stefania; Fanello, Silvia; Berardi, Rossana; Bittoni, Alessandro; Andrikou, Kalliopi; Cinquini, Michela; Torri, Valter; Scartozzi, Mario; Cascinu, Stefano

    2015-10-27

    Although lactate dehydrogenase (LDH) serum levels, indirect markers of angiogenesis, are associated with a worse outcome in several tumours, their prognostic value is not defined in pancreatic cancer. Moreover, high levels are associated even with a lack of efficacy of tyrosine kinase inhibitors, contributing to explain negative results in clinical trials. We assessed the role of LDH in advanced pancreatic cancer receiving sorafenib. Seventy-one of 114 patients included in the randomised phase II trial MAPS (chemotherapy plus or not sorafenib) and with available serum LDH levels, were included in this analysis. Patients were categorized according to serum LDH levels (LDH ≤ vs.> upper normal rate). A significant difference was found in progression free survival (PFS) and in overall survival (OS) between patients with LDH values under or above the cut-off (PFS: 5.2 vs. 2.7 months, p = 0.0287; OS: 10.7 vs. 5.9 months, p = 0.0021). After stratification according to LDH serum levels and sorafenib treatment, patients with low LDH serum levels treated with sorafenib showed an advantage in PFS (p = 0.05) and OS (p = 0.0012). LDH appears to be a reliable parameter to assess the prognosis of advanced pancreatic cancer patients, and it may be a predictive parameter to select patients candidate to receive sorafenib. PMID:26397228

  11. Loss of /sup 51/chromium, lactate dehydrogenase, and /sup 111/indium as indicators of endothelial cell injury

    SciTech Connect

    Chopra, J.; Joist, J.H.; Webster, R.O.

    1987-11-01

    Injury to endothelial cells appears to be an important initial event in the pathogenesis of many diseases such as acute lung injury, venous and arterial thromboembolism, and atherosclerosis. Different methods for detecting damage to cultured endothelial cells have been described. However, their relative sensitivity as markers of endothelial cell damage has not been adequately determined. We compared the loss of /sup 51/Chromium (/sup 51/Cr), the cytoplasmic enzyme lactate dehydrogenase (LDH), and /sup 111/Indium (/sup 111/In) from endothelial cells upon exposure to several injurious agents. Cultured bovine pulmonary artery endothelial cells in confluent monolayers were labeled with /sup 51/Cr or /sup 111/Inoxine and exposed to increasing concentrations of the nonionic detergent, Triton X-100 (0.2 to 1%), hydrogen peroxide (1 to 500 microM), or neutrophils stimulated with phorbol myristate acetate. With all forms of injury, loss of /sup 51/Cr occurred earlier and to a greater extent than LDH loss which in turn was greater than loss of /sup 111/In. Substantial loss of /sup 51/Cr was observed in the absence of appreciable ultrastructural damage to endothelial cell external membranes. The findings may reflect the relative ease with which small molecules such as adenine nucleotides (/sup 51/Cr-labeled) escape whereas larger molecules such as LDH and proteins binding /sup 111/In are retained intracellularly. Thus, /sup 51/Cr loss appears to be a more sensitive indicator of sublytic endothelial cell injury than either /sup 111/In or LDH release.

  12. Pyruvate dehydrogenase complex activity controls metabolic and malignant phenotype in cancer cells.

    PubMed

    McFate, Thomas; Mohyeldin, Ahmed; Lu, Huasheng; Thakar, Jay; Henriques, Jeremy; Halim, Nader D; Wu, Hong; Schell, Michael J; Tsang, Tsz Mon; Teahan, Orla; Zhou, Shaoyu; Califano, Joseph A; Jeoung, Nam Ho; Harris, Robert A; Verma, Ajay

    2008-08-15

    High lactate generation and low glucose oxidation, despite normal oxygen conditions, are commonly seen in cancer cells and tumors. Historically known as the Warburg effect, this altered metabolic phenotype has long been correlated with malignant progression and poor clinical outcome. However, the mechanistic relationship between altered glucose metabolism and malignancy remains poorly understood. Here we show that inhibition of pyruvate dehydrogenase complex (PDC) activity contributes to the Warburg metabolic and malignant phenotype in human head and neck squamous cell carcinoma. PDC inhibition occurs via enhanced expression of pyruvate dehydrogenase kinase-1 (PDK-1), which results in inhibitory phosphorylation of the pyruvate dehydrogenase alpha (PDHalpha) subunit. We also demonstrate that PDC inhibition in cancer cells is associated with normoxic stabilization of the malignancy-promoting transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) by glycolytic metabolites. Knockdown of PDK-1 via short hairpin RNA lowers PDHalpha phosphorylation, restores PDC activity, reverts the Warburg metabolic phenotype, decreases normoxic HIF-1alpha expression, lowers hypoxic cell survival, decreases invasiveness, and inhibits tumor growth. PDK-1 is an HIF-1-regulated gene, and these data suggest that the buildup of glycolytic metabolites, resulting from high PDK-1 expression, may in turn promote HIF-1 activation, thus sustaining a feed-forward loop for malignant progression. In addition to providing anabolic support for cancer cells, altered fuel metabolism thus supports a malignant phenotype. Correction of metabolic abnormalities offers unique opportunities for cancer treatment and may potentially synergize with other cancer therapies. PMID:18541534

  13. Dehydrogenase activity of forest soils depends on the assay used

    NASA Astrophysics Data System (ADS)

    Januszek, Kazimierz; Długa, Joanna; Socha, Jarosław

    2015-01-01

    Dehydrogenases are exclusively intracellular enzymes, which play an important role in the initial stages of oxidation of soil organic matter. One of the most frequently used methods to estimate dehydrogenase activity in soil is based on the use of triphenyltetrazolium chloride as an artificial electron acceptor. The purpose of this study was to compare the activity of dehydrogenases of forest soils with varied physicochemical properties using different triphenyltetrazolium chloride assays. The determination was carried out using the original procedure by Casida et al., a modification of the procedure which involves the use of Ca(OH)2 instead of CaCO3, the Thalmann method, and the assay by Casida et al. without addition of buffer or any salt. Soil dehydrogenase activity depended on the assay used. Dehydrogenase determined by the Casida et al. method without addition of buffer or any salt correlated with the pH values of soils. The autoclaved strongly acidic samples of control soils showed high concentrations of triphenylformazan, probably due to chemical reduction of triphenyltetrazolium chloride. There is, therefore, a need for a sterilization method other than autoclaving, ie a process that results in significant changes in soil properties, thus helping to increase the chemical reduction of triphenyltetrazolium chloride.

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

    PubMed

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

    2014-05-10

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

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

    PubMed Central

    2012-01-01

    Background NAD-independent l-lactate dehydrogenase (l-iLDH) from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids. Results Val-108 of l-iLDH was changed to Ala by rationally site-directed mutagenesis. The l-iLDH mutant exhibited much higher activity than wide-type l-iLDH towards l-mandelate, an aromatic 2-hydroxycarboxylic acid. Using the engineered Escherichia coli expressing the mutant l-iLDH as a biocatalyst, 40 g·L-1 of dl-mandelic acid was converted to 20.1 g·L-1 of d-mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g·L-1 of benzoylformic acid. Conclusions A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally re-design mutagenesis. Two building block intermediates (optically pure d-mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system. PMID:23176608

  16. Interaction of carbohydrates with alcohol dehydrogenase: Effect on enzyme activity.

    PubMed

    Jadhav, Swati B; Bankar, Sandip B; Granström, Tom; Ojamo, Heikki; Singhal, Rekha S; Survase, Shrikant A

    2015-09-01

    Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol dehydrogenase with oxidized carbohydrates was also confirmed by fluorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase.

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

    PubMed

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

    2016-01-01

    Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor. PMID:27266631

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    1993-01-01

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

  20. A Streamlined, Automated Protocol for the Production of Milligram Quantities of Untagged Recombinant Rat Lactate Dehydrogenase A Using ÄKTAxpressTM

    PubMed Central

    Nowicki, Matthew W.; Blackburn, Elizabeth A.; McNae, Iain W.; Wear, Martin A.

    2015-01-01

    We developed an efficient, automated 2-step purification protocol for the production of milligram quantities of untagged recombinant rat lactate dehydrogenase A (rLDHA) from E. coli, using the ÄKTAxpress™ chromatography system. Cation exchange followed by size exclusion results in average final purity in excess of 93% and yields ~ 14 milligrams per 50 ml of original cell culture in EnPresso B media, in under 8 hrs, including all primary sample processing and column equilibration steps. The protein is highly active and coherent biophysically and a viable alternative to the more problematic human homolog for structural and ligand-binding studies; an apo structure of untagged rLDHA was solved to a resolution 2.29 Å (PDB ID 5ES3). Our automated methodology uses generic commercially available pre-packed columns and simple buffers, and represents a robust standard method for the production of milligram amounts of untagged rLDHA, facilitating a novel fragment screening approach for new inhibitors. PMID:26717415

  1. Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors.

    PubMed

    Meng, Hengkai; Liu, Pi; Sun, Hongbing; Cai, Zhen; Zhou, Jie; Lin, Jianping; Li, Yin

    2016-04-25

    Engineering the cofactor specificity of a natural enzyme often results in a significant decrease in its activity on original cofactor. Here we report that a NADH-dependent dehydrogenase (d-LDH) from Lactobacillus delbrueckii 11842 can be rationally engineered to efficiently use both NADH and NADPH as cofactors. Point mutations on three amino acids (D176S, I177R, F178T) predicted by computational analysis resulted in a modified enzyme designated as d-LDH*. The Kcat/Km of the purified d-LDH* on NADPH increased approximately 184-fold while the Kcat/Km on NADH also significantly increased, showing for the first time that a rationally engineered d-LDH could exhibit comparable activity on both NADPH and NADH. Further kinetic analysis revealed that the enhanced affinity with NADH or NADPH and the significant increased Kcat of d-LDH* resulted in the significant increase of d-LDH* activity on both NADPH and NADH. This study thus demonstrated that the cofactor specificity of dehydrogenase can be broadened by using targeted engineering approach, and the engineered enzyme can efficiently function in NADH-rich, or NADPH-rich, or NADH and NADPH-rich environment.

  2. Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors

    PubMed Central

    Meng, Hengkai; Liu, Pi; Sun, Hongbing; Cai, Zhen; Zhou, Jie; Lin, Jianping; Li, Yin

    2016-01-01

    Engineering the cofactor specificity of a natural enzyme often results in a significant decrease in its activity on original cofactor. Here we report that a NADH-dependent dehydrogenase (d-LDH) from Lactobacillus delbrueckii 11842 can be rationally engineered to efficiently use both NADH and NADPH as cofactors. Point mutations on three amino acids (D176S, I177R, F178T) predicted by computational analysis resulted in a modified enzyme designated as d-LDH*. The Kcat/Km of the purified d-LDH* on NADPH increased approximately 184-fold while the Kcat/Km on NADH also significantly increased, showing for the first time that a rationally engineered d-LDH could exhibit comparable activity on both NADPH and NADH. Further kinetic analysis revealed that the enhanced affinity with NADH or NADPH and the significant increased Kcat of d-LDH* resulted in the significant increase of d-LDH* activity on both NADPH and NADH. This study thus demonstrated that the cofactor specificity of dehydrogenase can be broadened by using targeted engineering approach, and the engineered enzyme can efficiently function in NADH-rich, or NADPH-rich, or NADH and NADPH-rich environment. PMID:27109778

  3. Benefit from thoracic radiotherapy in patients with extensive-disease small-cell lung cancer with elevated lactate dehydrogenase

    PubMed Central

    Qin, Tao; Zhou, Ningning; Zeng, Yin-duo; Dinglin, Xiaoxiao; Zhao, Yuanyuan; Liu, Huai; Chen, Likun

    2016-01-01

    Background High lactate dehydrogenase (LDH) is associated with a large tumor burden in extensive-disease small-cell lung cancer (ED-SCLC). This study evaluated the benefit of additional thoracic radiotherapy (TRT) in patients with ED-SCLC with elevated LDH. Methods We analyzed 94 patients with ED-SCLC and evaluated LDH at Sun Yat-sen University Cancer Center during the period between January 2000 and March 2010. Patients were divided into two groups according to whether TRT was received. Survival was evaluated by the Kaplan–Meier method and Cox’s regression analysis. Results The median age of the 94 patients with ED-SCLC was 58.5 years. The main metastatic sites included the liver, bone, brain, and adrenal glands. The response rate in the TRT group was 46.9%. There were 32 patients (34.04%) receiving TRT and 5.3% receiving prophylactic cranial irradiation. The median survival time reached 10 months (95% confidence interval: 8.22, 11.78 months), and the 1-, 2-, and 5-year survival rates were 43.6%, 11.7%, and 2.1%, respectively. There was a significant difference in the median progression-free survival (PFS) and overall survival (OS) between the TRT group and the no TRT group (PFS: 9.0 months vs 6.0 months, P=0.018; OS: 13.0 months vs 9.0 months, P=0.006). Conclusion The use of TRT improves the survival of patients with ED-SCLC. Future studies should use the LDH level for categorizing patients for treatment. PMID:27042102

  4. Elevation of serum lactate dehydrogenase at posterior reversible encephalopathy syndrome onset in chemotherapy-treated cancer patients.

    PubMed

    Fitzgerald, Ryan T; Wright, Steven M; Samant, Rohan S; Kumar, Manoj; Ramakrishnaiah, Raghu H; Van Hemert, Rudy; Brown, Aliza T; Angtuaco, Edgardo J

    2014-09-01

    The pathophysiology of posterior reversible encephalopathy syndrome (PRES) is incompletely understood; however, an underlying state of immune dysregulation and endothelial dysfunction has been proposed. We examined alterations of serum lactate dehydrogenase (LDH), a marker of endothelial dysfunction, relative to the development of PRES in patients receiving chemotherapy. A retrospective Institutional Review Board approved database of 88 PRES patients was examined. PRES diagnosis was confirmed by congruent clinical diagnosis and MRI. Clinical features at presentation were recorded. Serum LDH values were collected at three time points: prior to, at the time of, and following PRES diagnosis. Student's t-test was employed. LDH values were available during the course of treatment in 12 patients (nine women; mean age 57.8 years [range 33-75 years]). Chemotherapy-associated PRES patients were more likely to be normotensive (25%) versus the non-chemotherapy group (9%). LDH levels at the time of PRES diagnosis were higher than those before and after (p=0.0263), with a mean difference of 114.8 international units/L. Mean time intervals between LDH measurement prior to and following PRES diagnosis were 44.8 days and 51.4 days, respectively. Mean elapsed time between last chemotherapy administration and PRES onset was 11.1days. In conclusion, serum LDH, a marker of endothelial dysfunction, shows statistically significant elevation at the onset of PRES toxicity in cancer patients receiving chemotherapy. Our findings support a systemic process characterized by endothelial injury/dysfunction as a factor, if not the prime event, in the pathophysiology of PRES.

  5. Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome

    PubMed Central

    Mohammad, Goran Hamid; Olde Damink, S. W. M.; Malago, Massimo; Dhar, Dipok Kumar; Pereira, Stephen P.

    2016-01-01

    Pancreatic cancer has a 5-year survival rate of less than 4%. Despite advances in diagnostic technology, pancreatic cancer continues to be diagnosed at a late and incurable stage. Accurate biomarkers for early diagnosis and to predict treatment response are urgently needed. Since alteration of glucose metabolism is one of the hallmarks of cancer cells, we proposed that pyruvate kinase type M2 (M2PK) and lactate dehydrogenase A (LDHA) enzymes could represent novel diagnostic markers and potential therapeutic targets in pancreatic cancer. In 266 tissue sections from normal pancreas, pancreatic cystic neoplasms, pancreatic intraepithelial neoplasia (PanIN) and cancer, we evaluated the expression of PKM2, LDHA, Ki-67 and CD8+ by immunohistochemistry and correlated these markers with clinicopathological characteristics and patient survival. PKM2 and LDHA expression was also assessed by Western blot in 10 human pancreatic cancer cell lines. PKM2 expression increased progressively from cyst through PanIN to cancer, whereas LDHA was overexpressed throughout the carcinogenic process. All but one cell line showed high expression of both proteins. Patients with strong PKM2 and LDHA expression had significantly worse survival than those with weak PKM2 and/or LDHA expression (7.0 months vs. 27.9 months, respectively, p = 0.003, log rank test). The expression of both PKM2 and LDHA correlated directly with Ki-67 expression, and inversely with intratumoral CD8+ cell count. PKM2 was significantly overexpressed in poorly differentiated tumours and both PKM2 and LDHA were overexpressed in larger tumours. Multivariable analysis showed that combined expression of PKM2 and LDHA was an independent poor prognostic marker for survival. In conclusion, our results demonstrate a high expression pattern of two major glycolytic enzymes during pancreatic carcinogenesis, with increased expression in aggressive tumours and a significant adverse effect on survival. PMID:26989901

  6. Prognostic value of combined preoperative lactate dehydrogenase and alkaline phosphatase levels in patients with resectable pancreatic ductal adenocarcinoma.

    PubMed

    Ji, Fei; Fu, Shun-Jun; Guo, Zhi-Yong; Pang, Hui; Ju, Wei-Qiang; Wang, Dong-Ping; Hua, Yun-Peng; He, Xiao-Shun

    2016-07-01

    Serum enzymes, including lactate dehydrogenase (LDH) and alkaline phosphatase (ALP), have recently been reported to play important roles in tumor growth. Increases in LDH and ALP have been confirmed to predict poor prognosis in patients with various cancers. However, their prognostic value in pancreatic cancer has not been well studied. Therefore, we reviewed the preoperative data on LDH and ALP in 185 pancreatic ductal adenocarcinoma (PDAC) patients who underwent surgery between July 2005 and December 2010 to explore the prognostic value of these markers. The cutoff points were determined based on the upper limit of their normal values. The Chi-square test was used to analyze the relationships between LDH/ALP and clinical characteristics. Univariate and multivariate analyses were performed to identify the predictive value of the above factors for disease-free survival (DFS) and overall survival (OS). We found that elevation of LDH was related to carbohydrate antigen 19-9 (CA19-9), lymph node involvement, tumor size, TNM, distant metastasis, and recurrence. Additionally, ALP was correlated to perineural invasion. After multivariate analysis, LDH and ALP were identified as independent prognostic factors for DFS and OS, and elevation of LDH/ALP was correlated with poor DFS and OS. Notably, there was a positive correlation between LDH and ALP. The predictive power of LDH combined with ALP was more sensitive than that of either one alone. Therefore, we conclude that the preoperative LDH and ALP values are prognostic factors for PADC, and the prognostic accuracy of testing can be enhanced by the combination of LDH and ALP. PMID:27399091

  7. Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome.

    PubMed

    Mohammad, Goran Hamid; Olde Damink, S W M; Malago, Massimo; Dhar, Dipok Kumar; Pereira, Stephen P

    2016-01-01

    Pancreatic cancer has a 5-year survival rate of less than 4%. Despite advances in diagnostic technology, pancreatic cancer continues to be diagnosed at a late and incurable stage. Accurate biomarkers for early diagnosis and to predict treatment response are urgently needed. Since alteration of glucose metabolism is one of the hallmarks of cancer cells, we proposed that pyruvate kinase type M2 (M2PK) and lactate dehydrogenase A (LDHA) enzymes could represent novel diagnostic markers and potential therapeutic targets in pancreatic cancer. In 266 tissue sections from normal pancreas, pancreatic cystic neoplasms, pancreatic intraepithelial neoplasia (PanIN) and cancer, we evaluated the expression of PKM2, LDHA, Ki-67 and CD8+ by immunohistochemistry and correlated these markers with clinicopathological characteristics and patient survival. PKM2 and LDHA expression was also assessed by Western blot in 10 human pancreatic cancer cell lines. PKM2 expression increased progressively from cyst through PanIN to cancer, whereas LDHA was overexpressed throughout the carcinogenic process. All but one cell line showed high expression of both proteins. Patients with strong PKM2 and LDHA expression had significantly worse survival than those with weak PKM2 and/or LDHA expression (7.0 months vs. 27.9 months, respectively, p = 0.003, log rank test). The expression of both PKM2 and LDHA correlated directly with Ki-67 expression, and inversely with intratumoral CD8+ cell count. PKM2 was significantly overexpressed in poorly differentiated tumours and both PKM2 and LDHA were overexpressed in larger tumours. Multivariable analysis showed that combined expression of PKM2 and LDHA was an independent poor prognostic marker for survival. In conclusion, our results demonstrate a high expression pattern of two major glycolytic enzymes during pancreatic carcinogenesis, with increased expression in aggressive tumours and a significant adverse effect on survival.

  8. Correlation between the Lactate Dehydrogenase Levels with Laboratory Variables in the Clinical Severity of Sickle Cell Anemia in Congolese Patients

    PubMed Central

    Mikobi, Tite Minga; Lukusa Tshilobo, Prosper; Aloni, Michel Ntetani; Mvumbi Lelo, Georges; Akilimali, Pierre Zalagile; Muyembe-Tamfum, Jean Jacques; Race, Valérie; Matthijs, Gert; Mbuyi Mwamba, Jean Marie

    2015-01-01

    Background Sickle cell anemia is an inflammatory disease and is characterized by chronic hemolysis. We sought to evaluate the association of lactate dehydrogenase levels with specific clinical phenotypes and laboratory variables in patients with sickle cell anemia. Methods The present cross-sectional study was conducted in Sickle Cell Centre of Yolo in Kinshasa, the Democratic Republic of Congo. Two hundred and eleven patients with Sickle Cell Anemia in steady state were recruited. Seventy-four participants with normal Hb (Hb-AA) were selected as a control group. Results The average rates of hemoglobin, hematocrit, and red blood cells tended to be significantly lower in subjects with Hb-SS (p<0.001). The average rates of white blood cells, platelets, reticulocytes and serum LDH were significantly higher in subjects with Hb-SS (p<0.001). The average rates of Hb, HbF, hematocrit and red blood cells of Hb-SS patients with asymptomatic clinical phenotype were significantly higher than those of the two other phenotypes. However, the average rates of white blood cells, platelets, reticulocytes, and LDH of Hb-SS patients with the severe clinical phenotype are higher than those of two other clinical phenotypes. Significant correlations were observed between Hb and white blood cell in severe clinical phenotype (r3 = -0.37 *) between Hb and red blood cells in the three phenotypes (r1 = 0.69 * r2 * = 0.69, r3 = 0.83 *), and finally between Hb and reticulocytes in the asymptomatic clinical phenotype and severe clinical phenotype (r1 = -0.50 * r3 = 0.45 *). A significant increase in LDH was observed in patients with leg ulcer, cholelithiasis and aseptic necrosis of the femoral head. Conclusion The increase in serum LDH is accompanied by changes in hematological parameters. In our midst, serum LDH may be considered as an indicator of the severity of the disease. PMID:25946088

  9. Impact of Pre-Treatment Lactate Dehydrogenase Levels on Prognosis and Bevacizumab Efficacy in Patients with Metastatic Colorectal Cancer

    PubMed Central

    Passardi, Alessandro; Scarpi, Emanuela; Tamberi, Stefano; Cavanna, Luigi; Tassinari, Davide; Fontana, Annalisa; Pini, Sara; Bernardini, Ilaria; Accettura, Caterina; Ulivi, Paola; Frassineti, Giovanni Luca; Amadori, Dino

    2015-01-01

    Background To investigate the impact of pre-treatment lactate dehydrogenase (LDH) levels on the outcome of patients with metastatic colorectal cancer treated with first-line chemotherapy with or without the anti-VEGF monoclonal antibody, bevacizumab, in a phase III prospective multicentre randomized ITACa (Italian Trial in Advanced Colorectal Cancer) trial. Methods Three hundred and seventy patients enrolled onto the ITACa first-line trial were considered for this study, 176 receiving chemotherapy (either FOLFIRI or FOLFOX) plus bevacizumab and 194 receiving chemotherapy only. Pre-treatment LDH levels were evaluated to identify a potential correlation with progression-free survival (PFS), overall survival (OS) and objective response rate. Results Information on pre-treatment LDH levels was available for 344 patients. High LDH levels were predictive of a lower median PFS (8.1 months vs. 9.2 months, p< 0.0001) and median OS (16.1 months vs. 25.2 months, p< 0.0001) in the overall population. In the chemotherapy plus bevacizumab group, median PFS was 9.1 and 9.8 months in patients with high LDH and low LDH, respectively (p= 0.073), whereas in the chemotherapy-only arm it was 6.9 and 9.1 months, respectively (p < 0.0001). In patients with high LDH, the addition of bevacizumab to chemotherapy led to a reduction in the rate of progressive disease (16.4 vs. 30.5%, p= 0.081) and to a prolonged PFS (p= 0.028). Conclusion A high LDH value was confirmed as a marker of poor prognosis. Bevacizumab reduced the progressive disease rate and improved PFS in the high-LDH subgroup, making serum LDH a potentially effective an easily available and marker to select patients who benefit from bevacizumab. Trial Registration NCT01878422 ClinicalTrials.gov PMID:26244985

  10. Spatial variability of the dehydrogenase activity in forest soils

    NASA Astrophysics Data System (ADS)

    Błońska, Ewa; Lasota, Jarosław

    2014-05-01

    The aim of this study was to assess the spatial variability of the dehydrogenase activity (DH) in forest soils using geostatistics. We have studied variability soil dehydrogenase and their relationship with variability of some physic-chemical properties. Two study areas (A and B) were set up in southern Poland in the Zlotoryja Forest District. Study areas were covered by different types of vegetation (A- broadleaf forest with beech, ash and sycamore), B- coniferous forest with Norway spruce). The soils were classified as Dystric Cambisols (WRB 2006). The samples for laboratory testing were collected from 49 places on each areas. 15 cm of surface horizon of soil were taken (with previously removed litter). Dehydrogenase activity was marked with Lenhard's method according to the Casida procedure. Soil pH, nitrogen (N) and soil organic carbon (C) content (by LECO CNS 2000 carbon analyzer) was marked. C/N ratio was calculated. Particle size composition was determined using laser diffraction. Statistical analysis were performed using STATISTICA 10 software. Geostatistical analysis and mapping were done by application of GS 9+ (Gamma Design) and Surfer 11 (Golden Software). The activity of DH ranged between 5,02 and 71,20 mg TPP• kg-1 •24 h-1 on the A area and between 0,94 and 16,47 mg TPP• kg-1 •24 h-1. Differences in spatial variability of the analised features were noted. The variability of dehydrogenase activity on the A study area was described by an exponential model, whereas on the B study area the spatial correlation has not been noted. The relationship of dehydrogenase activity with the remaining parameters of soil was noted only in the case of A study area. The variability of organic carbon content on the A and B study areas were described by an exponential model. The variability of nitrogen content on both areas were described by an spherical model.

  11. Effect of a Marathon Run on Serum Lipoproteins, Creatine Kinase, and Lactate Dehydrogenase in Recreational Runners

    ERIC Educational Resources Information Center

    Kobayashi, Yoshio; Takeuchi, Toshiko; Hosoi, Teruo; Yoshizaki, Hidekiyo; Loeppky, Jack A.

    2005-01-01

    The objective of this study was to determine the effect of a marathon run on serum lipid and lipoprotein concentrations and serum muscle enzyme activities and follow their recovery after the run. These blood concentrations were measured before, immediately after, and serially after a marathon run in 15 male recreational runners. The triglyceride…

  12. Bioactivity-Guided Identification and Cell Signaling Technology to Delineate the Lactate Dehydrogenase A Inhibition Effects of Spatholobus suberectus on Breast Cancer

    PubMed Central

    Wang, Zhiyu; Wang, Dongmei; Han, Shouwei; Wang, Neng; Mo, Feizhi; Loo, Tjing Yung; Shen, Jiangang; Huang, Hui; Chen, Jianping

    2013-01-01

    Aerobic glycolysis is an important feature of cancer cells. In recent years, lactate dehydrogenase A (LDH-A) is emerging as a novel therapeutic target for cancer treatment. Seeking LDH-A inhibitors from natural resources has been paid much attention for drug discovery. Spatholobus suberectus (SS) is a common herbal medicine used in China for treating blood-stasis related diseases such as cancer. This study aims to explore the potential medicinal application of SS for LDH-A inhibition on breast cancer and to determine its bioactive compounds. We found that SS manifested apoptosis-inducing, cell cycle arresting and anti-LDH-A activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cell. Oral herbal extracts (1 g/kg/d) administration attenuated tumor growth and LDH-A expression in both breast cancer xenografts. Bioactivity-guided fractionation finally identified epigallocatechin as a key compound in SS inhibiting LDH-A activity. Further studies revealed that LDH-A plays a critical role in mediating the apoptosis-induction effects of epigallocatechin. The inhibited LDH-A activities by epigallocatechin is attributed to disassociation of Hsp90 from HIF-1α and subsequent accelerated HIF-1α proteasome degradation. In vivo study also demonstrated that epigallocatechin could significantly inhibit breast cancer growth, HIF-1α/LDH-A expression and trigger apoptosis without bringing toxic effects. The preclinical study thus suggests that the potential medicinal application of SS for inhibiting cancer LDH-A activity and the possibility to consider epigallocatechin as a lead compound to develop LDH-A inhibitors. Future studies of SS for chemoprevention or chemosensitization against breast cancer are thus warranted. PMID:23457597

  13. Upregulation of lactate dehydrogenase a by 14-3-3ζ leads to increased glycolysis critical for breast cancer initiation and progression

    PubMed Central

    Chang, Chia-Chi; Zhang, Chenyu; Zhang, Qingling; Sahin, Ozgur; Wang, Hai; Xu, Jia; Xiao, Yi; Zhang, Jian; Rehman, Sumaiyah K.; Li, Ping; Hung, Mien-Chie; Behbod, Fariba; Yu, Dihua

    2016-01-01

    Metabolic reprogramming is a hallmark of cancer. Elevated glycolysis in cancer cells switches the cellular metabolic flux to produce more biological building blocks, thereby sustaining rapid proliferation. Recently, new evidence has emerged that metabolic dysregulation may occur at early-stages of neoplasia and critically contribute to cancer initiation. Here, our bioinformatics analysis of microarray data from early-stages breast neoplastic lesions revealed that 14-3-3ζ expression is strongly correlated with the expression of canonical glycolytic genes, particularly lactate dehydrogenase A (LDHA). Experimentally, increasing 14-3-3ζ expression in human mammary epithelial cells (hMECs) up-regulated LDHA expression, elevated glycolytic activity, and promoted early transformation. Knockdown of LDHA in the 14-3-3ζ-overexpressing hMECs significantly reduced glycolytic activity and inhibited transformation. Mechanistically, 14-3-3ζ overexpression activates the MEK-ERK-CREB axis, which subsequently up-regulates LDHA. In vivo, inhibiting the activated the MEK/ERK pathway in 14-3-3ζ-overexpressing hMEC-derived MCF10DCIS.COM lesions led to effective inhibition of tumor growth. Therefore, targeting the MEK/ERK pathway could be an effective strategy for intervention of 14-3-3ζ-overexpressing early breast lesions. Together, our data demonstrate that overexpression of 14-3-3ζ in early stage pre-cancerous breast epithelial cells may trigger an elevated glycolysis and transcriptionally up-regulating LDHA, thereby contributes to human breast cancer initiation. PMID:27150057

  14. sup 51 Cr loss and lactate dehydrogenase (LDH) release in irradiated human tumor cells

    SciTech Connect

    Ts'ao, C.; Molteni, A.; Hinz, J. )

    1991-03-11

    Much of what is known about tumor cell radiosensitivity in vitro derives from the colony formation assay. Other endpoints of cytotoxicity in irradiated tumor cells are rarely examined. The purpose of this study was to determine whether loss of {sup 51}Cr from prelabeled cells and release of LDH could be used to quantify radiation injury in two cultured human tumor cell lines: a prostate carcinoma and a melanoma. Bovine aortic endothelial cells (EC) known to release {sup 51}Cr and LDH following irradiation, were cotested. Radioactivity and LDH activity in the culture medium were determined after 0-40 Gy of {sup 60}CO {gamma} rays. Proliferation of irradiated tumor cells was also studied. EC exhibited a time- and radiation dose-dependent increase in {sup 51}Cr and LDH release. Both tumor cell lines showed a time-dependent increase in {sup 51}Cr release, but this baseline release was not elevated after irradiation. LDH release from the prostate cancer cell line was observed within 8 hr after 40 Gy, and at 48 hr by 10 Gy. Irradiated melanoma cells, in contrast, never release excess LDH into the culture medium. Melanoma cells continued to proliferate after 10 Gy, while proliferation of prostate cancer cells was totally arrested by this dose of exposure. While {sup 51}Cr loss and LDH release appear to be sensitive indicators of radiation-induced damage in EC, they have limited value in the assessment of radiation-induced cytotoxicity in human prostate cancer and melanoma cells.

  15. [The expression of the sperm-specific lactate dehydrogenase gene Ldh-c in plateau pika (Ochotona curzoniae) cardiac muscle and its effect on the anaerobic glycolysis].

    PubMed

    Li, Xiao; Wei, Lian; Wang, Yang; Xu, Li-Na; Wei, Lin-Na; Wei, Deng-Bang

    2015-06-25

    The plateau pika (Ochotona curzoniae) has a strong adaptability to hypoxic plateau environment. We found that the sperm-specific lactate dehydrogenase (LDH-C4) gene Ldh-c expressed in plateau pika cardiac muscle. In order to shed light on the effect of LDH-C4 on the anaerobic glycolysis in plateau pika cardiac muscle, 20 pikas were randomly divided into the inhibitor group and the control group, and the sample size of each group was 10. The pikas of inhibitor group were injected with 1 mL 1 mol/L N-isopropyl oxamate, a specific LDH-C4 inhibitor, in biceps femoris muscle of hind legs, each leg with 500 μL. The pikas of control group were injected with the same volume of normal saline (0.9% NaCl). The mRNA and protein expression levels of Ldh-c gene in plateau pika cardiac muscle were determined by real-time PCR and Western blot. The activities of LDH, and the contents of lactate (LD) and ATP in cardiac muscle were compared between the inhibitor group and the control group. The results showed that 1) the expression levels of Ldh-c mRNA and protein were 0.47 ± 0.06 and 0.68 ± 0.08, respectively; 2) 30 min after injection of 1 mL 1 mol/L N-isopropyl oxamate in biceps femoris muscle, the concentration of N-isopropyl oxamate in blood was 0.08 mmol/L; 3) in cardiac muscle of the inhibitor group and the control group, the LDH activities were (6.18 ± 0.48) U/mg and (9.08 ± 0.58) U/mg, the contents of LD were (0.21 ± 0.03) mmol/g and (0.26 ± 0.04) mmol/g, and the contents of ATP were (4.40 ± 0.69) nmol/mg and (6.18 ± 0.73) nmol/mg (P < 0.01); 5) the inhibition rates of N-isopropyl oxamate to LDH, LD and ATP were 31.98%, 20.90% and 28.70%, respectively. The results suggest that Ldh-c expresses in cardiac muscle of plateau pika, and the pika cardiac muscle may get at least 28% ATP for its activities by LDH-C4 catalyzed anaerobic glycolysis, which reduces the dependence on oxygen and enhances the adaptation to the hypoxic environments.

  16. Long term intensive exercise training leads to a higher plasma malate/lactate dehydrogenase (M/L) ratio and increased level of lipid mobilization in horses.

    PubMed

    Li, Gebin; Lee, Peter; Mori, Nobuko; Yamamoto, Ichiro; Arai, Toshiro

    2012-06-01

    Continuous high intensity training may induce alterations to enzyme activities related to glucose and lipid metabolism in horses. In our study, five Thoroughbred race horses (3 male and 2 female, avg age=5 yrs old) were compared against five riding horses (1 male, 1 female, 3 gelding, avg age=13 yrs old) in terms of energy metabolism, by examining plasma malate (MDH) and lactate (LDH) dehydrogenase activities and M/L ratio. MDH is involved in NADH and ATP generation, whereas LDH can convert NADH back into NAD(+) for ATP generation. An increase in plasma M/L ratio can reflect heightened energy metabolism in the liver and skeletal muscle of horses adapted to continuous intensive exercise. Moreover, plasma lipid metabolism analytes (adiponectin, NEFA, total cholesterol (T-Cho), and triglycerides (TG)) can reflect changes to lipolysis rate, which can also indicate a change in energy metabolism. Overall, race horses demonstrated increased MDH and LDH activity in plasma (4x and 2x greater, respectively), in addition to a plasma M/L ratio twice as high as that of riding horses (2.0 vs 1.0). In addition, race horses also demonstrated significantly higher levels of plasma NEFA (50% greater), TG (2x greater), and T-Cho (20% greater) as compared to riding horses. Therefore, race horse muscles may have adapted to prolonged high intensity endurance exercise by gaining a higher oxidative capacity and an increased capacity for fat utilization as an energy source, resulting in heightened energy metabolism and increased rate of lipid mobilization.

  17. D(--)-lactic acid and d(--)-lactate dehydrohgenase in octopus spermatozoa.

    PubMed

    Mann, T; Martin, A W; Thiersch, J B; Lutwak-Mann, C; Brooks, D E; Jones, R

    1974-08-01

    The spermatozoa of Octopus dofleini martini produce anaerobically D(-)-lactic acid and possess a very active D(-)-lactate dehydrogenase. In this respect, while resembling certain microorganisms, they differ strikingly from mammalian spermatozoa which produce L(+)-lactic acid and contain L(+)-lactate dehydrogenase. PMID:4366789

  18. Molecular characterization of CcpA and involvement of this protein in transcriptional regulation of lactate dehydrogenase and pyruvate formate-lyase in the ruminal bacterium Streptococcus bovis.

    PubMed

    Asanuma, Narito; Yoshii, Takahiro; Hino, Tsuneo

    2004-09-01

    A ccpA gene that encodes global catabolite control protein A (CcpA) in Streptococcus bovis was identified and characterized, and the involvement of CcpA in transcriptional control of a gene (ldh) encoding lactate dehydrogenase (LDH) and a gene (pfl) encoding pyruvate formate-lyase (PFL) was examined. The ccpA gene was shown to be transcribed as a monocistronic operon. A catabolite-responsive element (cre) was found in the promoter region of ccpA, suggesting that ccpA transcription in S. bovis is autogenously regulated. CcpA required HPr that was phosphorylated at the serine residue at position 46 (HPr-[Ser-P]) for binding to the cre site, but glucose 6-phosphate, fructose 1,6-bisphosphate, and NADP had no effect on binding. Diauxic growth was observed when S. bovis was grown in a medium containing glucose and lactose, but it disappeared when ccpA was disrupted, which indicates that CcpA is involved in catabolite repression in S. bovis. The level of ccpA mRNA was higher when cells were grown on glucose than when they were grown on lactose, which was in line with the level of ldh mRNA. When cells were grown on glucose, the ldh mRNA level was lower but the pfl mRNA level was higher in a ccpA-disrupted mutant than in the parent strain, which suggests that ldh transcription is enhanced and pfl transcription is suppressed by CcpA. The ccpA-disrupted mutant produced less lactate and more formate than the parent, probably because the mutant had reduced LDH activity and elevated PFL activity. In the upper region of both ldh and pfl, a cre-like sequence was found, suggesting that the complex consisting of CcpA and HPr-[Ser-P] binds to the possible cre sites. Thus, CcpA appears to be involved in the global regulation of sugar utilization in S. bovis. PMID:15345406

  19. Lactate dehydrogenase test

    MedlinePlus

    ... injury Muscle weakness and loss of muscle tissue ( muscular dystrophy ) New abnormal tissue formation (usually cancer) Pancreatitis Stroke ... test LDH isoenzyme blood test Liver disease Mononucleosis Muscular dystrophy Pernicious anemia Stroke Vitamin B12 deficiency anemia Update ...

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

    PubMed

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

    2014-12-01

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

  1. Cloning of the Staphylococcus aureus ddh gene encoding NAD+-dependent D-lactate dehydrogenase and insertional inactivation in a glycopeptide-resistant isolate.

    PubMed Central

    Boyle-Vavra, S; de Jonge, B L; Ebert, C C; Daum, R S

    1997-01-01

    The mechanism of low-level glycopeptide resistance among staphylococci is not known. A cytoplasmic protein, provisionally called Ddh (W. M. Milewski, S. Boyle-Vavra, B. Moreira, C. C. Ebert, and R. S. Daum, Antimicrob. Agents Chemother. 40:166-172, 1996), and the RNA transcript that contains the ddh gene, which encodes Ddh, are present in increased amounts in a vancomycin-resistant isolate, 523k, compared with the susceptible parent isolate, 523. Sequence analysis had previously revealed that Ddh is related to NAD+-dependent D-lactate dehydrogenase (D-nLDH) and VanH. This latter protein is essential for high-level glycopeptide resistance in Enterococcus faecium and Enterococcus faecalis by synthesizing the D-lactate needed for biosynthesis of D-lactate-terminating peptidoglycan precursors with low affinity for vancomycin. We now provide the direct evidence that the ddh gene product is Staphylococcus aureus D-nLDH and hereafter refer to the protein as D-nLDH. However, overproduction of this protein in isolate 523k did not result in production of D-lactate-containing peptidoglycan precursors, and susceptibility testing of ddh mutants of 523k demonstrated that S. aureus D-nLDH is not necessary for glycopeptide resistance in this isolate. We conclude that the mechanism of glycopeptide resistance in this isolate is distinct from that in enterococci. PMID:9352927

  2. E4F1-mediated control of pyruvate dehydrogenase activity is essential for skin homeostasis.

    PubMed

    Goguet-Rubio, Perrine; Seyran, Berfin; Gayte, Laurie; Bernex, Florence; Sutter, Anne; Delpech, Hélène; Linares, Laetitia Karine; Riscal, Romain; Repond, Cendrine; Rodier, Geneviève; Kirsh, Olivier; Touhami, Jawida; Noel, Jean; Vincent, Charles; Pirot, Nelly; Pavlovic, Guillaume; Herault, Yann; Sitbon, Marc; Pellerin, Luc; Sardet, Claude; Lacroix, Matthieu; Le Cam, Laurent

    2016-09-27

    The multifunctional protein E4 transcription factor 1 (E4F1) is an essential regulator of epidermal stem cell (ESC) maintenance. Here, we found that E4F1 transcriptionally regulates a metabolic program involved in pyruvate metabolism that is required to maintain skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetyltransferase (Dlat), a gene encoding the E2 subunit of the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) keratinocytes exhibited impaired PDH activity and a redirection of the glycolytic flux toward lactate production. The metabolic reprogramming of E4f1 KO keratinocytes associated with remodeling of their microenvironment and alterations of the basement membrane, led to ESC mislocalization and exhaustion of the ESC pool. ShRNA-mediated depletion of Dlat in primary keratinocytes recapitulated defects observed upon E4f1 inactivation, including increased lactate secretion, enhanced activity of extracellular matrix remodeling enzymes, and impaired clonogenic potential. Altogether, our data reveal a central role for Dlat in the metabolic program regulated by E4F1 in basal keratinocytes and illustrate the importance of PDH activity in skin homeostasis. PMID:27621431

  3. Galloflavin, a new lactate dehydrogenase inhibitor, induces the death of human breast cancer cells with different glycolytic attitude by affecting distinct signaling pathways.

    PubMed

    Farabegoli, F; Vettraino, M; Manerba, M; Fiume, L; Roberti, M; Di Stefano, G

    2012-11-20

    Galloflavin (GF), a recently identified lactate dehydrogenase inhibitor, hinders the proliferation of cancer cells by blocking glycolysis and ATP production. The aim of the present experiments was to study the effect of this compound on breast cancer cell lines reproducing different pathological subtypes of this tumor: MCF-7 (the well differentiated form), MDA-MB-231 (the aggressive triple negative tumor) and MCF-Tam (a sub-line of MCF-7 with acquired tamoxifen resistance). We observed marked differences in the energetic metabolism of these cell lines. Compared to MCF-7 cells, both MDA-MB-231 and MCF-Tam cells exhibited higher LDH levels and glucose uptake and showed lower capacity of oxygen consumption. In spite of these differences, GF exerted similar growth inhibitory effects. This result was explained by the finding of a constitutively activated stress response in MDA-MB-231 and MCF-Tam cells, which reproduce the poor prognosis tumor forms. As a further proof, different signaling pathways were found to be involved in the antiproliferative action of GF. In MCF-7 cells we observed a down regulation of the ERα-mediated signaling needed for cell survival. On the contrary, in MCF-Tam and MDA-MB-231 cells growth inhibition appeared to be contributed by an oxidative stress condition. The prevalent mechanism of cell death was found to be apoptosis induction. Because of the clinical relevance of breast cancer forms having the triple negative and/or chemoresistant phenotype, our results showing comparable effects of GF even on aggressively growing cells encourage further studies to verify the potential of this compound in improving the chemotherapy of breast cancer.

  4. Use of inline measures of l-lactate dehydrogenase for classification of posttreatment mammary Staphylococcus aureus infection status in dairy cows.

    PubMed

    Jørgensen, C Hildebrandt; Kristensen, A R; Østergaard, S; Bennedsgaard, T W

    2016-10-01

    An automated method for determining whether dairy cows with subclinical mammary infections recover after antibiotic treatment would be a useful tool in dairy production. For that purpose, inline l-lactate dehydrogenase (LDH) measurements was modeled using a dynamic linear model; the variance parameters were estimated using the expectation-maximization algorithm. The method used to classify cows as infected or uninfected was based on a multiprocess Kalman filter. Two learning data sets were created: infected and uninfected. The infected data set consisted of records from 48 cows with subclinical Staphylococcus aureus infection from 4 herds collected in 2010. The uninfected data set came from 35 uninfected cows collected during 2013 from 2 herds. Bacteriological culturing was used as gold standard. To test the model, we collected data from the 48 infected cows 50 d after antibiotic treatment. As a result of the treatment, this test data set consisted of 25 cows that still had a subclinical infection and 23 cows that were recovered. Model sensitivity was 36.0% and specificity was 82.6%. To a large extent, l-lactate dehydrogenase reflected the cow's immune response to the presence of pathogens in the udder. However, cows that were classified correctly before treatment had a better chance of correct classification after treatment. This indicated a variation between cows in immune response to subclinical mammary infection that may complicate the detection of subclinically infected cows and determination of recovery. PMID:27522431

  5. Screening and isolation of potential lactate dehydrogenase inhibitors from five Chinese medicinal herbs: Soybean, Radix pueraria, Flos pueraria, Rhizoma belamcandae, and Radix astragali.

    PubMed

    Tang, Ying; Li, Senlin; Li, Sainan; Yang, Xiaojing; Qin, Yao; Zhang, Yuchi; Liu, Chunming

    2016-06-01

    Stroke is among the leading causes of death and severe disability worldwide. Flavonoids have been extensively used in the treatment of ischemic stroke by reducing lactate dehydrogenase levels and thereby enhancing blood perfusion to the ischemic region. Here, we used ultrafiltration high-performance liquid chromatography coupled with diode array detection and mass spectrometry for the rapid screening and identification of flavonoids from five Chinese medicinal herbs: soybean, Radix pueraria, Flos pueraria, Rhizoma belamcandae, and Radix astragali. Using PC12 cells as a suitable in vitro model of toxicity, cell viability was quantitated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results showed that the extracts of soybean and the six major components, namely, acetyldaidzin, malonylgenistin, daidiain, glycitin, genistin, and acetylcitin; the extract of R. pueraria and its main component daidzein; the extract of F. pueraria and its three major components, tectorigenin, tectoridin, and tectorigenin-7-O-xylosylglucosid; and the extract of R. belamcandae and its main component, tectoridin, were strong lactate dehydrogenase inhibitors. Also, the components of R. astragali showed no bioactivity. These findings indicate that the ultrafltration high-performance liquid chromatography coupled with diode array detection and mass spectrometry method could be utilized in rapid screening and separation of bioactive compounds from a complex matrix. PMID:27059876

  6. Neurometabolic coupling between neural activity, glucose, and lactate in activated visual cortex.

    PubMed

    Li, Baowang; Freeman, Ralph D

    2015-11-01

    Neural activity is closely coupled with energy metabolism but details of the association remain to be identified. One basic area involves the relationships between neural activity and the main supportive substrates of glucose and lactate. This is of fundamental significance for the interpretation of non-invasive neural imaging. Here, we use microelectrodes with high spatial and temporal resolution to determine simultaneous co-localized changes in glucose, lactate, and neural activity during visual activation of the cerebral cortex in the cat. Tissue glucose and lactate concentration levels are measured with electrochemical microelectrodes while neural spiking activity and local field potentials are sampled by a microelectrode. These measurements are performed simultaneously while neurons are activated by visual stimuli of different contrast levels, orientations, and sizes. We find immediate decreases in tissue glucose concentration and simultaneous increases in lactate during neural activation. Both glucose and lactate signals return to their baseline levels instantly as neurons cease firing. No sustained changes or initial dips in glucose or lactate signals are elicited by visual stimulation. However, co-localized measurements of cerebral blood flow and neural activity demonstrate a clear delay in the cerebral blood flow signal such that it does not correlate temporally with the neural response. These results provide direct real-time evidence regarding the coupling between co-localized energy metabolism and neural activity during physiological stimulation. They are also relevant to a current question regarding the role of lactate in energy metabolism in the brain during neural activation. Dynamic changes in energy metabolites can be measured directly with high spatial and temporal resolution by use of enzyme-based microelectrodes. Here, to examine neuro-metabolic coupling during brain activation, we use combined microelectrodes to simultaneously measure

  7. Lactic acid-producing yeast cells having nonfunctional L- or D-lactate:ferricytochrome C oxidoreductase cells

    DOEpatents

    Miller, Matthew; Suominen, Pirkko; Aristidou, Aristos; Hause, Benjamin Matthew; Van Hoek, Pim; Dundon, Catherine Asleson

    2012-03-20

    Yeast cells having an exogenous lactate dehydrogenase gene ae modified by reducing L- or D-lactate:ferricytochrome c oxidoreductase activity in the cell. This leads to reduced consumption of lactate by the cell and can increase overall lactate yields in a fermentation process. Cells having the reduced L- or D-lactate:ferricytochrome c oxidoreductase activity can be screened for by resistance to organic acids such as lactic or glycolic acid.

  8. Functional response of the isolated, perfused normoxic heart to pyruvate dehydrogenase activation by dichloroacetate and pyruvate

    PubMed Central

    Jaimes, Rafael; Kuzmiak-Glancy, Sarah; Brooks, Daina M.; Swift, Luther M.; Posnack, Nikki G.; Kay, Matthew W.

    2015-01-01

    Dichloroacetate (DCA) and pyruvate activate pyruvate dehydrogenase (PDH), a key enzyme that modulates glucose oxidation and mitochondrial NADH production. Both compounds improve recovery after ischemia in isolated hearts. However, the action of DCA and pyruvate in normoxic myocardium is incompletely understood. We measured the effect of DCA and pyruvate on contraction, mitochondrial redox state, and intracellular calcium cycling in isolated rat hearts during normoxic perfusion. Normalized epicardial NADH fluorescence (nNADH) and left ventricular developed pressure (LVDP) were measured before and after administering DCA (5 mM) or pyruvate (5 mM). Optical mapping of Rhod-2AM was used to measure cytosolic calcium kinetics. DCA maximally activated PDH, increasing the ratio of active to total PDH from 0.48±0.03 to 1.03 ±0.03. Pyruvate sub-maximally activated PDH to a ratio of 0.75±0.02. DCA and pyruvate increased LVDP. When glucose was the only exogenous fuel, pyruvate increased nNADH by 21.4±2.9 % while DCA reduced nNADH by 21.4±6.1 % and elevated the incidence of premature ventricular contractions (PVCs). When lactate, pyruvate, and glucose were provided together as exogenous fuels, nNADH increased with DCA, indicating that PDH activation with glucose as the only exogenous fuel depletes PDH substrate. Calcium transient time-to-peak was shortened by DCA and pyruvate and SR calcium re-uptake was 30 % longer. DCA and pyruvate increased SR calcium load in myocyte monolayers. Overall, during normoxia when glucose is the only exogenous fuel, DCA elevates SR calcium, increases LVDP and contractility, and diminishes mitochondrial NADH. Administering DCA with plasma levels of lactate and pyruvate mitigates the drop in mitochondrial NADH and prevents PVCs. PMID:26142699

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

    NASA Technical Reports Server (NTRS)

    Casida, L. E., Jr.

    1977-01-01

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

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

    PubMed

    Singhvi, Mamata; Jadhav, Akanksha; Gokhale, Digambar

    2013-10-01

    The production of D-lactic acid by Lactobacillus lactis RM2-24 was investigated using modified media to increase the efficiency of the fermentation process. The results indicated that the addition of 5 g/l peptone and 1 g/l (NH4)2HPO4 enhanced D-lactic acid production by 32%, as compared to that obtained from non supplemented media, with a productivity of 3.0 g/l/h. Lactate dehydrogenase (LDH) expression profile in these different media was studied which resulted in appearance of additional LDH isoform produced by cells when they were grown in HSYE supplemented with (NH4)2HPO4. The additional LDH appears to be L-LDH contributing to production of L-lactic acid in the fermented broth. This is totally new information in the lactic acid fermentation and could be very useful to industries engaged in D-lactic acid production. PMID:23932744

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

    PubMed

    Singhvi, Mamata; Jadhav, Akanksha; Gokhale, Digambar

    2013-10-01

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

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

    PubMed

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

    2009-08-01

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

  13. Pyruvate dehydrogenase activity and quantity decreases after coronary artery bypass grafting: a prospective observational study

    PubMed Central

    Andersen, Lars W.; Liu, Xiaowen; Peng, Teng J.; Giberson, Tyler A.; Khabbaz, Kamal R.; Donnino, Michael W.

    2014-01-01

    Introduction Pyruvate dehydrogenase (PDH) is a key gatekeeper enzyme in aerobic metabolism. The main purpose of this study was to determine if PDH activity is affected by major stress in the form of coronary artery bypass grafting (CABG) which has previously been used as a model of critical illness. Methods We conducted a prospective, observational study of patients undergoing CABG at an urban, tertiary care hospital. We included adult patients undergoing CABG with or without concomitant valve surgery. Measurements of PDH activity and quantity and thiamine were obtained prior to surgery, at the completion of surgery, and 6 hours post-surgery. Results Fourteen patients were enrolled (age: 67 ± 10 years, 21 % female). Study subjects had a mean 41.7 % (SD: 27.7) reduction in PDH activity after surgery and a mean 32.0% (SD: 31.4) reduction 6 hours after surgery (p < 0.001). Eight patients were thiamine deficient (≤ 7 nmol/L) after surgery compared to none prior to surgery (p = 0.002). Thiamine level was a significantly associated with PDH quantity at all time points (p = 0.01). Post-surgery lactate levels were inversely correlated with post-surgery thiamine levels (r = −0.58 and p = 0.04). Conclusion The stress of major surgery causes decreased PDH activity and quantity, and depletion of thiamine levels. PMID:25526377

  14. Blood-derived proteins in milk at start of lactation: Indicators of active or passive transfer.

    PubMed

    Wall, Samantha K; Gross, Josef J; Kessler, Evelyne C; Villez, Kris; Bruckmaier, Rupert M

    2015-11-01

    Colostrum has a different composition compared with milk in established lactation. This difference is in part due to the partially open blood-milk barrier, which, when closed, is designed to prevent the interdiffusion of blood and milk components. In the first days of lactation, α-lactalbumin (α-LA), a milk protein, is typically present in blood and several blood-derived proteins are also present in milk, such as IgG1, IgG2, serum albumin (SA), and lactate dehydrogenase (LDH). With the exception of IgG1, which is known to be transferred by active transcellular transport, the other proteins are thought to pass paracellularly through the temporarily open barrier. Along with an exchange of blood and milk components, somatic cell count (SCC) is typically high in colostrum. The decline of these proteins and SCC can be used as indicators to determine transcellular or paracellular transport. Two hypotheses were tested. The first hypothesis was that the decline curve for a protein or SCC would be the same as IgG1, indicating transcellular transport, or the decline curve would be different than IgG1, indicating paracellular transport. The second hypothesis was that the decline curves of SCC and all proteins that are thought to have paracellular transport would be the same. Ten Holstein cows were milked at 4 h after parturition, the next 5 consecutive milkings, and the afternoon milking on d 5, 8, 10, and 14 of lactation for a total of 10 milking time points, and sequential jugular blood samples were also taken. Blood and milk samples were analyzed for the concentrations of LDH, SA, IgG1, IgG2, and α-LA and milk samples were measured for SCC. Protein concentration and SCC curves were generated from all 10 time points and were evaluated using the tau time constant model to determine the rate of decline of the slope of each protein. When examining the first hypothesis, the concentration of IgG1 declined significantly faster in the milk than the proteins IgG2 and LDH, but

  15. [Activity of NADP-dependent glycerol-3-phosphate dehydrogenase in skeletal muscles of animals].

    PubMed

    Epifanova, Iu E; Glushankov, E P; Kolotilova, A I

    1978-01-01

    The NADP-dependent glycerol-3-phosphate dehydrogenase activity was studied in sketetal muscles of the rat, rabbit and frog. The dehydrogenase activity in the skeletal muscles of the rat and rabbit was higher than that of the frog. The enzyme activity was found to depend upon the buffer, being higher in tris-HCl buffer than in triethanolamine buffer.

  16. Slow structural changes shown by the 3-nitrotyrosine-237 residue in pig heart [Tyr(3NO2)237] lactate dehydrogenase.

    PubMed Central

    Parker, D M; Jeckel, D; Holbrook, J J

    1982-01-01

    1. The pKa of the phenolic hydroxy group of the Tyr(3NO2)-237 residue in pig heart [Tyr(3NO2)237]lactate dehydrogenase is 7.2 in the apoenzyme, 7.4 in the enzyme-NADH complex and 7.8 in the enzyme-NADH-oxamate complex. The alkaline shift from apoenzyme to ternary complex is ascribed to the approach of the Glu-107 residue during the movement of the polypeptide loop residues 98-110. 2. The affinities of the nitrated enzyme for NADH and for oxamate (in the presence of NADH) are slightly less than those of the native enzyme. The turnover number for the nitrated enzyme in the pyruvate-to-lactate direction is about 0.75 of the value for the native enzyme. 3. Temperature-jump relaxation experiments of the enzyme saturated with NADH but fractionally saturated with oxamate are interpreted to show that the pKa of the nitrotyrosine residue responds to a protein rearrangement after oxamate binds to the binary enzyme-NADH complex. 4. Transient-kinetic experiments show the environment of the Tyr(3NO2)-237 residue in the enzyme-NADH-pyruvate complex of the steady state to be similar to that in the enzyme-NADH-oxamate inhibitor complex. Images Fig. 2. PMID:7092806

  17. Metabolic fate of lactate after vigorous activity in the leopard frog, Rana pipiens.

    PubMed

    Fournier, P A; Guderley, H

    1992-02-01

    Although the ability of isolated frog muscle to synthesize glycogen from lactate has long been known, it has never been demonstrated that this metabolic activity occurs in the intact frog. Our results clearly indicate that lactate glycogenesis in frog muscle occurs to a significant extent in vivo. During recovery from strenuous exercise, most of the lactate accumulated by frogs seems to be recycled into muscle glycogen because the lactate that disappears during recovery could account nearly stoichiometrically for the glycogen that accumulates in muscle. Furthermore, the decrease in body lactate and the increase in muscle glycogen follow corresponding time courses, suggesting a precursor-product relationship between lactate and glycogen. During recovery from intense exercise, hepatectomized and normal frogs have nearly identical extents of lactate elimination and glycogen synthesis. This suggests that muscle is the main tissue responsible for the recycling of lactate into muscle glycogen and that liver plays a negligible role in lactate disposal. The negligible hepatic contribution to lactate recycling results in part from the liver's incapacity to produce glucose from lactate. In support of this proposition, we show that frog liver perfused in vitro is unable to incorporate any detectable labeled lactate into glucose despite its excellent physiological integrity. Changes in dietary status, training state, season at which the experiments were done, exercise status, and composition of the perfusion media (pH, hormonal composition, physiological saline vs. culture medium) did not give rise to lactate gluconeogenesis. Because frog liver contains all the regulatory enzymes of the gluconeogenic pathway, its inability to synthesize glucose from lactate is not due to an absence of pyruvate carboxylase. A limited ability for lactate uptake may explain why frog liver cannot produce glucose from lactate. PMID:1539733

  18. Lactate Modulates the Activity of Primary Cortical Neurons through a Receptor-Mediated Pathway

    PubMed Central

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves

    2013-01-01

    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism. PMID:23951229

  19. Mitochondrial Respiratory Defect Causes Dysfunctional Lactate Turnover via AMP-activated Protein Kinase Activation in Human-induced Pluripotent Stem Cell-derived Hepatocytes.

    PubMed

    Im, Ilkyun; Jang, Mi-Jin; Park, Seung Ju; Lee, Sang-Hee; Choi, Jin-Ho; Yoo, Han-Wook; Kim, Seyun; Han, Yong-Mahn

    2015-12-01

    A defective mitochondrial respiratory chain complex (DMRC) causes various metabolic disorders in humans. However, the pathophysiology of DMRC in the liver remains unclear. To understand DMRC pathophysiology in vitro, DMRC-induced pluripotent stem cells were generated from dermal fibroblasts of a DMRC patient who had a homoplasmic mutation (m.3398T→C) in the mitochondrion-encoded NADH dehydrogenase 1 (MTND1) gene and that differentiated into hepatocytes (DMRC hepatocytes) in vitro. DMRC hepatocytes showed abnormalities in mitochondrial characteristics, the NAD(+)/NADH ratio, the glycogen storage level, the lactate turnover rate, and AMPK activity. Intriguingly, low glycogen storage and transcription of lactate turnover-related genes in DMRC hepatocytes were recovered by inhibition of AMPK activity. Thus, AMPK activation led to metabolic changes in terms of glycogen storage and lactate turnover in DMRC hepatocytes. These data demonstrate for the first time that energy depletion may lead to lactic acidosis in the DMRC patient by reduction of lactate uptake via AMPK in liver. PMID:26491018

  20. The lactate dehydrogenase--reduced nicotinamide--adenine dinucleotide--pyruvate complex. Kinetics of pyruvate binding and quenching of coeznyme fluorescence.

    PubMed

    Südi, J

    1974-04-01

    The stopped-flow kinetic studies described in this and the following paper (Südi, 1974) demonstrate that a Haldane-type description of the reversible lactate dehydrogenase reaction presents an experimentally feasible task. Combined results of these two papers yield numerical values for the six rate constants defined by the following equilibrium scheme, where E represents lactate dehydrogenase: [Formula: see text] The experiments were carried out at pH8.4 at a relatively low temperature (6.3 degrees C) with the pig heart enzyme. Identification of the above two intermediates and determination of the corresponding rate constants actually involve four series of independent observations in these studies, since (a) the reaction can be followed in both directions, and (b) both the u.v. absorption and the fluorescence of the coenzymes are altered in the reaction, and it is shown that these two spectral changes do not occur simultaneously. Kinetic observations made in the reverse direction are reported in this paper. It is demonstrated that the fluorescence of NADH can no longer be observed in the ternary complex E(NADH) (Pyr). Even though the oxidation-reduction reaction rapidly follows the formation of this complex, the numerical values of k(-4) (8.33x10(5)m(-1).s(-1)) and k(+4) (222s(-1)) are easily obtained from a directly observed second-order reaction step in which fluorescent but not u.v.-absorbing material is disappearing. U.v.-absorption measurements do not clearly resolve the subsequent oxidation-reduction step from the dissociation of lactate. It is shown that this must be due partly to the instrumental dead time, and partly to a low transient concentration of E(NAD+) (Lac) in the two-step sequential reaction in which the detectable disappearance of u.v.-absorbing material takes place. It is estimated that about one-tenth of the total change in u.v. absorption is due to a ;burst reaction' in which E(NAD+) (Lac) is produced, and this estimation yields, from k

  1. [Effect Of Polyelectrolytes on Catalytic Activity of Alcohol Dehydrogenase].

    PubMed

    Dubrovsky, A V; Musina, E V; Kim, A L; Tikhonenko, S A

    2016-01-01

    Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 M and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect. PMID:27266256

  2. RECIPIENT PRETRANSPLANT INOSINE MONOPHOSPHATE DEHYDROGENASE ACTIVITY IN NONMYELOABLATIVE HCT

    PubMed Central

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

    2014-01-01

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

  3. Hypoxic repression of pyruvate dehydrogenase activity is necessary for metabolic reprogramming and growth of model tumours.

    PubMed

    Golias, Tereza; Papandreou, Ioanna; Sun, Ramon; Kumar, Bhavna; Brown, Nicole V; Swanson, Benjamin J; Pai, Reetesh; Jaitin, Diego; Le, Quynh-Thu; Teknos, Theodoros N; Denko, Nicholas C

    2016-01-01

    Tumour cells fulfil the bioenergetic and biosynthetic needs of proliferation using the available environmental metabolites. Metabolic adaptation to hypoxia causes decreased mitochondrial function and increased lactate production. This work examines the biological importance of the hypoxia-inducible inhibitory phosphorylations on the pyruvate dehydrogenase E1α subunit. Pancreatic cancer cell lines were genetically manipulated to alter the net phosphorylation of PDH E1α through reduced kinase expression or enhanced phosphatase expression. The modified cells were tested for hypoxic changes in phosphorylated E1α, mitochondrial metabolism and growth as xenografted tumours. Even though there are four PDHK genes, PDHK1 is essential for inhibitory PDH phosphorylation of E1α at serine 232, is partially responsible for modification of serines 293 and 300, and these phosphorylations are necessary for model tumour growth. In order to determine the clinical relevance, a cohort of head and neck cancer patient biopsies was examined for phosphorylated E1α and expression of PDHK1. Patients with detectable 232 phosphorylation or expression of PDHK1 tend to have worse clinical outcome. These data show that PDHK1 activity is unique and non-redundant in the family of PHDK enzymes and a PDHK1 specific inhibitor would therefore have anti-cancer activity with reduced chance of side effects from inhibition of other PDHKs. PMID:27498883

  4. Hypoxic repression of pyruvate dehydrogenase activity is necessary for metabolic reprogramming and growth of model tumours

    PubMed Central

    Golias, Tereza; Papandreou, Ioanna; Sun, Ramon; Kumar, Bhavna; Brown, Nicole V.; Swanson, Benjamin J.; Pai, Reetesh; Jaitin, Diego; Le, Quynh-Thu; Teknos, Theodoros N.; Denko, Nicholas C.

    2016-01-01

    Tumour cells fulfil the bioenergetic and biosynthetic needs of proliferation using the available environmental metabolites. Metabolic adaptation to hypoxia causes decreased mitochondrial function and increased lactate production. This work examines the biological importance of the hypoxia-inducible inhibitory phosphorylations on the pyruvate dehydrogenase E1α subunit. Pancreatic cancer cell lines were genetically manipulated to alter the net phosphorylation of PDH E1α through reduced kinase expression or enhanced phosphatase expression. The modified cells were tested for hypoxic changes in phosphorylated E1α, mitochondrial metabolism and growth as xenografted tumours. Even though there are four PDHK genes, PDHK1 is essential for inhibitory PDH phosphorylation of E1α at serine 232, is partially responsible for modification of serines 293 and 300, and these phosphorylations are necessary for model tumour growth. In order to determine the clinical relevance, a cohort of head and neck cancer patient biopsies was examined for phosphorylated E1α and expression of PDHK1. Patients with detectable 232 phosphorylation or expression of PDHK1 tend to have worse clinical outcome. These data show that PDHK1 activity is unique and non-redundant in the family of PHDK enzymes and a PDHK1 specific inhibitor would therefore have anti-cancer activity with reduced chance of side effects from inhibition of other PDHKs. PMID:27498883

  5. Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

    PubMed Central

    Jean, Elise; Laoudj-Chenivesse, Dalila; Notarnicola, Cécile; Rouger, Karl; Serratrice, Nicolas; Bonnieu, Anne; Gay, Stéphanie; Bacou, Francis; Duret, Cédric; Carnac, Gilles

    2011-01-01

    Abstract Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56+ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non-human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H2O2)-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. PMID:19840193

  6. Analysis of rat cytosolic 9-cis-retinol dehydrogenase activity and enzymatic characterization of rat ADHII.

    PubMed

    Popescu, G; Napoli, J L

    2000-01-01

    We report the characterization of two enzymes that catalyze NAD(+)-dependent 9-cis-retinol dehydrogenase activity in rat liver cystol. Alcohol dehydrogenase class I (ADHI) contributes > 80% of the NA D+-dependent 9-cis-retinol dehydrogenase activity recovered, whereas alcohol dehydrogenase class II (ADHII), not identified previously at the protein level, nor characterized enzymatically in rat, accounts for approximately 2% of the activity. Rat ADHII exhibits properties different from those described for human ADHII. Moreover, rat ADHII-catalyzed rates of ethanol dehydrogenation are markedly lower than octanol or retinoid dehydrogenation rates. Neither ethanol nor 4-methylpyrazole inhibits the 9-cis-retinol dehydrogenase activity of rat ADHII. We propose that ADHII represents the previously observed additional retinoid oxidation activity of rat liver cytosol which occurred in the presence of either ethanol or 4-methylpyrazole. We also show that human and rat ADHII differ considerably in enzymatic properties. PMID:10606766

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

    PubMed

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

    2016-05-01

    Ionic liquids (ILs) were widely used in scientific and industrial application and have been reported to possess potential toxicity to the environment and human health. The effects of six typical N-methylimidazolium-based ILs ([Cnmim]X, n=4, 6, 8; X=Br(-), Cl(-), BF4(-), CF3SO3(-)) on the lactic dehydrogenase (LDH) activity and the molecular interaction mechanism of ILs and the LDH were investigated with the aid of spectroscopic techniques. Experimental results showed that the LDH activity was inhibited in the presence of ILs. For the ILs with the same anion but different cations, their inhibitory ability on the LDH activity increased with increasing the alkyl chain length on the IL cation. Thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of LDH with the addition of ILs. Both positive ΔH and ΔS suggested that hydrophobicity was the major driven force in the interaction process as expected. PMID:26802246

  8. 2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase activities in plant mitochondria: interaction via a common coenzyme a pool.

    PubMed

    Dry, I B; Wiskich, J T

    1987-08-15

    2-Oxoglutarate (2-OG)-dependent O2 uptake by washed or purified turnip (Brassica rapa L.) and pea (Pisum sativum L. cv. Massey Gem) leaf mitochondria, in the presence of malonate, was inhibited between 65 and 90% by micromolar levels of pyruvate. The inhibition was not observed in the absence of malonate and was reversed by alpha-cyano-4-hydroxycinnamic acid. The inhibition was also reversed by oxaloacetate or by malate, but not by any other tricarboxylic acid cycle intermediates. The stimulation of O2 uptake by oxaloacetate was half maximal at 8-9 microM and was transient, indicating its action was not mediated through the complete metabolic removal of pyruvate. Pyruvate had not effect on 2-OG oxidation under conditions in which pyruvate dehydrogenase was not active, indicating that pyruvate metabolism, rather than pyruvate itself, was responsible for producing the inhibition of 2-OG oxidation. Similar results were obtained with detergent-treated mitochondrial extracts with the exception that the inhibition of 2-OG oxidation by pyruvate could also be reversed by coenzyme A. The results suggest that pyruvate inhibits 2-oxoglutarate oxidation, in intact plant mitochondria, by sequestering intramitochondrial CoA as acetyl-CoA and, in the absence of citrate synthase activity, reduces the amount of free coenzyme A available for 2-oxoglutarate dehydrogenase. These results indicate that pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase share a common CoA pool within plant mitochondria and that the turnover of the acyl-CoA product of one enzyme will dramatically influence the activity of the other.

  9. Protein kinase A stimulates binding of multiple proteins to a U-rich domain in the 3'-untranslated region of lactate dehydrogenase A mRNA that is required for the regulation of mRNA stability.

    PubMed

    Tian, D; Huang, D; Brown, R C; Jungmann, R A

    1998-10-23

    We have explored the molecular basis of the cAMP-induced stabilization of lactate dehydrogenase A (LDH-A) mRNA and identified four cytoplasmic proteins of 96, 67, 52, and 50 kDa that specifically bind to a 30-nucleotide uridine-rich sequence in the LDH 3'-untranslated region with a predicted stem-loop structure. Mutational analysis revealed that specific protein binding is dependent upon an intact primary nucleotide sequence in the loop as well as integrity of the adjoining double-stranded stem structure, thus indicating a high degree of primary and secondary structure specificity. The critical stem-loop region is located between nucleotides 1473 and 1502 relative to the mRNA cap site and contains a previously identified cAMP-stabilizing region (CSR) required for LDH-A mRNA stability regulation by the protein kinase A pathway. The 3'-untranslated region binding activity of the proteins is up-regulated after protein kinase A activation, whereas protein dephosphorylation is associated with a loss of binding activity. These results imply a cause and effect relationship between LDH-A mRNA stabilization and CSR-phosphoprotein binding activity. We propose that the U-rich CSR is a recognition signal for CSR-binding proteins and for an mRNA processing pathway that specifically stabilizes LDH mRNA in response to activation of the protein kinase A signal transduction pathway.

  10. Interdependence of coenzyme-induced conformational work and binding potential in yeast alcohol and porcine heart lactate dehydrogenases: a hydrogen-deuterium exchange study.

    PubMed

    De Weck, Z; Pande, J; Kägi, J H

    1987-07-28

    Binding of NAD coenzymes to yeast alcohol dehydrogenase (YADH) and porcine heart lactate dehydrogenase (PHLDH) was studied by hydrogen-deuterium exchange with the infrared technique. Conformational changes in the enzymes specific to the coenzymes and their fragments were observed, and the pH dependence of the exchange reaction shows that it conforms to the EX-2 scheme. In both YADH and PHLDH the magnitude of the conformational change of measured by exchange retardation is considerably larger for NAD+ than for NADH. Studies with coenzyme fragments like ADP-ribose, ADP, and AMP also highlight the lack of rigorous correlation between structural features such as charge and size and their influence on exchange behavior. Ternary complexes such as YADH-NAD+-pyrazole, PHLDH-NAD+-oxalate, and PHLDH-NADH-oxamate, which mimic the transition state, have a significantly more pronounced effect on exchange rates than the corresponding binary complexes. The outstanding feature of this study is the demonstration that in the binary enzyme-coenzyme complexes the more loosely bound NAD+ is more effective in retarding exchange than the more firmly bound NADH. These differences are attributed to the unequal structural constraints exerted by the two coenzymes upon the enzymes, which translate to unequal expenditure of transconformational work in the formation of the two complexes. The opposing variation in the free energy of binding and the transconformational work expended can be viewed as an unequal partitioning of the net free energy gain resulting from the protein-ligand interaction into a binding term and that required for conformational change.

  11. Glycogen synthesis from lactate in a chronically active muscle

    SciTech Connect

    Talmadge, R.J.; Scheide, J.I.; Silverman, H.

    1989-05-01

    In response to neural overactivity (pseudomyotonia), gastrocnemius muscle fibers from C57Bl/6Jdy2J/dy2J mice have different metabolic profiles compared with normal mice. A population of fibers in the fast-twitch superficial region of the dy2J gastrocnemius stores unusually high amounts of glycogen, leading to an increased glycogen storage in the whole muscle. The dy2J muscle also contains twice as much lactate as normal muscle. A (/sup 14/C)lactate intraperitoneal injection leads to preferential /sup 14/C incorporation into glycogen in the dy2J muscle compared with normal muscle. To determine whether skeletal muscles were incorporating lactate into glycogen without body organ (liver, kidney) input, gastrocnemius muscles were bathed in 10 mM (/sup 14/C)lactate with intact neural and arterial supply but with impeded venous return. The contralateral gastrocnemius serves as a control for body organ input. By using this in situ procedure, we demonstrate that under conditions of high lactate both normal and dy2J muscle can directly synthesize glycogen from lactate. In this case, normal whole muscle incorporates (14C) lactate into glycogen at a higher rate than dy2J whole muscle. Autoradiography, however, suggests that the high-glycogen-containing muscle fibers in the dy2J muscle incorporate lactate into glycogen at nearly four times the rate of normal or surrounding muscle fibers.

  12. Furosemide and 11beta-hydroxysteroid dehydrogenase activity, in man.

    PubMed

    Palermo, M; Armanini, D; Shackleton, C H L; Sorba, G; Cossu, M; Roitman, E; Scaroni, C; Delitala, G

    2002-09-01

    Mineralocorticoid receptors possess the same affinity for aldosterone and for cortisol and preferential binding of aldosterone is modulated by the 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) enzyme, which converts cortisol to its inactive metabolite cortisone. Several endogenous or exogenous compounds able to inhibit the enzyme have been described and, as a consequence, produce the syndrome of apparent mineralocorticoid excess (AME) characterized by hypertension, hypokalemia, volume repletion and suppression of the renin-angiotensin-aldosterone system. High doses of furosemide, a diuretic that works in the luminal surface of the thick ascending limb of Henle's loop, have been reported to inhibit 11 beta-OHSD activity to the same extent as licorice in vivo and in vitro, in rat. The aim of our study was to verify the effect of the drug on 11 beta-OHSD activity in man at the doses currently used in clinical practice. We tested the activity of 11 beta-OHSD following both acute and protracted administration of furosemide. In the acute study, the drug was administered at low (40 mg i.v. in bolo) and high doses (infusion of 10 mg/kg bw i.v for six hours); the protracted furosemide administration consisted in 50 mg/day for 20 days, by mouth. The ratios between the cortisol metabolites tetrahydrocortisol plus allo-tetrahydrocortisol to tetra-hydrocortisone and urinary free cortisol to urinary free cortisone were used to measure the activity of 11 beta-OHSD. Urinary cortisol, cortisone and their metabolites were tested by a gas-chromatographic/mass spectrometric method. Neither acute nor prolonged administration of furosemide did affect the activity of 11 beta-OHSD although the drug was able to modify plasma aldosterone and PRA secretion and to determine hypokalemia. Our results suggest that furosemide does not play a significant role in 11 beta-OHSD modulation in humans, at least at the dosage used in clinical practice. PMID:12373630

  13. Utilization of Lactate Isomers by Propionibacterium freudenreichii subsp. shermanii: Regulatory Role for Intracellular Pyruvate

    PubMed Central

    Crow, Vaughan L.

    1986-01-01

    Five strains of Propionibacterium freudenreichii subsp. shermanii utilized the l-(+) isomer of lactate at a faster rate than they did the d-(−) isomer when grown with a mixture of lactate isomers under a variety of conditions. ATCC 9614, grown anaerobically in defined medium containing 160 mM dl-lactate, utilized only 4 and 15% of the d-(−)-lactate by the time 50 and 90%, respectively, of the l-(+)-lactate was used. The intracellular pyruvate concentration was high (>100 mM) in the initial stages of lactate utilization, when either dl-lactate or the l-(+) isomer was the starting substrate. The concentration of this intermediate dropped during dl-lactate fermentation such that when only d-(−)-lactate remained, the concentration was <20 mM. When only the d-(−) isomer was initially present, a similar relatively low concentration of intracellular pyruvate was present, even at the start of lactate utilization. The NAD+-independent lactate dehydrogenase activities in extracts showed different kinetic properties with regard to pyruvate inhibition, depending upon the lactate isomer present. Pyruvate gave a competitive inhibitor pattern with l-(+)-lactate and a mixed-type inhibitor pattern with d-(−)-lactate. It is suggested that these properties of the lactate dehydrogenases and the intracellular pyruvate concentrations explain the preferential use of the l-(+) isomer. PMID:16347134

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

    PubMed

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

    2015-01-01

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

  15. [Enzyme activity in the subcellular fractions of the liver of rats following a flight on board the Kosmos-1129 biosatellite].

    PubMed

    Tigranian, R A; Vetrova, E G; Abraham, S; Lin, C; Klein, H

    1983-01-01

    The activities of malate, isocitrate, and lactate dehydrogenases were measured in the liver mitochondrial and cytoplasmatic fractions of rats flown for 18.5 days onboard Cosmos-1129. The activities of the oxidative enzymes, malate and isocitrate dehydrogenases, in the mitochondrial fraction and those of the glycolytic enzyme, lactate dehydrogenase, in the cytoplasmatic fraction were found to decrease.

  16. Adult-onset multiple acyl CoA dehydrogenation deficiency associated with an abnormal isoenzyme pattern of serum lactate dehydrogenase.

    PubMed

    Sugai, Fuminobu; Baba, Kousuke; Toyooka, Keiko; Liang, Wen-Chen; Nishino, Ichizo; Yamadera, Misaki; Sumi, Hisae; Fujimura, Harutoshi; Nishikawa, Yoshiro

    2012-02-01

    We report a case of a 37 year-old male with multiple acyl-CoA dehydrogenation deficiency (MADD). The patient had suffered from exercise intolerance in his hip and thigh muscles for one year. Then, restriction of carbohydrates for a diet made his symptoms rapidly deteriorate. Blood test revealed compound heterozygosity for two novel missense mutations in the electron transfer flavoprotein dehydrogenase gene (ETFDH), and an abnormal LDH isoenzyme pattern: LDH-1 (60.0%) and LDH-2 (26.0%) predominated with abnormally elevated LDH-1/LDH-2 ratio (2.3), compared with muscle-derived LDH-5 (4.0%). Oral riboflavin treatment significantly improved his exercise intolerance and the LDH profile: LDH-1 (34.4%), LDH-2 (34.9%), LDH-5 (6.9%) and LDH-1/LDH-2 ratio (1.0). The abnormal LDH isoenzyme pattern may be one feature of adult-onset MADD selectively affecting type I muscle fibers with relatively high LDH-1 content. PMID:21907580

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

    PubMed

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

    2014-07-01

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

  18. D-lactate metabolism in the alga, Chlamydomonas Reinhardtii

    SciTech Connect

    Husic, D.W.; Tolbert, N.E.

    1986-05-01

    (/sup 14/C)D-lactate rapidly accumulates in Chlamydomonas cells under anaerobic conditions from the sugar-phosphate pools which are labeled during photosynthesis with /sup 14/CO/sub 2/. A soluble D-lactate dehydrogenase (30 ..mu..mol NADH oxidized/h/mg Chl), which functions only in the direction of pyruvate reduction, has been partially purified and characterized. The D-lactate is reoxidized in Chlamydomonas by a mitochondrial membrane-bound dehydrogenase. This enzyme is known in the plant literature as glycolate dehydrogenase, an enzyme of the oxidative photosynthetic carbon (C/sub 2/) cycle. This dehydrogenase may be linked to the mitochondrial electron transport chain, although the direct electron acceptor is unknown. Therefore, D-lactate accumulation may be, in part, due to the shut down of electron transport during anaerobiosis. In vivo chase experiments have shown that the D-lactate turns over rapidly when algal cells, which have been grown with air levels of CO/sub 2/ (0.04%), are returned to aerobic conditions in the light. Such turnover is not observed in cells which had been grown with 1 to 5% CO/sub 2/. Cells grown with high CO/sub 2/ have lower levels of glycolate dehydrogenase activity. They are currently using mutants of Chlamydomonas deficient in mitochondrial respiration to study the role of D-lactate oxidation in these algae.

  19. Decreased Hematocrit-To-Viscosity Ratio and Increased Lactate Dehydrogenase Level in Patients with Sickle Cell Anemia and Recurrent Leg Ulcers

    PubMed Central

    Connes, Philippe; Lamarre, Yann; Hardy-Dessources, Marie-Dominique; Lemonne, Nathalie; Waltz, Xavier; Mougenel, Danièle; Mukisi-Mukaza, Martin; Lalanne-Mistrih, Marie-Laure; Tarer, Vanessa; Tressières, Benoit; Etienne-Julan, Maryse; Romana, Marc

    2013-01-01

    Leg ulcer is a disabling complication in patients with sickle cell anemia (SCA) but the exact pathophysiological mechanisms are unknown. The aim of this study was to identify the hematological and hemorheological alterations associated with recurrent leg ulcers. Sixty-two SCA patients who never experienced leg ulcers (ULC-) and 13 SCA patients with a positive history of recurrent leg ulcers (ULC+) - but with no leg ulcers at the time of the study – were recruited. All patients were in steady state condition. Blood was sampled to perform hematological, biochemical (hemolytic markers) and hemorheological analyses (blood viscosity, red blood cell deformability and aggregation properties). The hematocrit-to-viscosity ratio (HVR), which reflects the red blood cell oxygen transport efficiency, was calculated for each subject. Patients from the ULC+ group were older than patients from the ULC- group. Anemia (red blood cell count, hematocrit and hemoglobin levels) was more pronounced in the ULC+ group. Lactate dehydrogenase level was higher in the ULC+ group than in the ULC- group. Neither blood viscosity, nor RBC aggregation properties differed between the two groups. HVR was lower and RBC deformability tended to be reduced in the ULC+ group. Our study confirmed increased hemolytic rate and anemia in SCA patients with leg ulcers recurrence. Furthermore, our data suggest that although systemic blood viscosity is not a major factor involved in the pathophysiology of this complication, decreased red blood cell oxygen transport efficiency (i.e., low hematocrit/viscosity ratio) may play a role. PMID:24223994

  20. Inhibition of Growth by Combined Treatment with Inhibitors of Lactate Dehydrogenase and either Phenformin or Inhibitors of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3.

    PubMed

    Lea, Michael A; Guzman, Yolanda; Desbordes, Charles

    2016-04-01

    Enhanced glycolysis in cancer cells presents a target for chemotherapy. Previous studies have indicated that proliferation of cancer cells can be inhibited by treatment with phenformin and with an inhibitor of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB) namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). In the present work, the action of two inhibitors that are effective at lower concentrations than 3PO, namely 1-(3-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PQP) and 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) were investigated. The inhibitors of lactate dehydrogenase (LDHA) studied in order of half-maximal inhibitory concentrations were methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2) < isosafrole < oxamate. In colonic and bladder cancer cells, additive growth inhibitory effects were seen with the LDHA inhibitors, of which NHI-2 was effective at the lowest concentrations. Growth inhibition was generally greater with PFK15 than with PQP. The increased acidification of the culture medium and glucose uptake caused by phenformin was blocked by combined treatment with PFKFB3 or LDHA inhibitors. The results suggest that combined treatment with phenformin and inhibitors of glycolysis can cause additive inhibition of cell proliferation and may mitigate lactic acidosis caused by phenformin when used as a single agent. PMID:27069123

  1. A comparison of the primary structures of lactate dehydrogenase isozymes M4 from giant panda, red panda, black bear and dog.

    PubMed

    Liang, S P; Zhang, L X

    1987-03-01

    Lactate dehydrogenase isozymes M4 have been isolated and purified from red panda (Ailurus fulgens), black bear (Selenarctos thibetanus) and dog (Canis familiars) by affinity chromatography and compared with that from giant panda (Ailuropoda melanoleuca). Experimental results have shown that the N-termini, C-termini and the molecular weights of LDH-M subunits of red panda, black bear and dog are the same as those of the LDH-M subunit of giant panda. Analysis and comparison of HPLC peptide maps from the tryptic digests of the isozymes of red panda, black bear and dog have shown that most of their peptide fragments had the same retention time and amino acid composition as the corresponding peptide fragments from giant panda. Fragments with different retention times and/or amino acid compositions were sequenced. Careful examination of those variant amino acid residues demonstrated clearly that the primary structure of giant panda LDH-M subunit is unique and it appears that the giant panda might be classified as an independent family.

  2. Baseline Serum Lactate Dehydrogenase Levels for Patients Treated With Intensity-Modulated Radiotherapy for Nasopharyngeal Carcinoma: A Predictor of Poor Prognosis and Subsequent Liver Metastasis

    SciTech Connect

    Zhou Guanqun; Tang Linglong; Mao Yanping; Chen Lei; Li Wenfei; Sun Ying; Liu Lizhi; Li Li; Lin Aihua; Ma Jun

    2012-03-01

    Purpose: To evaluate the prognostic value of baseline serum lactate dehydrogenase (LDH) levels in patients with nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiotherapy (IMRT). Methods and Materials: Cases of NPC (n = 465) that involved treatment with IMRT with or without chemotherapy were retrospectively analyzed. Results: The mean ({+-}SD) and median baseline serum LDH levels for this cohort were 172.77 {+-} 2.28 and 164.00 IU/L, respectively. Levels of LDH were significantly elevated in patients with locoregionally advanced disease (p = 0.016). Elevated LDH levels were identified as a prognostic factor for rates of overall survival (OS), disease-free survival (DFS), and distant metastasis-free survival (DMFS), with p values <0.001 in the univariate analysis and p < 0.001, p = 0.004, and p = 0.003, respectively, in the multivariate analysis. Correspondingly, the prognostic impact of patient LDH levels was found to be statistically significant for rates of OS, DFS, and DMFS (p = 0.028, 0.024, and 0.020, respectively). For patients who experienced subsequent liver failure after treatment, markedly higher pretreatment serum LDH levels were detected compared with patients experiencing distant metastasis events at other sites (p = 0.032). Conclusions: Elevated baseline LDH levels are associated with clinically advanced disease and are a poor prognosticator for OS, DFS, and DMFS for NPC patients. These results suggest that elevated serum levels of LDH should be considered when evaluating treatment options.

  3. Effects of repeated ischemia on release kinetics of troponin T, creatine kinase, and lactate dehydrogenase in coronary effluent from isolated rat hearts.

    PubMed

    Asayama, J; Yamahara, Y; Miyazaki, H; Ohta, B; Kobara, M; Tatsumi, T; Inoue, D; Nakagawa, M

    1994-04-01

    We studied the release kinetics of cardiac troponin T (TnT) from coronary effluent in a re-stenosis model of 13 isolated rat hearts. After a 20-min period of global ischemia, we reperfused the hearts for 60 min according to the method of Langendorff. A second period of global ischemia was then induced for 5 min (protocol A) or 20 min (protocol B), followed by a second 60-min period of reperfusion. Coronary flow was measured by a timed collection of the coronary effluent. Levels of TnT in the effluent were compared to those of creatine kinase (CK) and lactate dehydrogenase (LD). Levels of TnT increased after the second global ischemia, but no differences were found in the released levels of TnT between protocols A and B. However, the amounts of CK and LD released in protocol B were much greater than those released in protocol A. These studies indicate that the release kinetics of TnT are different from that of CK and LD during reperfusion. It appears that after the initial ischemic damage to TnT, subsequent ischemia causes damage to TnT regardless of the duration of the insult, whereas the damage to sarcolemma is dependent on the duration of the ischemia.

  4. Relationship of creatine kinase, aspartate aminotransferase, lactate dehydrogenase, and proteinuria to cardiomyopathy in the owl monkey (Aotus vociferans)

    SciTech Connect

    Gozalo, Alfonso S.; Chavera, Alfonso; Montoya, Enrique J.; Takano, Juan; Weller, Richard E.

    2008-02-01

    The purpose of this study was to determine serum reference values for crea- tine kinase (CK), aspartate aminotransferase (AST), and lactate dehydroge- nase (LDH) in captive-born and wild-caught owl monkeys to assess their usefulness for diagnosing myocardial disease. Urine samples were also collected and semi-quantitative tests performed. There was no statistically significant difference between CK, AST, and LDH when comparing both groups. However, when comparing monkeys with proteinuria to those without proteinuria, a statistically significant difference in CK value was observed (P = 0.021). In addition, the CK/AST ratio revealed that 29% of the animals included in this study had values suggesting cardiac infarction. Grossly, cardiac concentric hypertrophy of the left ventricle and small, pitted kidneys were the most common findings. Microscopically, myocardial fibrosis, contraction band necrosis, hypertrophy and hyperplasia of coronary arteries, medium-sized renal arteries, and afferent glomerular arteriolae were the most significant lesions, along with increased mesangial matrix and hypercellularity of glomeruli, Bowman’s capsule, and peritubular space fibroplasia. These findings suggest that CK, AST, and LDH along with urinalysis provide a reliable method for diagnosing cardiomyopathies in the owl monkey. In addition, CK/AST ratio, proteinuria, and the observed histological and ultrastructural changes suggest that Aotus vociferans suffer from arterial hypertension and chronic myocardial infarction.

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

    PubMed

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

    2009-12-01

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

  6. Activation of dehydrogenase activity and cardiac respiration: A sup 31 P-NMR study

    SciTech Connect

    Katz, L.A.; Koretsky, A.P.; Balaban, R.S. )

    1988-07-01

    {sup 31}P-NMR studies were performed to determine the tissue phosphate and oxygen consumption effects of known maneuvers on the activation of pyruvate dehydrogenase during work jumps in the perfused rat heart. In control studies of the glucose-perfused heart, work jumps, with pacing, resulted in a 32% increase in oxygen consumption ({dot char}Qo{sub 2}) from 1.72 {plus minus} 0.09 to 2.29 {plus minus} 0.12 mmol O{sub 2}{center dot}h{sup {minus}1}{center dot}g dry wt{sup {minus}1}. During this transition no significant change in the high energy phosphates were detected. In contrast, work jumps did cause changes in the phosphates when the activation of pyruvate dehydrogenase was blocked with 2.5 {mu}g of ruthenium red per milliliter or maximally stimulated with 11 mM pyruvate before the increase in work. The observed increase in {dot char}Qo{sub 2} and inorganic phosphate and calculated increase in ADP are consistent with these phosphates controlling mitochondrial respiration under these conditions. These results suggest that the activation of pyruvate dehydrogenase and/or other dehydrogenases may be an important step in the orchestration of work and {dot char}Qo{sub 2}.

  7. Single mutation in Shine-Dalgarno-like sequence present in the amino terminal of lactate dehydrogenase of Plasmodium effects the production of an eukaryotic protein expressed in a prokaryotic system.

    PubMed

    Cicek, Mustafa; Mutlu, Ozal; Erdemir, Aysegul; Ozkan, Ebru; Saricay, Yunus; Turgut-Balik, Dilek

    2013-06-01

    One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5'-GGAGGC-3' sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5'-GGAGGA-3', by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.

  8. Blending foundry sands with soil: Effect on dehydrogenase activity.

    PubMed

    Dungan, Robert S; Kukier, Urzsula; Lee, Brad

    2006-03-15

    Each year U.S. foundries landfill several million tons of sand that can no longer be used to make metalcasting molds and cores. A possible use for these materials is as an ingredient in manufactured soils; however, potentially harmful metals and resin binders (used to make cores) may adversely impact the soil microbial community. In this study, the dehydrogenase activity (DHA) of soil amended with molding sand (clay-coated sand known as "green sand") or core sands at 10%, 30%, and 50% (dry wt.) was determined. The green sands were obtained from iron, aluminum, and brass foundries; the core sands were made with phenol-formaldehyde or furfuryl alcohol based resins. Overall, incremental additions of these sands resulted in a decrease in the DHA which lasted throughout the 12-week experimental period. A brass green sand, which contained high concentrations of Cu, Pb, and Zn, severely impacted the DHA. By week 12 no DHA was detected in the 30% and 50% treatments. In contrast, the DHA in soil amended with an aluminum green sand was 2.1 times higher (all blending ratios), on average, at week 4 and 1.4 times greater (30% and 50% treatments only) than the controls by week 12. In core sand-amended soil, the DHA results were similar to soils amended with aluminum and iron green sands. Increased activity in some treatments may be a result of the soil microorganisms utilizing the core resins as a carbon source. The DHA assay is a sensitive indicator of environmental stress caused by foundry sand constituents and may be useful to assess which foundry sands are suitable for beneficial use in the environment. PMID:15975632

  9. Separation of NADH-fumarate reductase and succinate dehydrogenase activities in Trypanosoma cruzi.

    PubMed

    Christmas, P B; Turrens, J F

    2000-02-15

    A recent review suggested that the activity of NADH-fumarate reductase from trypanosomatids could be catalyzed by succinate dehydrogenase working in reverse (Tielens and van Hellemond, Parasitol. Today 14, 265-271, 1999). The results reported in this study demonstrate that the two activities can easily be separated without any loss in either activity, suggesting that fumarate reductase and succinate dehydrogenase are separate enzymes.

  10. Insufficient filling of vacuum tubes as a cause of microhemolysis and elevated serum lactate dehydrogenase levels. Use of a data-mining technique in evaluation of questionable laboratory test results.

    PubMed

    Tamechika, Yoshie; Iwatani, Yoshinori; Tohyama, Kaoru; Ichihara, Kiyoshi

    2006-01-01

    Experienced physicians noted unexpectedly elevated concentrations of lactate dehydrogenase in some patient samples, but quality control specimens showed no bias. To evaluate this problem, we used a "latent reference individual extraction method", designed to obtain reference intervals from a laboratory database by excluding individuals who have abnormal results for basic analytes other than the analyte in question, in this case lactate dehydrogenase. The reference interval derived for the suspected year was 264-530 U/L, while that of the previous year was 248-495 U/L. The only change we found was the introduction of an order entry system, which requests precise sampling volumes rather than complete filling of vacuum tubes. The effect of vacuum persistence was tested using ten freshly drawn blood samples. Compared with complete filling, 1/5 filling resulted in average elevations of lactate dehydrogenase, aspartic aminotransferase, and potassium levels of 8.0%, 3.8%, and 3.4%, respectively (all p<0.01). Microhemolysis was confirmed using a urine stick method. The length of time before centrifugation determined the degree of hemolysis, while vacuum during centrifugation did not affect it. Microhemolysis is the probable cause of the suspected pseudo-elevation noted by the physicians. Data-mining methodology represents a valuable tool for monitoring long-term bias in laboratory results.

  11. Lactate Effectively Covers Energy Demands during Neuronal Network Activity in Neonatal Hippocampal Slices

    PubMed Central

    Ivanov, Anton; Mukhtarov, Marat; Bregestovski, Piotr; Zilberter, Yuri

    2011-01-01

    Although numerous experimental data indicate that lactate is efficiently used for energy by the mature brain, the direct measurements of energy metabolism parameters during neuronal network activity in early postnatal development have not been performed. Therefore, the role of lactate in the energy metabolism of neurons at this age remains unclear. In this study, we monitored field potentials and contents of oxygen and NAD(P)H in correlation with oxidative metabolism during intense network activity in the CA1 hippocampal region of neonatal brain slices. We show that in the presence of glucose, lactate is effectively utilized as an energy substrate, causing an augmentation of oxidative metabolism. Moreover, in the absence of glucose lactate is fully capable of maintaining synaptic function. Therefore, during network activity in neonatal slices, lactate can be an efficient energy substrate capable of sustaining and enhancing aerobic energy metabolism. PMID:21602909

  12. Aerobic Production and Utilization of Lactate Satisfy Increased Energy Demands Upon Neuronal Activation in Hippocampal Slices and Provide Neuroprotection Against Oxidative Stress

    PubMed Central

    Schurr, Avital; Gozal, Evelyne

    2012-01-01

    Ever since it was shown for the first time that lactate can support neuronal function in vitro as a sole oxidative energy substrate, investigators in the field of neuroenergetics have been debating the role, if any, of this glycolytic product in cerebral energy metabolism. Our experiments employed the rat hippocampal slice preparation with electrophysiological and biochemical methodologies. The data generated by these experiments (a) support the hypothesis that lactate, not pyruvate, is the end-product of cerebral aerobic glycolysis; (b) indicate that lactate plays a major and crucial role in affording neural tissue to respond adequately to glutamate excitation and to recover unscathed post-excitation; (c) suggest that neural tissue activation is accompanied by aerobic lactate and NADH production, the latter being produced when the former is converted to pyruvate by mitochondrial lactate dehydrogenase (mLDH); (d) imply that NADH can be utilized as an endogenous scavenger of reactive oxygen species (ROS) to provide neuroprotection against ROS-induced neuronal damage. PMID:22275901

  13. D-Lactate transport and metabolism in rat liver mitochondria.

    PubMed Central

    de Bari, Lidia; Atlante, Anna; Guaragnella, Nicoletta; Principato, Giovanni; Passarella, Salvatore

    2002-01-01

    In the present study we investigated whether isolated rat liver mitochondria can take up and metabolize D-lactate. We found the following: (1) externally added D-lactate causes oxygen uptake by mitochondria [P/O ratio (the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation)=2] and membrane potential (Delta(psi)) generation in processes that are rotenone-insensitive, but inhibited by antimycin A and cyanide, and proton release from coupled mitochondria inhibited by alpha-cyanocinnamate, but not by phenylsuccinate; (2) the activity of the putative flavoprotein (D-lactate dehydrogenase) was detected in inside-out submitochondrial particles, but not in mitochondria and mitoplasts, as it is localized in the matrix phase of the mitochondrial inner membrane; (3) three novel separate translocators exist to mediate D-lactate traffic across the mitochondrial inner membrane: the D-lactate/H(+) symporter, which was investigated by measuring fluorimetrically the rate of endogenous flavin reduction, the D-lactate/oxoacid antiporter (which mediates both the D-lactate/pyruvate and D-lactate/oxaloacetate exchanges) and D-lactate/malate antiporter studied by monitoring photometrically the appearance of the D-lactate counteranions outside mitochondria. The D-lactate translocators, in the light of their different inhibition profiles separate from the monocarboxylate carrier, were found to differ from each other in the V(max) values and in the inhibition and pH profiles and were shown to regulate mitochondrial D-lactate metabolism in vitro. The D-lactate translocators and the D-lactate dehydrogenase could account for the removal of the toxic methylglyoxal from cytosol, as well as for D-lactate-dependent gluconeogenesis. PMID:11955284

  14. Hepatic alcohol dehydrogenase activity in alcoholic subjects with and without liver disease.

    PubMed Central

    Vidal, F; Perez, J; Morancho, J; Pinto, B; Richart, C

    1990-01-01

    Alcohol dehydrogenase activity was measured in samples of liver tissue from a group of alcoholic and non-alcoholic subjects to determine whether decreased liver alcohol dehydrogenase activity is a consequence of ethanol consumption or liver damage. The alcoholic patients were classified further into the following groups: control subjects with no liver disease (group 1), subjects with non-cirrhotic liver disease (group 2), and subjects with cirrhotic liver disease (group 3). The non-alcoholic subjects were also divided, using the same criteria, into groups 4, 5, and 6, respectively. The analysis of the results showed no significant differences when mean alcohol dehydrogenase activities of alcoholic and non-alcoholic patients with similar degrees of liver pathology were compared (groups 1 v 4, 2 v 5, and 3 v 6. Alcohol dehydrogenase activity was, however, severely reduced in patients with liver disease compared with control subjects. Our findings suggest that alcohol consumption does not modify hepatic alcohol dehydrogenase activity. The reduction in specific alcohol dehydrogenase activity in alcoholic liver disease is a consequence of liver damage. PMID:2379876

  15. Control of rat mammary-gland pyruvate dehydrogenase by insulin and prolactin.

    PubMed Central

    Field, B; Coore, H G

    1976-01-01

    Withdrawal of prolactin or of insulin from the circulation of lactating rats leads, within 3h, to increased inactivation by phosphorylation of mammary-gland pyruvate dehydrogenase. Prolactin may act by priming the tissue to respond directly to normal concentrations of circulating insulin and by this means be responsible for the increased activation of the enzyme during the course of normal lactation. PMID:133680

  16. The primary pathway for lactate oxidation in Desulfovibrio vulgaris

    PubMed Central

    Vita, Nicolas; Valette, Odile; Brasseur, Gaël; Lignon, Sabrina; Denis, Yann; Ansaldi, Mireille; Dolla, Alain; Pieulle, Laetitia

    2015-01-01

    The ability to respire sulfate linked to lactate oxidation is a key metabolic signature of the Desulfovibrio genus. Lactate oxidation by these incomplete oxidizers generates reductants through lactate dehydrogenase (LDH) and pyruvate-ferredoxin oxidoreductase (PFOR), with the latter catalyzing pyruvate conversion into acetyl-CoA. Acetyl-CoA is the source of substrate-level phosphorylation through the production of ATP. Here, we show that these crucial steps are performed by enzymes encoded by a nonacistronic transcriptional unit named now as operon luo (for lactate utilization operon). Using a combination of genetic and biochemical techniques, we assigned a physiological role to the operon genes DVU3027-28 and DVU3032-33. The growth of mutant Δ26-28 was highly disrupted on D-lactate, whereas the growth of mutant Δ32-33 was slower on L-lactate, which could be related to a decrease in the activity of D-lactate or L-lactate oxidase in the corresponding mutants. The DVU3027-28 and DVU3032-33 genes thus encode functional D-LDH and L-LDH enzymes, respectively. Scanning of the genome for lactate utilization revealed several lactate permease and dehydrogenase homologs. However, transcriptional compensation was not observed in any of the mutants except for lactate permease. Although there is a high degree of redundancy for lactate oxidase, it is not functionally efficient in LDH mutants. This result could be related to the identification of several operon enzymes, including LDHs, in the PFOR activity bands, suggesting the occurrence of a lactate-oxidizing supermolecular structure that can optimize the performance of lactate utilization in Desulfovibrio species. PMID:26167158

  17. The primary pathway for lactate oxidation in Desulfovibrio vulgaris.

    PubMed

    Vita, Nicolas; Valette, Odile; Brasseur, Gaël; Lignon, Sabrina; Denis, Yann; Ansaldi, Mireille; Dolla, Alain; Pieulle, Laetitia

    2015-01-01

    The ability to respire sulfate linked to lactate oxidation is a key metabolic signature of the Desulfovibrio genus. Lactate oxidation by these incomplete oxidizers generates reductants through lactate dehydrogenase (LDH) and pyruvate-ferredoxin oxidoreductase (PFOR), with the latter catalyzing pyruvate conversion into acetyl-CoA. Acetyl-CoA is the source of substrate-level phosphorylation through the production of ATP. Here, we show that these crucial steps are performed by enzymes encoded by a nonacistronic transcriptional unit named now as operon luo (for lactate utilization operon). Using a combination of genetic and biochemical techniques, we assigned a physiological role to the operon genes DVU3027-28 and DVU3032-33. The growth of mutant Δ26-28 was highly disrupted on D-lactate, whereas the growth of mutant Δ32-33 was slower on L-lactate, which could be related to a decrease in the activity of D-lactate or L-lactate oxidase in the corresponding mutants. The DVU3027-28 and DVU3032-33 genes thus encode functional D-LDH and L-LDH enzymes, respectively. Scanning of the genome for lactate utilization revealed several lactate permease and dehydrogenase homologs. However, transcriptional compensation was not observed in any of the mutants except for lactate permease. Although there is a high degree of redundancy for lactate oxidase, it is not functionally efficient in LDH mutants. This result could be related to the identification of several operon enzymes, including LDHs, in the PFOR activity bands, suggesting the occurrence of a lactate-oxidizing supermolecular structure that can optimize the performance of lactate utilization in Desulfovibrio species. PMID:26167158

  18. Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria

    PubMed Central

    Land, John M.; Clark, John B.

    1973-01-01

    1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of 14CO2 from [1-14C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a Ki of 100μm. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria. PMID:16742815

  19. The prognostic role of lactate dehydrogenase serum levels in patients with hepatocellular carcinoma who are treated with sorafenib: the influence of liver fibrosis

    PubMed Central

    Yada, Masayoshi; Miyazaki, Masayuki; Motomura, Kenta; Masumoto, Akihide; Nakamuta, Makoto; Kohjima, Motoyuki; Sugimoto, Rie; Aratake, Yoshifusa; Higashi, Nobuhiko; Morizono, Shusuke; Takao, Shinichiro; Yamashita, Naoki; Satoh, Takeaki; Yamashita, Shinsaku; Kuniyoshi, Masami

    2016-01-01

    Background Serum lactate dehydrogenase (LDH) levels could be a prognostic factor for sorafenib-treated patients with several types of solid tumor because it reflects hypoxic circumstances in aggressive tumors. For hepatocellular carcinoma (HCC), however, the prognostic role of LDH has been controversial. Liver fibrosis can potentially cause hypoxia in the liver, which has not been previously studied in the patients with advanced HCC. Thus, we aimed to analyze the prognostic role of LDH based on the degree of fibrosis. Methods Eighty-nine consecutive patients with HCC (Child-Pugh class A) who were treated using sorafenib were enrolled into this study. Pretreatment characteristics and changes in hepatic functional tests based on early response to sorafenib and serum LDH levels were analyzed. The degree of fibrosis was estimated using the aspartate aminotransferase (AST) to platelet ratio index (APRI), and the tumor response was evaluated after 3 months of sorafenib treatment. Results Overall, five patients discontinued sorafenib within 4 weeks. For the other 84 patients, those with progressive disease (PD) had significantly high pretreatment LDH levels, which correlated with the APRI score but not with the tumor stage. Multivariate logistic analysis revealed that older age and lower pretreatment LDH levels were independent prognostic factors for a better response to sorafenib. In patients who discontinued sorafenib early, three experienced acute liver failure accompanied with an increase in serum LDH. Conclusions We demonstrated that baseline serum LDH levels in HCC patients were affected by liver fibrosis but not by the tumor stage, and these LDH levels could be a marker for early response to sorafenib. A marked increase in serum LDH levels during sorafenib administration might also indicate subsequent acute liver failure. Close observation of serum LDH levels before and during sorafenib treatment could be useful in managing treatment of patients receiving this

  20. Poliomyelitis in MuLV-infected ICR-SCID mice after injection of basement membrane matrix contaminated with lactate dehydrogenase-elevating virus.

    PubMed

    Carlson Scholz, Jodi A; Garg, Rohit; Compton, Susan R; Allore, Heather G; Zeiss, Caroline J; Uchio, Edward M

    2011-10-01

    The arterivirus lactate dehydrogenase-elevating virus (LDV) causes life-long viremia in mice. Although LDV infection generally does not cause disease, infected mice that are homozygous for the Fv1(n) allele are prone to develop poliomyelitis when immunosuppressed, a condition known as age-dependent poliomyelitis. The development of age-dependent poliomyelitis requires coinfection with endogenous murine leukemia virus. Even though LDV is a common contaminant of transplantable tumors, clinical signs of poliomyelitis after inadvertent exposure to LDV have not been described in recent literature. In addition, LDV-induced poliomyelitis has not been reported in SCID or ICR mice. Here we describe the occurrence of poliomyelitis in ICR-SCID mice resulting from injection of LDV-contaminated basement membrane matrix. After exposure to LDV, a subset of mice presented with clinical signs including paresis, which was associated with atrophy of the hindlimb musculature, and tachypnea; in addition, some mice died suddenly with or without premonitory signs. Mice presenting within the first 6 mo after infection had regions of spongiosis, neuronal necrosis and astrocytosis of the ventral spinal cord, and less commonly, brainstem. Axonal degeneration of ventral roots prevailed in more chronically infected mice. LDV was identified by RT-PCR in 12 of 15 mice with typical neuropathology; positive antiLDV immunolabeling was identified in all PCR-positive animals (n = 7) tested. Three of 8 mice with neuropathology but no clinical signs were LDV negative by RT-PCR. RT-PCR yielded murine leukemia virus in spinal cords of all mice tested, regardless of clinical presentation or neuropathology.

  1. Karnofsky Performance Status and Lactate Dehydrogenase Predict the Benefit of Palliative Whole-Brain Irradiation in Patients With Advanced Intra- and Extracranial Metastases From Malignant Melanoma

    SciTech Connect

    Partl, Richard; Richtig, Erika; Avian, Alexander; Berghold, Andrea; Kapp, Karin S.

    2013-03-01

    Purpose: To determine prognostic factors that allow the selection of melanoma patients with advanced intra- and extracerebral metastatic disease for palliative whole-brain radiation therapy (WBRT) or best supportive care. Methods and Materials: This was a retrospective study of 87 patients who underwent palliative WBRT between 1988 and 2009 for progressive or multiple cerebral metastases at presentation. Uni- and multivariate analysis took into account the following patient- and tumor-associated factors: gender and age, Karnofsky performance status (KPS), neurologic symptoms, serum lactate dehydrogenase (LDH) level, number of intracranial metastases, previous resection or stereotactic radiosurgery of brain metastases, number of extracranial metastasis sites, and local recurrences as well as regional lymph node metastases at the time of WBRT. Results: In univariate analysis, KPS, LDH, number of intracranial metastases, and neurologic symptoms had a significant influence on overall survival. In multivariate survival analysis, KPS and LDH remained as significant prognostic factors, with hazard ratios of 3.3 (95% confidence interval [CI] 1.6-6.5) and 2.8 (95% CI 1.6-4.9), respectively. Patients with KPS ≥70 and LDH ≤240 U/L had a median survival of 191 days; patients with KPS ≥70 and LDH >240 U/L, 96 days; patients with KPS <70 and LDH ≤240 U/L, 47 days; and patients with KPS <70 and LDH >240 U/L, only 34 days. Conclusions: Karnofsky performance status and serum LDH values indicate whether patients with advanced intra- and extracranial tumor manifestations are candidates for palliative WBRT or best supportive care.

  2. Methylmalonic semialdehyde dehydrogenase deficiency: demonstration of defective valine and beta-alanine metabolism and reduced malonic semialdehyde dehydrogenase activity in cultured fibroblasts

    SciTech Connect

    Gray, R.G.; Pollitt, R.J.; Webley, J.

    1987-08-01

    Intact cultured fibroblasts from a child with a new metabolic disorder, thought to be due to a deficiency of methylmalonic semialdehyde dehydrogenase, produced labeled CO/sub 2/ normally from (1-/sup 14/C)valine but not from (2-/sup 14/C)valine. CO/sub 2/ production from labeled beta-alanine was also much reduced, confirming the suspicion that malonic semialdehyde dehydrogenase is also deficient in this condition. An assay for malonic semialdehyde dehydrogenase in cell homogenates showed low activity but it was impossible to assess the degree of reduction.

  3. Lactate metabolism in the fetal rabbit lung

    SciTech Connect

    Engle, M.J.; Brown, D.J.; Dooley, M.

    1986-05-01

    Lactate is frequently overlooked as a potential substrate for the fetal lung, even though it is present in the fetal circulation in concentrations as high as 8 mM. These high concentrations, coupled with the relatively low levels of glucose in the fetal blood, may indicate that lactate can substitute for glucose in pulmonary energy generation and phospholipid synthesis. A series of experiments was therefore undertaken in order to investigate the role of lactate in perinatal pulmonary development. Explants from 30 day gestation fetal rabbit lungs were incubated in Krebs-Ringer bicarbonate buffer supplemented with 3 mM (U-/sup 14/C)-glucose and varying levels of lactate. In the absence of medium lactate, fetal rabbit lung explants were capable of producing lactate at a rate of approximately 200 etamoles/mg protein/hour. The addition of lactate to the bathing medium immediately reduced net lactate production and above 4 mM, fetal rabbit lung explants became net utilizers of lactate. Media lactate concentrations of 2.5 mM, 5 mM and 10 mM also decreased glucose incorporation into total tissue disaturated phosphatidylcholine by approximately 20%, 35%, and 45%, respectively. Glucose incorporation into surfactant phosphatidylcholine was also reduced by approximately 50%, when lactate was present in the incubation medium at a concentration of 5 mM. Additional experiments also revealed that fetal lung lactate dehydrogenase activity was almost twice that found in the adult rabbit lung. These data indicate that lactate may be an important carbon source for the developing lung and could be a significant component in the manufacture of surfactant phosphatidylcholine during late gestation.

  4. Hemichannel-mediated release of lactate

    PubMed Central

    Karagiannis, Anastassios; Sylantyev, Sergiy; Hadjihambi, Anna; Hosford, Patrick S; Kasparov, Sergey

    2015-01-01

    In the central nervous system lactate contributes to the extracellular pool of readily available energy substrates and may also function as a signaling molecule which mediates communication between glial cells and neurons. Monocarboxylate transporters are believed to provide the main pathway for lactate transport across the membranes. Here we tested the hypothesis that lactate could also be released via opening of pannexin and/or functional connexin hemichannels. In acute slices prepared from the brainstem, hippocampus, hypothalamus and cortex of adult rats, enzymatic amperometric biosensors detected significant tonic lactate release inhibited by compounds, which block pannexin/connexin hemichannels and facilitated by lowering extracellular [Ca2+] or increased PCO2. Enhanced lactate release triggered by hypoxia was reduced by ∼50% by either connexin or monocarboxylate transporter blockers. Stimulation of Schaffer collateral fibers triggered lactate release in CA1 area of the hippocampus, which was facilitated in conditions of low extracellular [Ca2+], markedly reduced by blockade of connexin hemichannels and abolished by lactate dehydrogenase inhibitor oxamate. These results indicate that lactate transport across the membranes may occur via mechanisms other than monocarboxylate transporters. In the central nervous system, hemichannels may function as a conduit of lactate release, and this mechanism is recruited during hypoxia and periods of enhanced neuronal activity. PMID:26661210

  5. Increased lactate/pyruvate ratio augments blood flow in physiologically activated human brain

    NASA Astrophysics Data System (ADS)

    Mintun, Mark A.; Vlassenko, Andrei G.; Rundle, Melissa M.; Raichle, Marcus E.

    2004-01-01

    The factors regulating cerebral blood flow (CBF) changes in physiological activation remain the subject of great interest and debate. Recent experimental studies suggest that an increase in cytosolic NADH mediates increased blood flow in the working brain. Lactate injection should elevate NADH levels by increasing the lactate/pyruvate ratio, which is in near equilibrium with the NADH/NAD+ ratio. We studied CBF responses to bolus lactate injection at rest and in visual stimulation by using positron-emission tomography in seven healthy volunteers. Bolus lactate injection augmented the CBF response to visual stimulation by 38-53% in regions of the visual cortex but had no effect on the resting CBF or the whole-brain CBF. These lactate-induced CBF increases correlated with elevations in plasma lactate/pyruvate ratios and in plasma lactate levels but not with plasma pyruvate levels. Our observations support the hypothesis that an increase in the NADH/NAD+ ratio activates signaling pathways to selectively increase CBF in the physiologically stimulated brain regions.

  6. The risk of thrombo-embolic events is increased in patients with germ-cell tumours and can be predicted by serum lactate dehydrogenase and body surface area

    PubMed Central

    Piketty, A-C; Fléchon, A; Laplanche, A; Nouyrigat, E; Droz, J-P; Théodore, C; Fizazi, K

    2005-01-01

    The aim of this study was to evaluate the risk of thrombo-embolic events (TEE) in patients with germ-cell tumours (GCT) who receive cisplatin-based chemotherapy, to compare this risk to that of a matched control group of non-GCT cancer patients, and to identify risk factors of TEE. The rate of TEE during the 6 months following the initiation of chemotherapy was assessed in 100 consecutive patients with GCT and in 100 controls with various neoplasms who were matched on sex and age, and who received first-line cisplatin-based chemotherapy during the same period of time at Institut Gustave Roussy, Villejuif, France. Data were subsequently tested on a validation group of 77 GCT patients treated in Lyon, France. A total of 19 patients (19%) (95% confidence interval (CI): 13–28) and six patients (6%) (95% CI: 3–13) had a TEE in the GCT group and the non-GCT control group, respectively (relative risk (RR): 3.4; P<0.01). Three patients from the GCT group died of pulmonary embolism. In multivariate analysis, two factors had independent predictive value for TEE: a high body surface area (>1.9 m2) (RR: 5 (1.8–13.9)) and an elevated serum lactate dehydrogenase (LDH) (RR: 6.4 (2.3–18.2)). Patients with no risk factor (n=26) and those with at least one risk factor (n=71) had a probability of having a TEE of 4% (95% CI: 1–19) and 26% (95% CI: 17–37), respectively. In the GCT validation set, 10 (13%) patients had a TEE; patients with no risk factor and those with at least one risk factor had a probability of having a TEE of 0 and 17% (95% CI: 10–29), respectively. Patients with GCT are at a higher risk for TEE than patients with non-GCT cancer while on cisplatin-based chemotherapy. This risk can be accurately predicted by serum LDH and body surface area. This predictive index may help to study prospectively the impact of thromboprophylaxis in GCT patients. PMID:16205699

  7. (1-3)-beta-D-glucan in association with lactate dehydrogenase as biomarkers of Pneumocystis pneumonia (PcP) in HIV-infected patients.

    PubMed

    Esteves, F; Lee, C-H; de Sousa, B; Badura, R; Seringa, M; Fernandes, C; Gaspar, J F; Antunes, F; Matos, O

    2014-07-01

    Pneumocystis pneumonia (PcP) is a major HIV-related illness caused by Pneumocystis jirovecii. Definitive diagnosis of PcP requires microscopic detection of P. jirovecii in pulmonary specimens. The objective of this study was to evaluate the usefulness of two serum markers in the diagnosis of PcP. Serum levels of (1-3)-beta-d-glucan (BG) and lactate dehydrogenase (LDH) were investigated in 100 HIV-positive adult patients and 50 healthy blood donors. PcP cases were confirmed using indirect immunofluorescence with monoclonal anti-Pneumocystis antibodies and nested-PCR to amplify the large subunit mitochondrial rRNA gene of P. jirovecii in pulmonary specimens. BG and LDH levels in serum were measured using quantitative microplate-based assays. BG and LDH positive sera were statistically associated with PcP cases (P ≤ 0.001). Sensitivity, specificity, positive/negative predictive values (PPV/NPV), and positive/negative likelihood ratios (PLR/NLR) were 91.3 %, 61.3 %, 85.1 %, 79.2 %, 2.359, and 0.142, respectively, for the BG kit assay, and 91.3 %, 35.5 %, 75.9 %, 64.7 %, 1.415 and 0.245, respectively, for the LDH test. Serologic markers levels combined with the clinical diagnostic criteria for PcP were evaluated for their usefulness in diagnosis of PcP. The most promising cutoff levels for diagnosis of PcP were determined to be 400 pg/ml of BG and 350 U/l of LDH, which combined with clinical data presented 92.8 % sensitivity, 83.9 % specificity, 92.8 % PPV, 83.9 % NPV, 5.764 PLR and 0.086 NLR (P < 0.001). This study confirmed that BG is a reliable indicator for detecting P. jirovecii infection. The combination between BG/LDH levels and clinical data is a promising alternative approach for PcP diagnosis.

  8. Discovery of 2-((3-cyanopyridin-2-yl)thio)acetamides as human lactate dehydrogenase A inhibitors to reduce the growth of MG-63 osteosarcoma cells: Virtual screening and biological validation.

    PubMed

    Cui, Wei; Lv, Wei; Qu, Ying; Ma, Rui; Wang, Yi-Wei; Xu, Yong-Jun; Wu, Di; Chen, Xuanhuang

    2016-08-15

    Lactate dehydrogenase A (LDHA) has emerged as an attractive target in the oncology field. In this paper, we present the identification of 2-((3-cyanopyridin-2-yl)thio)acetamide-containing compounds as LDHA inhibitors. The in vitro enzymatic assay suggested that inhibitor 9 had good inhibitory potency against LDHA with IC50 value as 1.24μM. Cytotoxicity assay showed that inhibitor 9 strongly inhibited the proliferation of cancer cell MG-63 (EC50=0.98μM). These findings indicated that inhibitor 9 could be employed as a lead for developing more potent LDHA inhibitor with anti-proliferative potency. PMID:27406795

  9. Effects of aluminum on activity of krebs cycle enzymes and glutamate dehydrogenase in rat brain homogenate.

    PubMed

    Zatta, P; Lain, E; Cagnolini, C

    2000-05-01

    Aluminum is a neurotoxic agent for animals and humans that has been implicated as an etiological factor in several neurodegenerative diseases and as a destabilizer of cell membranes. Due to its high reactivity, Al3+ is able to interfere with several biological functions, including enzymatic activities in key metabolic pathways. In this paper we report that, among the enzymes that constitute the Krebs cycle, only two are activated by aluminum: alpha-ketoglutarate dehydrogenase and succinate dehydrogenase. In contrast, aconitase, shows decreased activity in the presence of the metal ion. Al3+ also inhibits glutamate dehydrogenase, an allosteric enzyme that is closely linked to the Krebs cycle. A possible correlation between aluminum, the Krebs cycle and aging processes is discussed.

  10. IL-4 and IL-13 induce protection from complement and melittin in endothelial cells despite initial loss of cytoplasmic proteins: membrane resealing impairs quantifying cytotoxicity with the lactate dehydrogenase permeability assay.

    PubMed

    Benson, Barbara A; Vercellotti, Gregory M; Dalmasso, Agustin P

    2015-01-01

    Endothelial cell activation and injury by the terminal pathway of complement is important in various pathobiological processes, including xenograft rejection. Protection against injury by human complement can be induced in porcine endothelial cells (ECs) with IL-4 and IL-13 through metabolic activation. However, despite this resistance, the complement-treated ECs were found to lose membrane permeability control assessed with the small molecule calcein. Therefore, to define the apparent discrepancy of permeability changes vis-à-vis the protection from killing, we now investigated whether IL-4 and IL-13 influence the release of the large cytoplasmic protein lactate dehydrogenase (LDH) in ECs incubated with complement or the pore-forming protein melittin. Primary cultures of ECs were pre-treated with IL-4 or IL-13 and then incubated with human serum as source of antibody and complement or melittin. Cell death was assessed using neutral red. Membrane permeability was quantitated measuring LDH release. We found that IL-4-/IL-13-induced protection of ECs from killing by complement or melittin despite loss of LDH in amounts similar to control ECs. However, the cytokine-treated ECs that were protected from killing rapidly regained effective control of membrane permeability. Moreover, the viability of the protected ECs was maintained for at least 2 days. We conclude that the protection induced by IL-4/IL-13 in ECs against lethal attack by complement or melittin is effective and durable despite severe initial impairment of membrane permeability. The metabolic changes responsible for protection allow the cells to repair the membrane injury caused by complement or melittin.

  11. Cloning of the rat pyruvate dehydrogenase kinase 4 gene promoter: activation of pyruvate dehydrogenase kinase 4 by the peroxisome proliferator-activated receptor gamma coactivator.

    PubMed

    Ma, Ke; Zhang, Yi; Elam, Marshall B; Cook, George A; Park, Edwards A

    2005-08-19

    The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the metabolism of glucose to acetyl-CoA. Phosphorylation of pyruvate dehydrogenase by the pyruvate dehydrogenase kinases (PDK) inhibits pyruvate dehydrogenase complex activity. There are four PDK isoforms, and expression of PDK4 and PDK2 genes is elevated in starvation and diabetes, allowing glucose to be conserved while fatty acid oxidation is increased. In these studies we have investigated the transcriptional mechanisms by which the expression of the PDK4 gene is increased. The peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of genes involved in hepatic gluconeogenesis and mitochondrial fatty acid oxidation. We have found that PGC-1alpha will induce the expression of both the PDK2 and PDK4 genes in primary rat hepatocytes and ventricular myocytes. We cloned the promoter for the rat PDK4 gene. Hepatic nuclear factor 4 (HNF4), which activates many genes in the liver, will induce PDK4 expression. Although HNF4 and PGC-1alpha interact to stimulate several genes encoding gluconeogenic enzymes, the induction of PDK4 does not involve interactions of PGC-1alpha with HNF4. Using the chromatin immunoprecipitation assay, we have demonstrated that HNF4 and PGC-1alpha are associated with the PDK4 gene in vivo. Our data suggest that by inducing PDK genes PGC-1alpha will direct pyruvate away from metabolism into acetyl-CoA and toward the formation of oxaloacetate and into the gluconeogenic pathway. PMID:15967803

  12. Measuring the Impact of Microenvironmental Conditions on Mitochondrial Dehydrogenase Activity in Cultured Cells.

    PubMed

    Sun, Ramon C; Koong, Albert; Giaccia, Amato; Denko, Nicholas C

    2016-01-01

    Mitochondria are powerhouses of a cell, producing much of the cellular ATP. However, mitochondrial enzymes also participate in many cellular biosynthetic processes. They are responsible for helping to maintain NAD(P)/H and redox balance, supplying metabolic intermediates for cell growth, and regulating several types of programed cell death. Several mitochondrial enzymes have even been shown to participate in the oncogenic process such as isocitrate dehydrogenase, succinate dehydrogenase, and fumarate hydratase. Recent advances have identified significant metabolic changes in the mitochondria that are regulated by malignant transformation and environmental stimuli. Understanding the biological activity and regulation of mitochondrial enzymes can provide insight into how they participate in the process of oncogenic transformation and work to sustain malignant growth. This chapter describes a technique to measure mitochondrial dehydrogenase activities that is faster and more cost effective which can also be scaled up for high throughput. PMID:27325264

  13. Immune response to lactate dehydrogenase-elevating virus: isolation of infectious virus-immunoglobulin G complexes and quantitation of specific antiviral immunoglobulin G response in wild-type and nude mice.

    PubMed Central

    Cafruny, W A; Plagemann, P G

    1982-01-01

    Lactate dehydrogenase-elevating virus (LDV) causes a normally benign persistent infection of mice, resulting in a life-long viremia characterized by the presence of circulating infectious immune complexes, impaired clearance of certain enzymes from the blood, and modification of the host immune response to various heterologous antigens. In this study, we isolated infectious immunoglobulin G (IgG)-LDV complexes in the plasma of persistently infected mice by adsorption to and elution from protein A-Sepharose CL-4B. We found that practically all infectious LDV in the plasma of persistently infected mice is complexed to IgG. LDV infectivity in these complexes was partially neutralized, but could be reactivated by treatment with 2-mercaptoethanol. We also quantitated total plasma IgG and anti-LDV IgG in wild-type and nude Swiss and BALB/c mice as a function of the time after infection with LDV by radial immunodiffusion and an enzyme-linked immunosorbent assay, respectively. Total plasma IgG levels nearly doubled in BALB/c mice during 150 days of infection. IgG levels in uninfected nude mice were only 20% of those in uninfected BALB/c mice, but during infection with LDV increased to approximately those found in uninfected BALB/c mice. Anti-LDV IgG levels were almost as high in nude mice as in normal BALB/c mice. Isoelectric focusing of purified IgG from BALB/c mice showed that LDV infection resulted in the enhanced synthesis of all 16 normal IgG fractions that we could separate by this method, which suggests that LDV infection results in polyclonal activation of IgG-producing lymphocytes. PMID:7129626

  14. Decreased number and bactericidal activity against Staphylococcus aureus of the resident cells in milk of dairy cows during early lactation.

    PubMed

    Dosogne, H; Vangroenweghe, F; Barrio, B; Rainard, P; Burvenich, C

    2001-11-01

    Phagocytic and bactericidal activity of polymorphonuclear neutrophil leukocytes (PMN) isolated from blood and milk, against Staphylococcus aureus, was compared between groups of six healthy dairy cows in early, mid- and late lactation using a bacteriological assay. PMN were isolated from blood with a high degree of purity, but the cells isolated from milk contained variable amounts of macrophages (Mphi) and lymphocytes (L). The results were therefore calculated using the percentage PMN in order to evaluate phagocytosis and killing by PMN only. Blood PMN phagocytosed 82% Staph. aureus and milk PMN 43% on average and there was no significant difference between the different stages of lactation. The bactericidal activity of blood PMN against Staph. aureus was 36+/-8% in early lactation (significantly different from mid lactation, P < 0.05), 64+/-10% in mid lactation and 53+/-6% in late lactation. Milk PMN killed only 6+/-3% Staph. aureus in early lactation (significantly different from mid lactation, P < 0.01), 27+/-3% in mid lactation and 20+/-9% Staph. aureus in late lactation. The ratio of the bactericidal activity of milk to blood PMN was 0.08, 0.43 and 0.22 in early, mid- and late lactation, respectively. In addition to the decreased function. the number of cells in milk (somatic cell count, SCC) was also 60% lower in early lactation than in mid lactation cows (P < 0.01). Our results suggest an impairment of blood and milk-resident PMN bactericidal activity against Staph. aureus and a decreased number of milk-resident PMN in dairy cows at the onset of lactation.

  15. Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775.

    PubMed

    Snoep, J L; Teixeira de Mattos, M J; Postma, P W; Neijssel, O M

    1990-01-01

    Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are discussed. In particular attention is paid to the NADH/NAD ratio (redox potential) and the fructose-1,6-bisphosphate-dependent lactate dehydrogenase activity as possible regulatory factors.

  16. Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase.

    PubMed

    Herdendorf, Timothy J; Plapp, Bryce V

    2011-05-30

    The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD(+) and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 Å resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.

  17. Amino ketone formation and aminopropanol-dehydrogenase activity in rat-liver preparations

    PubMed Central

    Turner, J. M.; Willetts, A. J.

    1967-01-01

    1. Rat tissue homogenates convert dl-1-aminopropan-2-ol into aminoacetone. Liver homogenates have relatively high aminopropanol-dehydrogenase activity compared with kidney, heart, spleen and muscle preparations. 2. Maximum activity of liver homogenates is exhibited at pH9·8. The Km for aminopropanol is approx. 15mm, calculated for a single enantiomorph, and the maximum activity is approx. 9mμmoles of aminoacetone formed/mg. wet wt. of liver/hr.at 37°. Aminoacetone is also formed from l-threonine, but less rapidly. An unidentified amino ketone is formed from dl-4-amino-3-hydroxybutyrate, the Km for which is approx. 200mm at pH9·8. 3. Aminopropanol-dehydrogenase activity in homogenates is inhibited non-competitively by dl-3-hydroxybutyrate, the Ki being approx. 200mm. EDTA and other chelating agents are weakly inhibitory, and whereas potassium chloride activates slightly at low concentrations, inhibition occurs at 50–100mm. 4. It is concluded that aminopropanol-dehydrogenase is located in mitochondria, and in contrast with l-threonine dehydrogenase can be readily solubilized from mitochondrial preparations by ultrasonic treatment. 5. Soluble extracts of disintegrated mitochondria exhibit maximum aminopropanol-dehydrogenase activity at pH9·1 At this pH, Km values for the amino alcohol and NAD+ are approx. 200 and 1·3mm respectively. Under optimum conditions the maximum velocity is approx. 70mμmoles of aminoacetone formed/mg. of protein/hr. at 37°. Chelating agents and thiol reagents appear to have little effect on enzyme activity, but potassium chloride inhibits at all concentrations tested up to 80mm. dl-3-Hydroxybutyrate is only slightly inhibitory. 6. Dehydrogenase activities for l-threonine and dl-4-amino-3-hydroxybutyrate appear to be distinct from that for aminopropanol. 7. Intraperitoneal injection of aminopropanol into rats leads to excretion of aminoacetone in the urine. Aminoacetone excretion proportional to the amount of the amino alcohol

  18. Utilization of d-Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans

    PubMed Central

    Sheng, Binbin; Xu, Jing; Zhang, Yingxin; Jiang, Tianyi; Deng, Sisi; Kong, Jian; Ma, Cuiqing; Xu, Ping

    2015-01-01

    d-Lactate was identified as one of the few available organic acids that supported the growth of Gluconobacter oxydans 621H in this study. Interestingly, the strain used d-lactate as an energy source but not as a carbon source, unlike other lactate-utilizing bacteria. The enzymatic basis for the growth of G. oxydans 621H on d-lactate was therefore investigated. Although two putative NAD-independent d-lactate dehydrogenases, GOX1253 and GOX2071, were capable of oxidizing d-lactate, GOX1253 was the only enzyme able to support the d-lactate-driven growth of the strain. GOX1253 was characterized as a membrane-bound dehydrogenase with high activity toward d-lactate, while GOX2071 was characterized as a soluble oxidase with broad substrate specificity toward d-2-hydroxy acids. The latter used molecular oxygen as a direct electron acceptor, a feature that has not been reported previously in d-lactate-oxidizing enzymes. This study not only clarifies the mechanism for the growth of G. oxydans on d-lactate, but also provides new insights for applications of the important industrial microbe and the novel d-lactate oxidase. PMID:25862219

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

    PubMed

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

    2013-07-01

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

  20. Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

    SciTech Connect

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.; Kakar, S. N.; Stevens, F. J.; Donnelly, M. I.; Nebraska Wesleyan Univ.

    1995-09-10

    Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the native enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the mutant

  1. Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases.

    PubMed

    Sugden, Mary C; Holness, Mark J

    2006-07-01

    The mechanisms that control mammalian pyruvate dehydrogenase complex (PDC) activity include its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDKs 1 - 4). Here we review new developments in the regulation of the activities and expression of the PDKs, in particular PDK2 and PDK4, in relation to glucose and lipid homeostasis. This review describes recent advances relating to the acute and long-term modes of regulation of the PDKs, with particular emphasis on the regulatory roles of nuclear receptors including peroxisome proliferator-activated receptor (PPAR) alpha and Liver X receptor (LXR), PPAR gamma coactivator alpha (PGC-1alpha) and insulin, and the impact of changes in PDK activity and expression in glucose and lipid homeostasis. Since PDK4 may assist in lipid clearance when there is an imbalance between lipid delivery and oxidation, it may represent an attractive target for interventions aimed at rectifying abnormal lipid as well as glucose homeostasis in disease states. PMID:17132539

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

    PubMed

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

    2010-10-01

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

  3. Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth

    PubMed Central

    2014-01-01

    L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD+-dependent L-lactate dehydrogenase. Naturally, Escherichia coli does not produce L-lactate in noticeable amounts, but can catabolize it via a dehydrogenation reaction mediated by an FMN-dependent L-lactate dehydrogenase. In aims to make the E. coli strain to produce L-lactate, three L-lactate dehydrogenase genes from different bacteria were cloned and expressed. The L-lactate producing strains, 090B1 (B0013-070, ΔldhA::diflldD::Pldh-ldhLca), 090B2 (B0013-070, ΔldhA::diflldD::Pldh-ldhStrb) and 090B3 (B0013-070, ΔldhA::diflldD::Pldh-ldhBcoa) were developed from a previously developed D-lactate over-producing strain, E. coli strain B0013-070 (ack-ptappspflBdldpoxBadhEfrdA) by: (1) deleting ldhA to block D-lactate formation, (2) deleting lldD to block the conversion of L-lactate to pyruvate, and (3) expressing an L-lactate dehydrogenase (L-LDH) to convert pyruvate to L-lactate under the control of the ldhA promoter. Fermentation tests were carried out in a shaking flask and in a 25-l bioreactor. Strains 090B1, 090B2 or 090B3 were shown to metabolize glucose to L-lactate instead of D-lactate. However, L-lactate yield and cell growth rates were significantly different among the metabolically engineered strains which can be attributed to a variation between temperature optimum for cell growth and temperature optimum for enzymatic activity of individual L-LDH. In a temperature-shifting fermentation process (cells grown at 37°C and L-lactate formed at 42°C), E. coli 090B3 was able to produce 142.2 g/l of L-lactate with no more than 1.2 g/l of by-products (mainly acetate, pyruvate and succinate) accumulated. In conclusion, the production of lactate by E. coli is limited by the competition relationship between cell growth and lactate synthesis. Enzymatic properties, especially the thermodynamics of an L

  4. The dihydrolipoamide dehydrogenase of Aeromonas caviae ST exhibits NADH-dependent tellurite reductase activity.

    PubMed

    Castro, Miguel E; Molina, Roberto; Díaz, Waldo; Pichuantes, Sergio E; Vásquez, Claudio C

    2008-10-10

    Potassium tellurite (K(2)TeO(3)) is extremely toxic for most forms of life and only a limited number of organisms are naturally resistant to the toxic effects of this compound. Crude extracts prepared from the environmental isolate Aeromonas caviae ST catalize the in vitro reduction of TeO32- in a NADH-dependent reaction. Upon fractionation by ionic exchange column chromatography three major polypeptides identified as the E1, E2, and E3 components of the pyruvate dehydrogenase (PDH) complex were identified in fractions exhibiting tellurite-reducing activity. Tellurite reductase and pyruvate dehydrogenase activities co-eluted from a Sephadex gel filtration column. To determine which component(s) of the PDH complex has tellurite reductase activity, the A. caviae ST structural genes encoding for E1 (aceE), E2 (aceF), and E3 (lpdA) were independently cloned and expressed in Escherichia coli and their gene products purified. Results indicated that tellurite reductase activity lies almost exclusively in the E3 component, dihydrolipoamide dehydrogenase. The E3 component of the PDH complex from E. coli, Zymomonas mobilis, Streptococcus pneumoniae, and Geobacillus stearothermophilus also showed NADH-dependent tellurite reductase in vitro suggesting that this enzymatic activity is widely distributed among microorganisms. PMID:18675788

  5. Activity patterns, blood lactate concentrations and ratings of perceived exertion during a professional singles tennis tournament

    PubMed Central

    Mendez‐Villanueva, Alberto; Fernandez‐Fernandez, Jaime; Bishop, David; Fernandez‐Garcia, Benjamin; Terrados, Nicolas

    2007-01-01

    Objective To examine the game characteristics and physiological (ie, blood lactate concentration) and perceptual (ie, rating of perceived exertion, RPE) responses during actual tennis competition in professional performers. Methods Eight trained and internationally ranked (Association of Tennis Professionals rankings) male tennis players were studied during singles matches (best of three sets) played on an outdoor clay court surface during a professional, invitational tournament. Blood lactate concentrations (n = 53) and RPE (n = 113) were determined at selected changeovers during the game. The variables describing the characteristics of the matches, (a) duration of rallies (DRs); (b) rest time (RT); (c) effective playing time (EPT); and (d) shots per rally (SR), were determined from video recordings. Results The mean (SD) values for the match‐play activity variables were DR 7.5 (7.3) s, RT 16.2 (5.2) s, EPT 21.5 (4.9%), SR 2.7 (2.2) shots. Average blood lactate concentration and RPE values were 3.8 (2.0) mmol/l and 13 (2). Blood lactate concentrations and RPE values were significantly higher (p<0.01) in service games than in receiving games. Both blood lactate concentration and RPE values were significantly correlated with SR and DR (r = 0.80 to 0.28; p<0.001). Conclusions Blood lactate concentrations and RPE were found to be influenced by the characteristics of the match and the playing situation (ie, serving or returning). These specific situations might be used to alter the overload training stimulus during tennis on‐court practice. PMID:17237121

  6. Stability and activity of alcohol dehydrogenases in W/O-microemulsions: enantioselective reduction including cofactor regeneration.

    PubMed

    Orlich, B; Berger, H; Lade, M; Schomäcker, R

    2000-12-20

    Microemulsions provide an interesting alternative to classical methods for the conversion of less water-soluble substrates by alcohol dehydrogenase, but until now stability and activity were too low for economically useful processes. The activity and stability of the enzymes are dependent on the microemulsion composition, mostly the water and the surfactant concentration. Therefore, it is necessary to know the exact phase behavior of a given microemulsion reaction system and the corresponding enzyme behavior therein. Because of their economic and ecologic suitability polyethoxylated fatty alcohols were investigated concerning their phase behavior and their compatibility with enzymes in ternary mixtures. The phase behavior of Marlipal O13-60 (C13EO6 in industrial quality)/cyclohexane/water and its effect on the activity and stability of alcohol dehydrogenase from Yeast (YADH) and horse liver (HLADH) and the carbonyl reductase from Candida parapsilosis (CPCR) is presented in this study. Beside the macroscopic phase behavior of the reaction system, the viscosity of the system indicates structural changes of aggregates in the microemulsion. The changes of the enzyme activities with the composition are discussed on the basis of transitions from reverse micelles to swollen reverse micelles and finally, the transition to the phase separation. The formate dehydrogenase from Candida boidinii was used for the NADH-regeneration during reduction reactions. While the formate dehydrogenase did not show any kinetic effect on the microemulsion composition, the other enzymes show significant changes of activity and stability varying the water or surfactant concentration of the microemulsion. Under certain conditions, stability could be maintained with HLADH for several weeks. Successful experiments with semi-batch processes including cofactor regeneration and product separation were performed.

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

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

  8. Removal of CO dehydrogenase from Pseudomonas carboxydovorans cytoplasmic membranes, rebinding of CO dehydrogenase to depleted membranes, and restoration of respiratory activities.

    PubMed Central

    Jacobitz, S; Meyer, O

    1989-01-01

    In Pseudomonas carboxydovorans, CO dehydrogenase and hydrogenase were found in association with the cytoplasmic membrane in a weakly bound and a tightly bound pool. The pools could be experimentally distinguished on the basis of resistance to removal by washes in low-ionic-strength buffer. The tightly bound pool of the enzymes could be differentially solubilized under conditions leaving the electron transport system intact and with the nondenaturing zwitterionic detergent 3-(3-cholamidopropyl) dimethylammonio 1-propane-sulfonic acid (CHAPS) and the nonionic detergent dodecyl beta-D-maltoside. In vitro reconstitution of depleted membranes with the corresponding supernatants containing CO dehydrogenase led to binding of the enzyme and to reactivation of respiratory activities with CO. The reconstitution reaction required cations with effectiveness which increased with increasing ionic charge: monovalent (Li+), divalent (Mg2+, Mn2+), or trivalent (Cr3+, La3+). Reconstitution of depleted membranes with CO dehydrogenase was specific for CO-grown bacteria. Cytoplasmic membranes from H2- or heterotrophically grown Pseudomonas carboxydovorans had no affinity for CO dehydrogenase at all, indicating the absence of the physiological electron acceptor of the enzyme, which presumably is cytochrome b561, or another membrane anchor. PMID:2808305

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  13. Evolutionary origins of retinoid active short-chain dehydrogenases/reductases of SDR16C family

    PubMed Central

    Belyaeva, Olga V.; Chang, Chenbei; Berlett, Michael C; Kedishvili, Natalia Y.

    2014-01-01

    Vertebrate enzymes that belong to the 16C family of short-chain dehydrogenases/reductases (SDR16C) were shown to play an essential role in the control of retinoic acid (RA) levels during development. To trace the evolution of enzymatic function of SDR16C family, and to examine the origins of the pathway for RA biosynthesis from vitamin A, we identified putative SDR16C enzymes through the extensive search of available genome sequencing data in a subset of species representing major metazoan phyla. The phylogenetic analysis revealed that enzymes from protostome, non-chordate deuterostome and invertebrate chordate species are found in three clades of SDR16C family containing retinoid active enzymes, which are retinol dehydrogenase 10 (RDH10), retinol dehydrogenases E2 (RDHE2) and RDHE2-similar, and dehydrogenase reductase (SDR family) member 3 (DHRS3). For the initial functional analysis, we cloned RDH10- and RDHE2-related enzymes from the early developmental stages of a non-chordate deuterostome, green sea urchin Lytechinus variegatus, and an invertebrate chordate, sea squirt Ciona intestinalis. In situ hybridization revealed that these proteins are expressed in a pattern relevant to development, while assays performed on proteins expressed in mammalian cell culture showed that they possess retinol-oxidizing activity as their vertebrate homologs. The existence of invertebrate homologs of DHRS3 was inferred from the analysis of phylogeny and cofactor-binding residues characteristic of preference for NADP(H). The presence of invertebrate homologs in the DHRS3 group of SDR16C is interesting in light of the complex mutually activating interaction, which we have recently described for human RDH10 and DHRS3 enzymes. Further functional analysis of these homologs will establish whether this interaction evolved to control retinoid homeostasis only in vertebrates, or is also conserved in pre-vertebrates. PMID:25451586

  14. Escherichia coli d-Malate Dehydrogenase, a Generalist Enzyme Active in the Leucine Biosynthesis Pathway*

    PubMed Central

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

    2014-01-01

    The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of d-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on d-malate as a carbon source, the d-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB− strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (l(+)-tartrate, d-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution. PMID:25160617

  15. [The pentose phosphate pathway and NADP-dependent glycerol-3-phosphate dehydrogenase activity in some tissues of albino rat].

    PubMed

    Glushankov, E P; Epifanova, Iu E; Kolotilova, A I

    1976-10-01

    The NADP-dependent glycerol-3-phosphate dehydrogenase activity in liver, heart and skeletal muscle of rat was studied. The activity is found when glyceraldehyde-3-phosphate or ribose-5-phosphate in the presence of ATP are taken as substrates. The data obtained confirm that NADP-dependent glycerol-3-phosphate dehydrogenase exists in skeletal muscle and demonstrate that it is found in heart muscle as well.

  16. Complete Deficiency of Leukocyte Glucose-6-Phosphate Dehydrogenase with Defective Bactericidal Activity

    PubMed Central

    Cooper, M. Robert; DeChatelet, Lawrence R.; McCall, Charles E.; La Via, Mariano F.; Spurr, Charles L.; Baehner, Robert L.

    1972-01-01

    A 52 yr old Caucasian female (F. E.) had hemolytic anemia, a leukemoid reaction, and fatal sepsis due to Escherichia coli. Her leukocytes ingested bacteria normally but did not kill catalase positive Staphylococcus aureus, Escherichia coli, and Serratia marcescens. An H2O2-producing bacterium, Streptococcus faecalis, was killed normally. Granule myeloperoxidase, acid and alkaline phosphatase, and beta glucuronidase activities were normal, and these enzymes shifted normally to the phagocyte vacuole (light and electron microscopy). Intravacuolar reduction of nitroblue tetrazolium did not occur. Moreover, only minimal quantities of H2O2 were generated, and the hexose monophosphate shunt (HMPS) was not stimulated during phagocytosis. These observations suggested the diagnosis of chronic granulomatous disease. However, in contrast to control and chronic granulomatous disease leukocytes, glucose-6-phosphate dehydrogenase activity was completely absent in F. E. leukocytes whereas NADH oxidase and NADPH oxidase activities were both normal. Unlike chronic granulomatous disease, methylene blue did not stimulate the hexose monophosphate shunt in F. E. cells. Thus, F. E. and chronic granulomatous disease leukocytes appear to share certain metabolic and bactericidal defects, but the metabolic basis of the abnormality differs. Chronic granulomatous disease cells lack oxidase activity which produces H2O2; F. E. cells had normal levels of oxidase activity but failed to produce NADPH due to complete glucose-6-phosphate dehydrogenase deficiency. These data indicate that a complete absence of leukocyte glucose-6-phosphate dehydrogenase with defective hexose monophosphate shunt activity is associated with low H2O2 production and inadequate bactericidal activity, and further suggest an important role for NADPH in the production of H2O2 in human granulocytes. Images PMID:4401271

  17. Mitochondrial complex I, aconitase, and succinate dehydrogenase during hypoxia-reoxygenation: modulation of enzyme activities by MnSOD.

    PubMed

    Powell, Charles S; Jackson, Robert M

    2003-07-01

    Both NADH dehydrogenase (complex I) and aconitase are inactivated partially in vitro by superoxide (O2-.) and other oxidants that cause loss of iron from enzyme cubane (4Fe-4S) centers. We tested whether hypoxia-reoxygenation (H-R) by itself would decrease lung epithelial cell NADH dehydrogenase, aconitase, and succinate dehydrogenase (SDH) activities and whether transfection with adenoviral vectors expressing MnSOD (Ad.MnSOD) would inhibit oxidative enzyme inactivation and thus confirm a mechanism involving O2-. Human lung carcinoma cells with alveolar epithelial cell characteristics (A549 cells) were exposed to <1% O2-5% CO2 (hypoxia) for 24 h followed by air-5% CO2 for 24 h (reoxygenation). NADH dehydrogenase activity was assayed in submitochondrial particles; aconitase and SDH activities were measured in cell lysates. H-R significantly decreased NADH dehydrogenase, aconitase, and SDH activities. Ad.MnSOD increased mitochondrial MnSOD substantially and prevented the inhibitory effects of H-R on enzyme activities. Addition of alpha-ketoglutarate plus aspartate, but not succinate, to medium prevented cytotoxicity due to 2,3-dimethoxy-1,4-naphthoquinone. After hypoxia, cells displayed significantly increased dihydrorhodamine fluorescence, indicating increased mitochondrial oxidant production. Inhibition of NADH dehydrogenase, aconitase, and SDH activities during reoxygenation are due to excess O2-. produced in mitochondria, because enzyme inactivation can be prevented by overexpression of MnSOD. PMID:12665464

  18. A bifunctional enzyme from Rhodococcus erythropolis exhibiting secondary alcohol dehydrogenase-catalase activities.

    PubMed

    Martinez-Rojas, Enriqueta; Kurt, Tutku; Schmidt, Udo; Meyer, Vera; Garbe, Leif-Alexander

    2014-11-01

    Alcohol dehydrogenases have long been recognized as potential biocatalyst for production of chiral fine and bulk chemicals. They are relevant for industry in enantiospecific production of chiral compounds. In this study, we identified and purified a nicotinamide adenine dinucleotide (NAD)-dependent secondary alcohol dehydrogenase (SdcA) from Rhodococcus erythropolis oxidizing γ-lactols into γ-lactones. SdcA showed broad substrate specificity on γ-lactols; secondary aliphatic alcohols with 8 and 10 carbon atoms were also substrates and oxidized with (2S)-stereospecificity. The enzyme exhibited moderate stability with a half-life of 5 h at 40 °C and 20 days at 4 °C. Mass spectrometric identification revealed high sequence coverage of SdcA amino acid sequence to a highly conserved catalase from R. erythropolis. The corresponding encoding gene was isolated from genomic DNA and subsequently overexpressed in Escherichia coli BL21 DE3 cells. In addition, the recombinant SdcA was purified and characterized in order to confirm that the secondary alcohol dehydrogenase and catalase activity correspond to the same enzyme.

  19. Adaptation of methods for glutamate dehydrogenase and alcohol dehydrogenase activities to a centrifugal analyser: assessment of their clinical use in anoxic states of the liver.

    PubMed Central

    Shephard, M D; Penberthy, L A; Berry, M N

    1987-01-01

    Sensitive, precise, and rapid methods for the measurement of alcohol dehydrogenase (ADH) and glutamate dehydrogenase (GDH) were developed on the Cobas Bio centrifugal analyser. The optimal pH for ADH in caucasians was 9.8. Non-linearity of ADH enzyme activity was observed when samples were diluted in saline; linearity was restored when inactivated serum was used as diluent. ADH was shown to be a sensitive index of liver anoxia due to cardiorespiratory disturbance (clinical sensitivity 90%) and generalised anoxia. GDH exhibited sensitivity equal to that of alanine aminotransferase (ALT) but was inferior to gamma-glutamyltransferase (GGT) in the detection of specific liver disease. Both ADH and GDH were sensitive indicators of alcoholic liver disease. PMID:2890662

  20. Differential effects of acute and chronic fructose administration on pyruvate dehydrogenase activity and lipogenesis

    SciTech Connect

    Wilson, L.

    1988-01-01

    These studies were undertaken to distinguish between the acute and chronic effects of fructose administration. In vivo, liver lipogenesis, as measured by {sup 3}H{sub 2}O incorporation, was greater in rats fed 60% fructose than in their glucose fed controls. Both fructose feeding, and fructose feeding plus intraperitoneal fructose injection increased the activities of 6-phosphogluconate dehydrogenase and malic enzyme. Liver PDH activity was increased by fructose feeding, and was increased even more by fructose feeding and injection of fructose, but this was not associated with any changes in hepatic ATP concentrations.

  1. Induction of triglyceride accumulation and mitochondrial maintenance in muscle cells by lactate.

    PubMed

    Sun, Jingquan; Ye, Xin; Xie, Minhao; Ye, Jianping

    2016-01-01

    Muscle exercise induces intramuscular triglyceride (TG) accumulation and promotes mitochondrial maintenance in myotubes. However, the mechanism underlying exercise effects remains unknown. In this study, lactic acid was tested as a signaling molecule in C2C12 myotubes to understand the mechanism. Intracellular TG storage was induced in the cells by sodium lactate. The lactate activity was observed with an inhibition of the cAMP-PKA pathway as indicated by a reduction in the phosphorylation status of CREB (pCREB). Induction of pCREB signal by forskolin was blocked by pretreatment of cells with lactate. The impact of lactate on mitochondrial function was examined with a focus on the activities of two enzymes, MCAT (malonylCoA:ACP transferase) and PDH (pyruvate dehydrogenase). The enzyme activities were induced in the cells by lactate. Expression of the lactate receptor (GPR81) and lactate transporters (MCT1/4) were induced as well by lactate. The lactate activities were observed at concentrations between 4-64 mM, and were not dependent on the increase in intracellular pyruvate. Pyruvate treatment did not generate the same effects in the cells. Those results suggest that lactate may induce intramuscular TG storage and mitochondrial maintenance in myotubes through inhibition of the cAMP pathway by activation of GPR81 in a positive feedback manner. PMID:27645401

  2. Induction of triglyceride accumulation and mitochondrial maintenance in muscle cells by lactate

    PubMed Central

    Sun, Jingquan; Ye, Xin; Xie, Minhao; Ye, Jianping

    2016-01-01

    Muscle exercise induces intramuscular triglyceride (TG) accumulation and promotes mitochondrial maintenance in myotubes. However, the mechanism underlying exercise effects remains unknown. In this study, lactic acid was tested as a signaling molecule in C2C12 myotubes to understand the mechanism. Intracellular TG storage was induced in the cells by sodium lactate. The lactate activity was observed with an inhibition of the cAMP-PKA pathway as indicated by a reduction in the phosphorylation status of CREB (pCREB). Induction of pCREB signal by forskolin was blocked by pretreatment of cells with lactate. The impact of lactate on mitochondrial function was examined with a focus on the activities of two enzymes, MCAT (malonylCoA:ACP transferase) and PDH (pyruvate dehydrogenase). The enzyme activities were induced in the cells by lactate. Expression of the lactate receptor (GPR81) and lactate transporters (MCT1/4) were induced as well by lactate. The lactate activities were observed at concentrations between 4–64 mM, and were not dependent on the increase in intracellular pyruvate. Pyruvate treatment did not generate the same effects in the cells. Those results suggest that lactate may induce intramuscular TG storage and mitochondrial maintenance in myotubes through inhibition of the cAMP pathway by activation of GPR81 in a positive feedback manner. PMID:27645401

  3. Induction of triglyceride accumulation and mitochondrial maintenance in muscle cells by lactate.

    PubMed

    Sun, Jingquan; Ye, Xin; Xie, Minhao; Ye, Jianping

    2016-09-20

    Muscle exercise induces intramuscular triglyceride (TG) accumulation and promotes mitochondrial maintenance in myotubes. However, the mechanism underlying exercise effects remains unknown. In this study, lactic acid was tested as a signaling molecule in C2C12 myotubes to understand the mechanism. Intracellular TG storage was induced in the cells by sodium lactate. The lactate activity was observed with an inhibition of the cAMP-PKA pathway as indicated by a reduction in the phosphorylation status of CREB (pCREB). Induction of pCREB signal by forskolin was blocked by pretreatment of cells with lactate. The impact of lactate on mitochondrial function was examined with a focus on the activities of two enzymes, MCAT (malonylCoA:ACP transferase) and PDH (pyruvate dehydrogenase). The enzyme activities were induced in the cells by lactate. Expression of the lactate receptor (GPR81) and lactate transporters (MCT1/4) were induced as well by lactate. The lactate activities were observed at concentrations between 4-64 mM, and were not dependent on the increase in intracellular pyruvate. Pyruvate treatment did not generate the same effects in the cells. Those results suggest that lactate may induce intramuscular TG storage and mitochondrial maintenance in myotubes through inhibition of the cAMP pathway by activation of GPR81 in a positive feedback manner.

  4. Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor, GPR81.

    PubMed

    Liu, Changlu; Wu, Jiejun; Zhu, Jessica; Kuei, Chester; Yu, Jingxue; Shelton, Jonathan; Sutton, Steven W; Li, Xiaorong; Yun, Su Jin; Mirzadegan, Taraneh; Mazur, Curt; Kamme, Fredrik; Lovenberg, Timothy W

    2009-01-30

    Lactic acid is a well known metabolic by-product of intense exercise, particularly under anaerobic conditions. Lactate is also a key source of energy and an important metabolic substrate, and it has also been hypothesized to be a signaling molecule directing metabolic activity. Here we show that GPR81, an orphan G-protein-coupled receptor highly expressed in fat, is in fact a sensor for lactate. Lactate activates GPR81 in its physiological concentration range of 1-20 mM and suppresses lipolysis in mouse, rat, and human adipocytes as well as in differentiated 3T3-L1 cells. Adipocytes from GPR81-deficient mice lack an antilipolytic response to lactate but are responsive to other antilipolytic agents. Lactate specifically induces internalization of GPR81 after receptor activation. Site-directed mutagenesis of GPR81 coupled with homology modeling demonstrates that classically conserved key residues in the transmembrane binding domains are responsible for interacting with lactate. Our results indicate that lactate suppresses lipolysis in adipose tissue through a direct activation of GPR81. GPR81 may thus be an attractive target for the treatment of dyslipidemia and other metabolic disorders.

  5. Gene algD coding for GDPmannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa.

    PubMed Central

    Deretic, V; Gill, J F; Chakrabarty, A M

    1987-01-01

    Transcriptional regulation of alginate biosynthesis by Pseudomonas aeruginosa was studied. A DNA region complementing the alg-5 mutation within the alginate gene cluster was found by RNA-DNA dot blot and Northern hybridization to be transcriptionally activated in mucoid P. aeruginosa. This region was subcloned as a 3.2-kilobase BglII-ClaI DNA fragment on the broad-host-range controlled transcription vector pMMB24, and gene products were analyzed by expression from the tac promoter. A 48-kilodalton polypeptide was detected in extracts of P. aeruginosa and 35S-labeled Escherichia coli maxicells. By using the same expression system, GDPmannose dehydrogenase activity was detected in both P. aeruginosa and E. coli. Thus, gene algD coding for this enzyme was found to be present in the transcriptionally active DNA area. Insertion of the xylE gene within the BglII-ClaI fragment disrupted the induction of the 48-kilodalton polypeptide, GDPmannose dehydrogenase activity, and alg-5 complementing ability. With the algD-xylE transcription fusion, activation of algD gene expression was shown to occur in mucoid P. aeruginosa of different origins. In addition, regulation of the algD promoter activity was demonstrated to be mediated by a diffusible factor. Images PMID:3025179

  6. Corneal aldehyde dehydrogenase and glutathione S-transferase activity after excimer laser keratectomy in guinea pigs

    PubMed Central

    Bilgihan, K.; Bilgihan, A.; Turkozkan, N.

    1998-01-01

    BACKGROUND—The free radical balance of the eye may be changed by excimer laser keratectomy. Previous studies have demonstrated that excimer laser keratectomy increases the corneal temperature, decreases the superoxide dismutase activity of the aqueous, and induces lipid peroxidation in the superficial corneal stroma. Aldehyde dehydrogenase (ALDH) and glutathione S-transferase (GST) are known to play an important role in corneal metabolism, particularly in detoxification of aldehydes, which are generated from free radical reactions.
METHODS—In three groups of guinea pigs mechanical corneal de-epithelialisation was performed in group I, superficial corneal photoablation in group II, and deep corneal photoablation in group III, and the corneal ALDH and GST activities measured after 48 hours.
RESULTS—The mean ALDH and GST activities of group I and II showed no differences compared with the controls (p>0.05). The corneal ALDH activities were found to be significantly decreased (p<0.05) and GST activities increased (p<0.05) in group III.
CONCLUSION—These results suggest that excimer laser treatment of high myopia may change the ALDH and GST activities, metabolism, and free radical balance of the cornea.

 Keywords: excimer laser keratectomy; aldehyde dehydrogenase; glutathione S-transferase PMID:9602629

  7. Effect of dimethylbenzanthracene (DMBA) on spontaneous activity in mice during pregnancy and lactation

    SciTech Connect

    Pitkow, H.S.; Rainieri, J.; Dwyer, P.

    1986-03-01

    Our laboratory has reported that DMBA, a carcinogenic polycyclic aromatic hydrocarbon, administered during pregnancy significantly depressed neonatal cerebrum growth and synthesis which was manifested by a retarded learning ability on day 32 post-partum. In order to determine the effects of DMBA on spontaneous movements during pregnancy and lactation, female CF-1C mice (23-25 g; 10 animals/group) were subcutaneously injected with 250 ..mu..g DMBA in 0.1 ml sesame oil on alternate days starting with day 8 of pregnancy to day 7 post-partum (i.e., day 27). On day 9 of pregnancy and on alternate days each female's cage, with pups removed, was individually placed on the stage of a Stoelting Electronic Activity Monitor for ten minutes and spontaneous movements per minute (mv/min) recorded. No significant differences were observed between control (64.1 +/- 7.3 mv/min) and DMBA (52.4 +/- 6.8 mv/min) groups during pregnancy. After parturition the spontaneous movements of the control mice significantly increased averaging 86.4 +/- 13.3 mv/min for days 1 to 7 post-partum, whereas, the DMBA group value decreased significantly averaging 41.8 +/- 3.7 mv/min during this time. Our data suggests that the effects of DMBA were manifested during lactation where it significantly depressed the lactating mother's nervous system and subsequent behavioral activity.

  8. Laboratory Prototype of Bioreactor for Oxidation of Toxic D-Lactate Using Yeast Cells Overproducing D-Lactate Cytochrome c Oxidoreductase.

    PubMed

    Karkovska, Maria; Smutok, Oleh; Gonchar, Mykhailo

    2016-01-01

    D-lactate is a natural component of many fermented foods like yogurts, sour milk, cheeses, and pickles vegetable products. D-lactate in high concentrations is toxic for children and people with short bowel syndrome and provokes encephalopathy. These facts convincingly demonstrate a need for effective tools for the D-lactate removal from some food products. The main idea of investigation is focused on application of recombinant thermotolerant methylotrophic yeast Hansenula polymorpha "tr6," overproducing D-lactate: cytochrome c oxidoreductase (EC 1.1.2.4, D-lactate cytochrome c oxidoreductase, D-lactate dehydrogenase (cytochrome), DLDH). In addition to 6-fold overexpression of DLDH under a strong constitutive promoter (prAOX), the strain of H. polymorpha "tr6" (gcr1 catX/Δcyb2, prAOX_DLDH) is characterized by impairment in glucose repression of AOX promoter, devoid of catalase and L-lactate-cytochrome c oxidoreductase activities. Overexpression of DLDH coupling with the deletion of L-lactate-cytochrome c oxidoreductase activity opens possibility for usage of the strain as a base for construction of bioreactor for removing D-lactate from fermented products due to oxidation to nontoxic pyruvate. A laboratory prototype of column-type bioreactor for removing a toxic D-lactate from model solution based on permeabilized cells of the H. polymorpha "tr6" and alginate gel was constructed and efficiency of this process was tested. PMID:27446952

  9. Laboratory Prototype of Bioreactor for Oxidation of Toxic D-Lactate Using Yeast Cells Overproducing D-Lactate Cytochrome c Oxidoreductase

    PubMed Central

    Karkovska, Maria

    2016-01-01

    D-lactate is a natural component of many fermented foods like yogurts, sour milk, cheeses, and pickles vegetable products. D-lactate in high concentrations is toxic for children and people with short bowel syndrome and provokes encephalopathy. These facts convincingly demonstrate a need for effective tools for the D-lactate removal from some food products. The main idea of investigation is focused on application of recombinant thermotolerant methylotrophic yeast Hansenula polymorpha “tr6,” overproducing D-lactate: cytochrome c oxidoreductase (EC 1.1.2.4, D-lactate cytochrome c oxidoreductase, D-lactate dehydrogenase (cytochrome), DLDH). In addition to 6-fold overexpression of DLDH under a strong constitutive promoter (prAOX), the strain of H. polymorpha “tr6” (gcr1 catX/Δcyb2, prAOX_DLDH) is characterized by impairment in glucose repression of AOX promoter, devoid of catalase and L-lactate-cytochrome c oxidoreductase activities. Overexpression of DLDH coupling with the deletion of L-lactate-cytochrome c oxidoreductase activity opens possibility for usage of the strain as a base for construction of bioreactor for removing D-lactate from fermented products due to oxidation to nontoxic pyruvate. A laboratory prototype of column-type bioreactor for removing a toxic D-lactate from model solution based on permeabilized cells of the H. polymorpha “tr6” and alginate gel was constructed and efficiency of this process was tested. PMID:27446952

  10. Efficient reduction of the formation of by-products and improvement of production yield of 2,3-butanediol by a combined deletion of alcohol dehydrogenase, acetate kinase-phosphotransacetylase, and lactate dehydrogenase genes in metabolically engineered Klebsiella oxytoca in mineral salts medium.

    PubMed

    Jantama, Kaemwich; Polyiam, Pattharasedthi; Khunnonkwao, Panwana; Chan, Sitha; Sangproo, Maytawadee; Khor, Kirin; Jantama, Sirima Suvarnakuta; Kanchanatawee, Sunthorn

    2015-07-01

    Klebsiella oxytoca KMS005 (∆adhE∆ackA-pta∆ldhA) was metabolically engineered to improve 2,3-butanediol (BDO) yield. Elimination of alcohol dehydrogenase E (adhE), acetate kinase A-phosphotransacetylase (ackA-pta), and lactate dehydrogenase A (ldhA) enzymes allowed BDO production as a primary pathway for NADH re-oxidation, and significantly reduced by-products. KMS005 was screened for the efficient glucose utilization by metabolic evolution. KMS005-73T improved BDO production at a concentration of 23.5±0.5 g/L with yield of 0.46±0.02 g/g in mineral salts medium containing 50 g/L glucose in a shake flask. KMS005-73T also exhibited BDO yields of about 0.40-0.42 g/g from sugarcane molasses, cassava starch, and maltodextrin. During fed-batch fermentation, KMS005-73T produced BDO at a concentration, yield, and overall and specific productivities of 117.4±4.5 g/L, 0.49±0.02 g/g, 1.20±0.05 g/Lh, and 27.2±1.1 g/gCDW, respectively. No acetoin, lactate, and formate were detected, and only trace amounts of acetate and ethanol were formed. The strain also produced the least by-products and the highest BDO yield among other Klebsiella strains previously developed. PMID:25895450

  11. Novel mutations in dihydrolipoamide dehydrogenase deficiency in two cousins with borderline-normal PDH complex activity.

    PubMed

    Cameron, Jessie M; Levandovskiy, Valeriy; Mackay, Neviana; Raiman, Julian; Renaud, Deborah L; Clarke, Joe T R; Feigenbaum, Annette; Elpeleg, Orly; Robinson, Brian H

    2006-07-15

    We have diagnosed dihydrolipoamide dehydrogenase (DLD) deficiency in two male second cousins, who presented with markedly different clinical phenotypes. Patient 1 had a recurrent encephalopathy, and patient 2 had microcephaly and lactic acidosis. Their presentation is unusual, in that the DLD subunit deficiency had little effect on pyruvate dehydrogenase complex activity, but caused a severe reduction in the activities of other enzymes that utilize this subunit. We have identified two mutations in the DLD gene in each patient. The second cousins have one novel mutation in common resulting in a substitution of isoleucine for threonine (I47T), which has not been previously reported in the literature. Patient 1 has a second mutation that has been reported to be common in the Ashkenazi Jewish population, G229C. Patient 2 has a second mutation, E375K, which has also been previously reported in the literature. Enzyme kinetic measurements on patient fibroblasts show that under certain conditions, one heteroallelic mutation may have a higher K(m). This may account for the differing clinical phenotypes. These findings have important repercussions for other patients with similar clinical phenotypes, as DLD activity is not normally measured in cases with normal PDHc activity.

  12. Lactic dehydrogenase isozyme patterns and alpha-hydroxybutyrate dehydrogenase activities in serum from newborns, patients with ovarian cancer or myocardial infarction.

    PubMed

    Kikuchi, Y; Kita, T; Furuya, K; Kato, K

    1988-11-01

    Lactic dehydrogenase (LDH) and alpha-hydroxybutyrate dehydrogenase (HBD) and LDH isozyme patterns were studied in serum from newborns and patients with ovarian cancer or myocardial infarction. LDH and HBD activities from newborns and patients with ovarian cancer or myocardial infarction were significantly increased, compared with those from patients with benign ovarian tumor. These increases were accompanied with a decrease of LDH-H and an increase of LDH-M in serum from newborns and patients with ovarian cancer, while an increase of LDH-H in serum from patients with myocardial infarction was dominant. However, the raised HBD activities in serum from patients with benign ovarian tumor did not affect the LDH isozyme patterns. From analysis of linear regression, a negative correlation between LDH-1 or -2 and HBD activity in serum from patients with ovarian cancer was observed while there was a positive correlation between LDH-4 and HBD activity. Similar patterns in serum from newborns were observed. On the other hand, a positive correlation between LDH-1 and HBD activity and a negative correlation between LDH-4 and HBD activity were found in serum from patients with myocardial infarction.

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

    PubMed

    Voloshchuk, O N; Kopylchuk, G P

    2016-01-01

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

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

    PubMed

    Voloshchuk, O N; Kopylchuk, G P

    2016-01-01

    Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage. PMID:27143375

  15. [Effects of Light Near-Infrared Radiation on Rats Assessed by Succinate Dehydrogenase Activity in Lymphocytes on Blood Smears].

    PubMed

    Khunderyakova, N V; Zakharchenko, A V; Zakharchenko, M V; Muller, H; Fedotcheva, I; Kondrashova, M N

    2015-01-01

    Biological effects of light near infrared radiation (850 nm), with modulation acoustic frequency of 101 Hz, was studied. The study was conducted on rats, the effect was recorded by succinate dehydrogenase activity in lymphocytes on the blood smear after administration of the activating dose of adrenaline, which simulates the state of the organism in the early stages of the pathogenic effects (stress). A pronounced regulating effect of infrared radiation on the activity of succinate dehydrogenase in animals activated by adrenaline was shown. Infrared radiation has a normalizing effect reducing the degree of inhibition or activation of the enzyme induced by adrenaline and had no effect on the control animals. Thus, by modulating the activity of succinate dehydrogenase infrared radiation regulates energy production in the mitochondria supported by the most powerful oxidation substrate--succinic acid, which is especially pronounced under stress. PMID:26841503

  16. [Effects of Light Near-Infrared Radiation on Rats Assessed by Succinate Dehydrogenase Activity in Lymphocytes on Blood Smears].

    PubMed

    Khunderyakova, N V; Zakharchenko, A V; Zakharchenko, M V; Muller, H; Fedotcheva, I; Kondrashova, M N

    2015-01-01

    Biological effects of light near infrared radiation (850 nm), with modulation acoustic frequency of 101 Hz, was studied. The study was conducted on rats, the effect was recorded by succinate dehydrogenase activity in lymphocytes on the blood smear after administration of the activating dose of adrenaline, which simulates the state of the organism in the early stages of the pathogenic effects (stress). A pronounced regulating effect of infrared radiation on the activity of succinate dehydrogenase in animals activated by adrenaline was shown. Infrared radiation has a normalizing effect reducing the degree of inhibition or activation of the enzyme induced by adrenaline and had no effect on the control animals. Thus, by modulating the activity of succinate dehydrogenase infrared radiation regulates energy production in the mitochondria supported by the most powerful oxidation substrate--succinic acid, which is especially pronounced under stress.

  17. Breast Milk Concentration of Rubidium in Lactating Mothers by Instrumental Neutron Activation Analysis Method

    PubMed Central

    Khatami, Seyedeh-Fatemeh; Parvaresh, Pouya; Parvaresh, Parviz; Madani Kouchak, Sara Sadat; Khorsandi, Jamshid

    2014-01-01

    Objective: Relatively little is known about the trace elements content of human milk from different countries. This has not been fully investigated especially among Iranian women. This study aimed to assess the concentration of Rubidium (Rb) as a poisonous trace element in transitional breast milk of lactating mothers living in Mashhad. Methods: Forty nursing mothers in early lactation 3 days to 15 days postpartum, free from any medical disorder and/or medication were randomly selected. We have applied Instrumental Neutron Activation Analysis (INAA) to assess the long-lived isotope trace element Rb in transitional milk of these economically moderate 18–39 year old Iranian women. Findings: The average concentration level of Rb was 32.176 ppm dry weight (min 8.660, max 107.210 ppm). No significant correlation was observed between Rb concentration and maternal weight and age (P=0.06, P=0.05 respectively) and newborns’ weight, age and sex (P=0.07, P=0.2, P=0.2 respectively). Conclusion: Although the Rubidium concentration found in this study is among the highest reported in the literature, it could not be compared to other studies because of differences in analytical performance, state of lactation, and unavailable reference ranges, so this finding needs further investigations. PMID:26019773

  18. Alcohol and polyol dehydrogenases are both divided into two protein types, and structural properties cross-relate the different enzyme activities within each type.

    PubMed Central

    Jörnvall, H; Persson, M; Jeffery, J

    1981-01-01

    Sorbitol dehydrogenase from sheep liver shows similarities to mammalian and yeast alcohol dehydrogenases. Comparisons based on peptides from segments of sorbitol dehydrogenase reveal that homologous regions with 38% identity include two ligands to the active site zinc atom in liver alcohol dehydrogenase, as well as further important residues. Similarities in in other regions are less extensive, exactly as they are between different alcohol dehydrogenases. In all aspects, sorbitol dehydrogenase appears as a typical member of the alcohol dehydrogenase family. On the other hand, alcohol dehydrogenase from Drosophila, which has a shorter subunit, is not closely related to either of these enzymes, except for a region that probably corresponds to the first part of the coenzyme binding domain in many dehydrogenases. Instead, Drosophila alcohol dehydrogenase in its supposed catalytic region shows similarities toward Klebsiella ribitol dehydrogenase, which also has a small subunit. It may be concluded that both alcohol and polyol dehydrogenases show two types of protein subunit, reflecting an early subdivision of polypeptide types into "long" and "short" subunits rather than into different enzymatic specificities or quaternary structures. The relationships explain known properties of all these enzymes and provide insight into functional mechanisms and evolutionary interpretations. PMID:7027257

  19. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

    ... control the activity of the complex: pyruvate dehydrogenase phosphatase turns on (activates) the complex, while pyruvate dehydrogenase ... binding protein (the PDHX gene), and pyruvate dehydrogenase phosphatase (the PDP1 gene) have been identified in people ...

  20. Correlation of loss of activity of human aldehyde dehydrogenase with reaction of bromoacetophenone with glutamic acid-268 and cysteine-302 residues. Partial-sites reactivity of aldehyde dehydrogenase.

    PubMed Central

    Abriola, D P; MacKerell, A D; Pietruszko, R

    1990-01-01

    Bromoacetophenone (2-bromo-1-phenylethanone) has been characterized as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) [MacKerell, MacWright & Pietruszko (1986) Biochemistry 25, 5182-5189], and has been shown to react specifically with the Glu-268 residue [Abriola, Fields, Stein, MacKerell & Pietruszko (1987) Biochemistry 26, 5679-5684] with an apparent inactivation stoichiometry of two molecules of bromoacetophenone per molecule of enzyme. The specificity of bromoacetophenone for reaction with Glu-268, however, is not absolute, owing to the extreme reactivity of this reagent. When bromo[14C]acetophenone was used to label the human cytoplasmic E1 isoenzyme radioactively and tryptic fragmentation was carried out, peptides besides that containing Glu-268 were found to have reacted with reagent. These peptides were purified by h.p.l.c. and analysed by sequencing and scintillation counting to quantify radioactive label in the material from each cycle of sequencing. Reaction of bromoacetophenone with the aldehyde dehydrogenase molecule during enzyme activity loss occurs with two residues, Glu-268 and Cys-302. The activity loss, however, appears to be proportional to incorporation of label at Glu-268. The large part of incorporation of label at Cys-302 occurs after the activity loss is essentially complete. With both Glu-268 and Cys-302, however, the incorporation of label stops after one molecule of bromoacetophenone has reacted with each residue. Reaction with other residues continues after activity loss is complete. PMID:1968743

  1. Decreased succinate dehydrogenase activity of gamma and alpha motoneurons in mouse spinal cords following 13 weeks of exposure to microgravity.

    PubMed

    Ishihara, Akihiko; Nagatomo, Fumiko; Fujino, Hidemi; Kondo, Hiroyo; Ohira, Yoshinobu

    2013-10-01

    Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 μm(2)) gamma and medium-sized (300-700 μm(2)) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 μm(2)) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity. PMID:23943522

  2. Cytoplasm-to-myonucleus ratios and succinate dehydrogenase activities in adult rat slow and fast muscle fibers

    NASA Technical Reports Server (NTRS)

    Tseng, B. S.; Kasper, C. E.; Edgerton, V. R.

    1994-01-01

    The relationship between myonuclear number, cellular size, succinate dehydrogenase activity, and myosin type was examined in single fiber segments (n = 54; 9 +/- 3 mm long) mechanically dissected from soleus and plantaris muscles of adult rats. One end of each fiber segment was stained for DNA before quantitative photometric analysis of succinate dehydrogenase activity; the other end was double immunolabeled with fast and slow myosin heavy chain monoclonal antibodies. Mean +/- S.D. cytoplasmic volume/myonucleus ratio was higher in fast and slow plantaris fibers (112 +/- 69 vs. 34 +/- 21 x 10(3) microns3) than fast and slow soleus fibers (40 +/- 20 vs. 30 +/- 14 x 10(3) microns3), respectively. Slow fibers always had small volumes/myonucleus, regardless of fiber diameter, succinate dehydrogenase activity, or muscle of origin. In contrast, smaller diameter (< 70 microns) fast soleus and plantaris fibers with high succinate dehydrogenase activity appeared to have low volumes/myonucleus while larger diameter (> 70 microns) fast fibers with low succinate dehydrogenase activity always had large volume/myonucleus. Slow soleus fibers had significantly greater numbers of myonuclei/mm than did either fast soleus or fast plantaris fibers (116 +/- 51 vs. 55 +/- 22 and 44 +/- 23), respectively. These data suggest that the myonuclear domain is more limited in slow than fast fibers and in the fibers with a high, compared to a low, oxidative metabolic capability.

  3. Decreased succinate dehydrogenase activity of gamma and alpha motoneurons in mouse spinal cords following 13 weeks of exposure to microgravity.

    PubMed

    Ishihara, Akihiko; Nagatomo, Fumiko; Fujino, Hidemi; Kondo, Hiroyo; Ohira, Yoshinobu

    2013-10-01

    Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 μm(2)) gamma and medium-sized (300-700 μm(2)) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 μm(2)) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity.

  4. Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles.

    PubMed

    Sarcar, S; Jain, T K; Maitra, A

    1992-02-20

    The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w(0), pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w(0) = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307 degrees C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.

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

    SciTech Connect

    Durand, Fabien; Stines-Chaumeil, Claire; Flexer, Victoria; Andre, Isabelle; Mano, Nicolas

    2010-11-26

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

  6. Plasma lactic dehydrogenase activities in men during bed rest with exercise training

    NASA Technical Reports Server (NTRS)

    Greenleaf, J. E.; Juhos, L. T.; Young, H. L.

    1985-01-01

    Peak oxygen uptake and the activity of lactic dehydrogenase (LDH-T) and its five isoenzymes were measured by spectrophotometer in seven men before, during, and after bed rest and exercise training. Exercise training consisted of isometric leg exercises of 250 kcal/hr for a period of one hour per day. It is found that LDH-T was reduced by 0.05 percent in all three regimens by day 10 of bed rest, and that the decrease occurred at different rates. The earliest reduction in LDH-T activity in the no-exercise regimen was associated with a decrease in peak oxygen uptake of 12.3 percent. It is concluded that isometric (aerobic) muscular strength training appear to maintain skeletal muscle integrity better during bed rest than isotonic exercise training. Reduced hydrostatic pressure during bed rest, however, ultimately counteracts the effects of both moderate isometric and isotonic exercise training, and may result in decreased LDH-T activity.

  7. Basolateral active uptake of nitrofurantoin in the CIT3 cell culture model of lactation.

    PubMed

    Gerk, Phillip M; Moscow, Jeffrey A; McNamara, Patrick J

    2003-06-01

    Nitrofurantoin and other agents are actively transported into human and rodent milk. The purpose of this study was to determine whether nitrofurantoin active transport across mammary epithelia occurs basolaterally or apically, using the CIT3 cell culture model of lactation. The CIT3 model actively transports nitrofurantoin in the basolateral to apical direction. Basolateral to apical permeability [92.9 +/- 6.6 (microl/h)/cm(2)] was differentially decreased by unlabeled nitrofurantoin (250 microM) on the basolateral, apical, or both sides [49.5 +/- 1.8, 57.9 +/- 1.4, or 48.5 +/- 1.6 (microl/h)/cm(2), respectively]. Apical to basolateral permeability [27.6 +/- 1.8 (microl/h)/cm(2)] was increased in the presence of unlabeled nitrofurantoin (250 microM) on the basolateral, apical, or both sides [36.4 +/- 1.5, 39.9 +/- 0.7, 42.4 +/- 1.1 (microl/h)/cm(2), respectively]. These data indicate a basolateral active uptake mechanism for nitrofurantoin, which remains to be identified. This mechanism may influence the exposure of suckling infants to xenobiotics, as well as having potentially toxic effects on the lactating mammary epithelium and possibly altering the nutritional quality of the milk.

  8. Hindbrain medulla catecholamine cell group involvement in lactate-sensitive hypoglycemia-associated patterns of hypothalamic norepinephrine and epinephrine activity.

    PubMed

    Shrestha, P K; Tamrakar, P; Ibrahim, B A; Briski, K P

    2014-10-10

    Cell-type compartmentation of glucose metabolism in the brain involves trafficking of the oxidizable glycolytic end product, l-lactate, by astrocytes to fuel neuronal mitochondrial aerobic respiration. Lactate availability within the hindbrain medulla is a monitored function that regulates systemic glucostasis as insulin-induced hypoglycemia (IIH) is exacerbated by lactate repletion of that brain region. A2 noradrenergic neurons are a plausible source of lactoprivic input to the neural gluco-regulatory circuit as caudal fourth ventricular (CV4) lactate infusion normalizes IIH-associated activation, e.g. phosphorylation of the high-sensitivity energy sensor, adenosine 5'-monophosphate-activated protein kinase (AMPK), in these cells. Here, we investigated the hypothesis that A2 neurons are unique among medullary catecholamine cells in directly screening lactate-derived energy. Adult male rats were injected with insulin or vehicle following initiation of continuous l-lactate infusion into the CV4. Two hours after injections, A1, C1, A2, and C2 neurons were collected by laser-microdissection for Western blot analysis of AMPKα1/2 and phosphoAMPKα1/2 proteins. Results show that AMPK is expressed in each cell group, but only a subset, e.g. A1, C1, and A2 neurons, exhibit increased sensor activity in response to IIH. Moreover, hindbrain lactate repletion reversed hypoglycemic augmentation of pAMPKα1/2 content in A2 and C1 but not A1 cells, and normalized hypothalamic norepinephrine and epinephrine content in a site-specific manner. The present evidence for discriminative reactivity of AMPK-expressing medullary catecholamine neurons to the screened energy substrate lactate implies that that lactoprivation is selectively signaled to the hypothalamus by A2 noradrenergic and C1 adrenergic cells.

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  10. Efficient production of optically pure D-lactic acid from raw corn starch by using a genetically modified L-lactate dehydrogenase gene-deficient and alpha-amylase-secreting Lactobacillus plantarum strain.

    PubMed

    Okano, Kenji; Zhang, Qiao; Shinkawa, Satoru; Yoshida, Shogo; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-01-01

    In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 alpha-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch.

  11. Inhibition of cellulolytic activity by lactate in a Cellulomonas uda species

    SciTech Connect

    Chosson, J.

    1987-04-20

    According to Stackebrandt and Kandler, Cellulomonas are aerobic, mesophilic, nonsporeforming, cellulose-utilizing bacteria which produce acid from glucose. Bacteria belonging to this genus secrete three types of enzyme involved in cellulolysis: endoglucanases, exoglucanases, and ..beta..-glucosidases or cellobiose phosphorylases. Inhibition of cellulolytic activity by glucose and cellobiose was evidenced in several Cellulomonas species. Particularly, Stoppok and coworkers reported in Cellulomonas uda a complete repression of endoglucanases synthesis by 4.8 and 2.7 g/l glucose and cellobiose, respectively. In addition, 9 g/l glucose had no effect on the endoglucanase activity, but cellobiose at the same concentration reduced it by 50%. During batch culture of C. uda on cellulose (20 g/l), degradation of the polymer remained incomplete in spite of very low concentrations of reducing sugars (0.4 g/l) in the culture supernatant. Furthermore, a careful analysis of organic acids evidenced an accumulation of L(+)-lactate up to 0.8 g/l. In this communication, the author reports the inhibitory effect of lactate on the cellulolytic activity of C. uda strains JC 3. 12 references.

  12. Oxygen control of breathing by an olfactory receptor activated by lactate

    PubMed Central

    Chang, Andy J.; Ortega, Fabian E.; Riegler, Johannes; Madison, Daniel V.; Krasnow, Mark A.

    2015-01-01

    Summary Animals have evolved homeostatic responses to changes in oxygen availability that act on different time scales. Although the hypoxia-inducible factor (HIF) transcriptional pathway that controls long term responses to low oxygen (hypoxia) has been established1, the pathway that mediates acute responses to hypoxia in mammals is not well understood. Here we show that the olfactory receptor Olfr78 is highly and selectively expressed in oxygen-sensitive glomus cells of the carotid body, a chemosensory organ at the carotid artery bifurcation that monitors blood oxygen and stimulates breathing within seconds when oxygen declines2. Olfr78 mutants fail to increase ventilation in hypoxia but respond normally to hypercapnia. Glomus cells are present in normal numbers and appear structurally intact, but hypoxia-induced carotid body activity is diminished. Lactate, a metabolite that rapidly accumulates in hypoxia and induces hyperventilation3–6, activates Olfr78 in heterologous expression experiments, induces calcium transients in glomus cells, and stimulates carotid sinus nerve activity through Olfr78. We propose that in addition to its role in olfaction, Olfr78 acts as a hypoxia sensor in the breathing circuit by sensing lactate produced when oxygen levels decline. PMID:26560302

  13. Early lactate clearance for predicting active bleeding in critically ill patients with acute upper gastrointestinal bleeding: a retrospective study.

    PubMed

    Wada, Tomoki; Hagiwara, Akiyoshi; Uemura, Tatsuki; Yahagi, Naoki; Kimura, Akio

    2016-08-01

    Not all patients with upper gastrointestinal bleeding (UGIB) require emergency endoscopy. Lactate clearance has been suggested as a parameter for predicting patient outcomes in various critical care settings. This study investigates whether lactate clearance can predict active bleeding in critically ill patients with UGIB. This single-center, retrospective, observational study included critically ill patients with UGIB who met all of the following criteria: admission to the emergency department (ED) from April 2011 to August 2014; had blood samples for lactate evaluation at least twice during the ED stay; and had emergency endoscopy within 6 h of ED presentation. The main outcome was active bleeding detected with emergency endoscopy. Classification and regression tree (CART) analyses were performed using variables associated with active bleeding to derive a prediction rule for active bleeding in critically ill UGIB patients. A total of 154 patients with UGIB were analyzed, and 31.2 % (48/154) had active bleeding. In the univariate analysis, lactate clearance was significantly lower in patients with active bleeding than in those without active bleeding (13 vs. 29 %, P < 0.001). Using the CART analysis, a prediction rule for active bleeding is derived, and includes three variables: lactate clearance; platelet count; and systolic blood pressure at ED presentation. The rule has 97.9 % (95 % CI 90.2-99.6 %) sensitivity with 32.1 % (28.6-32.9 %) specificity. Lactate clearance may be associated with active bleeding in critically ill patients with UGIB, and may be clinically useful as a component of a prediction rule for active bleeding.

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

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.

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

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression. PMID:24594397

  16. Pyruvate dehydrogenase kinase isoform 2 activity stimulated by speeding up the rate of dissociation of ADP.

    PubMed

    Bao, Haiying; Kasten, Shane A; Yan, Xiaohua; Hiromasa, Yasuaki; Roche, Thomas E

    2004-10-26

    Pyruvate dehydrogenase kinase 2 (PDK2) activity is stimulated by NADH and NADH plus acetyl-CoA via the reduction and reductive acetylation of the lipoyl groups of the dihydrolipoyl acetyltransferase (E2) component. Elevated K(+) and Cl(-) were needed for significant stimulation. Stimulation substantially increased both k(cat) and the K(m) for ATP; the fractional stimulation increased with the level of ATP. With an E2 structure lacking the pyruvate dehydrogenase (E1) binding domain, stimulation of PDK2 was retained, the K(m) for E1 decreased, and the equilibrium dissociation constant for ATP increased but remained much lower than the K(m) for ATP. Stimulation of PDK2 activity greatly reduced the fraction of bound ADP. These results fit an ordered reaction mechanism with ATP binding before E1 and stimulation increasing the rate of dissociation of ADP. Conversion of all of the lipoyl groups in the E2 60mer to the oxidized form (E2(ox)) greatly reduced k(cat) and the K(m) of PDK2 for ATP. Retention over an extended period of time of a low portion of reduced lipoyl groups maintains E2 in a state that supported much higher PDK2 activity than short-term (5 min) reduction of a large portion of lipoyl groups of E2(ox), but reduction of E2(ox) produced a larger fold stimulation. Reduction and to a greater extent reductive acetylation increased PDK2 binding to E2; conversion to E2(ox) did not significantly hinder binding. We suggest that passing even limited reducing equivalents among lipoyl groups maintains E2 lipoyl domains in a conformation that aids kinase function. PMID:15491151

  17. Activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates.

    PubMed Central

    Alp, P R; Newsholme, E A; Zammit, V A

    1976-01-01

    1. The activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were measured in muscles from a large number of animals, in order to provide some indication of the importance of the citric acid cycle in these muscles. According to the differences in enzyme activities, the muscles can be divided into three classes. First, in a number of both vertebrate and invertebrate muscles, the activities of all three enzymes are very low. It is suggested that either the muscles use energy at a very low rate or they rely largely on anaerobic glycolysis for higher rates of energy formation. Second, most insect flight muscles contain high activities of citrate synthase and NAD+-linked isocitrate dehydrogenase, but the activities of the NADP+-linked enzyme are very low. The high activities indicate the dependence of insect flight on energy generated via the citric acid cycle. The flight muscles of the beetles investigated contain high activities of both isocitrate dehydrogenases. Third, other muscles of both vertebrates and invertebrates contain high activities of citrate synthase and NADP+-liniked isocitrate dehydrogenase. Many, if not all, of these muscles are capable of sustained periods of mechanical activity (e.g. heart muscle, pectoral muscles of some birds). Consequently, to support this activity fuel must be supplied continually to the muscle via the circulatory system which, in most animals, also transports oxygen so that energy can be generated by complete oxidation of the fuel. It is suggested that the low activities of NAD+-linked isocitrate dehydrogenase in these muscles may be involved in oxidation of isocitrate in the cycle when the muscles are at rest. 2. A comparison of the maximal activities of the enzymes with the maximal flux through the cycle suggests that, in insect flight muscle, NAD+-linked isocitrate dehydrogenase catalyses a non-equilibrium reaction and citrate synthease catalyses a near-equilibrium reaction. In other muscles

  18. Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia.

    PubMed Central

    Jonas, S. K.; Benedetto, C.; Flatman, A.; Hammond, R. H.; Micheletti, L.; Riley, C.; Riley, P. A.; Spargo, D. J.; Zonca, M.; Slater, T. F.

    1992-01-01

    The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected]. Images Figure 1 PMID:1637668

  19. Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity

    DOE PAGES

    Kim, Youngchang; Makowska-Grzyska, Magdalena; Gorla, Suresh Kumar; Gollapalli, Deviprasad R.; Cuny, Gregory D.; Joachimiak, Andrzej; Hedstrom, Lizbeth

    2015-04-21

    Inosine 5´-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment of Cryptosporidium infections. Here, the structure of C. parvum IMPDH (CpIMPDH) in complex with inosine 5´-monophosphate (IMP) and P131, an inhibitor with in vivo anticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO2 moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compounds to replace these water molecules is a new strategymore » for the further optimization of C. parvum inhibitors for both antiparasitic and antibacterial applications.« less

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

    PubMed

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

    2015-11-02

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

  1. Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity.

    PubMed

    Kim, Youngchang; Makowska-Grzyska, Magdalena; Gorla, Suresh Kumar; Gollapalli, Deviprasad R; Cuny, Gregory D; Joachimiak, Andrzej; Hedstrom, Lizbeth

    2015-05-01

    Inosine 5'-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment of Cryptosporidium infections. Here, the structure of C. parvum IMPDH (CpIMPDH) in complex with inosine 5'-monophosphate (IMP) and P131, an inhibitor with in vivo anticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO2 moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compounds to replace these water molecules is a new strategy for the further optimization of C. parvum inhibitors for both antiparasitic and antibacterial applications. PMID:25945705

  2. Chaperones rejuvenate folding and activity of 3-β hydroxysteroid dehydrogenase 2.

    PubMed

    Rajapaksha, Maheshinie; Prasad, Manoj; Thomas, James L; Whittal, Randy M; Bose, Himangshu S

    2013-05-17

    The steroidogenic enzyme 3-β hydroxysteroid dehydrogenase 2 (3βHSD2) mediates the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione through both its dehydrogenase and isomerase activities, making it necessary for the protein to undergo a reversible conformational change. We hypothesized that chaperones assist 3βHSD2 in switching between the conformations to initiate, enhance, and maintain activity. In the presence of the chaperone lauryl maltoside (LM), 3βHSD2 immediately converted pregnenolone to progesterone, with a 6.4-fold increase in synthesis. Using far-UV circular dichroism (CD), we found that addition of LM increased 3βHSD2's α-helical content, which over time reverted to control levels, suggesting the formation of a stable but reversible conformation possibly due to hydrophobic interactions of the protein with LM micelles. We also found that LM increased fluorescence resonance energy transfer (FRET) about 11-fold between 3βHSD2 and fluorescing ANS molecules. This observation supports the idea that detergent(s) act as chaperones to assist 3βHSD2 in forming stable complexes, which in turn promotes proper folding. Mass spectrometric fingerprinting illustrated that LM incubation resulted in an ordered fragmentation of molecular mass from 39 to 13 kDa, as compared to limited or no proteolysis in the absence of LM. In addition, space-filling modeling demonstrated that 3βHSD2 association with detergents likely exposed the hydrophobic region, leading to its proteolysis. We conclude that detergents help 3βHSD2 to refold in order to rejuvenate, contributing to the ability of cells to rapidly produce steroids when needed.

  3. The carboxy-terminal tail of pyruvate dehydrogenase kinase 2 is required for the kinase activity.

    PubMed

    Klyuyeva, Alla; Tuganova, Alina; Popov, Kirill M

    2005-10-18

    Pyruvate dehydrogenase kinase 2 (PDK2) is a prototypical mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase complex. Recent structural studies have established that PDK2 consists of a catalytic core built of the B and K domains and the relatively long amino and carboxyl tails of unknown function. Here, we show that the carboxy-terminal truncation variants of PDK2 display a greatly diminished capacity for phosphorylation of holo-PDC. This effect is due largely to the inability of the transacetylase component of PDC to promote the phosphorylation reaction catalyzed by the truncated PDK2 variants. Furthermore, the truncated forms of PDK2 bind poorly to the lipoyl-bearing domain(s) provided by the transacetylase component. Taken together, these data strongly suggest that the carboxyl tails of PDK isozymes contribute to the lipoyl-bearing domain-binding site of the kinase molecule. We also show that the carboxyl tails derived from isozymes PDK1, PDK3, and PDK4 are capable of supporting the kinase activity of the kinase core derived from PDK2 as well as binding of the respective PDK2 chimeras to the lipoyl-bearing domain. Furthermore, the chimera carrying the carboxyl tail of PDK3 displays a stronger response to the addition of the transacetylase component along with a better binding to the lipoyl-bearing domain, suggesting that, at least in part, the differences in the amino acid sequences of the carboxyl tails account for the differences between PDK isozymes. PMID:16216081

  4. Reduced activity of 11β-hydroxysteroid dehydrogenase in patients with cholestasis

    PubMed Central

    Quattropani, Cristiana; Vogt, Bruno; Odermatt, Alex; Dick, Bernhard; Frey, Brigitte M.; Frey, Felix J.

    2001-01-01

    Enhanced renal sodium retention and potassium loss in patients with cirrhosis is due to activation of mineralocorticoid receptors (MRs). Increased aldosterone concentrations, however, do not entirely explain the activation of MR in cirrhosis. Here, we hypothesize that cortisol activates MRs in patients with cholestasis. We present evidence that access of cortisol to MRs is a result of bile acid−mediated inhibition of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), an MR-protecting enzyme that converts cortisol to cortisone. Twelve patients with biliary obstruction and high plasma bile acid levels were studied before and after removal of the obstruction. The urinary ratio of (tetrahydrocortisol + 5α-tetrahydrocortisol)/tetrahydrocortisone, a measure of 11β-HSD2 activity, decreased from a median of 1.91 during biliary obstruction to 0.78 at 4 and 8 weeks after removal of the obstruction and normalization of plasma bile acid concentrations. In order to demonstrate that bile acids facilitate access of cortisol to the MR by inhibiting 11β-HSD2, an MR translocation assay was performed in HEK-293 cells transfected with human 11β-HSD2 and tagged MR. Increasing concentrations of chenodeoxycholic acid led to cortisol-induced nuclear translocation of MR. In conclusion, 11β-HSD2 activity is reduced in cholestasis, which results in MR activation by cortisol. PMID:11696574

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

    PubMed

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

    2011-08-01

    Pyruvate dehydrogenase (PDH) is a mitochondrial enzyme responsible for regulating the conversion of pyruvate to acetyl-CoA for use in the tricarboxylic acid cycle. PDH is regulated through phosphorylation and inactivation by PDH kinase (PDK) and dephosphorylation and activation by PDH phosphatase (PDP). The effect of endurance training on PDK in humans has been investigated; however, to date no study has examined the effect of endurance training on PDP in humans. Therefore, the purpose of this study was to examine differences in PDP activity and PDP1 protein content in human skeletal muscle across a range of muscle aerobic capacities. This association is important as higher PDP activity and protein content will allow for increased activation of PDH, and carbohydrate oxidation. The main findings of this study were that 1) PDP activity (r(2) = 0.399, P = 0.001) and PDP1 protein expression (r(2) = 0.153, P = 0.039) were positively correlated with citrate synthase (CS) activity as a marker for muscle aerobic capacity; 2) E1α (r(2) = 0.310, P = 0.002) and PDK2 protein (r(2) = 0.229, P =0.012) are positively correlated with muscle CS activity; and 3) although it is the most abundant isoform, PDP1 protein content only explained ∼ 18% of the variance in PDP activity (r(2) = 0.184, P = 0.033). In addition, PDP1 in combination with E1α explained ∼ 38% of the variance in PDP activity (r(2) = 0.383, P = 0.005), suggesting that there may be alternative regulatory mechanisms of this enzyme other than protein content. These data suggest that with higher muscle aerobic capacity (CS activity) there is a greater capacity for carbohydrate oxidation (E1α), in concert with higher potential for PDH activation (PDP activity). PMID:21596918

  6. Activity of soil dehydrogenases, urease, and acid and alkaline phosphatases in soil polluted with petroleum.

    PubMed

    Wyszkowska, Jadwiga; Wyszkowski, Mirosław

    2010-01-01

    This study was undertaken to (1) determine the effects of petroleum pollution on changes in the biochemical properties of soil and (2) demonstrate whether the application of compost, bentonite, and calcium oxide is likely to restore biological balance. Petroleum soil pollution at a dose ranging from 2.5 to 10 cm(3)/kg disturbed the biochemical balance as evidenced by inhibition of the activities of soil dehydrogenases (SDH), urease (URE), and acid phosphatase (ACP). The greatest change was noted in the activity of SDH, whereas the least change occurred in URE. Petroleum significantly increased the activity of soil alkaline phosphatase (ALP) in soil used for spring rape, whereas in soil used for oat harvest there was decreased ALP activity. The application of compost, bentonite, and calcium oxide to soil proved effective in mitigating the adverse effects of petroleum on the activities of soil enzymes. Soil enrichment with compost, bentonite, and calcium oxide was found to stimulate the activities of URE and ALP and inhibit the activity of ACP. The influence of bentonite and calcium oxide was greater than that of compost. Calcium oxide and, to a lesser extent, compost were found to increase the activity of SDH, whereas bentonite exerted the opposite effect, especially in the case of the main crop, spring rape. The activities of SDH, URE, and ACP were higher in soil used for rape than that for oats. In contrast the activity of ALP was higher in soil used for oats. Data thus indicate that compost and especially bentonite and calcium oxide exerted a positive effect on activities of some enzymes in soil polluted with petroleum. Application of neutralizing additives to soil restored soil biological balance by counteracting the negative influence of petroleum on activities of URE and ALP. PMID:20706945

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

    PubMed

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

    2015-09-01

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

  8. Expression of 20 alpha-hydroxysteroid dehydrogenase activity in human lymphoid and non lymphoid cells.

    PubMed Central

    Carbone, A; Piantelli, M; Musiani, P; Larocca, L M; Revoltella, R P; Ranelletti, F O

    1986-01-01

    Expression of 20-alpha-hydroxysteroid dehydrogenase (20 alpha-SDH), a putative T cell marker in the murine system, has been examined in human haematopoietic cells. Higher levels of enzymatic activity were expressed by human peripheral blood mononuclear cells (PBMC) in comparison with thymocytes. When PBMC were fractionated into T and non T cell subsets, the greatest values of 20 alpha-SDH activity were associated with the non T cell population. Cells from bone marrow exhibited lower levels of 20 alpha-SDH than PBMC and thymocytes. Both myeloid and lymphoid leukaemic cells expressed significant 20 alpha-SDH activity. In addition our data in U-937 and CM-S human cell lines indicate that cells belonging to the myelomonocytic precursor compartment constitutively expressed 20 alpha-SDH activity. Furthermore, this activity was less expressed when these cells were induced to monocyte-macrophage differentiation. In conclusion, our data in the human system indicate that 20 alpha-SDH should not be viewed as a lymphoid lineage-restricted marker enzyme. PMID:3485485

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

    PubMed

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

    2014-10-01

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

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

    PubMed Central

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

    2014-01-01

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

  11. Cytochrome b5 augments 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase activity.

    PubMed

    Goosen, Pierre; Storbeck, Karl-Heinz; Swart, Amanda C; Conradie, Riaan; Swart, Pieter

    2011-11-01

    During adrenal steroidogenesis the competition between 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3βHSD) and cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1) for Δ(5) steroid intermediates greatly influences steroidogenic output. Cytochrome-b(5) (Cyt-b(5)), a small electron transfer hemoprotein, known to augment the lyase activity of CYP17A1, has been shown to alter the steroidogenic outcome of this competition. In this study, the influence of Cyt-b(5) on 3βHSD activity was investigated. In COS-1 cells, Cyt-b(5) was shown to significantly increase the activity of both caprine and ovine 3βHSD towards pregnenolone, 17-OH pregnenolone and dehydroepiandrosterone in a substrate and species specific manner. Furthermore, kinetic studies revealed Cyt-b(5) to have no influence on the K(m) values while significantly increasing the V(max) values of ovine 3βHSD for all its respective substrates. In addition, the activity of ovine 3βHSD in microsomal preparations was significantly influenced by the addition of either purified Cyt-b(5) or anti-Cyt-b(5) IgG. The results presented in this study indicate that Cyt-b(5) augments 3βHSD activity and represents the first documentation of such augmentation in any species. PMID:21930205

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

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

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

    PubMed Central

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

    2014-01-01

    Malate dehydrogenase (MDH) plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH) of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA) to L-malate (hereon referred to as MDH activity) was analyzed. Michaelis Constant (Km) and Maximum Reaction Velocity (Vmax) of the reaction were determined to be 6.45 × 10−3 M and 0.87 mM L−1 min−1, respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu2+, Zn2+, and Pb2+, respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH's roles in B. abortus metabolism. PMID:24895685

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

    PubMed

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

    2014-10-01

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

  15. Periplasmic nitrate reductase and formate dehydrogenase: similar molecular architectures with very different enzymatic activities.

    PubMed

    Cerqueira, Nuno M F S A; Gonzalez, Pablo J; Fernandes, Pedro A; Moura, José J G; Ramos, Maria João

    2015-11-17

    It is remarkable how nature has been able to construct enzymes that, despite sharing many similarities, have simple but key differences that tune them for completely different functions in living cells. Periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) from the DMSOr family are representative examples of this. Both enzymes share almost identical three-dimensional protein foldings and active sites, in terms of coordination number, geometry and nature of the ligands. The substrates of both enzymes (nitrate and formate) are polyatomic anions that also share similar charge and stereochemistry. In terms of the catalytic mechanism, both enzymes have a common activation mechanism (the sulfur-shift mechanism) that ensures a constant coordination number around the metal ion during the catalytic cycle. In spite of these similarities, they catalyze very different reactions: Nap abstracts an oxygen atom from nitrate releasing nitrite, whereas FdH catalyzes a hydrogen atom transfer from formate and releases carbon dioxide. In this Account, a critical analysis of structure, function, and catalytic mechanism of the molybdenum enzymes periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) is presented. We conclude that the main structural driving force that dictates the type of reaction, catalyzed by each enzyme, is a key difference on one active site residue that is located in the top region of the active sites of both enzymes. In both enzymes, the active site is centered on the metal ion of the cofactor (Mo in Nap and Mo or W in Fdh) that is coordinated by four sulfur atoms from two pyranopterin guanosine dinucleotide (PGD) molecules and by a sulfido. However, while in Nap there is a Cys directly coordinated to the Mo ion, in FdH there is a SeCys instead. In Fdh there is also an important His that interacts very closely with the SeCys, whereas in Nap the same position is occupied by a Met. The role of Cys in Nap and SeCys in FdH is similar in both

  16. Effect of feeding and of DDT on the activity of hepatic glucose 6- phosphate dehydrogenase in two salmonids

    USGS Publications Warehouse

    Buhler, Donald R.; Benville, P.

    1969-01-01

    The specific activity of liver glucose 6-phosphate dehydrogenase in yearling rainbow trout remained unchanged when the fish were starved for periods as long as 8 weeks and when starved animals were fed diets of various compositions. Injection of insulin concurrently with refeeding also failed to alter the specific activity of the enzyme in trout. The absence of a dietary or insulin influence on the teleost enzyme system is to be contrasted with studies in mammals in which the activity of hepatic glucose 6-P dehydrogenase was markedly stimulated after refeeding starved animals or injection of insulin.Ingestion of the pesticide DDT by juvenile coho salmon or adult rainbow trout also had no effect on the specific activity of liver glucose 6-P dehydrogenase and DDT failed to inhibit the rainbow trout enzyme in vitro. These results also differ considerably from those found in higher animals.These results suggest that the glucose 6-P dehydrogenase enzyme in teleosts may be under a different type of regulatory control from that found in mammals.

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

    PubMed

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

    2014-03-01

    This work evaluated the roles of the low-molecular-weight organic acids (LMWOAs) from root exudates and the dehydrogenase activity in the rhizosphere sediments of three mangrove plant species on the removal of mixed PAHs. The results showed that the concentrations of LMWOAs and dehydrogenase activity changed species-specifically with the levels of PAH contamination. In all plant species, the concentration of citric acid was the highest, followed by succinic acid. For these acids, succinic acid was positively related to the removal of all the PAHs except Chr. Positive correlations were also found between the removal percentages of 4-and 5-ring PAHs and all LMWOAs, except citric acid. LMWOAs enhanced dehydrogenase activity, which positively related to PAH removal percentages. These findings suggested that LMWOAs and dehydrogenase activity promoted the removal of PAHs. Among three mangrove plants, Bruguiera gymnorrhiza, the plant with the highest root biomass, dehydrogenase activity and concentrations of LMWOAs, was most efficient in removing PAHs. PMID:24287262

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

    PubMed

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

    2014-03-01

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

  19. Two mitochondrial alcohol dehydrogenase activities of Kluyveromyces lactis are differently expressed during respiration and fermentation.

    PubMed

    Saliola, M; Falcone, C

    1995-12-20

    The lactose-utilizing yeast Kluyveromyces lactis is an essentially aerobic organism in which both respiration and fermentation can coexist depending on the sugar concentration. Despite a low fermentative capacity as compared to Saccharomyces cerevisiae, four structural genes encoding alcohol dehydrogenase (ADH) activities are present in this yeast. Two of these activities, namely K1ADH III and K1ADH IV, are located within mitochondria and their presence is dependent on the carbon sources in the medium. In this paper we demonstrate by transcription and activity analysis that KlADH3 is expressed in the presence of low glucose concentrations and in the presence of respiratory carbon sources other than ethanol. Indeed ethanol acts as a strong repressor of this gene. On the other hand, KlADH4 is induced by the presence of ethanol and not by other respiratory carbon sources. We also demonstrate that the presence of KLADH III and KLADH IV in K. lactis cells is dependent on glucose concentration, glucose uptake and the amount of ethanol produced. As a consequence, these activities can be used as markers for the onset of respiratory and fermentative metabolism in this yeast.

  20. Identification of Active Retinaldehyde Dehydrogenase Isoforms in the Postnatal Human Eye

    PubMed Central

    Harper, Angelica R.; Wiechmann, Allan F.; Moiseyev, Gennadiy; Ma, Jian-Xing; Summers, Jody A.

    2015-01-01

    Background/Objectives Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid (atRA) synthesis in response to visual signals in animal models of myopia. To explore the potential role of retinaldehyde dehydrogenase (RALDH) enzymes and atRA in human postnatal ocular growth, RALDH activity, along with the distribution of RALDH1, RALDH2, and RALDH3 in the postnatal eye was determined. Methodology Retina, retinal pigment epithelium (RPE), choroid, and sclera were isolated from donor human eyes. RALDH catalytic activity was measured in tissue homogenates using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Homogenates were compared by western blotting for RALDH1, RALDH2, and RALDH3 protein. Immunohistochemistry was used to determine RALDH1 and RALDH2 localization in posterior fundal layers of the human eye. Principal Findings In the postnatal human eye, RALDH catalytic activity was detected in the choroid (6.84 ± 1.20 pmol/hr/ug), RPE (5.46 ± 1.18 pmol/hr/ug), and retina (4.21 ± 1.55 pmol/hr/ug), indicating the presence of active RALDH enzymes in these tissues. RALDH2 was most abundant in the choroid and RPE, in moderate abundance in the retina, and in relatively low abundance in sclera. RALDH1 was most abundant in the choroid, in moderate abundance in the sclera, and substantially reduced in the retina and RPE. RALDH3 was undetectable in human ocular fundal tissues. In the choroid, RALDH1 and RALDH2 localized to slender cells in the stroma, some of which were closely associated with blood vessels. Conclusions/Significance Results of this study demonstrated that: 1) Catalytically active RALDH is present in postnatal human retina, RPE, and choroid, 2) RALDH1 and RALDH2 isoforms are present in these ocular tissues, and 3) RALDH1 and RALDH2 are relatively abundant in the choroid and/or RPE. Taken together, these results suggest that RALDH1 and 2 may play a role in the regulation of

  1. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

    PubMed Central

    Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

    2015-01-01

    ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the

  2. The Comparative Effects of Sports Massage, Active Recovery, and Rest in Promoting Blood Lactate Clearance After Supramaximal Leg Exercise

    PubMed Central

    Martin, Nancy A.; Zoeller, Robert F.; Robertson, Robert J.; Lephart, Scott M.

    1998-01-01

    Objective: To determine the comparative effect of sports massage, active recovery, and rest on promoting blood lactate clearance after maximal anaerobic (supramaximal) leg exercise. Design and Setting: A counterbalanced experimental design with repeated measures was used. The repeated measures were the three treatment conditions. The order of the conditions was determined by random assignment to a counterbalanced test sequence. All data were collected in the Human Energy Research Laboratory at the University of Pittsburgh. Subjects: Ten male competitive cyclists volunteered for this investigation. Measurements: Serial venous blood samples were drawn and analyzed for blood lactate concentration for each test condition. Results: There were significant main effects for both absolute and relative values of blood lactate concentration between the three treatment groups and across time within groups. Conclusions: After supramaximal leg exercise, active recovery produced significant decreases in both absolute and relative measures of blood lactate concentration when compared with the sports massage and rest conditions. No significant difference was found between sports massage and rest for either absolute or relative changes in blood lactate concentration. PMID:16558481

  3. Activity against Listeria monocytogenes of human milk during lactation. A preliminary study.

    PubMed

    López-Expósito, Iván; Manso, María Asunción; López-Fandiño, Rosina; Recio, Isidra

    2008-02-01

    Human milk samples from three healthy donors were investigated in order to evaluate the antibacterial activity during lactation against Escherichia coli ATCC 25922 and Listeria monocytogenes. The concentration of the main human-milk antimicrobial proteins (lactoferrin (LF), lysozyme (LZ) and secretory immunoglobulin A (sIgA)) was determined by ELISA. Results showed that human milk exhibited antibacterial activity against List. monocytogenes, although it was weakly active against Esch. coli ATCC 25922. The observed antilisterial activity was positively correlated with LZ concentration. In addition, the effect of gastrointestinal proteases, at different pH conditions, that prevail in the stomach of infants (pH 2.0-6.5), on antilisterial activity and protein degradation was evaluated. Hydrolysis with pepsin at pH 4.0-6.5, followed by treatment with pancreatic enzymes, resulted in a decreased hydrolysis of LZ, LF and sIgA and an enhanced antibacterial activity against List. monocytogenes. It is suggested that partial degradation of certain milk proteins at the gastrointestinal level may produce peptides that could act synergistically with the remnant intact proteins.

  4. Activity and Conformation of Yeast Alcohol Dehydrogenase (YADH) Entrapped in Reverse Micelles.

    PubMed

    Das; Mozumdar; Maitra

    2000-10-15

    Yeast alcohol dehydrogenase (YADH) solubilized in reverse micelles of aerosol OT (i.e., AOT or sodium bis (2-ethyl hexyl) sulfosuccinate) in isooctane has been shown to be catalytically more active than that in aqueous buffer under optimum conditions of pH, temperature, and water content in reverse micelles. Studies of the secondary structure conformational changes of the enzyme in reverse micelles have been made from circular dichroism spectroscopy. It has been seen that the conformation of YADH in reverse micelles is extremely sensitive to pH, temperature, and water content. A comparison has been made between the catalytic activity of the enzyme and the alpha-helix content in the conformation and it has been observed that the enzyme is most active at the maximum alpha-helix content. While the beta-sheet content in the conformation of the entrapped enzyme was found to be dependent on the enzyme-micelle interface interaction, the alpha-helix and random coil conformations are governed by the degree of entrapment and the extent of rigidity provided by the micelle core to the enzyme structure. Copyright 2000 Academic Press.

  5. Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity

    PubMed Central

    Leopold, Jane A.; Dam, Aamir; Maron, Bradley A.; Scribner, Anne W.; Liao, Ronglih; Handy, Diane E.; Stanton, Robert C.; Pitt, Bertram; Loscalzo, Joseph

    2013-01-01

    Hyperaldosteronism is associated with impaired vascular reactivity; however, the mechanism by which aldosterone promotes endothelial dysfunction remains unknown. Glucose-6-phosphate dehydrogenase (G6pd), the principal source of Nadph, modulates vascular function by limiting oxidant stress to preserve bioavailable nitric oxide (NO•). In these studies, we show that aldosterone (10−9-10−7 mol/l) decreases endothelial G6pd expression and activity in vitro resulting in increased oxidant stress and decreased cGMP levels similar to what is observed in G6pd-deficient cells. Aldosterone decreases G6pd expression by protein kinase A activation to increase expression of Crem, which interferes with Creb binding to the G6pd promoter. In vivo, infusion of aldosterone decreases vascular G6pd expression and impairs vascular reactivity. These effects are abrogated by spironolactone or vascular gene transfer of G6pd. These studies demonstrate that aldosterone induces a G6pd-deficient phenotype to impair endothelial function; aldosterone antagonism or gene transfer of G6pd improves vascular reactivity by restoring G6pd activity. PMID:17273168

  6. Serratia marcescens Quinoprotein Glucose Dehydrogenase Activity Mediates Medium Acidification and Inhibition of Prodigiosin Production by Glucose

    PubMed Central

    Fender, James E.; Bender, Cody M.; Stella, Nicholas A.; Lahr, Roni M.; Kalivoda, Eric J.

    2012-01-01

    Serratia marcescens is a model organism for the study of secondary metabolites. The biologically active pigment prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine), like many other secondary metabolites, is inhibited by growth in glucose-rich medium. Whereas previous studies indicated that this inhibitory effect was pH dependent and did not require cyclic AMP (cAMP), there is no information on the genes involved in mediating this phenomenon. Here we used transposon mutagenesis to identify genes involved in the inhibition of prodigiosin by glucose. Multiple genetic loci involved in quinoprotein glucose dehydrogenase (GDH) activity were found to be required for glucose inhibition of prodigiosin production, including pyrroloquinoline quinone and ubiquinone biosynthetic genes. Upon assessing whether the enzymatic products of GDH activity were involved in the inhibitory effect, we observed that d-glucono-1,5-lactone and d-gluconic acid, but not d-gluconate, were able to inhibit prodigiosin production. These data support a model in which the oxidation of d-glucose by quinoprotein GDH initiates a reduction in pH that inhibits prodigiosin production through transcriptional control of the prodigiosin biosynthetic operon, providing new insight into the genetic pathways that control prodigiosin production. Strains generated in this report may be useful in large-scale production of secondary metabolites. PMID:22752173

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

    PubMed Central

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

    2011-01-01

    Total glucose-6-phosphate dehydrogenase (G6PDH) activity, protein abundance, and transcript levels of G6PDH isoforms were measured in response to exogenous abscisic acid (ABA) supply to barley (Hordeum vulgare cv Nure) hydroponic culture. Total G6PDH activity increased by 50% in roots treated for 12 h with exogenous 0.1 mM ABA. In roots, a considerable increase (35%) in plastidial P2-G6PDH transcript levels was observed during the first 3 h of ABA treatment. Similar protein variations were observed in immunoblotting analyses. In leaves, a 2-fold increase in total G6PDH activity was observed after ABA treatment, probably related to an increase in the mRNA level (increased by 50%) and amount of protein (increased by 85%) of P2-G6PDH. Together these results suggest that the plastidial P2-isoform plays an important role in ABA-treated barley plants. PMID:21464159

  8. Serratia marcescens quinoprotein glucose dehydrogenase activity mediates medium acidification and inhibition of prodigiosin production by glucose.

    PubMed

    Fender, James E; Bender, Cody M; Stella, Nicholas A; Lahr, Roni M; Kalivoda, Eric J; Shanks, Robert M Q

    2012-09-01

    Serratia marcescens is a model organism for the study of secondary metabolites. The biologically active pigment prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine), like many other secondary metabolites, is inhibited by growth in glucose-rich medium. Whereas previous studies indicated that this inhibitory effect was pH dependent and did not require cyclic AMP (cAMP), there is no information on the genes involved in mediating this phenomenon. Here we used transposon mutagenesis to identify genes involved in the inhibition of prodigiosin by glucose. Multiple genetic loci involved in quinoprotein glucose dehydrogenase (GDH) activity were found to be required for glucose inhibition of prodigiosin production, including pyrroloquinoline quinone and ubiquinone biosynthetic genes. Upon assessing whether the enzymatic products of GDH activity were involved in the inhibitory effect, we observed that d-glucono-1,5-lactone and d-gluconic acid, but not d-gluconate, were able to inhibit prodigiosin production. These data support a model in which the oxidation of d-glucose by quinoprotein GDH initiates a reduction in pH that inhibits prodigiosin production through transcriptional control of the prodigiosin biosynthetic operon, providing new insight into the genetic pathways that control prodigiosin production. Strains generated in this report may be useful in large-scale production of secondary metabolites.

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

    PubMed

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

    2009-12-01

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

  10. Affinity chromatography of nicotinamide-adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide.

    PubMed

    Barry, S; O'Carra, P

    1973-12-01

    1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems

  11. Expression of constitutively activated Akt in the mammary gland leads to excess lipid synthesis during pregnancy and lactation.

    PubMed

    Schwertfeger, Kathryn L; McManaman, James L; Palmer, Carol A; Neville, Margaret C; Anderson, Steven M

    2003-06-01

    Expression of constitutively activated Akt in the mammary glands of transgenic mice results in a delay in post-lactational involution. We now report precocious lipid accumulation in the alveolar epithelium of mouse mammary tumor virus-myr-Akt transgenic mice accompanied by a lactation defect that results in a 50% decrease in litter weight over the first 9 days of lactation. Although ductal structures and alveolar units develop normally during pregnancy, cytoplasmic lipid droplets appeared precociously in mammary epithelial cells in early pregnancy and were accompanied by increased expression of adipophilin, which is associated with lipid droplets. By late pregnancy the lipid droplets had become significantly larger than in nontransgenic mice, and they persisted into lactation. The fat content of milk from lactating myr-Akt transgenic mice was 65-70% by volume compared to 25-30% in wild-type mice. The diminished growth of pups nursed by transgenic mothers could result from the high viscosity of the milk and the inability of the pups to remove sufficient quantities of milk by suckling. Transduction of the CIT3 mammary epithelial cell line with a recombinant human adenovirus encoding myr-Akt resulted in an increase in glucose transport and lipid biosynthesis, suggesting that Akt plays an important role in regulation of lipid metabolism. PMID:12700340

  12. Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers

    PubMed Central

    Nemeria, Natalia S.; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-01-01

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4′-aminopyrimidine N1′ atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu571, Glu235, and Glu237) and Arg606 resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu235 makes no direct contact with the cofactor. The role of the conserved Glu571 residue in both catalysis and cofactor orientation is revealed by the combined results for the first time. PMID:20106967

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

    PubMed

    Deng, Qingfang; Zhou, Xin; Chen, Huaguo

    2014-10-13

    In the present study, enzyme assisted extraction of Fructus Mori polysaccharides (FMPS) from F. mori using four kinds of enzymes and three compound enzymes were examined. Research found that glucose oxidase offered a better performance in enhancement of the extraction yields of FMPS, antioxidant and activate alcohol dehydrogenase activities. The glucose oxidase assisted extraction process was further optimized by using response surface method (RSM) to obtain maximum yield of crude FMPS. The results showed that optimized extraction conditions were ratio of enzyme amount 0.40%, enzyme treated time 38 min, treated temperature 58 °C and liquid-solid radio 11.0. Under these conditions, the mean experimental value of extraction yield (16.16 ± 0.14%) corresponded well with the predicted values and increased 160% than none enzyme treated ones. Pharmacological verification tests showed that F. mori crude polysaccharides had good antioxidant and activate alcohol dehydrogenase activities in vitro. PMID:25037415

  14. Differential inhibition of PDKs by phenylbutyrate and enhancement of pyruvate dehydrogenase complex activity by combination with dichloroacetate.

    PubMed

    Ferriero, Rosa; Iannuzzi, Clara; Manco, Giuseppe; Brunetti-Pierri, Nicola

    2015-09-01

    Pyruvate dehydrogenase complex (PDHC) is a key enzyme in metabolism linking glycolysis to tricarboxylic acid cycle and its activity is tightly regulated by phosphorylation catalyzed by four pyruvate dehydrogenase kinase (PDK) isoforms. PDKs are pharmacological targets for several human diseases including cancer, diabetes, obesity, heart failure, and inherited PDHC deficiency. We investigated the inhibitory activity of phenylbutyrate toward PDKs and found that PDK isoforms 1-to-3 are inhibited whereas PDK4 is unaffected. Moreover, docking studies revealed putative binding sites of phenylbutyrate on PDK2 and 3 that are located on different sites compared to dichloroacetate (DCA), a previously known PDK inhibitor. Based on these findings, we showed both in cells and in mice that phenylbutyrate combined to DCA results in greater increase of PDHC activity compared to each drug alone. These results suggest that therapeutic efficacy can be enhanced by combination of drugs increasing PDHC enzyme activity. PMID:25601413

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

    PubMed

    Deng, Qingfang; Zhou, Xin; Chen, Huaguo

    2014-10-13

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

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

    PubMed

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

    2015-07-01

    Mitochondrial dihydrolipoyl dehydrogenase (mtLPD; L-protein) is an integral component of several multienzyme systems involved in the tricarboxylic acid (TCA) cycle, photorespiration, and the degradation of branched-chain α-ketoacids. The majority of the mtLPD present in photosynthesizing tissue is used for glycine decarboxylase (GDC), necessary for the high-flux photorespiratory glycine-into-serine conversion. We previously suggested that GDC activity could be a signal in a regulatory network that adjusts carbon flux through the Calvin-Benson cycle in response to photorespiration. Here, we show that elevated GDC L-protein activity significantly alters several diagnostic parameters of cellular metabolism and leaf gas exchange in Arabidopsis thaliana. Overexpressor lines displayed markedly decreased steady state contents of TCA cycle and photorespiratory intermediates as well as elevated NAD(P)(+)-to-NAD(P)H ratios. Additionally, increased rates of CO2 assimilation, photorespiration, and plant growth were observed. Intriguingly, however, day respiration rates remained unaffected. By contrast, respiration was enhanced in the first half of the dark phase but depressed in the second. We also observed enhanced sucrose biosynthesis in the light in combination with a lower diel magnitude of starch accumulation and breakdown. These data thus substantiate our prior hypothesis that facilitating flux through the photorespiratory pathway stimulates photosynthetic CO2 assimilation in the Calvin-Benson cycle. They furthermore suggest that this regulation is, at least in part, dependent on increased light-capture/use efficiency. PMID:26116608

  17. Aldehyde Dehydrogenase Activity Identifies a Population of Human Skeletal Muscle Cells With High Myogenic Capacities

    PubMed Central

    Vauchez, Karine; Marolleau, Jean-Pierre; Schmid, Michel; Khattar, Patricia; Chapel, Alain; Catelain, Cyril; Lecourt, Séverine; Larghéro, Jérôme; Fiszman, Marc; Vilquin, Jean-Thomas

    2009-01-01

    Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM), umbilical cord blood (UCB), and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study, we have identified ALDH+ cells within human skeletal muscles, and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH, Aldefluor, identified brightly stained (ALDHbr) cells with low side scatter (SSClo), in enzymatically dissociated muscle biopsies, thereafter abbreviated as SMALD+ (for skeletal muscle ALDH+) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD+/CD34− and SMALD+/CD34+ cells. These sub-populations did not initially express endothelial (CD31), hematopoietic (CD45), and myogenic (CD56) markers. Upon sorting, however, whereas SMALD+/CD34+ cells developed in vitro as a heterogeneous population of CD56− cells able to differentiate in adipoblasts, the SMALD+/CD34− fraction developed in vitro as a highly enriched population of CD56+ myoblasts able to form myotubes. Moreover, only the SMALD+/CD34− population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy. PMID:19738599

  18. Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

    PubMed Central

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

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

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

    PubMed

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

    2015-07-01

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

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

    PubMed Central

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

    2015-01-01

    Mitochondrial dihydrolipoyl dehydrogenase (mtLPD; L-protein) is an integral component of several multienzyme systems involved in the tricarboxylic acid (TCA) cycle, photorespiration, and the degradation of branched-chain α-ketoacids. The majority of the mtLPD present in photosynthesizing tissue is used for glycine decarboxylase (GDC), necessary for the high-flux photorespiratory glycine-into-serine conversion. We previously suggested that GDC activity could be a signal in a regulatory network that adjusts carbon flux through the Calvin-Benson cycle in response to photorespiration. Here, we show that elevated GDC L-protein activity significantly alters several diagnostic parameters of cellular metabolism and leaf gas exchange in Arabidopsis thaliana. Overexpressor lines displayed markedly decreased steady state contents of TCA cycle and photorespiratory intermediates as well as elevated NAD(P)+-to-NAD(P)H ratios. Additionally, increased rates of CO2 assimilation, photorespiration, and plant growth were observed. Intriguingly, however, day respiration rates remained unaffected. By contrast, respiration was enhanced in the first half of the dark phase but depressed in the second. We also observed enhanced sucrose biosynthesis in the light in combination with a lower diel magnitude of starch accumulation and breakdown. These data thus substantiate our prior hypothesis that facilitating flux through the photorespiratory pathway stimulates photosynthetic CO2 assimilation in the Calvin-Benson cycle. They furthermore suggest that this regulation is, at least in part, dependent on increased light-capture/use efficiency. PMID:26116608

  1. Biophysical and mutagenic analysis of Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase activity and specificity.

    PubMed Central

    Burdette, D S; Secundo, F; Phillips, R S; Dong, J; Scott, R A; Zeikus, J G

    1997-01-01

    The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (secondary ADH) was overexpressed in Escherichia coli at more than 10% of total protein. The recombinant enzyme was purified in high yield (67%) by heat-treatment at 85 degrees C and (NH4)2SO4 precipitation. Site-directed mutants (C37S, H59N, D150N, D150Eand D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding. X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS1(imid)1(N,O)3 co-ordination sphere. Inductively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type secondary ADH subunit. The C37S, H59N and D150N mutant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectively,suggesting that these residues are involved in Zn liganding. The D150E and D150C mutants retained 0.47 and 1.2 Zn atoms per subunit, indicating that an anionic side-chain moiety at this position preserves the bound Zn. All five mutant enzymes had activity, suggesting that the T. ethanolicus secondary ADH requires a properly co-ordinated catalytic Zn atom. The His-59 and Asp-150 mutations also altered secondary ADH affinity for propan-2-ol over a 140-fold range, whereas the overall change in affinity for ethanol spanned a range of only 7-fold, supporting the importance of the metal in secondary ADH substrate binding. The lack of significant changes in cofactor affinity as a result of these catalytic Zn ligand mutations suggested that secondary ADH substrate-and cofactor-binding sites are structurally distinct. Altering Gly198 to Asp reduced the enzyme specific activity 2.7-fold, increased the Km(app) for NADP+ 225-fold, and decreased the Km(app) for NAD+ 3-fold, supporting the prediction that the enzyme binds nicotinamide cofactor in a Rossmann fold. Our data indicate therefore that, unlike the liver primary ADH

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

    PubMed Central

    Fu, Bo; Ren, Liang; Liu, Di; Ma, Jian-Zhang; An, Tie-Zhu; Yang, Xiu-Qin; Ma, Hong; Zhang, Dong-Jie; Guo, Zhen-Hua; Guo, Yun-Yun; Zhu, Meng; Bai, Jing

    2015-01-01

    The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (−) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes. PMID:26580437

  3. The regulation of adipose tissue pyruvate dehydrogenase activity of dietary fiber.

    PubMed

    Ogunwole, J O; Knight, E M; Adkins, J S; Thomaskutty, K G; Pointer, R H

    1987-05-01

    In vitro studies have established that insulin enhances the oxidation of pyruvate to acetyl CoA by the stimulation of mitochondrial pyruvate dehydrogenase (PDH) activity through plasma membrane binding response (Jarett and Seals 1979; Kiechle, Jarett, Dennis and Kotagal 1980). In the present study adipose tissue PDH activity was utilized as a marker for insulin responsiveness. The metabolic response of this enzyme to exogenous insulin was employed to test the hypothesis that dietary fiber enhances tissue responsiveness to insulin using adipose tissue from male weanling Sprague Dawley rats. Eight groups of rats (n = 5 per group) were fed ad libitum various diets containing different levels of cellulose and protein as already reported elsewhere (Ogunwole, Knight, Adkins, Thomaskutty and Pointer 1985). Percent insulin stimulation of PDH from basal activity (PDS) was utilized as an index of insulin responsiveness. Compared to all fiber treated groups, both basal (PDB) and insulin stimulated (PDI) activities were significantly lower (P less than 0.05) in the fiber free groups at both low (10%) and high (20%) protein levels. At all fiber levels tested (0, 5, 15 and 30%) protein intake resulted in a significant increase in both PDB and PDI. Gradual increase in cellulose intake resulted in a biphasic increase in PDS in both protein groups at the 5% and 30% fiber levels. PDS was higher (P less than 0.05) in the 10% protein groups than the 20% protein group at all fiber levels tested. A significant interaction effect of protein and fiber was observed on PDB (P less than 0.001) and PDI (P less than 0.04) when caloric intake was held constant as a covariate.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Postnatal Chick Choroids Exhibit Increased Retinaldehyde Dehydrogenase Activity During Recovery From Form Deprivation Induced Myopia

    PubMed Central

    Harper, Angelica R.; Wang, Xiang; Moiseyev, Gennadiy; Ma, Jian-Xing; Summers, Jody A.

    2016-01-01

    Purpose Increases in retinaldehyde dehydrogenase 2 (RALDH2) transcript in the chick choroid suggest that RALDH2 may be responsible for increases observed in all-trans-retinoic acid (atRA) synthesis during recovery from myopic defocus. The purpose of the present study was to examine RALDH2 protein expression, RALDH activity, and distribution of RALDH2 cells in control and recovering chick ocular tissues. Methods Myopia was induced in White Leghorn chicks for 10 days, followed by up to 15 days of unrestricted vision (recovery). Expression of RALDH isoforms in chick ocular tissues was evaluated by Western blot. Catalytic activity of RALDH was measured in choroidal cytosol fractions using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Distribution of RALDH2 cells throughout the choroid was evaluated by immunohistochemistry. Results RALDH2 was expressed predominately in the chick choroid (P < 0.001) and increased after 24 hours and 4 days of recovery (76%, 74%, and 165%, respectively; P < 0.05). Activity of RALDH was detected solely in the choroid and was elevated at 3 and 7 days of recovery compared to controls (70% and 48%, respectively; P < 0.05). The number of RALDH2 immunopositive cells in recovering choroids was increased at 24 hours and 4 to 15 days of recovery (P < 0.05) and were concentrated toward the RPE side compared to controls. Conclusions The results of this study suggest that RALDH2 is the major RALDH isoform in the chick choroid and is responsible for the increased RALDH activity seen during recovery. PMID:27654415

  5. Lack of Skeletal Muscle IL-6 Affects Pyruvate Dehydrogenase Activity at Rest and during Prolonged Exercise

    PubMed Central

    Gudiksen, Anders; Schwartz, Camilla Lindgren; Bertholdt, Lærke; Joensen, Ella; Knudsen, Jakob G.; Pilegaard, Henriette

    2016-01-01

    Pyruvate dehydrogenase (PDH) plays a key role in the regulation of skeletal muscle substrate utilization. IL-6 is produced in skeletal muscle during exercise in a duration dependent manner and has been reported to increase whole body fatty acid oxidation, muscle glucose uptake and decrease PDHa activity in skeletal muscle of fed mice. The aim of the present study was to examine whether muscle IL-6 contributes to exercise-induced PDH regulation in skeletal muscle. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and floxed littermate controls (control) completed a single bout of treadmill exercise for 10, 60 or 120 min, with rested mice of each genotype serving as basal controls. The respiratory exchange ratio (RER) was overall higher (P<0.05) in IL-6 MKO than control mice during the 120 min of treadmill exercise, while RER decreased during exercise independent of genotype. AMPK and ACC phosphorylation also increased with exercise independent of genotype. PDHa activity was in control mice higher (P<0.05) at 10 and 60 min of exercise than at rest but remained unchanged in IL-6 MKO mice. In addition, PDHa activity was higher (P<0.05) in IL-6 MKO than control mice at rest and 60 min of exercise. Neither PDH phosphorylation nor acetylation could explain the genotype differences in PDHa activity. Together, this provides evidence that skeletal muscle IL-6 contributes to the regulation of PDH at rest and during prolonged exercise and suggests that muscle IL-6 normally dampens carbohydrate utilization during prolonged exercise via effects on PDH. PMID:27327080

  6. The activity of liver alcohol dehydrogenase with nicotinamide–adenine dinucleotide phosphate as coenzyme

    PubMed Central

    Dalziel, K.; Dickinson, F. M.

    1965-01-01

    1. The separation of nucleotide impurities from commercial NADP preparations by chromatography is described. All the preparations studied contained 0·1–0·2% of NAD. 2. The activity of pure crystalline liver alcohol dehydrogenase with NADP as coenzyme has been confirmed. Initial-rate data are reported for the reaction at pH 6·0 and 7·0 with ethanol and acetaldehyde as substrates. With NADP and NADPH2 of high purity, the maximal specific rates were similar to those obtained with NAD and NADH2, but the Michaelis constants for the former coenzymes were much greater than those for the latter. 3. The oxidation of ethanol by NADP is greatly inhibited by NADH2, and this accounts for low values of certain initial-rate parameters obtained with commercial NADP preparations containing NAD. The kinetics of the inhibition are consistent with competitive inhibition in a compulsory-order mechanism. 4. Initial-rate data with NAD and NADPH2 do not conform to the requirements of the mechanism proposed by Theorell & Chance (1951), in contrast with results previously obtained with NAD and NADH2. The possibility that the deviations are due to competing nucleotide impurity in the oxidized coenzyme cannot be excluded. The data show that the enzyme reacts more slowly with, and has a smaller affinity for, NADP and NADPH2 than NAD and NADH2. 5. Phosphate behaves as a competitive inhibitor towards NADP. PMID:14340079

  7. Bactericidal activity of macrophages against Streptococcus uberis is different in mammary gland secretions of lactating and drying off cows.

    PubMed

    Denis, Michel; Parlane, Natalie A; Lacy-Hulbert, S Jane; Summers, Emma L; Buddle, Bryce M; Wedlock, D Neil

    2006-11-15

    to exert significant bactericidal activity against S. uberis. There were no significant differences in the bactericidal activity of milk macrophages obtained from lactating cows with low somatic cell counts (SCC; < 10(5) ml(-1)) compared with those with a mildly elevated SCC (> 10(5) ml(-1)) (P > 0.05). In contrast, mammary gland secretion macrophages isolated from the same cows in the mid-dry period killed a significant proportion of phagocytosed S. uberis (50-65% of ingested S. uberis killed, P < 0.01) although cytokine production in response to in vitro bacterial infection was low. We conclude that the bactericidal activity of mammary gland secretion macrophages against a virulent strain of S. uberis is low during the lactation period. In addition, our data indicate that S. uberis is not a strong inducer of NO and TNF-alpha in macrophages from the milk or mammary gland secretions of cows during the drying off period. Finally, IFN-gamma does not activate milk macrophages or macrophages from cows during the lactating period or mammary gland secretions during the drying off period.

  8. Lactate Chemical Exchange Saturation Transfer (LATEST) Imaging in vivo A Biomarker for LDH Activity

    PubMed Central

    DeBrosse, Catherine; Nanga, Ravi Prakash Reddy; Bagga, Puneet; Nath, Kavindra; Haris, Mohammad; Marincola, Francesco; Schnall, Mitchell D.; Hariharan, Hari; Reddy, Ravinder

    2016-01-01

    Non-invasive imaging of lactate is of enormous significance in cancer and metabolic disorders where glycolysis dominates. Here, for the first time, we describe a chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) method (LATEST), based on the exchange between lactate hydroxyl proton and bulk water protons to image lactate with high spatial resolution. We demonstrate the feasibility of imaging lactate with LATEST in lactate phantoms under physiological conditions, in a mouse model of lymphoma tumors, and in skeletal muscle of healthy human subjects pre- and post-exercise. The method is validated by measuring LATEST changes in lymphoma tumors pre- and post-infusion of pyruvate and correlating them with lactate determined from multiple quantum filtered proton magnetic resonance spectroscopy (SEL-MQC 1H-MRS). Similarly, dynamic LATEST changes in exercising human skeletal muscle are correlated with lactate determined from SEL-MQC 1H-MRS. The LATEST method does not involve injection of radioactive isotopes or labeled metabolites. It has over two orders of magnitude higher sensitivity compared to conventional 1H-MRS. It is anticipated that this technique will have a wide range of applications including diagnosis and evaluation of therapeutic response of cancer, diabetes, cardiac, and musculoskeletal diseases. The advantages of LATEST over existing methods and its potential challenges are discussed. PMID:26794265

  9. Substitution of Tyr254 with Phe at the active site of flavocytochrome b2: consequences on catalysis of lactate dehydrogenation

    SciTech Connect

    Dubois, J.; Chapman, S.K.; Mathews, F.S.; Reid, G.A.; Lederer, F. )

    1990-07-10

    A role for Tyr254 in L-lactate dehydrogenation catalyzed by flavocytochrome b2 has recently been proposed on the basis of the known active-site structure and of studies that had suggested a mechanism involving the initial formation of a lactate carbanion. This role is now examined after replacement of Tyr254 with phenylalanine. The kcat is decreased about 40-fold, Km for lactate appears unchanged, and the mainly rate-limiting step is still alpha-hydrogen abstraction, as judged from the steady-state deuterium isotope effect. Modeling studies with lactate introduced into the active site indicate two possible substrate conformations with different hydrogen-bonding partners for the substrate hydroxyl. If the hydrogen bond is formed with Tyr254, as was initially postulated, the mechanism must involve removal by His373 of the C2 hydrogen, with carbanion formation. If, in the absence of the Tyr254 phenol group, the hydrogen bond is formed with His373 N3, the substrate is positioned in such a way that the reaction must proceed by hydride transfer. Therefore the mechanism of the Y254F enzyme was investigated so as to distinguish between the two mechanistic possibilities. 2-Hydroxy-3-butynoate behaves with the mutant as a suicide reagent, as with the wild-type enzyme. Similarly, the mutant protein also catalyzes the reduction and the dehydrohalogenation of bromopyruvate under transhydrogenation conditions.

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

    PubMed

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

    2014-01-01

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

  11. Display of Bombyx mori Alcohol Dehydrogenases on the Bacillus subtilis Spore Surface to Enhance Enzymatic Activity under Adverse Conditions

    PubMed Central

    Wang, Nan; Chang, Cheng; Yao, Qin; Li, Guohui; Qin, Lvgao; Chen, Liang; Chen, Keping

    2011-01-01

    Alcohol dehydrogenases (ADHs) are oxidoreductases catalyzing the reversible oxidation of alcohols to corresponding aldehydes or ketones accompanied by nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. ADHs attract major scientific and industrial interest for the evolutionary perspectives, afforded by their wide occurrence in nature, and for their use in industrial synthesis. However, the low activity of ADHs under extremes of pH and temperature often limits their application. To obtain ADH with high activity, in this study, we used Bombyx mori alcohol dehydrogenases (BmADH) as foreign gene and constructed a recombinant integrative plasmid pJS700-BmADH. This pJS700-BmADH was transformed into Bacillus subtilis by double cross-over and produced an amylase inactivated mutant. The fusion protein containing BmADH was expressed on the spore surface and recognized by BmADH-specific antibody. We also assayed the alcohol dehydrogenase activity of the fusion protein together with the native BmADH at different pH and temperature levels, which indicated the recombinant enzyme exhibits activity over wider ranges of temperature and pH than its native form, perhaps due to the resistance properties of B. subtilis spores against adverse conditions. PMID:21738670

  12. Effect of dietary strong ions on chewing activity and milk production in lactating dairy cows.

    PubMed

    Mooney, C S; Allen, M S

    2007-12-01

    The objective of this experiment was to determine effects of strong ions on chewing activity and short-term lactational performance of dairy cows. Forty multiparous Holstein cows were used in a replicated 5 x 5 Latin square design with a 2 x 2 factorial arrangement of equimolar treatments for cations (sodium and potassium), anions (chloride and bicarbonate), plus a control diet. Periods were 14 d in length with the last 4 d for data and sample collection. Diets were formulated to 29% neutral detergent figer and 17.5% crude protein. Sodium bicarbonate was included at 1% of dry matter in one treatment diet, and other treatments (sodium chloride, potassium chloride, and potassium bicarbonate) were added to be equimolar to sodium bicarbonate in their respective diets. Chewing activity was recorded every 5 min for the last 24 h of each period. Dry matter intake was not affected by treatment (mean = 27.9 kg/d). Bicarbonate treatments increased yields of milk, milk fat, and fat- and solids-corrected milk compared with chloride treatments, but cation treatments did not affect any measured variable. The 4 ion treatments reduced ruminating time per day when compared with control by decreasing the length of rumination bouts. This effect was not specific to cations or anions suggesting a mechanism related to increased ruminal osmolality.

  13. Effect of metals and other inorganic ions on soil microbial activity: soil dehydrogenase assay as a simple toxicity test

    SciTech Connect

    Rogers, J.E.; Li, S.W.

    1985-06-01

    The purpose of this report is to illustrate the utility of the soil dehydrogenase assay as an effective primary test for assessing the potential toxicity of chemicals to soil microbial activity. In this manuscript the authors describe their use of the soil dehydrogenase assay in determining the effects of a number of potential toxic inorganic ions on soil microbial activity. The ions include Cu/sup 2 +/, Mg/sup 2 +/, Ni/sup 2 +/, Zn/sup 2 +/, NH/sub 4//sup +/, Cd/sup 2 +/, Cr/sup 32/, F/sup -/, AsO/sub 4//sup 3 -/, BO/sub 3//sup 3 -/, and SO/sub 4//sup 2 -/.

  14. Allelochemical L-DOPA induces quinoprotein adducts and inhibits NADH dehydrogenase activity and root growth of cucumber.

    PubMed

    Mushtaq, Muhammad Naeem; Sunohara, Yukari; Matsumoto, Hiroshi

    2013-09-01

    Allelochemical L-DOPA (l-3,4-dihydroxyphenylalanine) inhibits growth of several plant species. However, its mode of action is not well clarified in plants. The present studies were conducted to explore the action mechanism of L-DOPA in cucumber roots. The results revealed that L-DOPA suppressed the root growth of cucumber and induced quinoprotein and melanin formation in the roots. Moreover, L-DOPA not only decreased mitochondrial viability and NADH dehydrogenase (complex I) activity but also increased quinoprotein formation in vitro in isolated mitochondria from cucumber roots. Strong correlations were observed between quinoprotein formation and root growth inhibition, quinoprotein formation and NADH dehydrogenase activity, after L-DOPA treatment. The results suggest that quinoprotein formation and mitochondrial impairment might be involved in growth-inhibition mechanism of L-DOPA in cucumber roots. PMID:23831820

  15. Therapeutic activity of lactate 12% lotion in the treatment of ichthyosis. Active versus vehicle and active versus a petrolatum cream.

    PubMed

    Buxman, M; Hickman, J; Ragsdale, W; Stretcher, G; Krochmal, L; Wehr, R F

    1986-12-01

    Lactate 12% lotion was significantly more effective than both its vehicle and a petrolatum-based cream in the treatment of ichthyosis. The treatment regimen was twice-daily application for 4 weeks with evaluations weekly during the treatment period and for 2 weeks after treatment was stopped. Vulgaris, lamellar, sex-linked, Netherton's, and epidermolytic hyperkeratotic forms of ichthyosis were significantly improved by treatment with lactate 12% lotion. This new therapeutic modality expands the scope and extent of ichthyotic conditions that may now be successfully treated.

  16. Structure-Activity Relationship Studies and Biological Characterization of Human NAD+-dependent 15-Hydroxyprostaglandin Dehydrogenase Inhibitors

    PubMed Central

    Duveau, Damien Y.; Yasgar, Adam; Wang, Yuhong; Hu, Xin; Kouznetsova, Jennifer; Brimacombe, Kyle R.; Jadhav, Ajit; Simeonov, Anton; Thomas, Craig J.; Maloney, David J.

    2014-01-01

    The structure-activity relationship (SAR) study of two chemotypes identified as inhibitors of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (HPGD, 15-PGDH) was conducted. Top compounds from both series displayed potent inhibition (IC50 <50 nM), demonstrate excellent selectivity towards HPGD and potently induce PGE2 production in A549 lung cancer and LNCaP prostate cancer cells. PMID:24360556

  17. Structure-activity relationship studies and biological characterization of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase inhibitors.

    PubMed

    Duveau, Damien Y; Yasgar, Adam; Wang, Yuhong; Hu, Xin; Kouznetsova, Jennifer; Brimacombe, Kyle R; Jadhav, Ajit; Simeonov, Anton; Thomas, Craig J; Maloney, David J

    2014-01-15

    The structure-activity relationship (SAR) study of two chemotypes identified as inhibitors of the human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (HPGD, 15-PGDH) was conducted. Top compounds from both series displayed potent inhibition (IC50 <50 nM), demonstrate excellent selectivity towards HPGD and potently induce PGE2 production in A549 lung cancer and LNCaP prostate cancer cells. PMID:24360556

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

    PubMed Central

    Clementi, Marisa A; Deis, Ricardo P; Telleria, Carlos M

    2004-01-01

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

  19. Combining parasite lactate dehydrogenase-based and histidine-rich protein 2-based rapid tests to improve specificity for diagnosis of malaria Due to Plasmodium knowlesi and other Plasmodium species in Sabah, Malaysia.

    PubMed

    Grigg, Matthew J; William, Timothy; Barber, Bridget E; Parameswaran, Uma; Bird, Elspeth; Piera, Kim; Aziz, Ammar; Dhanaraj, Prabakaran; Yeo, Tsin W; Anstey, Nicholas M

    2014-06-01

    Plasmodium knowlesi causes severe and fatal malaria in Malaysia. Microscopic misdiagnosis is common and may delay appropriate treatment. P. knowlesi can cross-react with "species-specific" parasite lactate dehydrogenase (pLDH) monoclonal antibodies used in rapid diagnostic tests (RDTs) to detect P. falciparum and P. vivax. At one tertiary-care hospital and two district hospitals in Sabah, we prospectively evaluated two combination RDTs for malaria diagnosis by using both a pan-Plasmodium-pLDH (pan-pLDH)/P. falciparum-specific-pLDH (Pf-pLDH) RDT (OptiMAL-IT) and a non-P. falciparum VOM-pLDH/Pf-HRP2 RDT (CareStart). Differential cross-reactivity among these combinations was hypothesized to differentiate P. knowlesi from other Plasmodium monoinfections. Among 323 patients with PCR-confirmed P. knowlesi (n = 193), P. falciparum (n = 93), and P. vivax (n = 37) monoinfections, the VOM-pLDH individual component had the highest sensitivity for nonsevere (35%; 95% confidence interval [CI], 27 to 43%) and severe (92%; CI, 81 to 100%) P. knowlesi malaria. CareStart demonstrated a P. knowlesi sensitivity of 42% (CI, 34 to 49%) and specificity of 74% (CI, 65 to 82%), a P. vivax sensitivity of 83% (CI, 66 to 93%) and specificity of 71% (CI, 65 to 76%), and a P. falciparum sensitivity of 97% (CI, 90 to 99%) and specificity of 99% (CI, 97 to 100%). OptiMAL-IT demonstrated a P. knowlesi sensitivity of 32% (CI, 25 to 39%) and specificity of 21% (CI, 15 to 29%), a P. vivax sensitivity of 60% (CI, 42 to 75%) and specificity of 97% (CI, 94 to 99%), and a P. falciparum sensitivity of 82% (CI, 72 to 89%) and specificity of 39% (CI, 33 to 46%). The combination of CareStart plus OptiMAL-IT for P. knowlesi using predefined criteria gave a sensitivity of 25% (CI, 19 to 32%) and specificity of 97% (CI, 92 to 99%). Combining two RDT combinations was highly specific for P. knowlesi malaria diagnosis; however, sensitivity was poor. The specificity of pLDH RDTs was decreased for P. vivax and P

  20. Measurement of lactate formation from glucose using (6- sup 3 H)- and (6- sup 14 C)glucose in humans

    SciTech Connect

    Virkamaeki, A.P.; Puhakainen, I.; Nurjhan, N.; Gerich, J.E.; Yki-Jaervinen, H. )

    1990-09-01

    To assess the validity of determining the origin of plasma lactate from the ratio of lactate and glucose specific activities (SA) during infusion of labeled glucose, normal subjects received infusions of (6-3H)- and (6-14C)glucose for 4 h after a 12 h fast, and, on another day, cold glucose labeled with both tracers during 4-6 h of hyperinsulinemia (approximately 650 microU/ml). Basally, less lactate was derived from plasma glucose when measured with (6-3H)glucose (27 +/- 2%) than with (6-14C)glucose (40 +/- 2%, P less than 0.001). Insulin did not increase the percent of lactate derived from plasma glucose when measured with (6-3H)glucose (29 +/- 2%) but did increase when measured with (6-14C)glucose (60 +/- 4%). The arterialized blood (A) (3H)lactate SA was 30-40% higher (P less than 0.01) than deep venous blood (V) (3H)lactate SA, whereas A and V (14C)lactate SA were similar. During conversion of alanine to lactate with glutamic-pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) in vitro, 32 +/- 2% of 3H in (3-3H)alanine was found in water and 68 +/- 2% in lactate. During infusion of (6-3H)- and (6-14C)glucose, the ratio of (14C)alanine to lactate SA (0.88 +/- 0.05) was less than the ratio of (3H)alanine to lactate SA (0.31 +/- 0.03, P less than 0.001). In conclusion (1) loss of 3H relative to 14C from position 6 in glucose occurs during lactate formation in extrahepatic tissues possibly due to the GPT reaction (alanine conversion to pyruvate), and (2) even under supraphysiologic hyperinsulinemic conditions not all of plasma lactate originates from plasma glucose.

  1. Inhibition of Cancer-Associated Mutant Isocitrate Dehydrogenases: Synthesis, Structure–Activity Relationship, and Selective Antitumor Activity

    PubMed Central

    2015-01-01

    Mutations of isocitrate dehydrogenase 1 (IDH1) are frequently found in certain cancers such as glioma. Different from the wild-type (WT) IDH1, the mutant enzymes catalyze the reduction of α-ketoglutaric acid to d-2-hydroxyglutaric acid (D2HG), leading to cancer initiation. Several 1-hydroxypyridin-2-one compounds were identified to be inhibitors of IDH1(R132H). A total of 61 derivatives were synthesized, and their structure–activity relationships were investigated. Potent IDH1(R132H) inhibitors were identified with Ki values as low as 140 nM, while they possess weak or no activity against WT IDH1. Activities of selected compounds against IDH1(R132C) were found to be correlated with their inhibitory activities against IDH1(R132H), as well as cellular production of D2HG, with R2 of 0.83 and 0.73, respectively. Several inhibitors were found to be permeable through the blood–brain barrier in a cell-based model assay and exhibit potent and selective activity (EC50 = 0.26–1.8 μM) against glioma cells with the IDH1 R132H mutation. PMID:25271760

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

    PubMed

    Deutch, Charles E

    2013-11-01

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

  3. Isolation of an enzyme complex with carbon monoxide dehydrogenase activity containing corrinoid and nickel from acetate-grown Methanosarcina thermophila.

    PubMed Central

    Terlesky, K C; Nelson, M J; Ferry, J G

    1986-01-01

    Fast protein liquid chromatography of cell extract from methanol- or acetate-grown Methanosarcina thermophila resolved two peaks of CO dehydrogenase activity. The activity of one of the CO dehydrogenases was sixfold greater in acetate-grown compared with methanol-grown cells. This CO dehydrogenase was purified to apparent homogeneity (70 mumol of methyl viologen reduced per min per mg of protein) and made up greater than 10% of the cellular protein of acetate-grown cells. The native enzyme (Mr 250,000) formed aggregates with an Mr of approximately 1,000,000. The enzyme contained five subunits (Mrs 89,000, 71,000, 60,000, 58,000, and 19,000), suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The UV-visible spectrum suggested the presence of iron-sulfur centers. The electron paramagnetic resonance spectrum contained g values of 2.073, 2.049, and 2.028; these features were broadened in enzyme that was purified from cells grown in the presence of medium enriched with 61Ni, indicating the involvement of this metal in the spectrum. The pattern of potassium cyanide inhibition indicated that cyanide binds at or near the CO binding site. The properties of the enzyme imply an involvement in the dissimilation of acetate to methane, possibly by cleavage of acetate or activated acetate. Images PMID:3023296

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

    PubMed

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

    2016-07-01

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

  5. Stearic acid induces proinflammatory cytokine production partly through activation of lactate-HIF1α pathway in chondrocytes.

    PubMed

    Miao, Hongming; Chen, Liang; Hao, Lijun; Zhang, Xuan; Chen, Yujuan; Ruan, Zhihua; Liang, Houjie

    2015-01-01

    The biomechanics stress and chronic inflammation in obesity are causally linked to osteoarthritis. However, the metabolic factors mediating obesity-related osteoarthritis are still obscure. Here we scanned and identified at least two elevated metabolites (stearic acid and lactate) from the plasma of diet-induced obese mice. We found that stearic acid potentiated LDH-a-dependent production of lactate, which further stabilized HIF1α protein and increased VEGF and proinflammatory cytokine expression in primary mouse chondrocytes. Treatment with LDH-a and HIF1α inhibitors notably attenuated stearic acid-or high fat diet-stimulated proinflammatory cytokine production in vitro and in vivo. Furthermore, positive correlation of plasma lactate, cartilage HIF1α and cytokine levels with the body mass index was observed in subjects with osteoarthritis. In conclusion, saturated free fatty acid induced proinflammatory cytokine production partly through activation of a novel lactate-HIF1α pathway in chondrocytes. Our findings hold promise of developing novel clinical strategies for the management of obesity-related diseases such as osteoarthritis.

  6. Influence of fat addition on the antimicrobial activity of sodium lactate, lauric arginate and methylparaben in minced meat.

    PubMed

    Magrinyà, Núria; Terjung, Nino; Loeffler, Myriam; Gibis, Monika; Bou, Ricard; Weiss, Jochen

    2015-12-23

    A minced meat model system containing three different fat levels (0, 15, and 50 wt.%) was used to evaluate the antimicrobial efficacy of three antimicrobials with different aqueous solubilities (sodium lactate>lauric arginate (Nα-lauroyl-L-arginine ethyl ester, LAE)>methylparaben). Various concentrations of sodium lactate (20, 40, and 60 mg/g), lauric arginate (0.5, 1, 1.5, 2.0, and 2.5 mg/g) and methylparaben (0.1, 0.5, 1.0, and 2.0 mg/g) were used to evaluate the antimicrobial activity against natural meat microbiota (total aerobic mesophilic colony counts, coliform bacteria, and lactic acid bacteria). The results indicate that the three antimicrobials tested are influenced at different strengths by the changes of the fat addition of the minced meat. The antimicrobial efficacy of LAE and methylparaben is increased by a higher fat content in the meat batter, whereas for lactate no clear lactate proportionality relationship can be seen. This structure sensitivity is most strongly pronounced with lauric arginate, which we attributed to the amphiphilic character of the molecule.

  7. Influence of fat addition on the antimicrobial activity of sodium lactate, lauric arginate and methylparaben in minced meat.

    PubMed

    Magrinyà, Núria; Terjung, Nino; Loeffler, Myriam; Gibis, Monika; Bou, Ricard; Weiss, Jochen

    2015-12-23

    A minced meat model system containing three different fat levels (0, 15, and 50 wt.%) was used to evaluate the antimicrobial efficacy of three antimicrobials with different aqueous solubilities (sodium lactate>lauric arginate (Nα-lauroyl-L-arginine ethyl ester, LAE)>methylparaben). Various concentrations of sodium lactate (20, 40, and 60 mg/g), lauric arginate (0.5, 1, 1.5, 2.0, and 2.5 mg/g) and methylparaben (0.1, 0.5, 1.0, and 2.0 mg/g) were used to evaluate the antimicrobial activity against natural meat microbiota (total aerobic mesophilic colony counts, coliform bacteria, and lactic acid bacteria). The results indicate that the three antimicrobials tested are influenced at different strengths by the changes of the fat addition of the minced meat. The antimicrobial efficacy of LAE and methylparaben is increased by a higher fat content in the meat batter, whereas for lactate no clear lactate proportionality relationship can be seen. This structure sensitivity is most strongly pronounced with lauric arginate, which we attributed to the amphiphilic character of the molecule. PMID:26344644

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

    PubMed Central

    2013-01-01

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

  9. Factors affecting expression of estrus measured by activity monitors and conception risk of lactating dairy cows.

    PubMed

    Madureira, A M L; Silper, B F; Burnett, T A; Polsky, L; Cruppe, L H; Veira, D M; Vasconcelos, J L M; Cerri, R L A

    2015-10-01

    The objective of this study was to determine risk-factors affecting increase in physical activity during estrus and pregnancy per artificial insemination (P/AI) in lactating dairy cows. Cows were monitored continuously by 2 automated activity monitors [a collar-mounted accelerometer (HT; Heatime, SCR Engineers, Netanya, Israel) and a leg-mounted pedometer (BO; Boumatic Heat-seeker-TX, Boumatic Dairy Equipment, Madison, WI)]. When an increase in activity was detected, body condition score (BCS) and blood samples were collected, ovaries were scanned by ultrasonography, and, if the cow was eligible for breeding, artificial insemination was performed. Milk production and health-related data were recorded throughout the experimental period. Pregnancy diagnosis was performed at 42 ± 7 d of gestation. Data were analyzed using Pearson correlation, ANOVA, and logistic regression. A total of 1,099 true events of estrus from 318 lactating Holstein cows were recorded, averaging 3.46 ± 1.1 events per cow. Positive predictive value for estrus episodes detected by the HT and BO systems were 89.6 and 85.5%, respectively. Mean peak activity at estrus (PA) recorded by the HT system was 71.6 ± 20.7 index-value, and 334.3 ± 155.7% relative increase by the BO system. Compared with primiparous, multiparous cows expressed estrus with lower PA (69.3 ± 0.8 vs. 75.9 ± 1.1 index for HT; 323.9 ± 6.0 vs. 354.8 ± 8.48% for BO) and shorter duration (DU; 10.7 ± 0.2 vs. 12.0 ± 0.3 h); DU was measured by HT only. Lower BCS was associated with decreased PA measured by both systems, estrus DU, and P/AI. Peak activity was weakly correlated with milk production on the day of artificial insemination (r = -0.20); however, when categorized into quartiles, the highest-yield cows had lower PA and DU. Follicle diameter was not correlated with PA or DU, but cows with greater concentrations of estradiol had higher PA. Cows with greater PA in both systems had greater P/AI than those with lower PA (36

  10. Factors affecting expression of estrus measured by activity monitors and conception risk of lactating dairy cows.

    PubMed

    Madureira, A M L; Silper, B F; Burnett, T A; Polsky, L; Cruppe, L H; Veira, D M; Vasconcelos, J L M; Cerri, R L A

    2015-10-01

    The objective of this study was to determine risk-factors affecting increase in physical activity during estrus and pregnancy per artificial insemination (P/AI) in lactating dairy cows. Cows were monitored continuously by 2 automated activity monitors [a collar-mounted accelerometer (HT; Heatime, SCR Engineers, Netanya, Israel) and a leg-mounted pedometer (BO; Boumatic Heat-seeker-TX, Boumatic Dairy Equipment, Madison, WI)]. When an increase in activity was detected, body condition score (BCS) and blood samples were collected, ovaries were scanned by ultrasonography, and, if the cow was eligible for breeding, artificial insemination was performed. Milk production and health-related data were recorded throughout the experimental period. Pregnancy diagnosis was performed at 42 ± 7 d of gestation. Data were analyzed using Pearson correlation, ANOVA, and logistic regression. A total of 1,099 true events of estrus from 318 lactating Holstein cows were recorded, averaging 3.46 ± 1.1 events per cow. Positive predictive value for estrus episodes detected by the HT and BO systems were 89.6 and 85.5%, respectively. Mean peak activity at estrus (PA) recorded by the HT system was 71.6 ± 20.7 index-value, and 334.3 ± 155.7% relative increase by the BO system. Compared with primiparous, multiparous cows expressed estrus with lower PA (69.3 ± 0.8 vs. 75.9 ± 1.1 index for HT; 323.9 ± 6.0 vs. 354.8 ± 8.48% for BO) and shorter duration (DU; 10.7 ± 0.2 vs. 12.0 ± 0.3 h); DU was measured by HT only. Lower BCS was associated with decreased PA measured by both systems, estrus DU, and P/AI. Peak activity was weakly correlated with milk production on the day of artificial insemination (r = -0.20); however, when categorized into quartiles, the highest-yield cows had lower PA and DU. Follicle diameter was not correlated with PA or DU, but cows with greater concentrations of estradiol had higher PA. Cows with greater PA in both systems had greater P/AI than those with lower PA (36

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

    PubMed Central

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

    2015-01-01

    Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains of Escherichia coli and report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enables E. coli to resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc, aceE. Resistance to CXCL10 also occurred following deletion of either aceF or lpdA, genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both the E. coli parent and aceE deletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine. PMID:26553462

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

    PubMed

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

    2016-01-01

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

  13. Activator Protein-1 Regulation of Murine Aldehyde Dehydrogenase 1a1

    PubMed Central

    Makia, N. L.; Amunom, I.; Falkner, K. C.; Conklin, D. J.; Surapureddi, S.; Goldstein, J. A.

    2012-01-01

    Previously we demonstrated that aldehyde dehydrogenase (ALDH) 1a1 is the major ALDH expressed in mouse liver and is an effective catalyst in metabolism of lipid aldehydes. Quantitative real-time polymerase chain reaction analysis revealed a ≈2.5- to 3-fold induction of the hepatic ALDH1A1 mRNA in mice administered either acrolein (5 mg/kg acrolein p.o.) or butylated hydroxylanisole (BHA) (0.45% in the diet) and of cytosolic NAD+-dependent ALDH activity. We observed ≈2-fold increases in ALDH1A1 mRNA levels in both Nrf2(+/+) and Nrf2(−/−) mice treated with BHA compared with controls, suggesting that BHA-induced expression is independent of nuclear factor E2-related factor 2 (Nrf2). The levels of activator protein-1 (AP-1) mRNA and protein, as well as the amount of phosphorylated c-Jun were significantly increased in mouse liver or Hepa1c1c7 cells treated with either BHA or acrolein. With use of luciferase reporters containing the 5′-flanking sequence of Aldh1a1 (−1963/+27), overexpression of c-Jun resulted in an ≈4-fold induction in luciferase activity, suggesting that c-Jun transactivates the Aldh1a1 promoter as a homodimer and not as a c-Jun/c-Fos heterodimer. Promoter deletion and mutagenesis analyses demonstrated that the AP-1 site at position −758 and possibly −1069 relative to the transcription start site was responsible for c-Jun-mediated transactivation. Electrophoretic mobility shift assay analysis with antibodies against c-Jun and c-Fos showed that c-Jun binds to the proximal AP-1 site at position −758 but not at −1069. Recruitment of c-Jun to this proximal AP-1 site by BHA was confirmed by chromatin immunoprecipitation analysis, indicating that recruitment of c-Jun to the mouse Aldh1a1 gene promoter results in increased transcription. This mode of regulation of an ALDH has not been described before. PMID:22740640

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

    SciTech Connect

    Doherty, R.E.; Haywood-Small, S.L.; Sisley, K.; Cross, N.A.

    2011-11-04

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

  15. The role of glycogen, glucose and lactate in neuronal activity during hypoxia in the hooded seal (Cystophora cristata) brain.

    PubMed

    Czech-Damal, N U; Geiseler, S J; Hoff, M L M; Schliep, R; Ramirez, J-M; Folkow, L P; Burmester, T

    2014-09-01

    The brains of diving mammals are repeatedly exposed to hypoxic conditions during diving. Brain neurons of the hooded seal (Cystophora cristata) have been shown to be more hypoxia tolerant than those of mice, but the underlying mechanisms are not clear. Here we investigated the roles of different metabolic substrates for maintenance of neuronal activity and integrity, by comparing the in vitro spontaneous neuronal activity of brain slices from layer V of the visual cortex of hooded seals with those in mice (Mus musculus). Studies were conducted by manipulating the composition of the artificial cerebrospinal fluid (aCSF), containing either 10 mM glucose, or 20 mM lactate, or no external carbohydrate supply (aglycemia). Normoxic, hypoxic and ischemic conditions were applied. The lack of glucose or the application of lactate in the aCSF containing no glucose had little effect on the neuronal activity of seal neurons in either normoxia or hypoxia, while neurons from mice survived in hypoxia only few minutes regardless of the composition of the aCSF. We propose that seal neurons have higher intrinsic energy stores. Indeed, we found about three times higher glycogen stores in the seal brain (∼4.1 ng per μg total protein in the seal cerebrum) than in the mouse brain. Notably, in aCSF containing no glucose, seal neurons can tolerate 20 mM lactate while in mouse neuronal activity vanished after few minutes even in normoxia. This can be considered as an adaptation to long dives, during which lactate accumulates in the blood. PMID:24959743

  16. The role of glycogen, glucose and lactate in neuronal activity during hypoxia in the hooded seal (Cystophora cristata) brain.

    PubMed

    Czech-Damal, N U; Geiseler, S J; Hoff, M L M; Schliep, R; Ramirez, J-M; Folkow, L P; Burmester, T

    2014-09-01

    The brains of diving mammals are repeatedly exposed to hypoxic conditions during diving. Brain neurons of the hooded seal (Cystophora cristata) have been shown to be more hypoxia tolerant than those of mice, but the underlying mechanisms are not clear. Here we investigated the roles of different metabolic substrates for maintenance of neuronal activity and integrity, by comparing the in vitro spontaneous neuronal activity of brain slices from layer V of the visual cortex of hooded seals with those in mice (Mus musculus). Studies were conducted by manipulating the composition of the artificial cerebrospinal fluid (aCSF), containing either 10 mM glucose, or 20 mM lactate, or no external carbohydrate supply (aglycemia). Normoxic, hypoxic and ischemic conditions were applied. The lack of glucose or the application of lactate in the aCSF containing no glucose had little effect on the neuronal activity of seal neurons in either normoxia or hypoxia, while neurons from mice survived in hypoxia only few minutes regardless of the composition of the aCSF. We propose that seal neurons have higher intrinsic energy stores. Indeed, we found about three times higher glycogen stores in the seal brain (∼4.1 ng per μg total protein in the seal cerebrum) than in the mouse brain. Notably, in aCSF containing no glucose, seal neurons can tolerate 20 mM lactate while in mouse neuronal activity vanished after few minutes even in normoxia. This can be considered as an adaptation to long dives, during which lactate accumulates in the blood.

  17. Direct evidence for activity-dependent glucose phosphorylation in neurons with implications for the astrocyte-to-neuron lactate shuttle.

    PubMed

    Patel, Anant B; Lai, James C K; Chowdhury, Golam M I; Hyder, Fahmeed; Rothman, Douglas L; Shulman, Robert G; Behar, Kevin L

    2014-04-01

    Previous (13)C magnetic resonance spectroscopy experiments have shown that over a wide range of neuronal activity, approximately one molecule of glucose is oxidized for every molecule of glutamate released by neurons and recycled through astrocytic glutamine. The measured kinetics were shown to agree with the stoichiometry of a hypothetical astrocyte-to-neuron lactate shuttle model, which predicted negligible functional neuronal uptake of glucose. To test this model, we measured the uptake and phosphorylation of glucose in nerve terminals isolated from rats infused with the glucose analog, 2-fluoro-2-deoxy-D-glucose (FDG) in vivo. The concentrations of phosphorylated FDG (FDG6P), normalized with respect to known neuronal metabolites, were compared in nerve terminals, homogenate, and cortex of anesthetized rats with and without bicuculline-induced seizures. The increase in FDG6P in nerve terminals agreed well with the increase in cortical neuronal glucose oxidation measured previously under the same conditions in vivo, indicating that direct uptake and oxidation of glucose in nerve terminals is substantial under resting and activated conditions. These results suggest that neuronal glucose-derived pyruvate is the major oxidative fuel for activated neurons, not lactate-derived from astrocytes, contradicting predictions of the original astrocyte-to-neuron lactate shuttle model under the range of study conditions.

  18. The role of nicotinamide–adenine dinucleotide phosphate-dependent malate dehydrogenase and isocitrate dehydrogenase in the supply of reduced nicotinamide–adenine dinucleotide phosphate for steroidogenesis in the superovulated rat ovary

    PubMed Central

    Flint, A. P. F.; Denton, R. M.

    1970-01-01

    1. Superovulated rat ovary was found to contain high activities of NADP–malate dehydrogenase and NADP–isocitrate dehydrogenase. The activity of each enzyme was approximately four times that of glucose 6-phosphate dehydrogenase and equalled or exceeded the activities reported to be present in other mammalian tissues. Fractionation of a whole tissue homogenate of superovulated rat ovary indicated that both enzymes were exclusively cytoplasmic. The tissue was also found to contain pyruvate carboxylase (exclusively mitochondrial), NAD–malate dehydrogenase and aspartate aminotransferase (both mitochondrial and cytoplasmic) and ATP–citrate lyase (exclusively cytoplasmic). 2. The kinetic properties of glucose 6-phosphate dehydrogenase, NADP–malate dehydrogenase and NADP–isocitrate dehydrogenase were determined and compared with the whole-tissue concentrations of their substrates and NADPH; NADPH is a competitive inhibitor of all three enzymes. The concentrations of glucose 6-phosphate, malate and isocitrate in incubated tissue slices were raised at least tenfold by the addition of glucose to the incubation medium, from the values below to values above the respective Km values of the dehydrogenases. Glucose doubled the tissue concentration of NADPH. 3. Steroidogenesis from acetate is stimulated by glucose in slices of superovulated rat ovary incubated in vitro. It was found that this stimulatory effect of glucose can be mimicked by malate, isocitrate, lactate and pyruvate. 4. It is concluded that NADP–malate dehydrogenase or NADP–isocitrate dehydrogenase or both may play an important role in the formation of NADPH in the superovulated rat ovary. It is suggested that the stimulatory effect of glucose on steroidogenesis from acetate results from an increased rate of NADPH formation through one or both dehydrogenases, brought about by the increases in the concentrations of malate, isocitrate or both. Possible pathways involving the two enzymes are discussed

  19. Behaviour of mesotrione in maize and soil system and its influence on soil dehydrogenase activity.

    PubMed

    Kaczynski, Piotr; Lozowicka, Bozena; Hrynko, Izabela; Wolejko, Elzbieta

    2016-11-15

    The aim of this study was to investigate the dissipation of mesotrione and effect on dehydrogenase activity (DHA) in maize and soil system. The paper for the first time describes behaviour of this herbicide applied at various doses (separately or in mixture with other herbicide) in acidic and alkaline environment. The experiments were conducted using the method randomized blocks in four repetition cycles. Chemical application in seven variants at recommended doses of herbicide were performed. The sample preparation was performed by a modified QuEChERS method and the concentrations of mesotrione in maize and soil were determined by the liquid chromatography with tandem mass spectrometry (LC-MS/MS). The limit of detection was 0.0005mgkg(-1) and quantification 0.001mgkg(-1). The dissipation of mesotrione were described according to first-order (FO) kinetics equation with R(2) were between 0.8794 and 0.9934. The initial deposit of herbicide in soil and maize was higher in an acidic environment (0.06-0.18mgkg(-1)). A positive correlation between an alkaline pH and the rate of dissipation in soil was observed. The results showed that the time after which 50% (DT50) of substance has been degraded was different for both plant and soil. DT50 for soil was within the range 3.2-6.0days and 2.9-4.4days, for the maize 3.9-4.8days and 3.4-4.5days in an alkaline and an acidic environment, respectively. Concentration of mesotrione at applicable MRL level of 0.05mgkg(-1) in maize was achieved at 0.5-5.9days and at proposed MRL of 0.01mgkg(-1) at 8.8-15.8days. The results indicate that the application of mesotrione affected on DHA in the soil. One day after application this herbicide, concentration of DHA in soil was lower than in control plots, but after 21days was observed trend of increasing DHA. PMID:27492351

  20. Liposomal encapsulation of yeast alcohol dehydrogenase with cofactor for stabilization of the enzyme structure and activity.

    PubMed

    Yoshimoto, Makoto; Sato, Mami; Yoshimoto, Noriko; Nakao, Katsumi

    2008-01-01

    Yeast alcohol dehydrogenase (YADH) with its cofactor nicotinamide adenine dinucleotide (NAD+) could be stably encapsulated in liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine). The YADH- and NAD+-containing liposomes (YADH-NADL) were 100 nm in mean diameter. The liposomal YADH and NAD+ concentrations were 2.3 mg/mL and 3.9 mM, respectively. A synergistic effect of the liposomal encapsulation and the presence of NAD+ was examined on the thermal stability of YADH at 45 and 50 degrees C. The enzyme stability of the YADH-NADL was compared to the stabilities of the liposomal YADH (YADHL) containing 3.3 mg/mL YADH without NAD+ as well as the free YADH with and without NAD+. Free YADH was increasingly deactivated during its incubation at 45 degrees C for 2 h with decrease of the enzyme concentration from 3.3 to 0.01 mg/mL because of the dissociation of tetrameric YADH into its subunits. At that temperature, the coexistence of free NAD+ at 3.9 mM improved the stability of free YADH at 2.3 mg/mL through forming their thermostable complex, although the stabilization effect of NAD+ was lowered at 50 degrees C. The turbidity measurements for the above free YADH solution with and without NAD+ revealed that the change in the enzyme tertiary structure was much more pronounced at 50 degrees C than at 45 degrees C even in the presence of NAD+. This suggests that YADH was readily deactivated in free solution due to a decrease in the inherent affinity of YADH with NAD+. On the other hand, both liposomal enzyme systems, YADH-NADL and YADHL, showed stabilities at both 45 and 50 degrees C much higher than those of the above free enzyme systems, YADH/NAD+ and YADH. These results imply that the liposome membranes stabilized the enzyme tertiary and thus quaternary structures. Furthermore, the enzyme activity of the YADH-NADL showed a stability higher than that of the YADHL with a more remarkable effect of NAD+ at 50 degrees C than at 45 degrees C. This was

  1. Temperature and enzyme activity in poikilotherms. Isocitrate dehydrogenases in rainbow-trout liver

    PubMed Central

    Moon, Thomas W.; Hochachka, P. W.

    1971-01-01

    1. The kinetics of the thermally induced enzyme variants of the supernatant NADP–isocitrate dehydrogenase from rainbow-trout liver are investigated. 2. Fish acclimatized to 2°C (cold-adapted enzyme) and 17°C (warm-adapted enzyme) show different relative distributions of the three NADP–isocitrate dehydrogenase isoenzymes; this has been demonstrated with electrophoresis and electrofocusing techniques. 3. Plots of Km versus temperature for the cold-adapted and warm-adapted enzyme variants are complex in nature with apparent maximal enzyme–substrate affinity corresponding to the temperature at which the trout is acclimatized. Both substrates, dl-isocitrate and NADP+, give similar curves although the magnitude of the Km change with temperature is much decreased in the case of NADP+. 4. Ea values of approx. 18kcal/mol were determined for both the cold-adapted and warm-adapted enzyme variants. 5. In an attempt to determine how velocities can be increased at low temperatures, cation, pH requirements, metabolite and enzyme concentrations were examined. 6. NAD–isocitrate dehydrogenase could not be detected in trout tissues. ImagesFig. 1. PMID:4399398

  2. [The effect of a water-soluble vitamins on the activity of some enzymes in diabetes].

    PubMed

    Petrov, S A; Danilova, A O; Karpov, L M

    2014-01-01

    Intramuscular injections of the vitamin complex containing: thiamine chloride (B1), riboflavin (B2), lipoic acid (N), calcium pantothenate (B5), pyridoxine hydrochloride (B6), folic acid (B9), ascorbic acid (C) can reduce the blood glucose level in serum of rats with alloxan diabetes, stabilize activity of some enzymes of energy metabolism, lactate dehydrogenase and pyruvate dehydrogenase complex. PMID:25552500

  3. Modulation of activity of Bacillus subtilis regulatory proteins GltC and TnrA by glutamate dehydrogenase.

    PubMed

    Belitsky, Boris R; Sonenshein, Abraham L

    2004-06-01

    The Bacillus subtilis gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression and is repressed by the global regulatory protein TnrA. The factor that controls TnrA activity, a complex of glutamine synthetase and a feedback inhibitor, such as glutamine, is known, but the signal for modulation of GltC activity